Citation
Development of an Extended Culture System for Bovine Blastocysts

Material Information

Title:
Development of an Extended Culture System for Bovine Blastocysts Effects of Oxygen Tension, Medium Type and Serum Supplementation on Embryo Development and Interferon-tau Secretion
Creator:
Rodina, Teresa M
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (116 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Animal Sciences
Committee Chair:
Ealy, Alan
Committee Members:
Risco, Carlos A.
Hansen, Peter J.
Graduation Date:
12/14/2007

Subjects

Subjects / Keywords:
Blastocyst ( jstor )
Cattle ( jstor )
Embryogenesis ( jstor )
Embryos ( jstor )
In vitro fertilization ( jstor )
Oxygen ( jstor )
Placenta ( jstor )
Pregnancy ( jstor )
Secretion ( jstor )
Sheep ( jstor )
Animal Sciences -- Dissertations, Academic -- UF
blastocyst, culture, interferon, vitro
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Animal Sciences thesis, M.S.

Notes

Abstract:
The bovine conceptus produces interferon-tau (IFN?), and this factor acts on the endometrium to block the pulsatile release of prostaglandin F2 alpha (PGF2?) and prevent luteolysis. Factors secreted from the uterus control IFN? gene expression in bovine and ovine conceptuses. The overall goal of this work was to develop a culture system that supports bovine embryonic development until day 11 post-in vitro fertilization (IVF) so it may be used as a model to investigate how uterine-derived factors regulate IFN? synthesis and secretion. All embryos were produced via IVF and evaluated for overall quality, percent apoptotic nuclei (not study 3 and 4), total cell number and IFN? content in the medium. In study 1, individual blastocysts were placed in one of two culture medium formulations, KSOM or M-199, containing 5% FBS and incubated in a 5 or 20% oxygen environment from day 8 to 11. Survival to day 11 post-IVF was greatest for blastocysts cultured in M-199/5% oxygen. None of the treatments affected percentage of apoptotic nuclei. IFN? concentrations were greater for embryos cultured in 20% oxygen than for those cultured in 5% oxygen regardless of medium type. In study 2, individual blastocysts were cultured in M-199 supplemented with different protein supplements at 5% or 20% oxygen from day 8 to 11 post-IVF. Blastocysts supplemented with serum substitute (ITS), 1% bovine serum albumin (BSA) or 1% fetal bovine serum (FBS) contained more degenerated embryos at day 11, fewer blastomeres, and increased percent apoptotic nuclei compared to blastocysts incubated with 2.5% and 5% FBS. IFN? was detected only in embryos incubated in 2.5 or 5% FBS. In study 3, effects of FGF-2 treatment and oxygen concentration on blastocyst cell number and IFN? secretion were evaluated. Individual blastocysts were cultured in M-199 supplemented with 2.5% FBS in 5% or 20% oxygen from day 8 to 11 and treated with or without 100 ng/mL FGF-2. Blastocysts in 5% oxygen, regardless of FGF-2 treatment, yielded embryos with greater cell numbers. Blastocysts incubated in 20% oxygen had greater IFN? secreted in culture medium than those incubated in 5% oxygen. FGF-2 treatment increased IFN? secretion in blastocysts incubated in 5% oxygen but not in blastocysts cultured in 20% oxygen. In study 4, blastocysts individually cultured in M-199 supplemented with 2.5% FBS at 5% oxygen and treated with 100 ng/mL FGF-2, 100 ng/mL GM-CSF, 100 ng/mL of both FGF-2 and GM-CSF, or no growth factor treatment (control). There were no effects of FGF-2 and GM-CSF treatment on IFN? secretion, although individual treatment comparisons showed greater IFN? secretion in FGF-2 treated blastocysts when compared to controls. GM-CSF treatment did not affect IFN? secretion in blastocysts compared to controls; however mean IFN? concentrations were slightly increased and not different from FGF-2 treatment. Co-supplementation did not increase IFN? secretion in blastocysts when compared with controls. In summary, a culture system for maintaining individual blastocyst development until day 11 post-IVF has been identified. These outcomes indicate that this culture system can be used to better understand how IFN? expression and embryo development is regulated by growth factors present in the uterine secretory milieu, such as such as FGF-2 and GM-CSF. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2007.
Local:
Adviser: Ealy, Alan.
Statement of Responsibility:
by Teresa M Rodina.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Rodina, Teresa M. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
663111997 ( OCLC )
Classification:
LD1780 2007 ( lcc )

Downloads

This item has the following downloads:

rodina_t.pdf

rodina_t_Page_028.txt

rodina_t_Page_020.txt

rodina_t_Page_106.txt

rodina_t_Page_096.txt

rodina_t_Page_062.txt

rodina_t_Page_114.txt

rodina_t_Page_095.txt

rodina_t_Page_070.txt

rodina_t_Page_043.txt

rodina_t_Page_044.txt

rodina_t_Page_104.txt

rodina_t_Page_099.txt

rodina_t_Page_037.txt

rodina_t_Page_109.txt

rodina_t_Page_041.txt

rodina_t_Page_110.txt

rodina_t_Page_011.txt

rodina_t_Page_092.txt

rodina_t_Page_068.txt

rodina_t_Page_017.txt

rodina_t_Page_088.txt

rodina_t_Page_022.txt

rodina_t_Page_055.txt

rodina_t_Page_024.txt

rodina_t_Page_084.txt

rodina_t_Page_100.txt

rodina_t_Page_074.txt

rodina_t_Page_113.txt

rodina_t_Page_067.txt

rodina_t_Page_030.txt

rodina_t_Page_040.txt

rodina_t_Page_042.txt

rodina_t_Page_075.txt

rodina_t_Page_078.txt

rodina_t_Page_021.txt

rodina_t_Page_086.txt

rodina_t_Page_061.txt

rodina_t_Page_027.txt

rodina_t_Page_059.txt

rodina_t_Page_097.txt

rodina_t_Page_063.txt

rodina_t_Page_083.txt

rodina_t_Page_093.txt

rodina_t_Page_047.txt

rodina_t_Page_112.txt

rodina_t_Page_046.txt

rodina_t_Page_054.txt

rodina_t_Page_032.txt

rodina_t_Page_085.txt

rodina_t_Page_052.txt

rodina_t_Page_107.txt

rodina_t_Page_089.txt

rodina_t_Page_050.txt

rodina_t_Page_057.txt

rodina_t_Page_058.txt

rodina_t_Page_081.txt

rodina_t_Page_077.txt

rodina_t_Page_007.txt

rodina_t_Page_094.txt

rodina_t_Page_065.txt

rodina_t_Page_010.txt

rodina_t_Page_039.txt

rodina_t_Page_018.txt

rodina_t_Page_060.txt

rodina_t_Page_016.txt

rodina_t_Page_105.txt

rodina_t_Page_098.txt

rodina_t_Page_002.txt

rodina_t_Page_053.txt

rodina_t_Page_049.txt

rodina_t_Page_066.txt

rodina_t_Page_014.txt

rodina_t_Page_038.txt

rodina_t_Page_036.txt

rodina_t_Page_076.txt

rodina_t_Page_012.txt

rodina_t_Page_087.txt

rodina_t_Page_006.txt

rodina_t_Page_013.txt

rodina_t_pdf.txt

rodina_t_Page_045.txt

rodina_t_Page_071.txt

rodina_t_Page_108.txt

rodina_t_Page_033.txt

rodina_t_Page_035.txt

rodina_t_Page_029.txt

rodina_t_Page_003.txt

rodina_t_Page_031.txt

rodina_t_Page_001.txt

rodina_t_Page_111.txt

rodina_t_Page_103.txt

rodina_t_Page_034.txt

rodina_t_Page_025.txt

rodina_t_Page_091.txt

rodina_t_Page_101.txt

rodina_t_Page_005.txt

rodina_t_Page_023.txt

rodina_t_Page_008.txt

rodina_t_Page_073.txt

rodina_t_Page_056.txt

rodina_t_Page_082.txt

rodina_t_Page_090.txt

rodina_t_Page_069.txt

rodina_t_Page_019.txt

rodina_t_Page_080.txt

rodina_t_Page_079.txt

rodina_t_Page_115.txt

rodina_t_Page_009.txt

rodina_t_Page_051.txt

rodina_t_Page_116.txt

rodina_t_Page_004.txt

rodina_t_Page_026.txt

rodina_t_Page_072.txt

rodina_t_Page_064.txt

rodina_t_Page_102.txt

rodina_t_Page_048.txt

rodina_t_Page_015.txt


Full Text





Bovine Embryo Culture

Today's bovine embryo culture systems are capable of generating blastocyst stage

embryos that can be transferred to recipients with fair pregnancy outcomes [195-199]. However,

in vitro culture systems continue to remain inferior to the maternal environment, and identifying

components provided by the maternal uterine environment that are not currently present in these

culture systems is a maj or focus surrounding research regarding in vitro embryo culture. As the

identification of more uterine-derived factors affecting embryonic development arise, our

understanding of embryo development continues to become more complete.

Early attempts of bovine embryo culture met with little success as embryos would arrest

at the 8- to 16-cell stage due to a lack of adequate support embryonic genome activation [200-

202]. Several culture systems have been devised to overcome this obstacle. Traditionally, two

types of culture systems were used: those utilizing somatic cells in the culture medium, or co-

culture systems, and those that do not depend on somatic cells or serum supplementation, or

"defined" culture systems [203]. Co-culturing embryos with somatic cells was once the preferred

method of bovine and ovine embryo culture [204]. However, the disadvantage of co-culturing

with somatic cells is that the identification of true embryotrophic compounds or other

components of the physical environment are masked by the interaction that exists between the

culture medium and somatic cells [205, 206]. This lead to the development of several defined

culture systems.

Defined In vitro Culture Requirements

There is much debate over an accurate definition of"defined" embryo culture system. A

definition given by J.G. Thompson (1996) is that a defined culture system is one implementing a

culture medium and physical environment using identifiable components prior to embryo culture.

One of the primary reasons for such debate over the definition of a "defined" culture system is









Percoll gradient [333]. COCs were fertilized with approximately 1 X 106 Spermatozoa/well.

Fertilization wells were spiked with 25 CIL of PHE (0.5 mM penicillamine, 0.25 mM

hypotaurine, and 25 CIM epinephrine) to promote fertilization and incubated at 38.50C in 5%

CO2/95% humidified air for 18-20 hours. Within each replicate, a different combination of three

bulls was used for fertilization.

After fertilization, prospective zygotes were vortexed in HEPES-TALP containing 1000

units/ml hyaluronidase (Sigma-Aldrich Co.) to remove cumulus layers. Denuded oocytes were

cultured in 50 CIL drops (n=25-3 5 zygotes/drop) of modified Potassium-Simplex-Optimized

Medium (KSOM; Caisson Laboratories) at 38.50C in 5% CO2 /5% 02/90% N2 [333]. This low

oxygen environment was provided by a tri-gas chamber. The proportion of cleaved embryos

assessed on day 3 post-in vitro fertilization (IVF) averaged 73.0 & 1.7% among studies and the

percentage of oocytes that developed to blastocysts on day 8 post-IVF was 16. 1 + 1.2%.

Individual Embryo Culture from Day 8 to 11 Post-IVF

On day 8 post-IVF, all blastocysts were removed from culture in KSOM and washed in

0.01M Phosphate buffered saline containing 1 mg/mL of Polyvinyl Pyrrolidone (PBS/PVP;

Fisher Scientific). The stage ofblastocyst development was recorded (early, expanded, hatched)

and embryos were evenly distributed across treatment groups according to the stage of blastocyst

development and placed in medium drops (30 Cll) covered in mineral oil.

Experiment 1: Determination of Whether Medium Type and Atmospheric Oxygen

Concentration Affects Embryo Development to Day 11 Post-IVF

Individual embryos were placed in 30 CIL drops of either M-199 containing 5% [v/v] fetal

bovine serum (FBS; Invitrogen Corp.), 0.011 mM gentamicin (Sigma-Aldrich Co.), non-essential

amino acids (Sigma-Aldrich Co.; 0.0001 mM of each L-alanine, L-asparagine*H20, L-aspartic










understanding the physiological events associated with bovine embryo development and IFNz

production.

The following research proj ect was conceived to develop an in vitro culture system that

extended bovine embryo culture past the initial formation of blastocysts on days 6 to 8 post-IVF

so that it could be used to improve our understanding of embryo development during this crucial

period of development. To follow is a culture scheme that was developed to sustain bovine

blastocysts in culture up to at least day 11 post-IVF. Several experiments were completed to

determine how medium formulation, atmospheric conditions, and putative embryotrophic factors

affect embryo quality, development, and IFNz production in bovine blastocysts. This work is

expected to be valuable for future research describing how blastocyst development progresses

and understanding how uterine-derived factors influence bovine concepts development.











4-17 Supplementation with FGF-2 but not GM-CSF increases IFNz production from
bovine embryos. .............. ...............80....









Future work examining embryotrophic factors FGF-2 and GM-CSF involve the transfer

of embryos treated with these growth factors into recipients to evaluate filamentous embryonic

development, placentation and pregnancy rates compared to in vivo embryos. The analysis of

growth factor treatment effects on in vivo derived embryos flushed from donors on day 8 post-

insemination and comparisons between their growth factor treated in vitro counterparts for

development and IFNz secretion will provide further insight on the research presented here.

Further interests include the effects of these growth factors at various stages of embryonic

development between in vitro and in vivo derived embryos beginning at the time of in vitro

fertilization to day 8 post-IVF, day 8-11 post-IVF and then day 11-14 post-IVF. Finally,

evaluating the effects of intrauterine infusion/inj section of these growth factors post-insemination

will bring this research full circle, completely evaluating FGF-2 and GM-CSF effects from a

strictly in vitro environment to an entirely in vivo one.

A more complete understanding of the events associated with embryonic development is

a vital component in the complex series of events surrounding the fetus and mother, which

results in either the gain or loss of a pregnancy. The work presented in this thesis is but one piece

to the puzzle and must continue so that methods for the ultimate resolution of embryonic loss and

reproductive failure may be devised.









variant may be detected locally as early as 33 days of gestation in cattle and is first detected in

peripheral circulation around day 70-100 of gestation. Its peak concentrations reach 5-30 nmol/L

from about day 265 of gestation through parturition [109-1 11]. The biological role of this

estrogen variant has yet to be discovered, however it is implicated in regulating myometrial

activity, placental maturation and softening of the birth canal [109-112].

Placentae from various species produce copious quantities of progesterone. However,

unlike the sheep and horse, where the placenta will take over as the dominant source of

progesterone at a particular stage of gestation, progesterone production by the bovine placenta is

not sufficient to maintain pregnancy. Instead, luteal-derived progesterone must also be available

throughout gestation to sustain a pregnant state [110].

One of the main protein hormones produced by the ruminant placenta is placental

lactogen (PL). PLs are produced by the binucleate cells [113-115]. This non-glycosylated,

single-chain, 23-kDa protein is structurally similar to prolactin and growth hormone. It can bind

and activate prolactin and growth hormone receptors [103, 113-120]. The onset of PL production

in the ewe occurs on day 16 of pregnancy [121, 122]. In the cow, there is a slow rise of PL

throughout pregnancy corresponding with a continuous fall in the fetal serum beginning at about

day 75 of pregnancy [123]. Throughout gestation in the cow and greater than two thirds of

gestation in the ewe, fetal PL concentrations remain consistently higher than those in maternal

blood [123, 124].

The precise functions of PL in the ruminant remains speculative, but recent work

implicates this hormone in regulating endometrial gland development. In utero biological effects

of ovine PL on endometrial differentiation and function has been demonstrated in ewes receiving

ovarian steroid replacement therapy, where intrauterine inj sections affected endometrial function










LIST OF TABLES


Table page

2-1 A comparison of medium components for M-199 and KSOM. ................ ................. .57

2-2 Medium components that are only included in M-199 ................. ......................._58










enzymes. Phospholipase A2 (PLA2) is an OTR-responsive enzyme that cleaves membrane-bound

phospholipids to generate arachidonic acid [166]. Arachidonic acid is then converted to PGH2 by

cyclooxygenase -1 and -2 (COX-1, COX-2), which are also known as prostaglandin

endoperoxide H synthases 1 and 2 (PGHS-1 and PGHS-2). PGH2 is then converted to various

other PGs by specific enzymes, including PGE and PGF synthases, which generate PGE2 and

PGF2a, TOSpectively. In the presence of progesterone, recombinant bovine IFNz can enhance the

secretion of PGE2, thereby increasing the PGE2/PGF2ae ratio [167]. A biphasic PG response to

IFNz treatment has been observed by several laboratories. Lower levels of IFNz similar to levels

found during early concepts develop at the time of pregnancy recognition suppress COX-2

expression in primary bovine endometrial cells or a bovine endometrial cell line (BEND cells)

[160, 168, 169]. Conversely, exposure to high concentrations of IFNz in these cell types,

reminiscent of levels in the uterus after maternal recognition of pregnancy, stimulates COX-2

expression [160, 169]. Collectively, IFNz appears to shift the predominate prostaglandin

production away from PGF2a and toward PGE2, thus permitting the putative luteotrophic actions

of this prostaglandin to favor CL maintenance [59, 170-172].

Other Activities of IFNz

Interferon-tau possesses the classical activities of Type I IFNs (antiviral,

immunomodulatory), and it is possible that these activities facilitate pregnancy in cattle, sheep

and other ruminants. IFNz inhibits the proliferation of cultured lymphocytes [173, 174] and

inhibits the proliferation of mitogen-stimulated or mixed population lymphocytes [173, 175-

178]. IFNz also activates natural killer cells indirectly by increasing granulocyte chemotactic

protein-2 production in the endometrium [132, 179]. Moreover, IFNz limits the expression of

maj or histocompatability complex (MHC) class I molecules in the luminal epithelium during










25



20
















1%6 FBS 2.5% FBS 5%X FBS 1% FBS 2.5%A FBS 5%L FBS
5% 02 20% 02

Figure 4-10. Effect of serum supplementation on percentage of TUNEL positive cells during
individual embryo culture from day 8 to 11 post-IVF. Embryos were cultured
individually from day 8 to 11 post-IVF in M-199 containing 1%, 2.5% or 5% FBS.
The percentage of TUNEL positive cells in each embryo was determined using a
fluorescent stain for DNA nicking followed by nuclear DNA counter-staining.
TUNEL positive and total nuclei were counted by epifluorescent microscopy. The
number of TUNEL positive cells was compared to the total cell numbers. Two
independent replicate studies were completed with a total of 6 to 12
blastocysts/treatment. Different superscripts above bars represent differences
(P<0.05). Error bars indicate mean standard error.










[232] Holm P, Walker SK, Seamark RF. Embryo viability, duration of gestation and birth
weight in sheep after transfer of in vitro matured and in vitro fertilized zygotes cultured
in vitro or in vivo. J. Reprod. Fertil. 1996; 107: 175-181.

[233] Papadopoulous S, Rizos D, Duffy P, Wade M, Quinn K, Boland MP, Lonergan P.
Embryo survival and recipient pregnancy rates after transfer of fresh or vitrified, in
vivo or in vitro produced ovine blastocysts. Anim. Reprod. Sci. 2002; 74: 35-44.

[234] Hasler JF. Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle.
Theriogenology 2001; 56: 1401-1415.

[235] Peterson AJ, Lee RSF. Improving successful pregnancies after embryo transfer.
Theriogenology 2003; 59: 687-697.

[236] Abd El Razek IM, Charpigny G, Kodja S, Marquant-Le Guienne B, Mermillod P,
Guyader-Joly C, Humblot P. Differences in lipid composition between in vivo- and in
vitro-produced bovine embryos. Theriogenology 2000; 53: 346 abstract.

[237] Duby RT, Hill JL, O'Callaghan D, Overstrom EW, Boland MP. Changes induced in the
bovine zona pellucida by ovine and bovine oviducts. Theriogenology 1997; 47: 332
ab stract.

[23 8] Pollard JW, Leibo SP. Chilling sensitivity of mammalian embryos. Theriogenology
1994; 41: 101-106.

[239] Van Soom A, Vlaenderen IV, Mahmoudzadeh AR, Deluyker H, de Kruif A.
Compaction rate of in vitro fertilized bovine embryos related to the interval from
insemination to first cleavage. Theriogenology 1992; 38: 905-919.

[240] Boni R, Tosti E, Roviello S, Dale B. Intracellular communication in in vivo- and in
vitro- produced bovine embryos. Biol. Reprod. 1999; 61: 1050-1055.

[241] Slimane W, Heyman Y, Lavergne Y, Humblot P, Renard JP. Assessing chromosomal
abnormalities in two-cell bovine in vitro fertilized embryos by using in situ
hybridization with three cloned probes. Biol. Reprod. 2000; 62: 628-635.

[242] Viuff D, Rickords L, Offenberg H, Hyttel P, Avery B, Greve T, Olsaker I, Williams JL,
Callesen H, Thomsen PD. High proportion of bovine blastocysts produced in vitro are
mixoploid. Biol. Reprod. 1999; 60: 1273-1278.

[243] Bordignon V, Morin N, Durocher J, Bousquet D, Smith LC. GnRH improves the
recovery rate and the in vitro developmental competence of oocytes obtained by
transvaginal follicular aspiration from superstimulated heifers. Theriogenology 1997;
48: -291.

[244] Greve T, Xu KP, Callesen H, Hyttel P. In vivo development of in vitro fertilized bovine
oocytes matured in vivo versus in vitro. J. In Vitro Fert. Embryo Transf. 1987; 4: 281-
285.









DEVELOPMENT OF AN EXTENDED CULTURE SYSTEM FOR BOVINE
BLASTOCYSTS: EFFECTS OF OXYGEN TENSION, MEDIUM TYPE AND SERUM
SUPPLEMENTATION ON EMBRYO DEVELOPMENT AND INTERFERON-TAU
SECRETION





















By

TERESA M. RODINA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007










improves proportion of embryos developing to the blastocyst stage [215, 228-231]. Presumably,

the results from these studies indicate that lowering the oxygen tension limits the production of

reactive oxygen species, thereby providing embryos with an environment that does not

compromise membrane integrity and basic intracellular functions.

Culture medium for early in vitro blastocyst development should be tailored to the stage

of development of the embryo. Earlier embryo development, up to day 5 post-fertilization,

requires a simple base medium containing either no serum or a low concentration of serum,

essential and non-essential amino acids (sometimes already in the base medium), pyruvate,

lactate, glutamine, and preferably no glucose. Embryo development after the late morula/early

blastocyst stage can be supported similarly by either a simple or complex medium type with

supplemented glucose. Throughout culture, it is preferable to utilize a 5% oxygen environment,

as this more closely replicates the oxygen environment an in vivo embryo is exposed to in utero.

Differences between In vivo and In vitro-Derived Embryos

Although healthy looking blastocysts are routinely obtained from in vitro maturation,

fertilization, and culture methods, these embryos remain somewhat inferior to their in vivo

derived counterparts. Most notably, pregnancy rates following transfer of in vitro-derived

embryos is lower than transfer of in vivo-derived embryos to recipients [232-23 5].

Morphologically, in vitro-derived bovine embryos contain a darker cytoplasm, greater lipid

content, and specifically more triglycerides, swollen blastomeres, and a more fragile zona

pellucidae [236-239]. Also, in vitro-derived bovine embryos have a greater incidence of

chromosomal abnormalities [236-242].

Blastocyst yield from in vitro produced embryos is affected by oocyte quality. While in

the dominant follicle, the oocyte undergoes maturation events that lead to developmental

competence for fertilization and subsequent embryo development. Several studies indicate that









One particularly well studied growth factor that plays a role in embryonic development is

insulin-like growth factor-I (IGF-I). Uterine and placental production of IGF-I occurs in several

species, including the rat [268], human [268, 269], pig [270] and sheep [271, 272]. Several

studies implicate IGF-I as an embryotrophic factor in porcine, murine, human and bovine

embryo development [273-276]. More recently IGF-I has been proposed to function primarily as

a survival factor, and decreases the rate of apoptosis during in vitro culture [277, 278]. Also,

IGF-I limits the deleterious effects of heat shock during bovine embryo culture [279, 280], and

supplementation with IGF-I increases pregnancy rates of in vitro produced bovine embryos

following transfer to recipients subj ected to environmental conditions that could induce heat

stress [281, 282].

Several other uterine-derived factors play vital roles in concepts development. In the

mouse, several factors, including transforming growth factor (TGF-a or TGFPl) platelet-

derived growth factor (PDGF), insulin-like growth factor (IGF-II), and epithelial growth factor

(EGF) promote early embryo development in vitro [283-285]. Transcripts for TGF-P2 and

insulin-like growth factor (IGF-II) and the receptors for PDGF and IGF-II are detectable

throughout preimplantation development in the bovine, indicating that these mRNAs are

products of both the maternal and the embryonic genome origin in the cow. Platelet derived

growth factor (PDGF) is produced in myometrial cells and promotes bovine embryo

development beyond the 16-cell stage when treated for 9 days after 24 hours post-fertilization,

although it does not increase blastocyst rate [286]. In that same study, TGFu was, in fact, able to

increase the percentage of embryos developing to blastocysts where PDGF could not.









from normal in vivo embryo development when embryos attached down to collagen gel matrix

and initiated (spread) putative trophectoderm cell growth [9].

Using agarose tunnels to promote elongation is an intriguing concept for extended

blastocyst culture. This method was first reported by Stringfellow and Thompson [265] and has

been investigated more recently by others [13, 266]. These agar gel tunnels are created with 3%

(w/v) agarose suspended in Dulbecco PB S with Ca2+ and Mg2+, pOured into a frame for vertical

gels with a comb containing glass capillaries. Once the agarose is set, the comb of capillaries is

removed and cut into 3 pieces, each containing four or five tunnels approximately 12-14 mm in

length and 1 mm in diameter. Using this agarose scaffold, 52% of blastocysts elongated to a

minimum of 1.6 mm by day 12 post-IVF. These embryos completely filled the space between the

tunnel walls, while blastocysts cultured without an agarose scaffold averaged 1.3mm in diameter

and possessed no signs of elongation. By day 14 post-IVF, 24% of the blastocysts remained

viable and continued to grow within the tunnels, reaching an average of 4.3 mm in length.

However, by day 16 post-IVF, only 2 embryos continued to grow in the tunnels, one reached 12

mm in length and the other 8 mm in length. In summary, this system successfully induced

elongation but few embryos remained viable by days 14 and 16.

Another extended culture system utilizing agarose gel tunnels that achieved success was

developed by Brandio and colleagues [12]. This group developed the Post Hatching

Development (PHD) system. In this system, a Petri dish was set up with two glass capillary

combs were placed in opposite directions from one another with the attached ends resting on the

top edge of the dish and the closed ends on the bottom of the dish. The Petri dish was filled with

2.4% agarose in PB S, supplemented with either 4.5% or 9% FBS. When the gel was set, 2 mL of

PHD medium (SOFaaci with 0.5% glucose and 10% FB S) was poured over the surface and the









CHAPTER 3
MATERIALS AND METHODS

In vitro Embryo Production

In vitro maturation, fertilization and culture procedures [333-335] were used to generate

the bovine embryos used in these studies. Bovine ovaries from dairy and beef breeds were

collected from Central Beef Packing Co. (Center Hill, FL) and stored in sterile saline (or 0.9%

[w/v] NaC1) with 100 units/mL penicillin and 100 Clg/mL streptomycin (Chemicon International)

at room temperature during transport. Ovaries were rinsed in warmed (380C) sterile saline

containing 100 units/mL penicillin and 100 Clg/mL streptomycin to remove blood and debris.

Follicles (2-10 mm) were slashed with a scalpel blade and rinsed in a beaker filled with 75 mL of

Oocyte Collection Medium [Tissue Culture Medium-199 (M-199) with Hank' s salts, without

phenol red; (Hyclone), 2% (v/v) bovine steer serum (BSS; Pel-Freez) containing 2 units/mL

heparin, 100 units/mL penicillin, 0.1 Clg/mL streptomycin, 1 mM glutamine] to liberate and

collect Cumulus Oocyte Complexes (COCs) containing one or more complete layers of compact

cumulus cells. COCs were transferred in groups of 10 to 50 CIL drops of Oocyte Maturation

Medium [OMM; M-199 with Earle's salts, 10% (v/v) BSS, 2 Clg/mL estradiol 17-P, 20 Clg/mL

bovine FSH (Folltropin-V; Bioniche), 22 Clg/mL sodium pyruvate, 50 Clg/mL gentamicin sulfate,

1 mM glutamine] equilibrated to 38.50C in 5% CO2 and 95% humidified air and covered with

mineral oil. COCs were incubated for 22-24 hours at 38.50C in 5% CO2 and 95% humidified air.

Following maturation, COCs were washed in 4-(2-hydroxyethyl)- 1-

piperazineethanesulfonic acid -Tyrode's Albumin Lactate Pyruvate solution (HEPES-TALP) and

placed into 600 CIL of In vitro Fertilization-Tyrode's Albumin Lactate Pyruvate solution (IVF-

TALP) (Cell and Molecular Technologies). Frozen and thawed sperm from three different

Holstein bulls (Genex Cooperative, Inc) were pooled and viable sperm were separated with a











70



60








10


20- a 1I


1% FBS 2.5%X FBS 5% FBS 1% FBS 2.5%A FBS 5%b FBS

5% 02 20% Oz
Figure 4-12. Serum supplementation and atmospheric oxygen conditions act independently to
mediate IFNz production. Blastocysts were cultured individually in M-199
supplemented with 1%, 2.5% or 5% FBS in either 5% or 20% oxygen from day 8 to
11 post-IVF. Concentration of bioactive IFNz in conditioned medium at day 11 post-
IVF was normalized by cell number. Two independent replicate studies were
completed with a total of 6 to 12 blastocysts/treatment. Different superscripts above
bars represent differences (P<0.05). Error bars indicate mean standard error.










[64] Tedeschi C, Hazum E, Kokia E, Ricciarelli E, Adashi EY, Payne DW. Endothelin-1
and a luteinization inhibitor: Inhibition of rat granulosa cell progesterone accumulation
via selective modulation of key steroidogenic steps affecting both progesterone
formation and degradation. Endocrinology 1992; 131: 2476-2478.

[65] Tedeschi C, Loham C, Hazum E, Ittop O, Ben-shlomo I, Resnick CE, Payne DW,
Adashi EY. Rat ovarian granulosa cell as a site of ET-1 reception and action:
attenuation of gonadotropin-stimulated steroidogenesis via perturbation of the A-kinase
signaling pathway. Biol. Reprod. 1994; 51: 1058-1065.

[66] Iwai M, Hasegawa M, Taii S, Sagawa M, Nakao K, Imura H, Nakanishi S, Mori T.
Endothelin inhibits luteinization of cultured porcine granulosa cells. Endocrinology
1991; 129: 1909-1914.

[67] Girsh E, Milvae RA, Wang W, Mcidan R. Effect of endothelin-1 on bovine luteal cell
function: role in prostaglandin F2alpha-induced antisteroidogenic action. Endocrinology
1996; 137: 1306-1312.

[68] Girsh E, Wang W, Arditi F, Friedman A, Milvae RA, Meidan R. Regulations of
endothelin-1 expression in the bovine corpus luteum: elevation by prostaglandin
F2alpha. Endocrinology 1996; 137: 5191-5196.

[69] Miyamoto A, Kobayashi S, Arata S, Ohtani M, Fukui Y, Schams D. Prostaglandin
F2alpha promotes the inhibitory action of endothelin-1 on the bovine luteal function in
vitro. J. of Endocrinol. 1997; 152: R7-R1 1.

[70] Ji I, Slaughter RG, Ellis JA, Ji TH, Murdoch WJ. Analysis of ovine corpora lutea for
tumor necrosis factor mRNA and bioactivity during prostaglandin-induced luteolysis.
Mol. Cell. Endocrinol. 1991; 81: 77-80.

[71] Shaw DW, Britt JH. Concentrations of tumor necrosis factor alpha and progesterone
within the bovine corpus luteum sampled by continuous-flow microdialysis during
luteolysis in vitro. Biol. Reprod. 1995; 53: 847-854.

[72] Juengel JL, Garverick HA, Johnson AL, Young-Quist RS, Smith MF. Apoptosis during
luteal regression in cattle. Endocrinology 1993; 132: 249-254.

[73] Sawyer HR, Niswender KD, Niswender GD. Nuclear changes in ovine luteal cells in
response to PGF2alpha. Domest. Anim. Endocrinol. 1990; 7: 229-238.

[74] Zheng J, Fricke PM, Reynolds LP, Redmer DA. Evaluation of growth, cell
proliferation, and cell death in bovine corpora lutea throughout the estrous cycle. 51
1994; 632.

[75] Hasumoto K, Sugimoto Y, Yamasaki A, Morimoto K, Negishi M, Ichikawa A.
Association of expression of mRNA encoding PGF2alpha receptor with luteal
apoptosis in ovaries of pseudopregnant mice. J. Reprod. Fertil. 1997; 109: 45-51.










[257] Thompson JG, Allen NW, McGowan LT, Bell ACS, Lambert MG, Tervit HR. Effect of
delayed supplementation of fetal calf serum to culture medium on bovine embryo
development in vitro and following transfer. Theriogenology 1998; 49: 1239-1249.

[258] Van Langendonckt A, Donnay I, Schuurbiers N, Auquier P, Carolan C, Massip A,
Dessy F. Effects of supplementation with fetal calf serum on development of bovine
embryos in synthetic oviduct fluid medium. J. Reprod. Fertil. 1997; 109: 87-93.

[259] Walker SK, Hartwich KM, Seamark RF. The production of unusually large offspring
following embryo manipulation: Concepts and challenges. Theriogenology 1996; 45:
111-120.

[260] Wrenzycki C, Herrmann D, Neimann H. Expression of the gap junction gene
connexin43 (C X 43) in preimplantation bovine embryos derived in vitro or in vivo. J.
Reprod. Fertil. 1996; 108: 17-24.

[261] Lonergan P, Fair T, Corcoran D, Evans ACO. Effect of culture environment on gene
expression and developmental chatactersitics in IVF-derived embryos. Theriogenology
2006; 65: 137-152.

[262] Corcoran D, Fair T, Park S, Rizos D, Patel OV, Smith GV, Coussens PM, Ireland JJ,
Boland MP, Evans ACO, Lonergan P. Suppressed expression of genes involved in
transcription and translation in in vitro compared to in vivo cultured bovine embryos.
Reproduction 2006; 131: 651-660.

[263] Alexopoulos NI, Maddox-Hyttel P, Vajta G. Effect of protein supplementation on
establishment of a hypoblast layer in IVP bovine embryos. Theriogenology 2002; 57:
213.

[264] Brackett BG, Zuelke KA. Analysis of factors involved in the in vitro production of
bovine embryos. Theriogenology 1993; 39: 43-64.

[265] Stringfellow DA, Thompson MS. Maintenance and development of bovine embryos in
vitro. Alabama Agricultural Experiment Station: Highlights of Agricultural Research
1986; 33: 11.

[266] Vajta G, Hyttel P, Trounson AO. Post-hatching development of in vitro produced
bovine embryos on agar and collagen gels. Anim Reprod. Sci. 2000; 60-61.

[267] Gray CA, Bartol FF, Tarleton BJ, Wiley AA, Johnson GA, Bazer FW, Spencer TE.
Dev. Biol. of uterine glands. Biol. Reprod. 2001; 65: 13 11-1323.

[268] Zhou J, Bondy C. Insulin-like growth factor-II and its binding proteins in placental
development. Endocrinology 1992; 131: 1230-1240.

[269] Han VKM, Bassett N, Walton J, Challis JRG. The expression of Insulin-like growth
factor (IGF) and IGF-binding protein (IGFBP) genes in the human placenta and









membranes: evidence for IGF-IGFBP interactions at the feto-maternal interface. J. Clin.
Endocrinol. Metab 1996; 81: 2680-2693.

[270] Simmen RCM, Ko Y, Simmen FA. Insulin-like growths factors and blastocyst
development. Theriogenology 1993; 39: 163-175.

[271] Reynolds TS, Stevenson KR, Wathes DC. Pregnancy-specific alterations in the
expression of the insulin-like growth factor system during early placental development
in the ewe. Endocrinology 1997; 138: 886-897.

[272] Stevenson KR, Gilmour RS, Wathes DC. Localization of insulin-like growth factor-I
and -II messenger ribonucleic acid and type 1 IGF receptors in the ovine uterus during
the estrous cycle and early pregnancy. Endocrinology 1994; 134: 1655-1664.

[273] Herrler A, Krusche CA, Beier HM. Insulin and insulin-like growth factor-1 promote
rabbit blastocyst development and prevent apoptosis. Biol. Reprod. 1998; 59: 1302-
1310.

[274] Matsui M, Takahashi Y, Hishinuma M, Kanagawa H. Insulin and insulin-like growth
factor-I (IGF-I) stimulate the development of bovine embryos fertilized in vitro. J. Vet.
Med. Sci. 1995; 57: 1109-1111.

[275] Mihalik M, Rehak P, Koppel J. The influence of insulin on the in vitro development of
mouse and bovine embryos. Physiol. Res. 2000; 49: 347-354.

[276] Xia P, Tekpetey FR, Armstrong DT. Effect of IGF-I on pig oocyte maturation,
fertilization, and early embryonic development in vitro, and on granulosa and cumulus
cell biosynthetic activity. Mol. Reprod. Dev. 1994; 38: 373-379.

[277] Makarevich AV, Markkula M. Apoptosis and cell proliferation potential of bovine
embryos stimulated with insulin-like growth factor I during in vitro maturation and
culture. Biol. Reprod. 2002; 66: 386-392.

[278] Spanos S, Becker DL, Winston RML, Hardy R. Anti-apoptotic action of insulin-like
growth factor-I during human preimplantation embryo development. Biol. Reprod.
2000; 63: 1413-1420.

[279] Jousan FD, Hansen PJ. Anti-apoptotic and growth-promoting actions of insulin-like
growth factor-1 in bovine preimplantation embryos are mediated through distinct
signaling pathways. [38]. 2005. The Society for the Study of Reproduction.

[280] Jousan FD, Hansen PJ. Insulin-like growth factor-I as a survival factor for bovine
embryos subjected to heat shock. [38]. 2005. The Society for the Study of
Reproduction.

[281] Block J, Hansen PJ. Interaction between season and culture with insulin-like growth
factor-1 on survival of in vitro produced embryos following transfer to lactating dairy
cows. Theriogenology 2007; 67: 1518-1529.












800J -

700 -
0 -

600 -

500 -

400 -
MO-

ZK-


O -C-


5% FBSI 1%FBS


1% FB5 2.5%A FBS

5%~ 02


2.5%C FBS 5%A FBS

20%6 02


Figure 4-9. Supplementation with FB S improves embryonic cell number after incubation in 5%
oxygen. Embryos were cultured in M-199 supplemented with 1%, 2.5% or 5% FBS
and incubated in 5% or 20% oxygen from day 8 to 11 post-IVF. Number of cells at
day 11 post-IVF was counted by epifluorescent microscopy after staining nuclear
DNA (Hoescht 33342 staining). Two independent replicate studies were completed
with a total of 6 to 12 blastocysts/treatment. Different superscripts above bars
represent differences (P<0.05). Error bars indicate mean standard error.










[133] Fuchs AR, Helmer H, Behrens O, Liu H-C, Antonian L, Chang SM, Fields MJ.
Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors
precedes onset of labor. Biol. Reprod. 1992; 47: 937-944.

[134] Moor RM, Rowson LE. The corpus luteum of the sheep: effect of the removal of
embryos on luteal function. J. Endocrinol. 1966; 34: 497-502.

[135] Moor RM, Rowson LE. The corpus luteum of the sheep: functional relationship
between the embryo and the corpus luteum. J. Endocrinol. 1966; 34: 233-239.

[136] Moor RM, Rowson LE, Hay MF, Caldwell BV. The corpus luteum of the sheep: effect
of the concepts on luteal function at several stages during pregnancy. J. Endocrinol.
1969; 43: 301-307.

[137] Rowson LE, Moor RM, Lawson RA. Fertility following egg transfer in the cow; effect
of method, medium and synchronization of oestrus. J. Reprod. Fertil. 1969; 18: 517-
523.

[138] Rowson LE, Lawson RA, Moor RM, Baker AA. Egg transfer in the cow:
synchronization requirements. J. Reprod. Fertil. 1972; 28: 427-431.

[139] Martal J, Lacroix MC, Loudes C, Saunier M, Wintenberger-Torres S. Trophoblastin, an
antiluteolytic protein present in early pregnancy in sheep. J. Reprod. Fertil. 1979; 56:
63-73.

[140] Godkin JD, Bazer FW, Thatcher WW, Roberts RM. Proteins released by cultured Day
15-16 conceptuses prolong luteal maintenance when introduced into the uterine lumen
of cyclic ewes. J. Reprod. Fertil. 1984; 71: 57-64.

[141] Knickerbocker JJ, Thatcher WW, Bazer FW, Drost M, Barron DH, Fincher KB,
Roberts RM. Proteins secreted by day-16 to -18 bovine conceptuses extend corpus
luteum function in cows. J. Reprod. Fertil. 1986; 77: 381-391.

[142] Imakawa K, Anthony RV, Kazemi M, Marotti KR, Polites HG, Roberts RM.
Interferon-like sequence of ovine trophoblast protein secreted by embryonic
trophectoderm. Nature 1987; 330: 377-379.

[143] Cross JC, Roberts RM. Constitutive and trophoblast-specific expression of a class of
bovine interferon genes. Proc. Natl. Acad. Sci. U. S. A 1991; 88: 3817-3821.

[144] Leaman DW, Cross JC, Roberts RM. Multiple regulatory elements are required to
direct trophoblast interferon gene expression in choriocarcinoma cells and
trophectoderm. Mol. Endocrinol. 1994; 8: 456-468.

[145] Farin CE, Imakawa K, Hansen TR, McDonnell JJ, Murphy CN, Farin PW, Roberts
RM. Expression of trophoblastic interferon genes in sheep and cattle. Biol. Reprod.
1990; 43: 210-218.









Over the next several days, the bovine concepts remains free floating in the uterine

lumen as embryonic and extraembryonic development continues [87]. Between days 8 and 10 of

development, endoderm emerges from the inner cell mass and lines blastocoel cavity underneath

the trophectoderm. This tissue eventually will form the inner border of the yolk sac. Between

days 12 to 14 of development, the outer cells of the inner cell mass begin to polarize and

differentiate into embryonic ectoderm. At this point the inner cell mass appears to be composed

of multiple cell layers [87]. Between days 14 to 16 of development, the mesoderm layer migrates

from the inner cell mass between the trophectoderm and endoderm to separate the yolk and

amniotic sacs, as well as forming the inner border of the chorion and eventually the umbilical

cord [93].

Approximately the same time that embryonic cell lineages are emerging (days 12-16), the

concepts begins to elongate first into an ovoid shape and soon thereafter into tubular and finally

a filamentous form [93]. After 17-18 days of development, the embryo may occupy two-thirds of

the uterine horn ipsilateral to the ovary bearing the ovulation and may be greater than 160 mm in

length [94]. Within 1-3 days later (day 18-20), the embryo usually occupies the entire gravid

uterine horn and encroaches into the contralateral horn. At this stage of the development the

amnion forms through encroachment of mesoderm and inversion of the trophectoderm. Finally, a

heartbeat is detectable between 20-22 days of development [95].

Implantation and Placental Development

Implantation is best described as a transitional stage during pregnancy where the

concepts assumes a fixed position on the endometrium and begins its intimate physiological

relationship with the uterus [96]. The extent to which placental invasion into the uterus is

dictated by the type of placenta utilized by that particular species. In the bovine and other

ruminants, a synepitheliochorial placenta forms between mother and fetus. The placental cells









CHAPTER 5
DISCUSSION

Reproductive failure is a considerable issue in the beef and dairy industries, with early

embryonic mortality being responsible for a substantial proportion of reproductive

insufficiencies. A more articulate understanding of the series of events of embryonic

development may lead to possible resolutions to these issues. The most valuable method of

studying embryonic development is through in vitro production and culture of embryos.

Conventional bovine embryo culture systems are not designed to adequately support blastocyst

development. This is because the full extent of components provided by the natural uterine

environment has yet to be defined, which proves to be problematic in developing embryo culture

systems, particularly during post-hatching development. The work presented here establishes that

blastocysts can be sustained in culture until at least day 11 post-IVF with the proper medium

formulation, by supplementing medium with serum, and by exposing embryos to a low oxygen

environment.

The medium formulation used in these experiments had a maj or impact on blastocyst

viability and development between days 8 and 11 post-IVF. A majority of the components in the

modified KSOM (with non-essential amino acids) and M-199 were fairly well balanced except

for their glucose levels (5.5 mM for M-199; 0.2 mM for KSOM). Glucose can be toxic to bovine

embryos, and especially female embryos, during initial cleavage stages [336-338]. As embryos

become more advanced, rate of glycolysis and glucose uptake increases [336, 337], and medium

containing glucose is required for propagating bovine embryos during and beyond the blastocyst

stage [6, 7, 10, 12, 13]. However, since glucose withdrawal and/or replacement studies were not

completed during the course of this work, it remains possible that minor differences between

media formulations also contributed to the superiority of M-199 as a blastocyst culture medium.









Insufficient quantities of the maternal recognition of pregnancy factor, interferon-tau

(IFNz), also are observed in ewes containing little to no glandular epithelium [4, 5]. Several

intrinsic and extrinsic factors affect IFNz expression during early pregnancy [15, 16], but length

of the developing concepts is the predominant determinant of overall IFNz production [17-19].

Therefore, it is imperative that the physiological events associated with early concepts

development and IFNz production be fully understood so that resolutions for pregnancy failures

can be conceived.

We examine several of the basic reproductive processes surrounding early embryonic and

concepts development in cattle and provide an update on our current knowledge of key

biological processes that influence fecundity. The literature review will begin with a brief

overview of the estrous cycle in cattle, followed by an overview of pregnancy where a

description of embryonic and concepts development is presented. Also, an overview of IFN O

expression and action will be provided. A brief review of past and present bovine embryo culture

systems and their similarity with in vivo derived embryos will be reviewed. The literature review

will end by discussing some uterine-derived factors that impact embryonic development. The

research focus of this thesis then will be presented. The overall goal of the research was to

develop a culture system that can be used to examine bovine blastocyst development. Through a

series of studies, a suitable system for maintaining blastocyst viability was created, and its

efficacy for use in examining how uterine-derived factors affect embryo development and IFNz

production was tested.










embryonic cells is the first in many differentiation events that the embryo proper and

extraembryonic membranes go through to generate a viable fetus and placenta.

It takes approximately 96 hours for the bovine embryo to travel through the oviduct and

into the uterine body. Normally, bovine embryos have reached the 16-cell stage in development,

which equates to developing through four cell divisions. After this point, subsequent mitosis

generates a mass of cells, termed a morula. Blastomeres will continue to undergo division and

will begin to compact. During compaction, tight junctions form between blastomeres lining the

outside of the embryo. Soon thereafter, the first signs of blastocoele formation become evident as

fluid begins to build up in the center of the morula [87, 88]. The timing of blastocoele formation

varies among individual embryos and is dependent on the rate of fluid entry into the newly

formed cavity and the extent of tight junction formation between blastomeres [87, 89, 90].

During the compaction phase of morula development, two cells types may be distinguished from

one another [88]. A population of flattened, outer cells will form the trophectoderm. This tissue

will give rise to outer layer of the placenta. The remaining inner cells will gather at one pole of

the embryo and form the inner cell mass, which also is termed the embryonic disk, or epiblast

[87]. These cells eventually will differentiate into various fetal and extraembryonic tissues.

By approximately day 9-10 of development, a small slit forms in the zona pellucida,

allowing the blastocyst to separate from the zona pellucida in a process termed "hatching" [91,

92]. Once hatched, the inner cell mass will bulge to the outside of the sphere-shaped blastocyst

and is clearly discernible as the embryonic disk, which is still covered by trophectoderm cells,

termed the Rauber' s layer. As hatched blastocyst development progresses, trophectoderm cells

within the Rauber's layer will be shed.








































Figure 4-1. Examples of degenerated embryos by day 11 post-IVF. Degeneration on day 11 of
development was determined by visual assessment of embryo integrity (see methods
section for detail). Panel A and B depict degenerated embryos stained with Hoescht
33342 nuclear stain. Overall visual appearance is poor with inner cellular contents
collapsed or absent, with presence of degenerated cellular debris and in some cases
the zona pellucida has ruptured.










follicular waves during the estrous cycle of the cow. J. of Reprod. and Dev. 1995; 41:
311-320.

[26] Fortune JE. Ovarian follicular growth and development if mammals. Biol. Reprod.
1994; 50: 225-232.

[27] Badinga L, Driancourt MA, Savio JD, Wolfenson D, Drost M, De La Sota RL,
Thatcher WW. Endocrine and ovarian responses associated with the first-wave
dominant follicle. Biol. Reprod. 1992; 47: 871-883.

[28] Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Selection of the dominant
follicle in cattle. Biol. Reprod. 1996; 55: 1187-1194.

[29] Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles
during the estrous cycle of heifers. J. Reprod. Fertil. 1988; 83: 663-671.

[30] Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous cycle in heifers
monitored by real-time ultrasonography. Biol. Reprod. 1988; 39: 308-317.

[31] Quirk SM, Cowman RG, Harman R.M., Hu C-L, Porter DA. Ovarian follicular growth
and atresia: The relationship between cell proliferation and survival. J. of Ani. Sci.
2007; 82: E40-E52.

[32] Webb R, Garnsworthy PC, Gong J-G, Armstrong DG. Control of follicular growth:
Local interactions and nutritional influences. J. of Ani. Sci. 2004; 82: E63-E74.

[33] Espey LL. Ovulation as an inflammatory reaction A hypothesis. Biol. Reprod. 1980;
22: 73-106.

[34] Fields MJ, Fields PA. Morphological characteristics of the bovine corpus luteum during
the estrous cycle and pregnancy. Theriogenology 1996; 45: 1295-1325.

[35] Barnes MA, Bierley ST, Halman RD, Henricks DM. Follicle stimulating hormone,
luteinizing hormone and estradiol-17beta response in GnRH treated prepubertal
Holstein heifers. Biol. Reprod. 1980; 22: 459-465.

[36] Kaltenbach CC, Dunn TG, Kiser TE, Corah LR, Akbar AM, Niswender GD. Release of
FSH and LH in beef heifers by synthetic gonadotropin releasing hormone. J. of Ani.
Sci. 1974; 38: 357-362.

[37] McCracken JA, Custer EE, Lamsa JC. Luteolysis: A neuroendocrine-mediated event.
Physiol Rev. 1999; 79: 263-323.

[38] Niswender GD, Juengel JL, McGuire WJ, Belflore CJ, Wiltbank MC. Luteal function:
the estrous cycle and early pregnancy. Biol. Reprod. 1994; 50: 239-247.










[76] Friedman A, Weiss S, Levy N, Meidan R. Role of tumor necrosis factor alpha and its
type I receptor in luteal regression: Induction of programmed cell death in bovine
corpus luteum-derived endothelial cells. Biol. Reprod. 2000; 63: 1905-1912.

[77] Evans JP, Floyd JG. The state of the union: the cell biology of fertilization. Nat. Cell.
Biol. 2002; 4: s57-s63.

[78] Suarez SS, Pacey AA. Sperm transport in the female reproductive tract. Hum. Reprod.
Update 2006; 12: 23-27.

[79] Florman HM, First NL. The regulation of acrosomal exocytosis. I. Sperm capacitation
is required for the induction of acrosome reactions by the bovine zona pellucidae in
vitro. Dev. Biol. 1988; 128: 453-463.

[80] Handrow R., Boehm SK, Lenz RW, Robinson JA, Ax RL. Specific binding of the
glycosaminoglycan 3H-heparin to bull, monkey, and rabbit spermatozoa in vitro. J.
Androl. 1984; 5: 51-63.

[81] Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by
heparin. Biol. Reprod. 1988; 35: 608-617.

[82] Parrish JJ, Susko-Parrish JL, Handrow R.H., Sims MM, First NL. Capacitation of
bovine spermatozoa by oviduct fluid. Biol. Reprod. 1989; 40: 1020-1025.

[83] Florman HM, Wassarman PM. O-linked oligosaccharides of mouse egg ZP3 account
for its sperm receptor activity. Cell 1985; 41: 313-324.

[84] Wassarman PM, Litscher ES. Towards the molecular basis of sperm and egg interaction
during mammalian fertilization. Cells Tissues Organs 2001; 168: 36-45.

[85] Schultz RM, Kopf GS. Molecular basis of of mammalian egg activation. Curr. Top.
Dev. Biol. 1995; 30: 21-62.

[86] Rossant J. Postimplantation development of blastomeres isolated from 4- and 8-cell
mouse eggs. J. of Embryol. Exp. Morphol. 1976; 36: 283-290.

[87] Betteridge KJ, Flechon J-E. The anatomy and physiology of pre-attachment bovine
embryos. Theriogenology 1988; 29: 155-187.

[88] Van Soom A, Boerj an M, Ysebaert M-T, de Kruif A. Cell allocation to the inner cell
mass and the trophectoderm in bovine embryos cultured in two different media. Mol.
Reprod. Dev. 1996; 45: 171-182.

[89] Garbutt CL, Chisholm JC, Johnson MH. The establishment of the embryonic-
abembryonic axis in the mouse embryo. Development 1987; 100: 125-134.

[90] Kidder GM, McLachlin JR. Timing of transcription and protein synthesis underlying
morphogenesis in preimplantation mouse embryos. Dev. Biol. 1985; 112: 265-275.










"700

600-b

m 500

-400 -1 a a



E 00-

00-

10

5%6 02 20%6 02 5%6 02 20% 02
KSOM M-199
Figure 4-4. Incubation in M-199 under 5% oxygen conditions yields greater total cell numbers on
day 11 post-IVF. Embryos were cultured individually in drops of KSOM or M-199
and 5% or 20% oxygen from day 8 to 11 post-IVF. Number of embryonic cells was
determined at day 11 post-IVF was determined by staining nuclear DNA (Hoescht
33342) and counting nuclei using epifluorescent microscopy. Four independent
replicate studies were completed with a total n=20 to 37 blastocysts/treatment.
Different superscripts above bars represent differences (P<0.05). Error bars indicate
mean standard error.










Table 2-2. Medium components that are only included in M-199.


MOLARITY
(mM)
0.000568
0.108
0.0763

0.153


0.0000142
0.000284
0.000041
0.00357
0.000021
0.0000227
0.000278
0.0000581
0.000205
0.000203
0.000365
0.000123
0.000121
0.0000266
0.0000297
0.000305
0.000252


MOLARITY
(mM)
0.00373
0.0248
0.000576

0.00165

0.000517
0.000163
0.0016
0.00294
0.0531
0.00333
0.61
0.00238
20 mg/ml
0.00268
0.00224


AMINO ACIDS


OTHER COMPONENTS

2-deoxy-D-ribose
Adenine sulfate
Adenosine 5'-phosphate

Adenosine 5'-triphosphate
Cholesterol
Glutathione (reduced)
Guanine hydrochloride
Hypoxanthine Na
Phenol red
Ribose
Sodium acetate
Thymine
Tween 80@
Uracil
Xanthine-Na


L-Cysteine hydrochloride-H20
L-Cystine 2HCI
L-Hydroxyproline
L-Tyrosine disodium salt
dihydrate
VITAMINS
Alpha-tocopherol phosphate
Ascorbic Acid
Biotin
Choline chloride
D-Calcium pantothenate
Folic Acid
i-Inositol
Menadione (Vitamin K3)
Niacinamide
Nicotinic acid (Niacin)
Para-Aminobenzoic Acid
Pyridoxal hydrochloride
Pyridoxine hydrochloride
Riboflavin
Thiamine hydrochloride
Vitamin A (acetate)
Vitamin D2 (CalCiferOl)
INORGANIC SALTS
Calcium chloride (CaCl2)
(anhyd.)
Ferric nitrate (Fe(NO3)-9H20)
Magnesium sulfate (MgSO4
(anhyd.)
Sodium phosphate monobasic
(NaH2PO4-H20)


1.8

0.00173

0.397

1.01










[106] Wooding FB, Morgan G, Adam CL. Structure and function in the ruminant
synepitheliochorial placenta: central role of the trophoblast binucleate cell in deer.
Microsc. Res. Tech. 1997; 38: 88-99.

[107] Lang CY, Hallack S, Leiser R, Pfarrer C. Cytoskeletal filaments and associated proteins
during resricted trophoblast invasion in bovine placentomes: light and transmission
electron microscopy and RT-PCR. Cell Tissue Res. 2004; 315: 339-348.

[108] Pfarrer C, Wirth C, Schuler G, Klisch K, Leiser R, Hoffman B. Frequency,
ultrastructural features, and relevance of apoptosis in the bovine placenta. Biol. Reprod.
1999; 60: 126.

[109] Eley RM, Thatcher WW, Bazer FW. Hormonal and physical changes associated with
concepts development. J. Reprod. Fertil. 1979; 55: 181-190.

[110] Hoffman B, Schuler G. The bovine placenta; a source and target of steroid hormones:
observations during the second half of gestation. Domest. Anim. Endocrinol. 2003; 23:
309-320.

[111] Hoffmann B, Goes de Pinho T, Schuler G. Determination of free and conjugated
oestrogens in peripheral blood plasma, feces and urine of cattle throughout pregnancy.
Exp. Clin. Endocrinol. Diabetes 1997; 105: 296-303.

[112] Birgel EH, Zerbe H, Grunert E. Untersuchugen uber Zusammenhange zwischen
Anzeichen der nahenden Abkalbung und Steroidhormonprofilen. Prakt Tierarzt 1996;
77: 627-630.

[113] Anthony RV, Liang R, Kayl EP, Pratt SL. The growth of hormone/prolactin gene
family in ruminant placentae. J. Reprod. Fertil. Suppl 1995; 49: 83-95.

[114] Duello TM, Byatt JC, Bremel RD. Immunohistochemical localization of placental
lactogen in binucleate cells of bovine placentomes. Endocrinology 1986; 119: 1351-
1355.

[115] Wooding FBP, Beckers JF. Trinucleate cells and the ultrastructural localization of
bovine placental lactogen. Cell Tissue Res. 1987; 247: 667-673.

[116] Anthony RV, Pratt SL, Liang R, Holland MD. Placental-fetal hormonal interactions:
impact on fetal growth. J. Anim Sci. 1995; 73: 1861-1871.

[117] Forsyth I.A., Wallis M. Growth hormone and prolactin-molecular and functional
evolution. J. Mammary Gland Biol. Neoplasia 2002; 7: 291-312.

[118] Gluckman PD, Pinal CS. Regulation of fetal growth by the somatotrophic axis. J. Nutr.
2003; 133: 1741S-1746S.

[119] Soares MJ, Muller H, Orwig KE, Peters TJ, Dai G. The uteroplacental prolactin family
and pregnancy. Biol. Reprod. 1998; 58: 273-284.
















.9 20


O100-


o a0

KSOM M-199 5% 02 20% 02

Figure 4-6. Incubation in atmospheric oxygen increases IFNz secretion from bovine blastocysts.
The quantity of bioactive IFNz present in conditioned medium on day 11 post-IVF
was determined for embryos cultured from day 8 to 11 post-IVF in KSOM or M-199
and 5% or 20% oxygen. Results depict the main effects of medium type (A.) and
oxygen concentration (B.) on IFNz concentrations in conditioned medium. Four
independent replicate studies were completed with a total n=20 to 37
blastocysts/treatment. Different superscripts above bars represent differences
(P=0.02). Error bars indicate mean standard error.

































69










[52] Robinson RS, Mann GE, Lamming GE, Wathes DC. The effect of pregnancy on the
expression of uterine oxytocin, oestrogen and progesterone receptors during early
pregnancy in the cow. J. Endocrinol. 1999; 160: 21-33.

[53] Robinson RS, Mann GE, Lamming GE, Wathes DC. Expression of oxytocin, oestrogen
and progesterone receptors in uterine biopsy samples throughout the oestrous cycle and
early pregnancy in cows. Reproduction 2001; 122: 965-979.

[54] Spencer TE, Ott TL, Bazer FW. Expression of interferon regulatory factors one and two
in the ovine endometrium: effects of pregnancy and ovine interferon tau. Biol. Reprod.
1998; 58: 1154-1162.

[55] Horn S, Bathgate R, Lioutas C, Bracken K, Ivell R. Bovine endometrial epithelial cells
as a model system to study oxytocin receptor regulation. Hum. Reprod. Update 1998; 4:
605-614.

[56] Leung ST, Wathes DC. Estradiol regulation of oxytocin receptor expression in cyclic
bovine endometrium. J. Reprod. Fertil. 2000; 119: 287-292.

[57] Sheldrick EL, Flick-Smith HC. Effect of ovarian hormones on oxytocin receptor
concentrations in explants of uterus from ovarectomised ewes. J. Reprod. Fertil. 1993;
97: 241-245.

[58] Wathes DC, Flick-Smith HC, Leung ST, Stevenson KR, Meier S, Jenkin G. Oxytocin
receptor development in the ovine uterus and cervix throughout pregnancy and at
parturition as determined by in situ hybridization analysis. J. Reprod. Fertil. 1996; 106:
25-31.

[59] Asselin E, Bazer FW, Fortier MA. Recombinant ovine and bovine interferons tau
regulate prostaglandin production and oxytocin response in cultured bovine endometrial
cells. Biol. Reprod. 1997; 56: 402-408.

[60] Bazer FW, Spencer TE, Ott TL, Ing NH. Regulation of endometrial responsiveness to
estrogen and progesterone by pregnancy recognition signals during the periimplantation
period. Dey, S. K. Molecular and cellular aspects of periimplantation processes. 27-47.
2000. Norwell, MA, Springer.

[61] Flint APF, Sheldrick EL, McCann TJ, Jones DSC. Luteal oxytocin: characteristics and
control of synchronous episodes of oxytocin and PGF2alpha secretion at luteolysis in
ruminants. Domest. Anim Endocrinol. 1990; 7: 111-124.

[62] Milvae RA. Inter-relationships between endothelium and prostaglandin fdalpha in
corpus luteum function. Rev. of Reprod. 2000; 5: 1-5.

[63] Ohtani M, Kobayashi S, Miyamoto A, Hayashi K, Fukui Y. Real-time relationships
between intraluteal and plasma concentrations of endothelin, oxytocin, and
progesterone during prostaglandin fdalpha-induced luteolysis in the cow. Biol. Reprod.
1998; 58: 103-108.









chemically defined medium (CDM), Charles Rosenkrans 1 (CR-1), Chatot/Ziomek/Bavi ster

medium (CZB), and Hamster embryo culture medium (HECM) [203, 214].

Carbohydrates are an important source of energy for the developing embryo. Prior to

compaction and morula development, the early bovine embryo does not readily utilize glucose as

its primary energy source. In fact, glucose can be toxic to early developing embryos [215, 216].

Supplementation with pyruvate and/or lactate are excellent sources of energy for pre-compacted

bovine embryos [203, 205, 217]. Work from Bavister and colleagues found that supplementing

culture medium with both essential and non-essential amino acids had an overall stimulatory

effect on embryo development, although some specific amino acids phenylalaninee, valine,

isoleucine, tyrosine, tryptophan, and arginine) inhibited development [218, 219]. Glutamine, in

particular, is a vital component of embryo culture media based on work in mice where glutamine

supports embryo development in mouse strains prone to arresting at the 2-cell stage [220].

Bovine embryos undergo a change in their requirements for essential and non-essential amino

acids as development progresses [221], although most culture procedures do not alter medium

formulations between the 1-cell and blastocyst stages.

Optimal embryo development in vitro also relies on other important considerations. The

optimal temperature for bovine in vitro maturation, fertilization, and development is between

38.5 and 39 oC [222, 223]. Maintenance of intracellular pH is critical for numerous cellular

mechanisms [224]. In the oviduct and uterine fluid, regulation of pH occurs through HCO3- and

its equilibration with CO2, which varies throughout the estrous cycle [225, 226]. Traditionally,

embryo culture was conducted in an atmospheric oxygen level (approximately 20%), which is

considerably greater than that found in the maternal environment [227]. Recent work in bovine

embryos note that reducing the oxygen levels during early development to between 5 and 7%













b~





*0 3 -










Control FGF-2 GM-CSF FGF-2fGM-CSF

Figure 4-17. Supplementation with FGF-2 but not GM-CSF increases IFNz production from
bovine embryos. Blastocysts were cultured individually in drops of M-199 containing
2.5% FBS and 100 ng/ml boFGF-2, 100 ng/ml poGM-CSF, both FGF-2 and GM-CSF
(100 ng/ml of each), or no growth factor treatment (control) from day 8 to 11 post-
IVF. Quantity of bioactive IFNz in conditioned medium at day 11 post-IVF was
determined and normalized for embryo cell number. Three independent replicate
studies were completed with a total of 24-28 blastocysts/treatment. Different
superscripts above bars represent differences (P<0.05). Error bars indicate mean
standard error.









and quality was assessed and embryos and medium samples were processed as described

previously. Two independent replicate studies were completed with 3 to 6 blastocysts per

treatment in each replicate (total n=6 to 16 blastocysts/treatment) .

Experiment 3: Determination of Whether FGF-2 Supplementation and Oxygen

Concentration Affects Embryo Development and IFNz Concentration to Day 11 Post-IVF

Day 8 blastocysts were cultured individually in 30 CIL drops of M-199 supplemented with

2.5% FBS, 0.011 mM gentamicin, non-essential amino acids (0.0001 mM of each L-alanine, L-

asparagine*H20, L-aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine), 0.019 mM

sodium pyruvate and either 0 or 100 ng/mL of recombinant bovine fibroblast growth factor-2

(boFGF-2; R&D Systems, Inc.) at 38.50C in either a 5% or 20% oxygen atmosphere. BoFGF-2

was purchased as a lyophilized powder and was reconstituted in M-199 containing 0. 1% [w/v]

BSA. Aliquots were stored at -200C. All treatments, including controls, were provided to cells

with identical amounts of BSA carrier (20 Clg BSA/ml medium). On day 11 post-IVF, blastocyst

development and quality was assesses and embryos and medium samples were processed as

described previously. Three independent replicate studies were completed with 8 to 10

blastocysts per treatment in each replicate (total n=25 to 30 blastocysts/treatment) .

Experiment 4: Determination of Whether FGF-2 and GM-CSF Co-supplementation

Affects Embryo Development and IFNz Concentration to Day 11 Post-IVF

Day 8 blastocysts were cultured individually in 30 CIL drops of M-199 supplemented with

2.5% FBS, 0.011 mM Gentamicin, Non-Essential Amino Acids (0.0001 mM of each L-alanine,

L-asparagine*H20, L-aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine), 0.019 mM

sodium pyruvate and either 100 ng/mL boFGF-2, 100 ng/mL of recombinant porcine GM-CSF

(poGM-CSF; R&D Systems, Inc.), both FGF-2 and GM-CSF (100 ng/ml of each), or no










[33]. Finally, PGF2a CauSCS Smooth muscle contraction within the ovary. Collectively, these

coordinated events result in follicle rupture [33].

Luteal Development and Function

Progesterone is the hormone responsible for preventing ovulation, preparing the uterus

for pregnancy, and maintaining a pregnant state in cattle. Progesterone is produced by the CL,

which is a mass of tissue created from differentiated follicle cells around the time of ovulation.

During ovulation, the follicle wall collapses and the basement membrane between the theca

internal and granulosa cells breaks down, giving way to the development of an extensive vascular

network where blood vessels invade the antral space of the follicle forming the corpus

hemorrhagicum (bloody body) and eventually the corpus luteum (CL; yellow body). In domestic

animals the maj or luteotropin is LH, which stimulates development of the CL and production of

progesterone. The CL is comprised of two types of luteal cells: large and small. Unlike the small

luteal cells, large luteal cells do not respond to LH stimulation, however, they are responsible for

a majority of the basal secretion of progesterone [34]. In cattle, LH pulses are required for the

development of a fully functional CL [3 5-3 9].

One of the important actions of progesterone is to prevent ovulation. Progesterone acts

on the hypothalamus to reduce GnRH pulse frequency and prevent the hypothalamic surge center

from generating an LH surge. These activities reduce LH and FSH pulse frequency but increase

pulse amplitude of LH and FSH; conditions that favor luteal function. Gonadotropin production

is sufficient to allow follicular development to occur, however ovulation is prevented by the

negative feedback actions of progesterone on the hypothalamus [40].

Copious progesterone secretion by the CL is vital for the maintenance of pregnancy.

Progesterone stimulates the endometrium to produce nutrients, growth factors,











Statistical Analy sis............... ...............56

4 RE SULT S .............. ...............59....

Experiment 1: Effect of Medium Type and Oxygen Atmosphere on Blastocyst Survival,
Development and IFNz Production............... .. ..... ..................5
Medium type did not affect the quantity of bioactive IFNz, as assessed with an antiviral
assay, in conditioned medium from blastocysts cultured individually from day 8 to 11
post-IVF (Fig 4-6). However, embryos incubated in 5% oxygen secreted more
(P=0.002) IFNz into medium than embryos incubated in 20% oxygen (Fig. 4-6). This
effect is evident in both medium types both before and after normalizing data
according to embryonic cell numbers on day 11 post-IVF (Fig. 4-7)............... ................60
Experiment 2: Serum Requirements for Extended Bovine Blastocyst Culture ................... ...60
Experiment 3: FGF-2-Mediated Induction of IFNz Production in Bovine Blastocysts
Depends on the Level of Atmospheric Oxygen ................. ............ .. ..... .. .......... .....6
Experiment 4: The Combined Effects of FGF-2 and GM-CSF on Blastocyst
Development and IFN-z Production .............. ...............62....

5 DI SCUS SSION ........... .......__ .............. 1...

LIST OF REFERENCES ........... .......__ ...............88..

BIOGRAPHICAL SKETCH ........... ......__ ...............116..









of nuclei (blue fluorescent stain) and number of TUNEL-positive nuclei (green fluorescent stain)

in each blastocyst.

Interferon-tau Production

The amount of biologically active IFNz present in conditioned medium from each bovine

blastocyst culture was determined by using antiviral assays [178]. Briefly, 20 Cll of conditioned

medium collected at day 11 post-IVF was diluted serially to determine when each sample was

able to prevent vesicular stomatitis virus-induced cell lysis by 50%. The activity of each sample

was compared with a bovine recombinant IFNz developed in the laboratory [151] that had been

standardized with recombinant human IFNot (EMD Biosciences Inc.; 3.84 x 10s IU/mg). The

average activity of the bovine recombinant IFNz standard was 8.03 x 10" IU/mg.

Statistical Analysis

All analyses were conducted using the general linear model of the Statistical

Analysis System (SAS Institute Inc.; Cary, NC). Differences in percentage of degenerated

embryos among treatments, total cell number per embryo, percentage of apoptotic cells per

embryo, and antiviral activity of spent medium were determined using least-squares analysis of

variance (LS-ANOVA). In most studies, the antiviral activity of IFNz in medium samples was

normalized for total cell number. Pair-wise comparisons were completed to further partition

effects among treatment groups. In all experiments, the results are presented as arithmetic means

+ SEM.










[282] Block J, Drost M, Monson RL, Rutledge JJ, Rivera RM, Paula-Lopes FF, Ocon OM,
Krininger CEI, Liu J, Hansen PJ. Use of insulin-like growth factor-I during embryo
culture and treatment of recipients with gonadotropin-releasing hormone to increase
pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed,
lactating cows. J.Anim Sci. 81, 1590-1602. 2003.

[283] Paria BC, Dey SK. Preimplantation embryo development in vitro: cooperative
interactions among embryos and role of growth factors. Proc. Natl. Acad. Sci. U. S. A
1990; 87: 4756-4760.

[284] Rappolee DA, Sturm KS, Behrendtsen O, Schulz GA, Pedersen RA, Werb Z. Insulin-
like growth factor II acts through an endogenous growth pathway regulated by
imprinting in early mouse embryos. Genes Dev. 1992; 6: 952.

[285] Watson AJ, Hogan A, Hahnel A, Schultz GA, Weirner KE. Expression of growth factor
ligand and receptor genes in the preimplantation bovine embryo. Mol. Reprod. Dev.
1992; 31: 87-95.

[286] Larson RC, Ignotz GG, Currie WB. Platelet derived growth factor (PDGF) stimulates
development of bovine embryos during the fourth cell cycle. Development 1992; 115:
821-826.

[287] Bottcher RT, Niehrs C. Fibroblast growth factor signaling during early vertebrate
development. Endocr. Rev. 2005; 26: 63-77.

[288] Burgess WH, Maciag T. The heparin-binding (fibroblast) growth factor family of
proteins. Annu. Rev. Biochem. 1989; 58: 575-606.

[289] Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and
signaling. Endocr. Relat Cancer 2000; 7: 165-197.

[290] Armelin HA. Pituitary extracts and steroid hormones in control of 3T3 cell growth.
Proc. Natl. Acad. Sci. U. S. A 1973; 70: 2702-2706.

[291] Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biol. 2001; 2: Reviews3005.

[292] Itoh N, Ornitz DM. Evolution of the Fgf and the Fgfr gene families. Trends Genet
2004; 20: 563-569.

[293] Feldman B, Poueymirou W, Papaioannou VE, DeChiara TM, Goldfarb M. Requirement
of FGF-4 for postimplantation mouse development. Science 1995; 267: 246-249.

[294] Leunda-Casi A, de Hertogh R, Pampfer S. Control of trophectoderm differentiation by
inner cell mass-derived fibroblast growth factor-4 in mouse blastocysts and corrective
effect of fgf-4 on high glucose-induced trophoblast disruption. Mol. Reprod. Dev.
2001; 60: 38-46.









This activity is mediated within the endometrium by two IFN-regulated transcription factors,

Interferon Regulatory Factor-1 (IRF-1) and Interferon Regulatory Factor-2 (IRF-2). IRF-1 is a

transcriptional activator of IRF-2, which is best known as a transcriptional repressor [164].

Luminal and glandular epithelial expression of both IRF-1 and IRF-2 increases in non-pregnant

ewes supplemented with IFNz [54]. The current working model of the antiluteolytic actions of

IFNz to limit oxytocin receptor expression, and thus luteolysis, is that IFNz stimulates IRF-1

gene transcription in endometrial epithelium, which in turn increases IRF-2 gene transcription.

IRF-2 represses ER gene expression by binding to an interferon regulatory factor element, and

this loss in ER expression prevents the up-regulation of OTR expression. Collectively, this loss

in OTR prevents oxytocin from inducing PGF2a Synthesis [54, 164, 165].

The estrogen receptor-mediated theory of oxytocin receptor gene expression regulation

appears to be well suited for the physiological events observed in ewes, but cattle are slightly

different in their mechanism of action. Most notably, in a non-pregnant state, increased

abundance of OTRs precedes that of increased ER expression in bovine endometrium [52, 53].

Based on this physiological difference between ewes and cows, it is possible that cows and ewes

differ in the mechanisms used to prevent oxytocin receptor gene expression. Perhaps IFNz acts

directly on oxytocin receptor expression in cattle and also acts indirectly by regulating estrogen

receptor expression during early pregnancy in both species. As mentioned earlier, there is

conflicting evidence in sheep as to which receptor is up-regulated first. Spencer and colleagues

found that ER concentrations increase before OTR [54] while observations by Wathes and

Hamilton contradicted these findings [49].

Interferon-tau also modifies prostaglandin metabolism in endometrium during early

pregnancy. The production of endometrial-derived prostaglandins is controlled by a series of










DU







.0

u10






5% 02 20% Oz 5%r 02 20% Oz
KSOM M-199

Figure 4-3. Incubating blastocysts from day 8 to 11 post-IVF in M-199 and 5% 02 prevents
degeneration. The incidence of embryo degeneration, as characterized by partial to
complete collapse or deterioration of cell membranes, more specifically the
trophectoderm, and/or rupturing of the zona pellucida, was determined for embryos
cultured from day 8 to 11 post-IVF in KSOM or M-199 and 5% or 20% oxygen.
Degeneration on day 11 was determined by visual assessment of embryo integrity
(see methods section for detail). Four independent replicate studies were completed
with a total n=20 to 46 blastocysts/treatment. Different superscripts above bars
represent differences (P<0.05). Error bars indicate mean standard error.









abundance and Bax, a cell death factor, is elevated within in vitro-derived bovine embryos

compared with in vivo derived embryos [260, 261]. A study using cDNA microarrays evaluated

the differential expression of 384 genes or expressed sequence tags between in vitro and in vivo

derived blastocysts. The results indicated that approximately 85% of these genes had reduced

expression for in vitro-derived blastocysts compared to their in vivo counterparts [262].

In summary, the in vitro production of bovine embryos is a powerful tool to describe the

events associated with normal embryo development in cattle. However, current in vitro systems

are inadequate in several regards and improvements in these systems or more stringent selection

and/or grading of embryos produced by these systems is required to obtain higher quality

embryos.

Extended Blastocyst Culture

The continuation of embryo culture during and beyond the blastocyst stage can be

accomplished, at least to a certain degree, but most experiments conducted with in vitro-derived

bovine embryos end on day 7 or 8 post-fertilization when they reach the blastocyst stage. One

method of extending the traditional culture systems is by simply placing the embryo into a new

medium drop. Using this type of conventional culture procedure, hatched blastocysts are inclined

to attach to the bottom of the culture dish and form trophectoderm outgrowths. Under these

culture conditions, trophectoderm cultures can be maintained for extended periods but the

remainder of the embryo (i.e. the embryonic disk) will degenerate on or after day 13-15 post-IVF

[263, 264]. More recently, Alexopoulos and colleagues successfully extended embryo culture to

day 27 post-fertilization when supplementing medium with Fetal Bovine Serum (FB S). Embryo

development reached a spherical stage and seemed to match up with typical in vivo embryonic

development. This was determined by evaluation of trophectoderm proliferation and whether or

not a hypoblast was established (Day 11). More frequently, however, there was a divergence










(PGF2a) is a prostaglandin produced by the uterus which is capable of terminating a pregnancy at

any time in the cow.

Throughout the estrous cycle, progesterone stimulates the accumulation of phospholipids

in the luminal and glandular epithelia of the endometrium, and these substrates are used to

generate arachidonic acid, which is required for synthesis and secretion of PGF2a [3 8, 46]. As

progesterone receptor (PR) abundance decreases in luminal and glandular epithelium, estrogens

acting through their receptors, termed estrogen receptors (ERs), stimulate the expression of

oxytocin receptors (OTR) [46, 47]. In sheep, OTRs first appear on day 14 and reach a peak at

estrus [48, 49], while in the cow OTRs appear between days 15 and 17, just prior to luteolysis

[50-52]. There is some debate as to how this up-regulation of OTRs occurs. It has been

hypothesized using ewes as the model species that estradiol is acting through its receptor to

cause the up-regulation on OTR. However if this were an estradiol-dependent event, then it

would be plausible to assume then that ER receptors would need to be up-regulated first. Two

studies conducted by Robinson and colleagues would argue otherwise in the cow. In one

experiment, luminal epithelium ER did not increase until between day 16 and 18 in the cow,

slightly after, approximately 1-2 days later, than the initial up-regulation of OTRs. Additionally,

OTR mRNA was detectable on day 14, while ER mRNA was not detectable until day 16 [53]. In

ewes, there is conflicting evidence as to which receptor is up-regulated first. Spencer and

colleagues found that ER concentrations increase before OTR [54] while Wathes and Hamilton

observed the exact opposite [49]. It remains plausible that an up-regulation of ER is not required

for estradiol to stimulate OTR expression in endometrium, or perhaps the OTR gene up-

regulation is mediated via estradiol acting in a paracrine manner in other cells, such as the deep










responsible for uterine invasion in cattle and other ruminants are termed binucleate cells or

multinucleated giant cells [97-100]. These cells are first detected around day 16-17 of pregnancy

and compose approximately 20% of the trophectoderm by day 25 of pregnancy in cows. They

remain abundant until just a few days before parturition, at which time there is a significant

decline in binucleate cell number [99, 101, 102]. The binucleate cells originate from

mononucleated trophectoderm and undergo a process known as acytokinesis, where nuclear

division occurs but there is no cytoplasmic division. These binucleate cells contain large

secretary granules which occupy about 50% of their volume [99, 100]. Once these cells have

transformed to binucleate cells they migrate into the maternal epithelial layer and fuse with the

maternal epithelial cells to form trinucleated hybrid cells. The lysis of these giant placental-

uterine cells creates the syncytium during early pregnancy, and their presence throughout

pregnancy is thought to facilitate uterine-placental communications and potentially promote

nutrient and gas exchange [97-99].

The trophoblast of the bovine concepts possesses areas of numerous microvilli that form

into structures known as the cotyledons, which attach to specific areas on the endometrium

known as caruncles. The finger-like proj sections on the fetal cotyledon fit into crypts of the

maternal caruncle and together they make up the unit known as a placentome [93, 99]. The

placentomes serve as the sites of nutrient, gas and waste exchange between the mother and fetus.

Additionally, they act as a barrier by preventing the migration of non-placental cells across the

two entities [103]. These placentomes can grow to a size of 10-12 cm long and 2-3 cm thick

towards the end of gestation in cattle. They are found throughout both uterine horns but are

larger in the horn containing the pregnancy. The contact surface area between the fetus and










[193] Evans SS, Collea RP, Leasure JA, Lee DB. IFN-alpha induces homotypic Leu-13
expression in human B lymphoid cells. J Immunol 1993; 150: 736-747.

[194] Chen YX, Welte K, Gebhard DH, Evans RL. Induction of T cell aggregation by
antibody to a 16kD human leukocyte surface antigen. J Immunol 1984; 133: 2496-
2501.

[195] Al-Katanani YM, Drost M, Monson RL, Rutledge JJ, Krininger CE, Block J, Thatcher
WW, Hansen PJ. Pregnancy rates following timed embryo transfer with fresh or
vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions.
Theriogenology 2002; 58: 171-182.

[196] Franco M, Block J, Jousan FD, de Castro e Paula LA, Brad AM, Franco JM, Grisel F,
Monson RL, Rutledge JJ, Hansen PJ. Pregnancy rates in heat-stressed dairy cattle
receiving one or two on vitro-produced embryos in a timed embryo transfer program.
Reprod.Fertil.Dev. 18[2], 202-203. 2006.

[197] Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD, Neely B, Shuey LS,
Stokes JE, Trimmer SA. Production, freezing and transfer of bovine IVF embryos and
subsequent calving results. Theriogenology 1995; 43: 141-152.

[198] Putney DJ, Drost M, Thatcher WW. Influence of Summer Heat-Stress on Pregnancy
Rates of Lactating Dairy-Cattle Following Embryo Transfer Or Artificial-Insemination.
Theriogenology 1989; 31: 765-778.

[199] Reichenbach HD, Liebrich J, Berg U, Brem G. Pregnancy rates and births after
unilateral or bilateral transfer of bovine embryos produced in vitro. J. Reprod. Fertil.
1992; 95: 363-370.

[200] Thibault C. In vitro culture of cow egg. Ann. Biol. Anim. Biochem. Biophys. 1966; 6:
159-164.

[201] Barnes FL, Eyestone WH. Early cleavage and the maternal zygotic transition in bovine
embryos. Theriogenology 1990; 33: 141-152.

[202] Frei RE, Schultz GA, Church RB. Qualitative and quantitative changes in protein
synthesis occur at the 8-16-cell stage of embryogenesis in the cow. J. Reprod. Fertil.
1989; 86: 637-641.

[203] Thompson JG. Defining the requirements for bovine embryo culture. Theriogenology
1996; 45: 27-40.

[204] Gandolfi F, Moor RM. Stimulation of early embryonic development in the sheep by co-
culture with oviduct cells. J. Reprod. Fertil. 1987; 81: 23-28.

[205] Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum. Reprod.
Update 1995; 1: 91-148.









mother can reach as much as 130 m2 due to this extensive interdigitation between the highly

villus cotyledon and plentiful crypts of the caruncle [93, 99].

Grosser originally defined the ruminant placenta as syndesmochorial because the

maternal epithelial layer degraded during early pregnancy [104]. His initial conclusions were that

the fetal epithelial layer lysed uterine epithelial cells so the placenta could be in direct contact

with the maternal connective tissue. Future work questioned this contention, notably because

during mid- and late-gestation a maj ority of the uterine epithelium is reformed in the

intercaruncular areas in cattle, sheep, deer and goats. In sheep and deer, caruncular areas remain

free of uterine epithelium throughout pregnancy, but epithelial re-growth is observed in

caruncular regions of the cow and doe (female goat) [99, 105, 106]. In all ruminant species,

limited placental cell migration and fusion persists in the intercaruncular zones throughout

pregnancy. Today, some researchers classify the ruminant placenta as epitheliochorial (or

desmochorial) under the misconception that the syncytial state is transient [107, 108]. However,

the synepitheliochorial or syndesmochorial classification remains the more precise terminology

for placentation in ruminants.

Placental Hormones

The placenta acts as a endocrine, paracrine, and autocrine organ and produces a wide

range of steroid and peptide hormones that facilitate fetal development and aid in altering the

maternal physiology to support pregnancy [103]. Several hormones are commonly produced by

mammalian placentae, including estrogens, progesterone, and placental lactogen. However,

ruminants also produce some unique secretary factors, including IFNz, the maternal recognition

of pregnancy factor in ruminants. This unique hormone will be discussed later in the review.

In ruminants and many other mammalian species, the placenta produces estrogens and

progesterone. The main placenta-derived estrogen product is estrone-3-sulfate. Production of this










A. B. 7











0~ 0
Control FGF-2 5% On 20%X O

Figure 4-14. Main effects of FGF-2 supplementation and oxygen concentrations on IFNz
secretion from bovine blastocysts. Blastocysts were cultured individually from day 8
to 11 post-IVF in M-199 supplemented with 0 or 100 ng/mL boFGF-2 under 5% or
20% atmospheric oxygen conditions. The quantity of bioactive IFNz in conditioned
medium on day 11 post-IVF was determined and normalized for embryo cell number.
Data represent the main effect of FGF-2 supplementation (A.) and oxygen condition
(B.) on IFNz concentrations in conditioned medium. Three independent replicate
studies were completed with a total of 25-30 blastocysts/treatment. Different
superscripts above bars represent differences (P<0.05). Error bars indicate mean
standard error.










[295] Taniguchi F, Harada T, Yoshida S, Iwabe T, Onohara Y, Tanikawa M, Terakawa N.
Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation.
Mol. Reprod. Dev. 1998; 50: 54-62.

[296] Ka H, Jaeger LA, Johnson GA, Spencer TE, Bazer FW. Keratinocyte growth factor is
up-regulated by estrogen in the porcine uterine endometrium and functions in
trophectoderm cell proliferation and differentiation. Endocrinology 2001; 142: 2303-
2310.

[297] Baird A, Walicke PA. Fibroblast growth-factors. Brit. Med. Bulletin 1989; 45: 438-452.

[298] Folkman J, Klagsbrun M. Angiogenic factors. Science 1987; 235: 442-447.

[299] Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G. Structural characterization
and biological functions of fibroblast growth-factor. Endocr. Rev. 1987; 8: 95-1 14.

[300] Sato Y, Rifkin DB. Autocrine activities of basic fibroblast growth factor regulation of
endothelial cell movement, plasminogen-activator synthesis, and DNA-synthesis. J.
Cell Biol. 1988; 107: 1199-1205.

[301] Carlone DL, Rider V. Embryonic modulation of basic fibroblast growth factor in the rat
uterus. Biol. Reprod. 1993; 49: 653-665.

[302] Grundker C, Kirchner C. Uterine fibroblast growth factor-2 and embryonic fibroblast
growth factor receptor-1 at the beginning of gastrulation in the rabbit. Anat. Embryol.
(Berl) 1996; 194: 169-175.

[303] Gupta A, Bazer FW, Jaeger LA. Immunolocalization of acidic and basic fibroblast
growth factors in porcine uterine and concepts tissues. Biol. Reprod. 1997; 56: 1527-
1536.

[304] Ortega S, Ittmann M, Tsang SH, Ehrlich M, Basilico C. Neuronal defects and delayed
wound healing in mice lacing fibroblast growth factor 2. Proc. Natl. Acad. Sci. U. S. A
1998; 95: 5672-5677.

[305] Zhou M, Sutliff RL, Paul RJ, Lorenz JN, Hoying JB, Haudenschild CC. Fibroblast
growth factor 2 control of vascular tone. Nat Med 1998; 4: 201-207.

[306] Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL.
Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol.
2006; 24: 185-187.

[307] Xu RH, Peck RM, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of
BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Meth
2005; 2: 185-190.









medium immediately prior to incubation at day 8 post-IVF. On day 11, embryos receiving GM-

CSF treatment had fewer (P=0.03) cells per embryo regardless of whether FGF-2 was co-

administered (data not shown). FGF-2 supplementation did not influence resulting cell numbers

when compared with controls, but cell numbers were greater (P<0.04) in FGF-2-supplemented

embryos on day 11 post-IVF than embryos supplemented with poGM-CSF (Fig. 4-16). The

incidence of apoptosis was not measured in this study.

There were no main effects of FGF-2 or GM-CSF supplementation on IFNz

concentrations in conditioned medium at day 11 post-IVF when data were normalized to account

for differences in day 11 cell counts. However, individual treatment comparisons determined that

IFNz concentrate on s i n conditi one d m edium were gre ater (P=0.02) i n b oFGF -2 -suppl em ented

cultures versus non-treated controls (Fig. 4-17). Interestingly, poGM-CSF supplementation did

not significantly affect IFNz concentrations when compared to control values, but the mean

concentrations of IFNz in medium were increased slightly and were not significantly different

from boFGF-2-supplemented samples. Co-supplementation with boFGF-2 and poGM-CSF did

not increase IFNz concentrations in conditioned medium when compared with controls.










three days later [6]. However, although early forming blastocysts produce less IFNz initially,

they produce much more IFNz after three to four days than blastocysts forming later in culture

[6]. Taken together, several genetic and environmental factors influence IFNz production in

bovine embryos, and these variables complicate the potential use of IFNz as a predictor of

pregnancy success in cattle. But, the contention that producing more IFNz could predict embryos

will be less able to produce pregnancies is a provocative concept that may explain why

blastocysts cultured in 20% oxygen produce more IFNz but have a greater incidence of

degeneration and fewer cell numbers by day 11 post-IVF.

In summary, studies presented here provide evidence that M-199 containing > 2.5% FBS

and a 5% oxygen environment is able to sustain bovine blastocyst viability until at least day 11

post-IVF. This system also permits blastocysts to respond to putative embryotrophic factors,

such as FGF-2. It is anticipated that this system will be used by others to resolve some of the key

issues relating to concepts wastage in cattle.

Implications: The extension of this research must continue and further expand upon

recent discoveries. This group continues to investigate this research area and limits of the culture

system developed. Utilizing slight modifications to this culture system resulted in successfully

lengthening its boundaries and obtaining healthy bovine blastocysts until day 14 post-IVF

(Cooke et al., Unpublished observations). Extension beyond this point met with little success.

Further expansion of this system will include utilizing these methods in conjunction with

treatment of other currently known uterine-derived factors and those that have yet to be

discovered, as well as developing an agarose tunnel mold, which will induce embryonic

elongation in culture, under treatment with FGF-2 and GM-CSF to evaluate for further progress

upon previous work.






















200







Control FGF-2 Control FGF-2

5% O, 20% Oz

Figure 4-13. Effect of FGF-2 supplementation on cell numbers in embryos on day 11 post-IVF.
Blastocysts were cultured individually from day 8 to 11 post-IVF in M-199
supplemented with 0 or 100 ng/mL boFGF-2 under 5% or 20% atmospheric oxygen
conditions. Cell number was determined at day 11 post-IVF by staining nuclear DNA
(Hoescht 33342) and counting nuclei using epifluorescent microscopy. Three
independent replicate studies were completed with a total of 25-30
blastocysts/treatment. Different superscripts above bars represent differences
(P<0.05). Error bars indicate mean standard error.









neuronal signals. One of the primary mediators of both hormones is a hypothalamic peptide

known as Gonadotropin Releasing Hormone (GnRH). This factor is released from hypothalamic

nuclei in specific regions of the hypothalamus and acts on LH and FSH-producing cells in the

anterior pituitary via transport through a closed portal hypothalamic-pituitary system to stimulate

hormone production and release [27, 28].

Ovulation of a dominant follicle depends on whether luteal-derived progesterone is

present. When circulating progesterone concentrations are high, which is the case throughout

80% of the estrous cycle, the dominant follicle will undergo atresia after 3-4 days of dominance.

The regression of the dominant follicle lifts the inhibin-mediated block in folliculogenesis and

permits a successive wave of follicle selection and dominance. Typically, two to three waves of

follicle growth/atresia exist during an estrous cycle, but as little as one follicular wave and as

many as four have been reported [29, 30].

Ovulation of the dominant follicle will occur in the absence of elevated luteal

progesterone. This process is driven by changes in the hypothalamic and pituitary responsiveness

to ovarian hormones. Notably, the loss of progesterone permits follicle-derived estradiol to

interact with the hypothalamic surge center, which generates the preovulatory surge of LH [31i,

32]. Between 24-36 hours after the LH surge, the dominant follicle will rupture, releasing the

oocyte. This rupturing is mediated by histamine and prostaglandin E2 (PGE2) prOduction, which

increases blood flow to the ovary after the LH surge, resulting in an increase in hydrostatic

pressure within the follicle [33]. Theca cells of the follicle begin producing progesterone, which

stimulates collagenase synthesis by the theca internal cells to breakdown collagen in the follicle.

Also, granulosa cells increase follicular fluid secretion, causing intrafollicular pressure build up









BIOGRAPHICAL SKETCH

Teresa Marie Rodina was born and raised in Ridge, New York on Long Island where she

grew up with her twin sister Kristin, older brother James and her parents, Barbara and Robert. At

an early age she developed a passionate interest in animals and sought to one day find a career in

the field. She spent her undergraduate career in Kingston, Rhode Island at the quaint campus of

the University of Rhode Island as an animal science and technology maj or. After graduating

Cum Laude in 2005, she accepted a position with Dr. Alan Ealy as a graduate research assistant

at the University of Florida. For the next two years she worked under Dr. Ealy pursuing her

Master of Science in Animal Sciences. She hopes to one day attend the College of Veterinary

Medicine at the University of Florida and conquer the final challenge in her ultimate goal of

developing a career as a veterinarian.










[169] Guzeloglu A, Michel F, Thatcher WW. Differential effects of interferon-tau on the
prostaglandin synthetic pathway in bovine endometrial cells treated with phorbol ester.
J. of Dairy Sci. 2004; 87: 2032-2041.

[170] Asselin E, Lacroix D, Fortier MA. IFN-tau increases PGE2 production and COX-2
gene expression in the bovine endometrium in vitro. Mol. Cell Endocrinol. 1997; 132:
117-126.

[171] Asselin E, Drolet P, Fortier MA. In vitro response to oxytocin and interferon-tau in
bovine endometrial cells from caruncular and intercaruncular areas. Biol. Reprod.
1998; 59: 241-247.

[172] Smith WL, Marnett LJ, DeWitt DL. Prostaglandin and thromboxane biosynthesis.
Pharmacol Ther 1991; 49: 153-179.

[173] Skopets B, Li J, Thatcher WW, Roberts RM, Hansen PJ. Inhibition of lymphocyte
proliferation by bovine trophoblast protein-1 (type I trophoblast interferon) and bovine
interferon-alpha II. Vet. Immunol. Immunopathol. 1992; 34: 81-96.

[174] Thatcher WW. Antiluteolytic signals between the concepts and endometrium.
Theriogenology 47, 131-140. 1997.

[175] Fillion C, Chaouat G, Reinaud P, Charpigny JC, Martal J. Immunoregulatory effects of
ovine trophoblastin protein (oTP): all five isoforms suppress PHA-induced lymphocyte
proliferation. J. Reprod. Immunol. 1991; 19: 237-249.

[176] Newton CR, Vallet JL, Hansen PJ, Bazer FW. Inhibition of lymphocyte proliferation by
ovine trophoblast protein-1 and a high molecular weight glycoprotein produced by the
peri-implantation sheep concepts. Am. J. Reprod. Immunol. 1989; 19: 99.

[177] Niwano Y, Hansen TR, Kazemi M, Malathy PV, Johnson HD, Roberts RM, Imakawa
K. Suppression of T-lymphocyte blastogenesis by ovine trophoblast protein-1 and
human interferon-alpha may be independent of interleukin-2 production. Am J Reprod
Immunol. 1989; 20: 21-26.

[178] Roberts RM, Imakawa K, Niwano Y, Kazemi M, Malathy PV, Hansen TR, Glass AA,
Kronenberg LH. Interferon production by the preimplantation sheep embryo. J.
Interferon Res. 1989; 9: 175-187.

[179] Proost P, Wuyts A, Conings R, Lenaerts JP, Billiau A, Opdenakker G, Van Damme J.
Human and bovine granulocyte chemotactic protein-2: complete amino acid sequence
and functional characterization as chemkines. Biochemistry 1993; 32: 10170-10177.

[180] Choi Y, Johnson GA, Spencer TE, Bazer FW. Pregnancy and interferon tau regulate
maj or histocompatibility complex class I and B2-microglobulin expression in the ovine
uterus. Biol. Reprod. 2003; 68: 1703-1710.










[320] Crainie M, Guilbert L, Wegmann TG. Expression of novel cytokine transcripts in the
murine placenta. Biol. Reprod. 1990; 43: 999-1005.

[321] Lea RG, Riley SC, Antipatis C, Hannah L, Ashworth CJ, Clark DA, Critchley HO.
Cytokines and the regulation of apoptosis in reproductive tissues: a review. Am. J.
Reprod. Immunol. 1999; 42: 100-109.

[322] de Moraes AA, Paula-Lopes FF, Chegini N, Hansen PJ. Localization of granulocyte-
macrophage colony-stimulating factor in the bovine reproductive tract. J. Reprod.
Immunol. 1999; 42: 135-145.

[323] McGuire WJ, Imakawa K, Tamura K, Meka CS, Christenson RK. Regulation of
endometrial granulocyte macrophage-colony stimulating factor (GM-CSF) in the ewe.
Domest. Anim Endocrinol. 2002; 23: 383-396.

[324] de Moraes AA, Hansen PJ. Granulocyte-macrophage colony-stimulating factor
promotes development of in vitro produced bovine embryos. Biol. Reprod. 1997; 57:
1060-1065.

[325] Sjoblom C, Wikland M, Robertson SA. Granulocyte-macrophage colony-stimulating-
factor promotes human blastocyst development in vitro. Hum. Reprod. 1999; 14: 3069-
3076.

[326] Imakawa K, Helmer SD, Nephew KP, Meka CS, Christenson RK. A novel role for
GM-CSF: enhancement of pregnancy specific interferon production, ovine trophoblast
protein-1. Endocrinology 1993; 132: 1869-1871.

[327] Imakawa K, Carlson KD, McGuire WJ, Christenson RK, Taylor A. Enhancement of
ovine trophoblast interferon by granulocyte macrophage- colony stimulating factor:
possible involvement of protein kinase C. J. Mol. Endocrinol. 1997; 19: 121-130.

[328] Michael DD, Wagner SK, Ocon OM, Talbot NC, Rooke JA, Ealy AD. Granulocyte-
macrophage colony stimulating factor increases interferon-tau protein secretion in
bovine trophectoderm cells. Am.J.Reprod.Immunol in press. 2006.

[329] de Moraes AA, Davidson JA, Fleming JG, Bazer FW, Edwards JL, Betts JG, Hansen
PJ. Lack of effect of granulocyte-macrophage colony-stimulating factor on secretion of
interferon-tau, other proteins, and prostaglandin E2 by the bovine and ovine concepts.
Domest. Anim Endocrinol. 1997; 193-197.

[330] Emond V, Fortier MA, Murphy BD, Lambert RD. Prostaglandin E-2 regulates both
interleukin-2 and granulocyte-macrophage colony-stimulating-factor gene expression in
bovine lymphocytes. Biol. Reprod. 1998; 58: 143-151.

[331] Fortin M, Ouellette MJ, Lambert RD. TGFbeta2 and PGE2 in rabbit blastocoelic fluid
can modulate GM-CSF production by human lymphocytes. Am J Reprod Immunol
1997; 38: 129-139.





5% 02
KSOM


M-199


20% Oz


5% On


20% Oz


M-1 99


Figure 4-7. Interaction of medium type and oxygen concentration on IFNz secretion on day 1 1
post-IVF. Concentrations of IFNz in conditioned medium are depicted for embryos
cultured in KSOM or M-199and 5% or 20% oxygen from day 8 to 11 post-IVF. Data
presented include IFNz concentrations in medium prior to (A.) or after (B.) data were
normalized to account for variation in embryo cell number. Four independent
replicate studies were completed with a total n=20 to 37 blastocysts/treatment.
Different superscripts above bars represent differences (P<0.05). Error bars indicate
mean standard error.


KSOM




























To all of my friends and family who have helped me through this time in my life and help shape
the person I am today









detected (P=0.006), and embryos cultured in TCM-199 in 5% oxygen contained greater (P<0.01)

cell numbers at day 11 post-IVF than other culture treatments (Fig. 4-2, 4-4). No medium type or

oxygen concentration effects, including interactions, were noted on the percentage of apoptotic

nuclei on day 11 post-IVF (Fig. 4-5). In each group, percent TUNEL positive nuclei ranged from

1.5-3%.

Medium type did not affect the quantity of bioactive IFNz, as assessed with an antiviral

assay, in conditioned medium from blastocysts cultured individually from day 8 to 11 post-IVF

(Fig 4-6). However, embryos incubated in 5% oxygen secreted more (P=0.002) IFNz into

medium than embryos incubated in 20% oxygen (Fig. 4-6). This effect is evident in both medium

types both before and after normalizing data according to embryonic cell numbers on day 11

post-IVF (Fig. 4-7).

Experiment 2: Serum Requirements for Extended Bovine Blastocyst Culture

In the previous study embryos were maintained in medium containing 5% [v/v] FBS

based on the laboratory's previous observations for group-cultured bovine blastocysts (Michael,

2006). The obj ective of this study was to define the minimum serum requirements for blastocyst

survival in vitro to day 11 post-IVF. Treatments included M-199 containing previously described

additives and 1% B SA (Negative control), Insulin-transferrin- selenium (IT S) containing 1%

BSA, or 1%, 2.5% and 5 % FBS. Embryos were incubated in either 5% or 20% oxygen. During

the experiment it became evident that embryos cultured with 1% BSA and ITS + 1% BSA had a

high incidence of degeneration (approximately 75%) regardless of oxygen exposure and these

treatments were not replicated. Rather, the remainder of the experiment was completed only with

1%, 2.5% and 5% FBS treatment groups. The incidence of degeneration was greater (P=0.04) for

embryos cultured in 20% oxygen than those cultured in 5% oxygen (Data not shown). Moreover,









the endothelial cells in the CL. The end result leads to both functional and structural luteolysis

[76].

Pregnancy

If copulation takes place during estrus, there is an opportunity for the sperm and egg to

unite and form a new organism, which initially is termed a zygote or embryo. This embryo will

develop first in the oviduct and then in the progesterone-dominated uterine environment. By a

particular period in development, the developing placenta must attach to the uterine wall to

create a direct line of communication between mother and fetus and allow for nutrient, gas and

waste exchange. The placenta will serve as this essential connection between the mother and

fetus throughout the duration of gestation until a critical series of signals are initiated to induce

parturition, or birth of the fetus.

Fertilization

Fertilization is the process by which sperm and egg unite and their haploid chromosomes

form a new, genetically distinct diploid organism [77]. Sperm travels through the female

reproductive tract in a regulated process that ensures those spermatozoa with normal morphology

and vigorous motility will have the greatest chance of fertilization [78]. Sperm that remain viable

through the arduous j ourney through the cervix and vagina enter the oviduct and travel to the

uterotubal junction where a single sperm will fuse with the oocyte and contribute its genetic

material to create a genetically distinct organism.

Upon insemination, spermatozoa in the female reproductive tract are not capable of

fertilizing an oocyte. Before fertilization can occur, three maj or phases of final spermatozoa

maturation and development must take place. The first phase involves an alteration of the sperm

membrane, an event termed capacitation. In the bovine, capacitation occurs in the oviduct of the

female reproductive tract by interactions with glucosaminoglycans [79-82]. This interaction









gland ERs [53]. Results from several studies indicate that estradiol is not required for the up-

regulation of OTRs, although it may enhance the process [55-58].

In the cow and other ruminant species, oxytocin is released by both the posterior pituitary

and the CL and can act on the endometrial epithelium to produce PGF2a. The synthesis and

release of PGF2a fTOm the uterus acts back on the CL to, amongst other things, induce further

oxytocin release, and this oxytocin stimulates further production of uterine PGF2a [59, 60]. This

positive feedback loop that is created continues until the CL is fully degraded and luteolysis is

complete [61].

PGF2a is considered a luteolysin and causes luteolysis by both abating progesterone

production and inducing the structural demise of the CL. While the exact mechanism remains

unclear, it is hypothesized that functional luteolysis by PGF2a is mediated by the endothelial cell-

derived vasoconstrictive peptide known as endothelin-1 (ET-1), which can alter the normal

pattern of progesterone synthesis [62]. PGF2a is thought to activate the ET-1 gene in luteal

endothelial cells, thus stimulating ET-1 production. ET-1 secretion then further stimulates luteal

PGF2a prOduction, which will act in a paracrine manner on the CL to further enhance ET-1

synthesis and secretion [37, 62, 63]. ET-1 binds to a specific receptors found on both small and

large luteal cells. This activation then decreases both basal and LH-induced production of

progesterone, possibly by interrupting the cAMP mediated pathway leading to progesterone

production [62]. This mechanism of action has been elucidated in the rat [64, 65] and pig [66],

although it still remains unclear if this same pathway interruption occurs in the cow and ewe. At

the time of structural luteolysis on the cow and ewe, it was shown that both ET-1 concentration

and mRNA encoding for ET-1 were greatest in luteal tissue at this time [62]. Further evidence









to day 11 post-IVF with or without 100 ng/mL FGF-2 in either 5% or 20% oxygen. No clearly

degenerate embryos were observed in this study. Also, TUNEL analysis was not completed since

previous studies did not detect any significant changes in percentage of apoptotic nuclei based on

oxygen atmosphere (see experiments 1 and 2).

As observed in previous work, embryos cultured in M-199 with 2.5% FBS in a 5%

oxygen atmosphere contained greater cell numbers (P<0.001) on day 11 post-IVF than embryos

cultured in 20% oxygen (Fig. 4-13). Supplementation with 100 ng/mL FGF-2 did not affect cell

numbers for embryos regardless of the oxygen environment during culture. IFNz concentration

in conditioned medium at day 11 post-IVF was not affected by FGF-2 supplementation (P=0.1)

and clearly increased by incubating embryos in 20% oxygen (P=0.01) (Fig. 4-14). When the

effects of FGF-2 supplementation and oxygen levels were partitioned further, it became evident

that FGF-2 supplementation was able to increase (P=0.06) IFNz concentrations in conditioned

medium when embryos were incubated in a 5% oxygen atmosphere but not when they were

incubated in 20% oxygen (Fig. 4-15).

Experiment 4: The Combined Effects of FGF-2 and GM-CSF on Blastocyst Development
and IFN-r Production

Several reports implicate GM-CSF as a mediator of IFNz production in bovine embryos

and trophectoderm cell lines [314, 323, 328], although this observation could not be observed in

some studies [329]. A final study was completed to determine if GM-CSF affects blastocyst

development and/or IFNz secretion in this newly developed embryo culture system and

determine if GM-CSF and FGF-2 act cooperatively to mediate embryo development and/or IFNz

secretion. All embryos were cultured individually in 30 Cll drops of M-199 containing 2.5% FBS

under a 5% oxygen atmosphere. Either boFGF-2 (100 ng/mL), poGM-CSF (100 ng/mL), both

FGF-2 and GM-CSF (100 ng/mL of each), or no growth factor treatment (Control) was added to









contained this antiluteolytic activity. When inj ected into uterine lumen of non-pregnant ewes or

cows during diestrus, concepts secretary proteins from day 15-16 ovine conceptuses and 16-18

bovine conceptuses successfully extended luteal function in cyclic ewes and cows [140, 141].

These proteins were termed trophoblast protein-1, or oTP-1 and bTP-1 for the ovine and bovine

counterparts, respectively. Sequencing of oTP-1 cDNA isolated from a day 13 ovine concepts

cDNA expression library determined that the protein product was structurally similar to Type I

interferons (IFN) [139, 142]. This newly discovered trophoblast-derived interferon was then

named interferon-tau (IFNz).

The IFNz Gene and its Expression

Expression of IFNz genes is distinct from many other Type I IFNs. Of particular note is

the lack of IFNz expression in response to a virus or pathogen challenge, which is a hallmark

feature of most other Type I and Type II (IFNy) IFNs [143, 144]. Also, IFNz is not expressed by

a variety of cell types, including immune cells, as are most other IFNs. Instead, IFNz genes are

transcribed solely in the trophectoderm for a specific period during early pregnancy [145, 146].

These unique features of IFNz expression likely were conceived as the ancestral IFNz

gene was created in percoran ruminant ancestors soon after their divergence from other

Artiodactyls, such as camels, llamas and pigs, approximately 36 millions years ago. It is

proposed that the ancestral IFNz gene was created by duplication of the IFNco gene. Coincident

or soon after this duplication event, the promoter region of the new IFN gene was replaced by

sequences that permitted placental-specific gene expression [16, 146, 147]. IFNz genes have

been identified in the Bovidae, Cervidae and Giraffidae families by Southern Blot analysis, but

comparable genes could not be found in other mammalian species [16, 148].














.0 400 ab



0..30 -I a,b












Control FGF-2 GM-CSF FGF-2IGM-CSF

Figure 4-16. Effect of FGF-2 and GM-CSF supplementation on embryo cell number.
Blastocysts were cultured individually in drops of M-199 containing 2.5% FB S and
100 ng/ml boFGF-2, 100 ng/ml poGM-CSF, both FGF-2 and GM-CSF (100 ng/ml of
each), or no growth factor treatment (control) from day 8 to 11 post-IVF. Cell number
was determined at day 11 post-IVF by staining nuclear DNA (Hoescht 33342) and
counting nuclei using epifluorescent microscopy. Three independent replicate studies
were completed with a total of 24-28 blastocysts/treatment. Different superscripts
above bars represent differences (P<0.05). Error bars indicate mean standard error.









serum concentration affected degeneration rate (P=0.04). This effect was most evident for

embryos cultured in 1% FBS versus those cultured in 2.5 and 5% FBS in 5% oxygen (Fig. 4-8).

Number of cells on day 11 post-IVF also was impacted by oxygen atmosphere (P=0.001)

and serum supplementation to a lesser degree (P=0.09). As shown in Figure 4-9, cell numbers at

day 11 post-IVF were increased (P<0.05) as serum concentrations in medium increased in

embryos incubated in 5% oxygen. However, this effect was not observed in embryos incubated

in 20% oxygen. There were no differences among serum and oxygen treatments in the proportion

of apoptotic nuclei in blastocysts on day 11 post-IVF (Fig. 4-10).

Serum concentration and oxygen concentration influenced the quantity of bioactive IFNz

produced by the individual embryos (Fig. 4-11). Embryos cultured in 1% FBS produced less

(P<0.05) IFNz per cell at day 11 post-IVF than embryos either 2.5% or 5% FBS treatments,

regardless of oxygen level. Moreover, IFNz levels were below the detection limit for the assay

(0. 1 ng/ml) in a maj ority of the medium samples derived from embryos cultured in medium

containing 1 % FBS. A clear increase (P=0.02) in IFNz concentrations was detected in embryos

cultured in 20% oxygen than those cultured in 5% oxygen. The effects of serum concentrations

in medium and oxygen atmosphere remained evident when individual treatment comparisons

were made (Fig. 4-12).

Experiment 3: FGF-2-Mediated Induction of IFNt Production in Bovine Blastocysts
Depends on the Level of Atmospheric Oxygen

Based on the work completed in experiments 1 and 2, incubating embryos individually in

drops of M-199 containing several additives, including 2.5% FBS, permits continued blastocyst

development and measurable IFNz production from day 8 to 11 post-IVF. This extended culture

system was used herein to determine if the level of atmospheric oxygen affects the ability of

FGF-2 to influence IFNz production. Bovine blastocysts were cultured individually from day 8










LIST OF FIGURES


Figure page

4-1 Examples of degenerated embryos by day 11 post-IVF. ................. .................6

4-2 Examples of Hoescht 33342 and TUNEL staining for total cell counts and incidence
of apoptosis. ............. ...............65.....

4-3 Incubating blastocysts from day 8 to 11 post-IVF in M-199 and 5% 02 prevents
degeneration. .............. ...............66....

4-4 Incubation in M-199 under 5% oxygen conditions yields greater total cell numbers
on day 11 post-IVF. ............. ...............67.....

4-5 Incidence of apoptosis is not affected by medium type or oxygen tension in bovine
embryos from day 8 to 11 post-IVF ............... ...............68.......... ..

4-6 Incubation in atmospheric oxygen increases IFNz secretion from bovine blastocysts. ....69

4-7 Interaction of medium type and oxygen concentration on IFNz secretion on day 1 1
post-IV F. ............. ...............70.....

4-8 Incubating embryos in M-199 containing 2.5 or 5% FBS with 5% oxygen limits
degeneration from day 8 to 11 post-IVF ....__ ......_____ ...... .... ............7

4-9 Supplementation with FBS improves embryonic cell number after incubation in 5%
oxygen. ................. ...............72....... ......

4-10 Effect of serum supplementation on percentage of TUNEL positive cells during
individual embryo culture from day 8 to 11 post-IVF .......................__ ................73

4-11 Degree of serum supplementation and oxygen concentrations during culture impact
IFNz secretion. .............. ...............74....

4-12 Serum supplementation and atmospheric oxygen conditions act independently to
mediate IFNz production. .............. ...............75....

4-13 Effect of FGF-2 supplementation on cell numbers in embryos on day 11 post-IVF.........76

4-14 Main effects of FGF-2 supplementation and oxygen concentrations on IFNz
secretion from bovine blastocysts. ................. ...............77........._....

4-15 Treatment with FGF-2 and 20% oxygen increases IFNz production from bovine
bl astocy sts. .............. ...............78....

4-16 Effect of FGF-2 and GM-CSF supplementation on embryo cell number. ........................79









The first FGF to be identified was FGF-2, or basic FGF. FGF-2 was first described as a

mitogen for mouse 3T3 fibroblasts [290] and is now best known as a mitogen and angiogenic

factor [297-300]. FGF-2 is produced by the uteri of several species during early pregnancy and

has been implicated in regulating placental development in several species [297, 299]. In pigs,

rodents and rabbits, FGF-2 is primarily localized to the luminal epithelial layer of the

endometrium during diestrus and early pregnancy [8, 301-303]. The loss of FGF-2 function in

mice does not have maj or impacts on embryonic and placental development although defects in

wound healing and heart contractility have been observed [304, 305]. Supplementation of FGF-2

in mouse embryo culture medium increased trophectoderm outgrowths [295]. FGF-2 also

promotes gastrulation in rabbits, limits differentiation in human embryonic stem cells in culture,

and improves bovine blastocyst development when used in conjunction with transforming

growth factor-p (TGF-P) [306-310]. Additionally, FGF-2 has been implicated in the regulation

of placental attachment and formation of the syncytium in sheep [3 11].

Most recently, FGF-2 has been implicated in influencing IFNz production in the bovine

blastocyst. In ruminants, FGF-2 is produced by both the endometrium and the concepts and in

the endometrium is localized primarily in the luminal and glandular epithelium throughout the

estrous cycle and early pregnancy [8, 312]. FGF-2 supplementation increased IFNz mRNA

concentrations in a bovine trophectoderm cell line (CT-1 cells) and increased IFNz protein

secretion from both CT-1 cells and bovine blastocysts [8].

Granulocyte-Macrophage Colony-Stimulating-Factor (GM-CSF)

This cytokine is best known for its ability to mediate inflammation and other immune

responses in macrophages, granulocytes and eosinophils. GM-CSF was first identified in

activated T cells for its role in proliferation and differentiation of myeloid haematopoietic cells









CHAPTER 1
INTTRODUCTION

The sustainability and profitability of livestock production systems relies on suitable

reproductive capacities. Reproductive problems preside in nearly all domesticated animals used

for food production, and numerous components of the reproductive process usually are involved

with reproductive insufficiencies. Problems associated with detecting estrus for artificial mating

and failure to maintain pregnancies to term are the predominant problems associated with

reproduction in dairy and beef cattle [1]. These issues will typically result in extended calving

intervals, decreased lifetime milk production, and an increase in the number of artificial

inseminations required to achieve a pregnancy [2]. Dairy producers stand to lose an average of

$550 in lifetime milk potential with each reproductive cycle that fails to produce a calf [3]. The

use of estrous synchronization and timed artificial insemination protocols has diminished some

of these issues related to fertility in cattle; however no solutions for embryonic mortality have

remained elusive to date.

It is estimated that 57% of all failed pregnancies occur between days 3 and 21 of

pregnancy when the newly formed embryo must develop, announce its presence to the maternal

system, and begin attaching to the uterine lining [1]. Several crucial developmental events occur

during this period of embryonic growth and proliferation. Newly formed embryonic cells divide

and begin differentiating into a variety of embryonic and extra-embryonic tissues that will give

rise to the fetus and placenta. The developing concepts, a term used to describe the embryonic

mass and its associated placental tissues, must also signal its presence within the maternal system

to sustain a progesterone-dominated environment conducive for pregnancy. In ruminants, this

crucial event occurs by placental production of a Type I interferon, termed interferon-tau (IFNz).









IFNz mRNA is expressed for a brief period of time during maternal recognition,

beginning early in bovine embryo development and reaching peak gene expression at day 14

coincident with the onset of concepts elongation in cattle. [145, 149-152]. IFNz mRNA can be

detected until approximately day 25 of development but is not expressed thereafter [145, 150,

151]. IFNz protein can be detected in conditioned medium as early as the late morula or early

blastocyst stage of development, approximately day 6-7 post-fertilization [153, 154]. Although

IFNz mRNA abundance reaches its peak at day 13-14 of development, the overall production of

IFNz protein continues to increase between days 13 and 19 of pregnancy due to the considerable

growth of trophectoderm over this period of development [149, 155-157].

Antiluteolytic Action of IFNz

The extension in CL function is considered as the primary activity of IFNz during early

pregnancy. IFNz accomplishes this task by acting on the uterus to limit the pulsatile secretion of

uterine-derived PGF2a HOrmally occurring during late diestrus. Recombinant forms of both ovine

and bovine IFNz are able to mimic the biological activities of native IFNz. Intrauterine inj sections

of recombinant bovine and ovine IFNz in cattle extend luteal function and repress the pulsatile

release of uterine PGF2a in TOSponse to oxytocin challenge [150, 15 8-160].

In a pregnant ewe and cow, IFNz limits PGF2a pulSatility by limiting expression of

oxytocin receptors within the endometrial epithelium. Flint and Sheldrick (1986) demonstrated

that oxytocin receptor concentrations are decreased in early pregnancy compared to the

concentrations of a normal estrous cycle in ewes [161]. This effect of pregnancy could be

mimicked by administering IFNz or ovine concepts secretary proteins to cyclic ewes [139].

Studies in ewes also revealed that a decrease in estrogen receptor abundance corresponded with

the decreased number and binding affinity of oxytocin receptors on the endometrium [162, 163].









embryo culture limits the production of reactive oxygen species that cause oxidative damage in

embryos [229, 230, 343]. Level of oxidative stress was not measured in these studies, but day 8

to 11 bovine blastocysts were sensitive to tert-butylhydroperoxide (tBH), an agent that oxidizes

various intracellular molecules, including glutathione, in mammalian embryos [344, 345]. This

oxidant produced a dose responsive increase in the proportion of degenerated blastocysts and

decrease in cell numbers but did not affect IFNz levels in conditioned medium [Cooke et al.,

Unpublished observations]. In related work, addition of hydrogen peroxide to culture medium

negatively affected developmental competence of bovine embryos but did not affect IFNz

production [346]. The agent tBH mimicked some but not all of the effects of 20% oxygen on

blastocysts, thereby suggesting that oxidative stress probably is at least partial responsible for the

inferior development of blastocysts cultured in 20% oxygen.

It was surprising that incubation in 20% oxygen increased IFNz concentrations in

conditioned medium. This oxygen tension compromised the overall fitness ofblastocysts, as

evidenced by reduced cell numbers and increased the incidence of degeneration, but increased

the amount of IFNz produced. Work by others did not detect differences in IFNz mRNA or

protein content in bovine blastocysts cultured in 5% versus 20% oxygen [215, 346]. In previous

studies, exposure to different oxygen environments began on the day following fertilization (1-

cell stage) [215] or on day 3 post-IVF (8-cell stage) [346] and IFNz levels were evaluated either

on day 7 post-IVF by measuring IFNz mRNA abundance [215] or after 24 or 48 h culture of

individual day 8 blastocysts by measuring the quantity of bioactive IFNz in conditioned medium

[346]. Early exposure to high oxygen also compromised the developmental potential of bovine

embryos in these studies. One explanation for why outcomes differ between this and previous

studies is that the early exposure to a high oxygen environment may provide embryos with an









During follicle recruitment, a group, or cohort, of follicles begins developing from the primordial

pool and mature over a 45 to 60 day period. A selection process occurs as cohorts of follicles

continue to increase in size and eventually only a single follicle will to continue develop. The

smaller, subordinate follicles will undergo a degenerative process, known as atresia, and fail to

ovulate. Greater than 99% of ovarian follicles will undergo atresia throughout the lifetime of the

cow [23]. The remaining follicle, which now is termed the dominant follicle, limits the ability of

other follicles to develop and is capable of ovulating if endocrine events associated with

hypothalamic-pituitary-ovarian axis permit it. During the first follicular wave of an estrous cycle,

luteal-derived progesterone levels do not permit ovulation. As a result, these dominant follicles

will also undergo atresia, allowing a new follicular wave to begin.

Folliculogenesis is controlled by hormones produced by the anterior pituitary, namely

luteinizing hormone (LH) and follicle stimulating hormone (FSH). As follicles develop, they

become responsive to gonadotropins, and primarily FSH, which mediates further development.

When the dominant follicle reaches a particular differentiated state it is hypothesized that its

growth can be sustained by lower levels of FSH which, in conjunction with inhibin, do not

facilitate the further recruitment and development of subordinate follicles. Inhibin is a 32-kDa

dimeric protein composed of a disulfide bonded co- and P-subunit that is produced by granulosa

cells under the control of FSH [24, 25]. Within this dominant follicle, theca cells produce

androgens in response to LH. The granulosa cells within the follicle then aromatize these

androgens to estradiol under the control of FSH [26]. As the dominant follicle emerges,

increased levels of estrogen and inhibin will feed back onto the pituitary and decrease FSH

secretion. The reduction in circulating FSH prevents most large follicles from continuing to

development. The production of both LH and FSH is controlled by numerous systemic and









CHAPTER 2
LITERATURE REVIEW

The Estrous Cycle

The estrous cycle consists of a series of predictable physiological events that occur

between successive periods of sexual receptivity, also known as estrus. These series of

reproductive events occur throughout the female's adult life until it is interrupted by pregnancy

and, in some species, season of the year. The estrous cycle provides the female with repeated

opportunities to copulate and become pregnant. Estrus is preceded by the development and

ovulation of a single follicle in cattle, and hormones secreted by this follicle drive female

receptivity to males and ovulation. Immediately following ovulation, the tissue mass that made

up the follicle differentiates into a body of tissue termed the corpus luteum (CL). As the CL ages,

copious amounts of progesterone are produced to support uterine secretary activities and

diminish uterine contractility, both of which sustain pregnancies in cattle. If there is no

pregnancy, the corpus luteum will undergo regression, or luteolysis, and the female will return to

estrus. In cattle, the interval between estrus activity, or the length of the estrous cycle, averages

from 20 to 23 days.

Follicular Development and Ovulation

Folliculogenesis is the process by which follicles capable of being ovulated are formed

on the ovary in the female from a pool of naive, or primordial follicles [20]. Folliculogenesis is

an ongoing process in cattle that begins prior to puberty, and cycles of follicle development and

death (or atresia) are seen repeatedly throughout estrous cycles and pregnancy. Follicles are

present on the ovary in a hierarchy according to their stage of development and functional status

[21]. These "waves" of follicle growth occur by the developmental progression of primordial

follicles through three primary processes: 1) recruitment, 2) selection, and 3) dominance [22].









shows that ET-1 blocks progesterone production in bovine and ovine luteal cell culture and

PGF2a treatment stimulates ET-1 expression and secretion in these luteal cells [67-69].

A variety of agents have been implicated as mediators in the PGFza-induced structural

demise of the CL in the bovine and ovine, but one of the maj or players is tumor necrosis factor-a

(TNF-u). Several groups have shown that PGF2a elevates TNF-a production by macrophages in

both the bovine and ovine CL [70, 71]. There is evidence to suggest that TNF-a induced

apoptosis plays a role in structural luteolysis. Several reports indicate that apoptosis occurs

during luteolysis in ruminants [72-74], while in rodents there is a strong correlation between

apoptosis and maximal expression for PGF2a receptor mRNA [75]. PGF2a elevates TNF-a levels

in both the bovine and ovine CL and demonstrated that endothelial cells express high levels of

TNFRI, a TNF-a receptor type, and that they are sensitive to TNF-a-induced apoptosis in vitro

[70, 71].

One group positively linked both PGF2a induced ET-1 and TNF-a in the resulting

functional and structural demise of the corpus luteum. As previously discussed, ET-1 plays a key

role in functional luteolysis through the inhibition of progesterone production by luteal cells and

TNF-a has been linked to apoptosis in luteal endothelial cells. It has been hypothesized that

during early and mid luteal phases, the high levels of progesterone that still persist, in

conjunction with low levels of TNF-a and a TNFR1, prevent apoptosis in endothelial cells. It

seems the progesterone producing cells are resistant to apoptosis and serves to protect luteal cells

[76]. However, at the time of luteolysis, PGF2a Stimulates ET-1 secretion by luteal cells and

TNF-a production by local macrophages up-regulate one another' s production via a positive

feedback loop, which synergize to inhibit progesterone production. These low progesterone

levels in conjunction with increased TNFR1 expression [70, 71] facilitate TNF-a apoptosis of









LIST OF REFERENCES


[1] Inskeep EK, Dailey RA. Embryonic death in cattle. Vet. Clin. Food Anim. 2005; 21:
437-461.

[2] Roche JF, Bolandl MP, McGeady TA. Reproductive wastage following artificial
insemination of heifers. Vet. Rec. 1981; 109: 401-404.

[3] De Vreis A. Economic value of pregnancy in dairy cattle. J. of Dairy Sci. 2006; 89:
3876-3885.

[4] Gray CA, Taylor KM, Ramsey WS, Hill JR, Bazer FW, Bartol FF, Spencer TE.
Endometrial glands are required for preimplantation concepts elongation and survival.
Biol. Reprod. 2001; 64: 1608-1613.

[5] Gray CA, Burghardt RC, Johnson GA, Bazer FW, Spencer TE. Evidence that absence
of endometrial gland secretions in uterine gland knockout ewes compromises concepts
survival and elongation. Reproduction 2002; 124: 289-300.

[6] Kubisch HM, Larson MA, Roberts RM. Relationship between age of blastocyst
formation and interferon-tau secretion by in vitro-derived bovine embryos. Mol.
Reprod. Dev. 1998; 49: 254-260.

[7] Kubisch HM, Larson MA, Kiesling DO. Control of interferon-tau secretion by in vitro-
derived bovine blastocysts during extended culture and outgrowth formation. Mol.
Reprod. Dev. 2001; 58: 390-397.

[8] Michael DD, Alvarez IM, Ocon OM, Powell AM, Talbot N, Johnson SE, Ealy AD.
Fibroblast Growth Factor-2 Is Expressed by the Bovine Uterus and Stimulates
Interferon-tau Production in Bovine Trophectoderm. Endocrinology 2006; 147: 3571-
3579.

[9] Alexopoulos NI, Vajta G, Maddox-Hyttel P, French AJ, Trounson AO.
Stereomicroscope and histochemical examination of bovine embryos following
extended in vitro culture. Reprod. Fertil. Dev. 2005; 17: 799-808.

[10] Talbot NC, Caperna TJ, Edwards JL, Garrett W, Wells KD, Ealy AD. Bovine
blastocyst-derived trophectoderm and endoderm cell cultures: interferon tau and
transferring expression as respective in vitro markers. Biol. Reprod. 2000; 62: 235-247.

[1l] Talbot NC, Powell AM, Camp M, Ealy AD. Establishment of a bovine blastocyst-
derived cell line collection for the comparative analysis of embryos created in vivo and
by in vitro fertilization, somatic cell nuclear transfer, or parthenogenetic activation. In
Vitro Cell Dev. Biol. 2007; 43: 59-71.

[12] Brandao DO, Maddox-Hyttel P, Lovendahl P, Rumpf R, Stringfellow D, Callesen H.
Post hatching development: a novel system for extended in vitro culture of bovine
embryos. Biol. Reprod. 2004; 71: 2048-2055.









due to the common usage of serum, which contains uncharacterized compounds as well as

defined compounds that vary in their concentration among preparations [205]. Serum is a

complex combination of both small and large molecules with growth-promoting and growth-

inhibiting activities, depending on the source of serum. The addition of serum is commonplace

during Einal oocyte maturation in vitro, and unknown agents in serum stimulate differentiation of

cumulus cells [207-209]. Bovine serum albumin (BSA) is a common medium supplement and is

the predominant protein in reproductive tract. Some of the other potential benefits of BSA in

culture systems include its potential role in chelation and blocking non-specifie binding sites on

surfaces [203]. There are reports of serum-free bovine oocyte maturation protocols available, but

others contend that these systems are not sufficient to mature oocytes to the point where they can

be fertilized in vitro and develop to the blastocyst stage [210-212].

Presently, a variety of defined media exist for culturing bovine embryos after

fertilization. Types of culture medium can be broken down into two main subtypes: complex and

simple. Complex culture medium, such as Tissue Culture Medium-199 (with Earle's salts) and

Menezo's B2 medium, contain various components including amino acids, vitamins, salts,

nucleotides and purines. These types of components typically reflect the needs of somatic cell

growth rather than for early embryo development, although the bovine embryo will develop in

these media [205, 213]. Simple medium formulations, which include most of the so-called

culture media designed specifically for embryos, typically are salt solutions supplemented with

some specific substrates and a supplemental protein source, usually BSA. Examples include

synthetic oviductal fluid (SOF; plus amino acids and BSA), which was formulated based on the

make-up of luminal oviduct components, potassium simplex optimized medium (KSOM),










[91] Flechon JE, Renard JP. A scanning electron microscope study of the hatching of bovine
blastocysts in vitro. J. Reprod. Fertil. 1978; 53: 9-12.

[92] Perry JS. The mammalian fetal membranes. J. Reprod. Fertil. 1981; 62: 321-335.

[93] Schlafer DH, Fisher PJ, Davies CJ. The bovine placenta before and after birth:
placental development and function in health and disease. Anim Reprod. Sci. 2000; 60-
61: 145-160.

[94] Chang MC. Development fo the bovine blastocyst with a note on implantation. Anat.
Rec. 1952; 113: 143-161.

[95] Curran S, Pierson RA, Ginther OJ. Ultrasonographic appearance of the bovine
concepts from days 20 through 60. JAVMA 1986; 189: 1295-1302.

[96] Schlafke S, Enders A.C. Cellular basis of interaction between trophoblast and uterus at
implantation. Biol. Reprod. 1975; 12: 41-65.

[97] Wango EO, Wooding FB, Heap RB. The role of trophoblastic binucleate cells in
implantation in the goat: a morphological study. J. Anat. 1990; 171: 241-257.

[98] Wooding FB. Role of binucleate cells in fetomaternal cell fusion at implantation in the
sheep. Am. J. Anat. 1984; 170: 233-250.

[99] Wooding FB. Current topic: the synepitheliochorial placenta of ruminants: binucleate
cell fusions and hormone production. Placenta 1992; 13: 101-113.

[100] Wooding FBP, Chambers SG, Perry JS, George M, Heap RB. Migration of binucleate
cells in the sheep placenta during normal pregnancy. Anat. Embryol. 1980; 158: 361-
370.

[101] Wooding FB, Flint AP, Heap RB, Morgan G, Buttle HL, Young IR. Control of
binucleate cell migration in the placenta of sheep and goats. J. Reprod. Fertil. 1986; 76:
499-512.

[102] Wooding FBP. Frequency and localisation of binucleate cells in the placentomes of
ruminants. Placenta 1983; 4: 527-540.

[103] Gootwine E. Placental hormones and fetal-placental development. Anim Reprod. Sci.
2004; 82-83: 551-566.

[104] Grosser O. Fruhentwicklung, Eihautbildung und Placentation des Menschen und der
Saugetiere. Munich: 1927.

[105] Wooding FB, Morgan G, Brandon MR, Camous S. Membrane dynamics during
migration of placental cells through trophectodermal tight junctions in sheep and goats.
Cell Tissue Res. 1994; 276: 387-397.









acid, L-glutamic acid, glycine, L-proline, and L-serine), and 0.019 mM sodium pyruvate (Sigma-

Aldrich Co.) or KSOM containing 5% [v/v] FBS, 0.011 mM gentamicin, and non-essential

amino acids. A comparison of M-199 and KSOM medium components is provided in Table 2-1

and 2-2. Embryos were incubated at 38.50C in either a 5%CO2/5%O2/90%/ N2 (5% oxygen

group) or a 5% CO2/95% air atmosphere (20% Oxygen group). On day 11 post-IVF the stage

and quality of embryo development was recorded. Embryo quality was determined by visual

observation for expansion and/or hatching and whether the embryo had degenerated.

Degenerated embryos were classified as having partial to complete collapse or deterioration of

cell membranes, more specifically the trophectoderm, and/or rupturing of the zona pellucida.

Embryos were washed once in PBS/PVP, fixed in 4% [w/v] paraformaldehyde (Polysciences

Inc.) for 10 minutes and stored in 500 CIL PBS/PVP with 0.02% [w/v] sodium azide (NaN3) until

further use. Spent medium (25 CIL) was collected from each drop and stored at -200C. Four

independent replicate studies were completed with 5 to 12 blastocysts per treatment in each

replicate (total n=20 to 46 blastocysts/treatment).

Experiment 2: Determination of Whether Serum Type or Concentration Affects Embryo

Development to Day 11 Post-IVF

Blastocysts collected on day 8 post-IVF were cultured individually in 30 CIL drops of M-

199 with 0.011 mM Gentamicin, Non-Essential Amino Acids (0.0001 mM of each L-alanine, L-

asparagine*H20, L-aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine), and 0.019

mM sodium pyruvate was supplemented with either insulin-transferrin-selenium (670 Clg/L

sodium selenite (anhydrous), 1.00 g/L insulin, and 0.55 g/L transferring) (ITS; Invitrogen Corp.),

1% [w/v] bovine serum albumin (BSA; Sigma-Aldrich Inc.), or 1%, 2.5%, or 5% [w/v] FBS at

38.50C in either a 5% or 20% oxygen atmosphere. On day 11 post-IVF, blastocyst development









CHAPTER 4
RESULTS

Experiment 1: Effect of Medium Type and Oxygen Atmosphere on Blastocyst Survival,
Development and IFNt Production

Bovine blastocysts were cultured individually in two medium types and under different

oxygen tensions to assess if embryo development could be extended to day 11 post-IVF in

culture. On day 11 post-IVF, a portion of blastocysts were considered degenerated. These

embryos possessed a partial to complete collapse or deterioration of cell membranes, more

specifically the trophectoderm, and/or rupturing of the zona pellucida. Those embryos which

appeared extremely unhealthy were small in size and did not seem to show any signs of having

an expanded trophectoderm. Embryos which appeared healthy upon visual observation were

large, rotund, and expanded, some extensively so, and most were hatched from their zona

pellucida by day 11 post-IVF (67.5 +2.8% overall mean and SEM). For a comparison of typical

degenerated and healthy embryos under nuclear fluorescent staining (Hoescht 33342), refer to

Fig. 4-1 and 4-2.

No main effects of medium type or oxygen concentration on rate of degeneration were

detected but a medium type by oxygen interaction was evident (P=0.05), where incubation in M-

199 in 5% oxygen prevented degeneration whereas from 23 to 26.5% of embryos incubated in

other medium and oxygen atmospheric conditions were degenerated at day 11 post-IVF (Fig. 4-

1, 4-3). Accurate cell counts could not be retrieved from these embryos and conditioned medium

from these embryos did not contain measurable antiviral activity. Therefore, only data collected

from non-degenerated embryos were used for subsequent analyses.

Total embryonic cell numbers on day 11 post-IVF were affected by the main effects of

medium (P=0.04) and oxygen concentration (P=0.03) with M-199 and 5% oxygen treatments

providing greater day 11 cell numbers (data not shown). A medium by oxygen interaction was














--.. b















Control FGF-2 Control FGF-2
5% Oz 20% Oz
Figure 4-15. Treatment with FGF-2 and 20% oxygen increases IFNz production from bovine
blastocysts. Embryos were cultured individually from day 8 to 11 post-IVF in M-
199 supplemented with 0 or 100 ng/mL boFGF-2 under 5% or 20% atmospheric
oxygen conditions. The quantity of bioactive IFNz in conditioned medium on day 11
post-IVF was determined and normalized for embryo cell number. Three independent
replicate studies were completed with a total of 25-30 blastocysts/treatment. Different
superscripts above bars represent differences (P<0.06). Error bars indicate mean
standard error.







































Figure 4-2. Examples of Hoescht 33342 and TUNEL staining for total cell counts and incidence
of apoptosis. The total number of embryonic cells and the percentage of embryonic
cells that were TUNEL positive at day 11 post-IVF was determined for embryos
cultured individually in KSOM or M-199 and 5% or 20% oxygen were recorded
using epifluorescent microscopy.










oocytes matured in vivo are more competent to become fertilized and develop to the blastocyst

stage than those matured in vitro [243-248]. Interestingly, work by Lonergan and colleagues

showed no differences in cleavage rate between in vivo and in vitro fertilized bovine oocytes.

This suggests that although fertilization is required to create embryos, it probably is not a

limiting factor in producing embryos in vitro. The in vivo fertilized group had a greater

proportion of oocytes reaching the blastocyst stage than in vitro fertilized oocytes, although there

was no difference in quality, as determined by vitrification and thawing. However, a higher

proportion of matured oocytes that developed to blastocysts after fertilization in vivo compared

with in vitro fertilization. This result indicates that events surrounding the time of fertilization

may play a role in developmental competence of oocytes, while the actual location of

fertilization has no effect on blastocyst quality [249].

Two related experiments conducted by Lonergan and colleagues exemplify how in vitro

development is suboptimal [249]. In their studies, in vivo and in vitro matured and fertilized

zygotes were placed in either an in vitro culture system or in the oviduct of ewes until day 7

post-fertilization. No differences in cleavage rates or the percentage of oocytes that developed to

blastocysts were observed, but blastocyst development and hatching rates increased in embryos

incubated in ovine oviducts regardless of whether oocytes were matured and fertilized in vivo or

in vitro [249]. Similarly, other work indicates that viability of in vitro produced embryos is 15-

25% less than their in vivo produced counterparts [250-252]. The addition of serum to in vitro

culture systems also is deleterious to post transfer development and promotes early blastulation,

decreases cell number, increases apoptosis, and reduces rates of glycolysis [253-259].

A subset of genes also is expressed differentially between in vitro and in vivo derived

bovine embryos. For example, mRNA for connexin 43, a gap junction protein, is lower in










placentation [180]. The physiological importance of IFNz's antiviral activity remains uncertain,

but it seems intuitive to contend that IFNz may act in a prophylactic capacity to limit viral

infections early in pregnancy.

IFNz also is implicated in orchestrating events to create a favorable uterine environment

for the maintenance of pregnancy. The expression of several uterine proteins is regulated by

IFNz. One maj or IFNz-induced uterine protein is ubiquitin cross reactive protein (URCP), which

also is known as IFN stimulated gene-15 or -17 (ISGl5 or ISGl7) [181-183]. UCRP conjugates

with cystolic proteins and prompts their degradation in cells. This activity of UCRP may be

integral to establishing and maintaining pregnancy, although this precise function of UCRP

remains uncertain. Granulocyte chemotactic protein-2 (GCP-2), which was discussed earlier for

its role in immunomodulatory activities, is induced by IFNz. Other IFNz-induced uterine

proteins include Mx proteins [184, 185], 2'5'-oligoadenylate synthetase [186], and a member of

the 1-8 family, termed Leul3 [187]. One well described function of Mx protein is in the antiviral

response, and it is unclear if it has a critical activity during early pregnancy or merely is

facilitative during early pregnancy in ruminants [185, 188, 189]. 2'5'-oligoadenylate synthetase

activates a constitutively expressed latent endonuclease, ribonuclease L (RNAse L). This enzyme

cleaves viral and cellular RNA and blocks viral replication and initiation of apoptosis in some

cell types [186, 190, 191]. Its importance during maternal recognition of pregnancy remains

uncertain. Leul3 was first described as a T-cell surface antigen implicated in inhibiting cell

growth [192, 193]. Leul3 is localized primarily in the glandular epithelium, and it was

hypothesized that this member of the 1-8 protein family, which is known for its involvement in

adhesion, wound healing, and inflammatory responses, plays a role in adhesion of the concepts

to the endometrium and in the development of the placentome [187, 192-194].










A. B.










1% FBS 2.5% FBS 5% FBS 5% On 20% Oa

Figure 4-11. Degree of serum supplementation and oxygen concentrations during culture impact
IFNz secretion. Blastocysts were cultured individually in M-199 supplemented with
1%, 2.5% or 5% FBS in either 5% or 20% oxygen from day 8 to 11 post-IVF.
Concentration of bioactive IFNz in conditioned medium at day 11 post-IVF was
normalized by cell number. Data represent the main effects of level of serum
supplementation (A.) and atmospheric oxygen concentration (B.) on IFNz secretion.
Two independent replicate studies were completed with a total of 6 to 12
blastocysts/treatment. Different superscripts above bars represent differences
(P<0.05). Error bars indicate mean standard error.










[345] Stover SK, Gushansky GA, Salmen JJ, Gardiner CS. Regulation of gamma-glutamate-
cysteine ligase expression by oxidative stress in the mouse preimplantation embryo.
Toxicol. Appl. Pharmacol. 2000; 168: 153-159.

[346] Kimura K, Spate LD, Green MP, Roberts RM. Effects of oxidative stress and inhibitors
of the pentose phosphate pathway on sexually dimorphic production of IFN-tau by
bovine blastocysts. Mol. Reprod. Dev. 2004; 68: 88-95.

[347] Kimura K, Spate LD, Green MP, Murphy CN, Seidel GE, Roberts RM. Sexual
dimorphism in interferon-tau production by in vivo-derived bovine embryos. Mol.
Reprod. Dev. 2004; 67: 193-199.

[348] Kubisch HM, Sirisathien S, Bosch P, Hernandez-Fonseca HJ, Clements G, Liukkonen
JR, Brackett BG. Effects of developmental stage, embryonic interferon-tau secretion
and recipient synchrony on pregnancy rate after transfer of in vitro produced bovine
blastocysts. Reprod. Domest. Anim. 2004; 39: 120-124.










[120] Gertler A, Djiane J. Mechanism of ruminant placental lactogen action: molecular and in
vivo studies. Mol. Genet. Metab. 2002; 75: 189-201.

[121] Johnson GA, Burghardt RC, Spencer TE, Newton GR, Ott TL, Bazer FW. Ovine
osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and
concepts during the periimplantation period. Biol. Reprod. 1999; 61: 892-899.

[122] Johnson GA, Spencer TE, Burghardt RC, Bazer FW. Ovine osteopontin: I. Cloning and
expression of messenger ribonucleic acid in the uterus during the periimplantation
period. Biol. Reprod. 1999; 61: 884-891.

[123] Beckers JF, Decoster R, Wouters-Ballman P, Fromont-Lienard CH, Van der Zwalmen
P, Ectors F. Dosage radioimmunologique de l'hormone placentaire somatotrope et
mammotrope bovine. Ann. Vet. Med. 1982; 126: 9-21.

[124] Chan JSD, Robertson HA, Friesen HG. Maternal and fetal concentrations of ovine
placental lactogen measured by radioimmunoassay. Endocrinology 1978; 102: 1606-
1613.

[125] Noel S, Herman A, Johnson GA, Gray CA, Stewart MD, Bazer FW, Gertler A, Spencer
TE. Ovine placental lactogen specifically binds to endometrial glands of the ovine
uterus. Biol. Reprod. 2003; 68: 772-780.

[126] Bole-Feysot C, Goffin V, Edery M, Kelly PA. Prolactin (PRL) and its receptor: actions,
signal transduction pathways and phenotypes observed in PRL receptor knockout mice.
Endocr. Rev. 1998; 19: 225-268.

[127] Igwebuike UM. Trophoblast cells of ruminant placentas-A minireview. Anim Reprod.
Sci. 2005.

[128] Goff JP, Horst RL. Physiological changes at parturition and their relationship to
metabolic disorders. J. of Dairy Sci. 1997; 80: 1260-1268.

[129] Chew BP, Keller HF, Erb RE, Malvern PV. Periparturient concentrations of prolactin,
progesterone, and estrogens in blood plasma of cows retaining and not retaining fetal
membranes. J. of Ani. Sci. 1977; 44: 1055.

[130] Challis JRG, Sloboda D, Matthews SG, Holloway A, Alfaidy N, Patel FA, Whittle W,
Fraser M, Moss TJM, Newnham J. The fetal hypothalamic-placental-adrenal (HPA)
axis, parturition and post natal health. Mol. Cell. Endocrinol. 2001; 185: 135-144.

[131] Bloomfield FH, Oliver MH, Hawkins P, Holloway AC, Campbell M, Gluckman PD,
Harding JE, Challis JRG. Periconceptional undernutrition in sheep accelerates
maturation of the fetal hypothalamic-pituitary-adrenal axis in late gestation.
Endocrinology 2004; 145: 4278-4285.

[132] Tuo W, Ott TL, Bazer FW. Natural killer cell activity of lymphocytes exposed to ovine,
type I, trophoblast interferon. Am J Reprod Immunol 1993; 29: 26-34.









via uterine milk protein mRNA and osteopontin mRNA levels and uterine gland density were

increased [125]. In addition to stimulating endometrial gland morphogenesis, placental lactogen

has also been implicated in regulating water and electrolyte balance, mediating fetal growth and

development, and potentially serving to regulate metabolism and behavior [126, 127].

Parturition

Parturition is a hormonal process where the fetus signals to the mother that it is ready to

be expelled from the uterus. Stress signals initiated by the fetus must lead to enormous changes

within the maternal environment to transition from a state conducive to maintaining a pregnancy

to one that facilitates removal of the fetus from the uterus. Progesterone produced by the CL

maintains its dominance as the main hormone of pregnancy in cattle, with a gestation of

approximately 280 days. By approximately 250 days of gestation, plasma progesterone

concentrations reach approximately 7-8 ng/mL and drop steadily afterwards to about 3-4 ng/mL.

The day before parturition progesterone drops to a nearly undetectable level [128]. Conversely,

estradiol concentrations remain fairly low throughout most of gestation (approximately 300

pg/mL by mid-gestation) but increase just before parturition (4000-6000 pg/mL) [129].

Current dogma implicates fetal stress signals as the initiator of parturition in many

mammals. In sheep and presumably cattle, fetal cortisol appears to be the initial parturition

signal. Cortisol, stimulated by corticotrophin releasing hormone (CRH) and produced by the

adrenal cortex, will act on the placenta to use progesterone as a substrate for estrogen production,

thereby reducing progesterone secretion by the placenta. This shift in steroid production

increases myometrial contractions [130]. During the cortisol surge at the onset of labor, a feed-

forward endocrine loop between cortisol, CRH, and ACTH is established in the fetus, and this

drives the endocrine events that produce active labor [131, 132]. It is hypothesized that increased

uterine activity is, in part, due to exogenous oxytocin production by the posterior pituitary of the





































O 2007 Teresa M. Rodina





40






10







5% O, 20% O, 5% O, 20% Oz
KSOM M-199
Figure 4-5. Incidence of apoptosis is not affected by medium type or oxygen tension in bovine
embryos from day 8 to 11 post-IVF. The percentage of embryonic cells that were
TUNEL positive at day 11 post-IVF was determined for embryos cultured
individually in KSOM or M-199 and 5% or 20% oxygen were recorded using
epifluorescent microscopy. Four independent replicate studies were completed with a
total n=20 to 37 blastocysts/treatment. Different superscripts above bars represent
differences (P<0.05). Error bars indicate mean standard error.









leads to altered flagellar motility and the development of the capacity for the sperm to fuse with

the oocyte [77]. The sperm then penetrates the cumulus oophorus, which consists of cumulus

cells and extracellular matrix. Once through the cumulus oophorus, the sperm comes in contact

with and binds to the zona pellucida, at which point the acrosome reaction is initiated by ZP3.

This constituent glycoprotein of the zone pellucida, called ZP3, binds to the receptors on the

anterior head of the acrosome-intact sperm [83, 84]. Once ZP3 binds, a chain of events occurs

through the activation of phospholipase C (PLC) and elevated cystolic calcium levels. This

directly drives the acrosome reaction by continuously pushing calcium into the plasma

membrane. Acrosome reacted sperm penetrate the zona pellucida where fusion of the outer

acrosome membrane to the plasma membrane takes place, resulting in sperm-egg adhesion and a

release of acrosomal contents [77]. Finally, an increase in cytosolic calcium instigated by sperm

egg fusion induces the oocyte to proceed through its final phases of meiosis. This activity also

induces cortical granules to expel their contents. This event alters the zona pellucida to limit

polyspermy [85].

Early Embryonic and Trophectoderm Development

Once fertilization has occurred, the male and female pronuclei of the zygote will undergo

synagmy within the cytoplasm. The newly formed zygote begins mitotic cellular divisions to

generate multiple embryonic cells, or blastomeres. This newly formed entity generally is referred

to as an embryo, but in reality this new entity is best referred to as a concepts since it also

contains the ability to form extraembryonic tissues (placenta). Both terms will be used in this

narrative. During the first few mitotic divisions, each blastomere has the capability to develop

into a completely formed individual [86], an event known as totipotency. As development

progresses, totipotency is lost in some blastomeres but not others. The initial segmentation of









become pregnant. Follicles grow and develop in waves, eventually allowing one to become

dominant and ovulate, forming a corpus luteum (CL). If the female is pregnant, the concepts

will signal its presence, via the maternal recognition of pregnancy signal Interferon-tau (IFNz),

maintaining the pregnant state. Otherwise, a complex series of events will occur, leading to

luteolysis and return to estrus for another breeding opportunity.

Once the zygote has become fertilized, it develops first in the oviduct and later in the

uterine lumen. At the blastocyst stage, trophectoderm is distinguishable from non-differentiated

embryonic cells in the inner cell mass, and proliferation of trophectoderm changes the size and

shape of the developing concepts into a filamentous structure that will begin attaching to the

uterine lining and forming the outer components of the placenta. Eventually, placentomes will be

created by the coupling of placental-derived cotyledons with uterine-derived caruncles.

Throughout the lifetime of the placenta, several placental hormones are produced and function in

various capacities to drive placental and fetal development and maintain the pregnant state until

parturition. One vital product of the early placenta is IFNz, which is a type I interferon expressed

solely by the trophectoderm. It is responsible for maintaining the uterus in a pregnancy-receptive

state during the second and third weeks of pregnancy.

Current bovine embryo culture systems are capable of producing transferable embryos that

result in fair pregnancy outcomes; however, they remain inferior to those created in the maternal

environment. The maternal uterine environment contains a complex array of components which

have yet to be fully characterized, and current culture systems cannot completely replicate that

environment. Extension of embryo culture beyond the hatched blastocyst stage of development is

particularly challenging, yet a few systems have met with success in inducing elongation.

Developing such extended embryo culture systems will certainly be a valuable tool for better










[146] Roberts RM, Liu L, Alexenko A. New and atypical families of type I interferons in
mammals: comparative functions, structures, and evolutionary relationships. Prog.
Nucleic Acid Res. Mol. Biol. 1997; 56: 287-325.

[147] Roberts RM, Liu L, Guo Q, Leaman D, Bixby J. The evolution of the type I interferons
[published erratum appears in J Interferon Cytokine Res 1999 Apr; 19(4):427]. J.
Interferon Cytokine Res. 1998; 18: 805-816.

[148] Leaman DW, Roberts RM. Genes for the trophoblast interferons in sheep, goat, and
musk ox and distribution of related genes among mammals. J. Interferon Res. 1992; 12:
1-11.

[149] Bartol FF, Roberts RM, Bazer FW, Lewis GS, Godkin JD, Thatcher WW.
Characterization of proteins produced in vitro by periattachment bovine conceptuses.
Biol. Reprod. 1985; 32: 681-693.

[150] Ealy AD, Green JA, Alexenko AP, Keisler DH, Roberts RM. Different ovine
interferon-tau genes are not expressed identically and their protein products display
different activities. Biol. Reprod. 1998; 58: 566-573.

[151] Ealy AD, Larson SF, Liu L, Alexenko AP, Winkelman GL, Kubisch HM, Bixby JA,
Roberts RM. Polymorphic forms of expressed bovine interferon-tau genes: relative
transcript abundance during early placental development, promoter sequences of genes
and biological activity of protein products. Endocrinology 2001; 142: 2906-2915.

[152] Stewart HJ, McCann SH, Northrop AJ, Lamming GE, Flint AP. Sheep antiluteolytic
interferon: cDNA sequence and analysis of mRNA levels. J. Mol. Endocrinol. 1989; 2:
65-70.

[153] Hernandez-Ledezma JJ, Sikes JD, Murphy CN, Watson AJ, Schultz GA, Roberts RM.
Expression of bovine trophoblast interferon in conceptuses derived by in vitro
techniques. Biol. Reprod. 1992; 47: 374-380.

[154] Hernandez-Ledezma JJ, Mathialagan N, Villanueva C, Sikes JD, Roberts RM.
Expression of bovine trophoblast interferons by in vitro-derived blastocysts is
correlated with their morphological quality and stage of development. Mol. Reprod.
Dev. 1993; 36: 1-6.

[155] Godkin JD, Bazer FW, Moffatt J, Sessions F, Roberts RM. Purification and properties
of a maj or, low molecular weight protein released by the trophoblast of sheep
blastocysts at day 13-21. J. Reprod. Fertil. 1982; 65: 141-150.

[156] Roberts RM. Minireview: Conceptus interferons and maternal recognition of
pregnancy. Biol. Reprod. 1989; 40: -449.

[157] Roberts RM, Cross JC, Leaman DW. Unique features of the trophoblast interferons.
Pharmacol Ther 1992; 51: 329-345.










[219] Carney EW, Bavister BD. Stimulatory and inhibiting effects of amino acids on the
development of hamster eight-cell embryos in vitro. J. In Vitro Fert. Embryo Transf.
1987; 4: 162-167.

[220] Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium
supports development of random-bred 1-cell mouse embryos in vitro. J. Reprod. Fertil.
1989; 86: 679-688.

[221] Steeves TE, Gardner DK. Temporal and differential effects of amino acids on bovine
embryo development in culture. Biol. Reprod. 1999; 61: 731-740.

[222] Ball GD, Liebfried ML, Lenz RW, Ax RL, Bavister BD, First NL. Factors effecting
successful in vitro fertilisation of bovine follicular oocytes. Biol. Reprod. 1983; 28:
717-725.

[223] Lenz RW, Ball GD, Liebfried ML, Ax RL, First NL. In-vitro maturation and
fertilisation of bovine oocytes are temperature dependent processes. Biol. Reprod.
1987; 29: 173-179.

[224] Busa WB, Nuccitelli R. Metabolic regulation via intracellular pH. Am J Physiol 1984;
246: R409-R438.

[225] Hunter RHF. "The Fallopian Tubes. Their role in fertility and in fertility". Berlin:
Springer-Verlag; 1988.

[226] Olds D, VanDemark NL. Luminal fluids of bovine female genitalia. J. Am. Vet. Med.
Assoc. 1957; 31: 555-556.

[227] Fischer B, Bavister BD. Oxygen tension in the oviduct and uterus of rhesus monkeys,
hamsters and rabbits. J. Reprod. Fertil. 1993; 99: 673-679.

[228] Guerin P, Mouatassim S, Menezo ElY. Oxidative stress and protection against reactive
oxygen species in the pre-implantation embryo and its surroundings. Hum. Reprod.
Update 2001; 7: 175-189.

[229] Kitagawa Y, Suzuki K, Yoneda A, Watanabe T. Effects of oxygen concentration and
antioxidants on the in vitro developmental ability, production of reactive oxygen
species (ROS), and DNA fragmentation in porcine embryos. Theriogenology 2004; 62:
1186-1197.

[230] Takahashi M, Keicho K, Takahashi H, Ogawa H, Schultz RM, Okano A. Effect of
oxidative stress on development and DNA change in in-vitro cultured bovine embryos
by comet assay. Theriogenology 2000; 54: 137-145.

[23 1] Yuan YQ, Van Soom A, Coopman FOJ, Mintiens K, Boerj an ML, Van Zeveren A, de
Kruif A, Peelman L.J. Influence of oxygen tension on apoptosis and hatching bovine
embryos cultured in vitro. Theriogenology 2003; 59: 1585-1596.










Adequate development of the initial placental tissue lineages and ample production of IFNz are

vital for pregnancy success in cattle and other ruminant species.

The predominate method of studying early bovine embryonic development is through the

utilization of in vitro production and in vitro culture of embryos. The production of bovine

embryos using in vitro maturation, fertilization, and culture technologies provides scientists with

a powerful tool for examining the initial events of embryogenesis in cattle and related species.

Establishing culture conditions that support bovine embryo development during the blastocyst

stage would offer considerable benefits. For example, extended culture could provide additional

end-point measurements that can be used to assess how media, supplements, and atmospheric

conditions impact the production of embryos suitable for recipient transfer. Also, a blastocyst-

based culture system could assist in describing how uterine-derived factors influence embryo

development. Uterine secretions are essential for proper concepts elongation. Ewes that fail to

develop uterine glandular epithelium, a model created by exposing neonatal ewes to progestins,

are unable to nourish a pregnancy past the hatched blastocyst stage [4, 5].

Extending bovine embryo culture beyond the blastocyst stage has been completed by

several groups. Culturing groups of embryos in microdrops of medium sustains blastocyst

viability to day 10 or 11 post-in vitro fertilization (IVF) [6-8]. Bovine blastocyst development

after hatching (i.e. protrusion from the zona pellucida) is completed, at least to some extent,

either by creating trophectoderm outgrowths on extracellular matrices [7, 9-11] or by provoking

elongation by placing bovine blastocysts in agarose tunnels [12-14]. Unfortunately, the

embryonic disk, or epiblast, fails to develop properly in most cases, and the resulting tissue mass

is comprised primarily of trophectoderm and primitive endoderm [9, 12-14].









cytokine/growth factor treatment (control) at 38.50C in a 5% oxygen atmosphere. Both boFGF-2

and poGM-CSF were purchased as a lyophilized powder and were reconstituted in M-199

containing 0.1 [w/v] BSA. Aliquots were stored at -200C. All treatments, including controls,

were provided to cells with identical amounts of BSA carrier (20 Clg BSA/ml medium). On day

11 post-IVF, blastocyst development and quality was assessed and embryos and medium

samples were processed as described previously. Three independent replicate studies were

completed with 8-10 blastocysts per treatment in each replicate (total n=24-28

blastocysts/treatment).

Evaluating Apoptosis and Total Cell Number

The Terminal dUTP Nick-End Labeling (TUNEL) procedure was performed on

individual embryos fixed in 4% [w/v] paraformaldehyde using the In Situ Cell Death Detection

Kit with a Fluorescein dye (Roche Diagnostics) [334]. Individual embryos were washed in 50 CIL

drops of PBS/PVP and permeabilized in 50 CIL drops composed of PBS with 0.5% [v/v] Triton

X-100 and 0.1% [w/v] sodium citrate for 15 minutes at room temperature. Positive controls were

incubated in a 50 C1L drop of RQ 1 RNase-free DNase (50 U/ml; New England BioLabs) diluted

in PB S/PVP at 37 OC for 1 h. Embryos were washed in PBS/PVP and incubated with 25 CIL drop

of TUNEL reaction mixture according to manufacturer instructions for 1 hour at 370C in a dark

and humidified environment. Negative controls were incubated in the absence of terminal

deoxynucleotidyltransferase. All embryos were then placed in 50 CIL drops of a 0.5 mg/mL

Hoescht 33342 (Molecular Probes). Embryos were washed 4 times in 500 CIL of PBS/PVP,

placed on microscope slides, and mounted with 10-12 CIL of Prolong Gold Antifade mounting

medium (Invitrogen Corp.). Epifluroescence microscopy was used to determine the total number










[181] Naivar KA, Ward SK, Austin KJ, Moore DW, Hansen TR. Secretion of bovine uterine
proteins in response to type I interferons. Biol. Reprod. 1995; 52: 848-854.

[182] Staggs KL, Austin KJ, Johnson GA, Teixeira MG, Talbott CT, Dooley VA, Hansen
TR. Complex induction of bovine uterine proteins by interferon-tau. Biol. Reprod.
1998; 59: 293-297.

[183] Teixeira MG, Austin KJ, Perry DJ, Dooley VD, Johnson GA, Francis BR, Hansen TR.
Bovine granulocyte chemotactic protein-2 is secreted by the endometrium in response
to interferon-tau (IFN-tau). Endocrine. 1997; 6: 31-37.

[184] Johnson GA, Joyce MM, Yankey SJ, Hansen TR, Ott TL. The Interferon Stimulated
Genes (ISG) 17 and Mx have different temporal and spatial expression in the ovine
uterus suggesting more complex regulation of the Mx gene. J. Endocrinol. 2002; 174:
R7-R11.

[185] Ott TL, Yin J, Wiley AA, Kim HT, Gerami-Naini B, Spencer TE, Bartol FF, Burghardt
RC, Bazer FW. Effects of the estrous cycle and early pregnancy on uterine expression
of Mx protein in sheep (Ovis aries). Biol. Reprod. 1998; 59: 784-794.

[186] Johnson GA, Stewart MD, Gray CA, Choi Y, Burghardt RC, Yu-Lee LY, Bazer FW,
Spencer TE. Effects of the estrous cycle, pregnancy, and interferon tau on 2',5'-
oligoadenylate synthetase expression in the ovine uterus. Biol. Reprod. 2001; 64: 1392-
1399.

[187] Pru JK, Austin KJ, Haas AL, Hansen TR. Pregnancy and interferon-tau upregulate gene
expression of members of the 1-8 family in the bovine uterus. Biol. Reprod. 2001; 65:
1471-1480.

[188] Charleston B, Stewart HJ. An interferon-induced Mx protein: cDNA sequence and
high-level expression in the endometrium of pregnant sheep. Gene 1993; 137: 327-33 1.

[189] Hicks BA, Etter SJ, Carnahan KG, Joyce MM, Assiri AM, Carling SJ, Kodali K,
Johnson GA, Hansen TR, Mirando MA, Woods GL, Vanderwall DK, Ott TL.
Expression of the uterine Mx protein in cyclic and pregnant cows, gilts and mares. J. of
Ani. Sci. 2003; 81: 1561.

[190] Horisberger MA, Stacheli P, Haller O. Interferon induces a unique protein in mouse
cells bearing a gene for resistance to influenza. Proc. Natl. Acad. Sci. U. S. A 1983; 80:
1914.

[191] Horisberger MA, Gunst MC. Interferon-induced proteins: identification of Mx proteins
in various mammalian species. Virology 1991; 180: 185-190.

[192] Deblandre GA, Marinx OP, Evans SS, Majjaj S, Leo O, Caput D, Huez GA, Wathelet
MG. Expression cloning of an interferon-inducible 17-kDa membrane protein
implicated in the control of cell growth. J. Biol. Chem. 1995; 270: 23860-23866.










[308] Seli E, Zeyneloglu HB, Senturk LM, Bahtiyar OM, Olive DL, Arici A. Basic fibroblast
growth factor: peritoneal and follicular fluid levels and its effect on early embryonic
development. Fertil. Steril. 1998; 69: 1145-1148.

[309] Larson RC, Ignotz GG, Currie WB. Transforming growth factor beta and basic
fibroblast growth factor synergistically promote early bovine embryo development
during the fourth cell cycle. Mol. Reprod. Dev. 1992; 33: 432-435.

[3 10] Lim JM, Hansel W. Roles of growth factors in the development of bovine embryos
fertilized in vitro and cultured singly in a defined medium. Reprod. Fertil. Dev. 1996;
8: 1199-1205.

[311] Reynolds LP, Redmer DA. Angiogenesis in the placenta. Biol. Reprod. 2001; 64: 1033-
1040.

[312] Ocon-Grove OM, Alvarez IM, Johnson SE, Ott TL, Ealy AD. Ovine endometrial
expression of fibroblast growth factor (FGF) 2 and concepts expression of FGF
receptors during early pregnancy. Domest. Anim. Endocrinol. 2007.

[313] Guthridge MA, Stomski FC, Thomas D, Woodcock JM, Bagley CJ, Berndt MC, Lopez
AF. Mechanism of activation of the GM-CSF, IL-3, and IL-5 family of receptors. Stem
Cells 1998; 16: 301-313.

[3 14] Rooke J, Ewen M, McEvoy T, Entrican G, Ashworth C. Effect of inclusion of serum
and granulocyte-macrophage colony stimulating factor on secretion of interferon-tau
during the in vitro culture of ovine embryos. Reprod. Fertil. Dev. 2005; 17: 513-521.

[315] Robertson SA, Roberts CT, Farr KL, Dunn AR, Seamark RF. Fertility impairment in
granulocyte-macrophage colony-stimulating factor-deficient mice. Biol. Reprod. 1999;
60: 251-261.

[3 16] Chaouat G, Menu E, Clark DA, Dy M, Minkowski M, Wegmann TG. Control of fetal
survival in CBA x DBA/2 mice by lymphokine therapy. J. Reprod. Fertil. 1990; 89:
447-458.

[3 17] Robertson SA, Sj oblom C, Jasper MJ, Norman RJ, Seamark RF. Granulocyte-
macrophage colony-stimulating factor promotes glucose transport and blastomere
viability in murine preimplantation embryos. Biol. Reprod. 2001; 64: 1206-1215.

[318] Sjoblom C, Wikland M, Robertson SA. Granulocyte-macrophage colony-stimulating-
factor (GM-CSF) acts independently of the beta common subunit of the GM-C SF
receptor to prevent inner cell mass apoptosis in human embryos. Biol. Reprod. 2002;
67: 1817-1823.

[319] Armstrong DT, Chaouat G. Effects of lymphokines and immune complexes on murine
placental cell growth in vitro. Biol. Reprod. 1989; 40: 466-474.









Serum supplementation, previously shown to be a required supplement in culture medium

[210, 21 1], limited the degeneration of bovine blastocysts between days 8 and 11 post-IVF in

these experiments. Attempts to supply M-199 with defined agents known to substitute for serum

in several somatic cell lines (i.e. insulin, transferring, selenium, BSA) failed to provide adequate

conditions for blastocyst development and survival. Others have noted the same requirement for

serum in extended bovine embryo cultures [9, 12, 13]. In one study [9], FBS was superior to

mature cow serum as a medium supplement and both serum types were better than media

formulations lacking serum at sustaining bovine embryonic outgrowths formed on collagen-

coated plates. In the present work, 2.5% FBS limited degeneration and sustained a modest

amount of embryonic growth during culture. An individual blastocyst culture system was used

throughout our studies to remove the confounding effects of group embryo culture on outcomes

and to permit using blastocyst cell numbers for normalizing IFNz production data. The minimum

serum requirement for blastocysts cultured in groups remains unknown, but serum

supplementation is routine during group culture [6-8]. Taken together, serum, and preferably

FBS supplementation is required during bovine blastocyst culture when using M-199 or a related

medium formulation.

Traditional embryo culture systems incubated embryos at an atmospheric oxygen level

(approximately 20%) is greater than that found in the uterus and oviducts (approximately 2-9%)

[227]. Recent work from several groups investigating bovine embryo development indicate that

reducing the oxygen levels during in vitro culture to between 5 and 7% improves proportion of

embryos developing to the blastocyst stage and in several species [215, 228-231, 339-342]. In

this work, a 5% oxygen atmosphere prevented degeneration and sustained cell proliferation

between days 8 and 11 post-IVF. It is generally accepted that reducing the oxygen tension during









Table 2-1. A comparison of medium components for M-199 and KSOM.
M-199 KSOM
COMPONENTS MOLARITY MOLARITY
(mM) (mM)
AMINO ACIDS
Glycine 0.667 0.050
L-Alanine 0.281 0.050
L-Arginine hydrochloride 0.332 0.299
L-Asparagine H20 0.050
L-Aspartic acid 0.226 0.051
L-Cystine 0.050
L-Glutamic acid 0.455 0.045
L-Glutamine 0.685 1.001
L-Histidine hydrochloride-H20014 .9
L-Isoleucine 0.305 0.200
L-Leucine 0.458 0.200
L-Lysine hydrochloride 0.383 0.199
L-Methionine 0.101 0.050
L-Phenylalanine 0.152 0.100
L-Proline 0.348 0.050
L-Serine 0.238 0.050
L-Threonine 0.252 0.200
L-Tryptophan 0.049 0.025
L-Tyrosine 0.100
L-Valine 0.214 0.200
INORGANIC SALTS
Calcium chloride (CaCl2) (dihyd.) 1.701
Magnesium sulfate (MgSO4*7H20) 0.200
Potassium chloride (KC1) 5.330 2.485
Monopotassium phosphate (KH2PO4) 0.353
Sodium bicarbonate (NaHCO3) 26.190 25.003
Sodium chloride (NaC1) 117.240 95.721
OTHER COMPONENTS
D-Glucose (Dextrose) 5.560 0.200
EDTA 0.013
Sodium lactate syrup 10.003
Sodium pyruvate 0.200










ACKNOWLEDGMENTS

First and foremost, I thank Dr. Alan Ealy. You have given me an incredible opportunity,

one which I will carry with me always. I have learned so much under your guidance and we were

even able to have some fun along the way! My experiences here working under you have only

allowed me to grow into a better person, and for that I am forever grateful.

I thank Dr. Peter Hansen and Dr. Carlos Risco who served on my committee. Thank you

both for your invaluable guidance and advice while reviewing my thesis and throughout my time

here. It has been an honor and privilege to have known you both. To Amber Brad, thank you for

your unfailing patience with me while teaching me all of the techniques required to complete my

research. Thank you to everyone in Dr. Hansen' s laboratory for all of their help and patience

with my incessant questions! Thank you to William Rembert for your dedication and providing

all of us IVF'ers with the means to conduct our research.

To all of my lab members, Idania Alvarez, Flavia Cooke, Krista DeRespino, Michelle

Eroh, Kathleen Pennington, Jessica Van Soyc and Qien Yang, thank you for all your help and

friendship these last two years. I will miss all of our morning chats before class and, well, our

chats that usually lasted throughout the day! I wish you all the best of luck in your future

endeavors .




Full Text

PAGE 1

1 DEVELOPMENT OF AN EXTENDED CULTURE SYSTEM FOR BOVINE BLASTOCYSTS: EFFECTS OF OXYGEN TENSION, MEDIUM TYPE AND SERUM SUPPLEMENTATION ON EMBRYO DEVE LOPMENT AND INTERFERON-TAU SECRETION By TERESA M. RODINA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Teresa M. Rodina

PAGE 3

3 To all of my friends and family who have helped me through this time in my life and help shape the person I am today

PAGE 4

4 ACKNOWLEDGMENTS First and foremost, I thank Dr. Alan Ealy. You have given me an incredible opportunity, one which I will carry with me always. I have le arned so much under your guidance and we were even able to have some fun along the way! My experiences here working under you have only allowed me to grow into a better pers on, and for that I am forever grateful. I thank Dr. Peter Hansen and Dr. Carlos Risco who served on my committee. Thank you both for your invaluable guidance and advice while reviewing my thesis and throughout my time here. It has been an honor and privilege to have known you both. To Amber Brad, thank you for your unfailing patience with me whil e teaching me all of the techni ques required to complete my research. Thank you to everyone in Dr. Hansens laboratory for all of their help and patience with my incessant questions! Thank you to William Rembert for your dedication and providing all of us IVFers with the means to conduct our research. To all of my lab members, Idania Alvar ez, Flavia Cooke, Krista DeRespino, Michelle Eroh, Kathleen Pennington, Jessica Van Soyc a nd Qien Yang, thank you for all your help and friendship these last two years. I will miss all of our morning ch ats before class and, well, our chats that usually lasted throughout the day! I wish you all the best of luck in your future endeavors.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 2 LITERATURE REVIEW.......................................................................................................15 The Estrous Cycle.............................................................................................................. .....15 Follicular Development and Ovulation...........................................................................15 Luteal Development and Function..................................................................................18 Luteolysis..................................................................................................................... ...19 Pregnancy...................................................................................................................... .........23 Fertilization.................................................................................................................. ....23 Early Embryonic and Trophectoderm Development.......................................................24 Implantation and Placen tal Development........................................................................26 Placental Hormones.........................................................................................................28 Parturition.................................................................................................................... ....30 Maternal Recognition of Pregnancy.......................................................................................31 Discovery of the Maternal Recognition of Pregnancy Factor in Ruminants...................31 The IFN Gene and its Expression..................................................................................32 Antiluteolytic Action of IFN .........................................................................................33 Other Activities of IFN ..................................................................................................35 Bovine Embryo Culture..........................................................................................................37 Defined In vitro Culture Requirements...........................................................................37 Differences between In vivo and In vitroDerived Embryos...........................................40 Extended Blastocyst Culture...........................................................................................42 Uterine Factors Affecting Embryo Development...................................................................44 Fibroblast Growth Factor-2 (FGF-2)...............................................................................46 Granulocyte-Macrophage ColonyStimulating-Factor (GM-CSF).................................47 Summary........................................................................................................................ .........48 3 MATERIALS AND METHODS...........................................................................................51 In vitro Embryo Production....................................................................................................51 Individual Embryo Culture from Day 8 to 11 Post-IVF.........................................................52 Evaluating Apoptosis a nd Total Cell Number........................................................................55 Interferon-tau Production...................................................................................................... ..56

PAGE 6

6 Statistical Analysis........................................................................................................... .......56 4 RESULTS........................................................................................................................ .......59 Experiment 1: Effect of Medium Type a nd Oxygen Atmosphere on Blastocyst Survival, Development and IFN Production.....................................................................................59 Medium type did not affect the quantity of bioactive IFN as assessed with an antiviral assay, in conditioned medium from blastocysts cultured individually from day 8 to 11 post-IVF (Fig 4-6). However, embryos in cubated in 5% oxygen secreted more (P=0.002) IFN into medium than embryos incuba ted in 20% oxygen (Fig. 4-6). This effect is evident in both medium type s both before and after normalizing data according to embryonic cell numbers on day 11 post-IVF (Fig. 4-7).................................60 Experiment 2: Serum Requirements for Extended Bovine Blastocyst Culture......................60 Experiment 3: FGF-2-Mediated Induction of IFN Production in Bovine Blastocysts Depends on the Level of Atmospheric Oxygen..................................................................61 Experiment 4: The Combined Effects of FGF-2 and GM-CSF on Blastocyst Development and IFNProduction...................................................................................62 5 DISCUSSION..................................................................................................................... ....81 LIST OF REFERENCES............................................................................................................. ..88 BIOGRAPHICAL SKETCH.......................................................................................................116

PAGE 7

7 LIST OF TABLES Table page 2-1 A comparison of medium co mponents for M-199 and KSOM.........................................57 2-2 Medium components that are only included in M-199......................................................58

PAGE 8

8 LIST OF FIGURES Figure page 4-1 Examples of degenerated embryos by day 11 post-IVF ...................................................64 4-2 Examples of Hoescht 33342 and TUNEL stai ning for total cell counts and incidence of apoptosis................................................................................................................... .....65 4-3 Incubating blastocysts from day 8 to 11 post-IVF in M-199 and 5% O2 prevents degeneration................................................................................................................... ....66 4-4 Incubation in M-199 under 5% oxygen conditions yields greater total cell numbers on day 11 post-IVF............................................................................................................67 4-5 Incidence of apoptosis is not affected by medium type or oxygen tension in bovine embryos from day 8 to 11 post-IVF...................................................................................68 4-6 Incubation in atmospheric oxygen increases IFN secretion from bovine blastocysts.....69 4-7 Interaction of medium type and oxygen concentration on IFN secretion on day 11 post-IVF....................................................................................................................... ......70 4-8 Incubating embryos in M-199 containi ng 2.5 or 5% FBS with 5% oxygen limits degeneration from day 8 to 11 post-IVF............................................................................71 4-9 Supplementation with FBS improves embryonic cell number after incubation in 5% oxygen......................................................................................................................... .......72 4-10 Effect of serum supplementation on pe rcentage of TUNEL positive cells during individual embryo culture fr om day 8 to 11 post-IVF.......................................................73 4-11 Degree of serum supplementation and oxygen concentrations during culture impact IFN secretion....................................................................................................................74 4-12 Serum supplementation and atmospheric oxygen conditions act independently to mediate IFN production ..................................................................................................75 4-13 Effect of FGF-2 supplementation on ce ll numbers in embryos on day 11 post-IVF.........76 4-14 Main effects of FGF-2 supplementation and oxygen concentrations on IFN secretion from bovine blastocysts .....................................................................................77 4-15 Treatment with FGF-2 and 20% oxygen increases IFN production from bovine blastocysts.................................................................................................................... ......78 4-16 Effect of FGF-2 and GM-CSF supplementation on embryo cell number.........................79

PAGE 9

9 4-17 Supplementation with FGF-2 but not GM-CSF increases IFN production from bovine embryos................................................................................................................. .80

PAGE 10

10 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DEVELOPMENT OF AN EXTENDED CULTURE SYSTEM FOR BOVINE BLASTOCYSTS: EFFECTS OF OXYGEN TENSION, MEDIUM TYPE AND SERUM SUPPLEMENTATION ON EMBRYO DEVE LOPMENT AND INTERFERON-TAU SECRETION By Teresa M. Rodina December 2007 Chair: Alan D. Ealy Major: Animal Sciences The bovine conceptus produces interferon-tau (IFN ), and this factor acts on the endometrium to block the pulsatile rele ase of prostaglandin F2 alpha (PGF2 ) and prevent luteolysis. Factors secreted from the uterus control IFN gene expression in bovine and ovine conceptuses. The overall goal of this work was to develop a culture system that supports bovine embryonic development until day 11 postin vitro fertilization (IVF) so it may be used as a model to investigate how uterin e-derived factors regulate IFN synthesis and secretion. All embryos were produced via IVF and evaluated fo r overall quality, percent apoptotic nuclei (not study 3 and 4), total cell number and IFN content in the medium. In study 1, individual blastocysts were placed in one of two culture medium fo rmulations, KSOM or M-199, containing 5% FBS and incubated in a 5 or 20% oxygen environment from day 8 to 11. Survival to day 11 post-IVF was greatest for blastocysts cultured in M-199/5% oxygen. None of the treatments affected percenta ge of apoptotic nuclei. IFN concentrations were greater for embryos cultured in 20% oxygen than for those cultured in 5% oxygen regardless of medium type. In study 2, individual blastocysts were cultured in M-199 supplemented w ith different protein

PAGE 11

11 supplements at 5% or 20% oxygen from day 8 to 11 post-IVF. Blastocysts supplemented with serum substitute (ITS), 1% bovine serum albu min (BSA) or 1% fetal bovine serum (FBS) contained more degenerated embryos at day 11, fewer blastomeres, and increased percent apoptotic nuclei compared to blastocyst s incubated with 2.5% and 5% FBS. IFN was detected only in embryos incubated in 2.5 or 5% FBS. In study 3, effects of FGF-2 treatment and oxygen concentration on blastocy st cell number and IFN secretion were ev aluated. Individual blastocysts were cultured in M-199 supplemente d with 2.5% FBS in 5% or 20% oxygen from day 8 to 11 and treated with or without 100 ng /mL FGF-2. Blastocysts in 5% oxygen, regardless of FGF-2 treatment, yielded embryos with grea ter cell numbers. Blastocysts incubated in 20% oxygen had greater IFN secreted in culture medium than those incubated in 5% oxygen. FGF-2 treatment increased IFN secretion in blastocyst s incubated in 5% oxygen but not in blastocysts cultured in 20% oxygen. In study 4, blastocysts individually cultured in M-199 supplemented with 2.5% FBS at 5% oxygen and treated with 100 ng/mL FGF-2, 100 ng/mL GM-CSF, 100 ng/mL of both FGF-2 and GM-CSF, or no growth factor treatment (control). There were no effects of FGF-2 and GM-CSF treatment on IFN secretion, although individual treatment comparisons showed greater IFN secretion in FGF-2 treated bl astocysts when compared to controls. GM-CSF treatment did not affect IFN secretion in blastocysts compared to controls; however mean IFN concentrations were slightly incr eased and not different from FGF-2 treatment. Co-supplementation did not increase IFN secretion in blastocysts when compared with controls. In summary, a culture system for maintaining individual blastocyst development until day 11 post-IVF has been identified. These out comes indicate that this culture system can be used to better understand how IFN expression and embryo development is regulated by growth factors present in the uterine secretory milieu, such as such as FGF-2 and GM-CSF.

PAGE 12

12 CHAPTER 1 INTRODUCTION The sustainability and profitability of liv estock production systems relies on suitable reproductive capacities. Reproductive problems preside in nearly a ll domesticated animals used for food production, and numerous components of th e reproductive process usually are involved with reproductive insufficiencies. Problems associat ed with detecting estrus for artificial mating and failure to maintain pregnancies to term are the predominant problems associated with reproduction in dairy and beef cattle [1]. These issues will typically result in extended calving intervals, decreased lifetime milk production, and an increase in the number of artificial inseminations required to achieve a pregnancy [2]. Dairy producers stand to lose an average of $550 in lifetime milk potentia l with each reproductiv e cycle that fails to produce a calf [3]. The use of estrous synchronization a nd timed artificial insemination protocols has diminished some of these issues related to fertility in cattle; however no solutions for embryonic mortality have remained elusive to date. It is estimated that 57% of all failed pr egnancies occur between days 3 and 21 of pregnancy when the newly formed embryo must develop, announce its presence to the maternal system, and begin attaching to th e uterine lining [1]. Several cruc ial developmental events occur during this period of embryonic growth and pro liferation. Newly formed embryonic cells divide and begin differentiating into a variety of embr yonic and extra-embryonic tissues that will give rise to the fetus and placenta. The developing conceptus, a term used to describe the embryonic mass and its associated placental tissues, must also signal its presence within the maternal system to sustain a progesterone-dominated environmen t conducive for pregnancy. In ruminants, this crucial event occurs by placental production of a Type I interferon, termed interferon-tau (IFN ).

PAGE 13

13 Adequate development of the initial placenta l tissue lineages and ample production of IFN are vital for pregnancy success in ca ttle and other ruminant species. The predominate method of studying early bov ine embryonic development is through the utilization of in vitro production and in vitro culture of embryos. The production of bovine embryos using in vitro maturation, fertilization, and culture technologies provides scientists with a powerful tool for examining the initial events of embryogenesis in cattle and related species. Establishing culture conditions that support bovine embryo deve lopment during the blastocyst stage would offer considerable benefits. For exam ple, extended culture could provide additional end-point measurements that can be used to assess how media, supplements, and atmospheric conditions impact the production of embryos suitabl e for recipient transfer. Also, a blastocystbased culture system could assi st in describing how uterine-de rived factors influence embryo development. Uterine secretions are essential for proper conceptus elongation. Ewes that fail to develop uterine glandular epithelium, a model cr eated by exposing neonatal ewes to progestins, are unable to nourish a pregnancy past th e hatched blastocyst stage [4, 5]. Extending bovine embryo culture beyond the bl astocyst stage has been completed by several groups. Culturing groups of embryos in microdrops of medium sustains blastocyst viability to day 10 or 11 postin vitro fertilization (IVF) [6-8]. Bovine blastocyst development after hatching (i.e. protrusion fr om the zona pellucida) is comp leted, at least to some extent, either by creating trophectoderm outgrowths on ex tracellular matrices [7, 9-11] or by provoking elongation by placing bovine blastocysts in agarose tunnels [12-14]. Unfortunately, the embryonic disk, or epiblast, fails to develop prop erly in most cases, and the resulting tissue mass is comprised primarily of trophectoderm and primitive endoderm [9, 12-14].

PAGE 14

14 Insufficient quantities of the maternal rec ognition of pregnancy factor, interferon-tau (IFN ), also are observed in ewes containing litt le to no glandular epith elium [4, 5]. Several intrinsic and extrinsi c factors affect IFN expression during early pre gnancy [15, 16], but length of the developing conceptus is the pr edominant determinant of overall IFN production [17-19]. Therefore, it is imperative that the physiologi cal events associated with early conceptus development and IFN production be fully understood so that resolutions for pregnancy failures can be conceived. We examine several of the basic reproduc tive processes surrounding early embryonic and conceptus development in cattle and provide an update on our current knowledge of key biological processes that influence fecundity. The literature review will begin with a brief overview of the estrous cycle in cattle, fo llowed by an overview of pregnancy where a description of embryonic and conceptus developm ent is presented. Also, an overview of IFN expression and action will be provided. A brief revi ew of past and presen t bovine embryo culture systems and their similarity with in vivo derived embryos will be reviewed. The literature review will end by discussing some uterine-derived fact ors that impact embryonic development. The research focus of this thesis then will be presented. The overall goal of the research was to develop a culture system that can be used to examine bovine blastocyst development. Through a series of studies, a suitable system for mainta ining blastocyst viabili ty was created, and its efficacy for use in examining how uterine-deri ved factors affect embryo development and IFN production was tested.

PAGE 15

15 CHAPTER 2 LITERATURE REVIEW The Estrous Cycle The estrous cycle consists of a series of predictable phys iological events that occur between successive periods of sexual receptiv ity, also known as estrus. These series of reproductive events occur throughout the females adult life until it is interrupted by pregnancy and, in some species, season of the year. The es trous cycle provides the female with repeated opportunities to copulate and become pregnant. Estrus is preceded by the development and ovulation of a single follicle in cattle, and hormones secreted by this follicle drive female receptivity to males and ovulation. Immediately following ovulation, the tissue mass that made up the follicle differentiates into a body of tissue te rmed the corpus luteum (CL). As the CL ages, copious amounts of progesterone are produced to support uterine secr etory activities and diminish uterine contractility, both of which sustain pregnancies in cattle. If there is no pregnancy, the corpus luteum will undergo regression, or luteolysis, and the female will return to estrus. In cattle, the interval be tween estrus activity, or the length of the estrous cycle, averages from 20 to 23 days. Follicular Development and Ovulation Folliculogenesis is the process by which follicl es capable of being ovulated are formed on the ovary in the female from a pool of nave, or primordial follicles [20]. Folliculogenesis is an ongoing process in cattle that begins prior to puberty, and cycl es of follicle development and death (or atresia) are seen re peatedly throughout estrous cycles and pregnancy. Follicles are present on the ovary in a hierarchy according to th eir stage of developmen t and functional status [21]. These waves of follicle growth occur by the developmental progression of primordial follicles through three primary processes: 1) recr uitment, 2) selection, and 3) dominance [22].

PAGE 16

16 During follicle recruitment, a group, or cohort, of follicles begins developing from the primordial pool and mature over a 45 to 60 day period. A sel ection process occurs as cohorts of follicles continue to increase in size and eventually only a single follicle will to continue develop. The smaller, subordinate follicles will undergo a degenerative process, known as atresia, and fail to ovulate. Greater than 99% of ovarian follicles w ill undergo atresia throughout the lifetime of the cow [23]. The remaining follicle, which now is term ed the dominant follicle, limits the ability of other follicles to develop and is capable of ovulating if endoc rine events associated with hypothalamic-pituitary-ovarian axis permit it. During the first follicular wave of an estrous cycle, luteal-derived progesterone levels do not perm it ovulation. As a result, these dominant follicles will also undergo atresia, allowing a new follicular wave to begin. Folliculogenesis is controlled by hormones produced by the anterior pituitary, namely luteinizing hormone (LH) and follicle stimulating hormone (FSH). As follicles develop, they become responsive to gonadotropins, and primar ily FSH, which mediates further development. When the dominant follicle reaches a particular differentiated state it is hypothesized that its growth can be sustained by lower levels of FSH which, in conjunction with inhibin, do not facilitate the further recruitment and developmen t of subordinate follicle s. Inhibin is a 32-kDa dimeric protein composed of a disulfide bonded and -subunit that is produced by granulosa cells under the control of FSH [24, 25]. Within this dominant follicle, theca cells produce androgens in response to LH. The granulosa ce lls within the follicle then aromatize these androgens to estradiol under th e control of FSH [26]. As th e dominant follicle emerges, increased levels of estrogen and inhibin will feed back onto the pituitary and decrease FSH secretion. The reduction in circulating FSH prev ents most large follicles from continuing to development. The production of both LH and FSH is controlled by numerous systemic and

PAGE 17

17 neuronal signals. One of the primary mediator s of both hormones is a hypothalamic peptide known as Gonadotropin Releasing Hormone (GnRH). This factor is released from hypothalamic nuclei in specific regions of the hypothalamus and acts on LH and FSH-producing cells in the anterior pituitary via tr ansport through a closed portal hypotha lamic-pituitary system to stimulate hormone production and release [27, 28]. Ovulation of a dominant follicle depends on whether luteal-derived progesterone is present. When circulating progesterone concentr ations are high, which is the case throughout 80% of the estrous cycle, the dominant follicle will undergo atresia after 3-4 days of dominance. The regression of the dominant follicle lifts the inhibin-mediated block in folliculogenesis and permits a successive wave of follicle selection and dominance. Typically, two to three waves of follicle growth/atresia exist during an estrous cycl e, but as little as one follicular wave and as many as four have been reported [29, 30]. Ovulation of the dominant follicle will occu r in the absence of elevated luteal progesterone. This process is driven by changes in the hypothalamic and pituitary responsiveness to ovarian hormones. Notably, the loss of progesterone permits follicle-derived estradiol to interact with the hypothalamic su rge center, which generates the preovulatory surge of LH [31, 32]. Between 24-36 hours after the LH surge, the dominant follicl e will rupture, releasing the oocyte. This rupturing is mediated by histamine and prostaglandin E2 (PGE2) production, which increases blood flow to the ovary after the LH su rge, resulting in an increase in hydrostatic pressure within the follicle [33]. Theca cells of the follicle begin producing progesterone, which stimulates collagenase synthesis by the theca inte rna cells to breakdown co llagen in the follicle. Also, granulosa cells increase follicular fluid s ecretion, causing intrafollicular pressure build up

PAGE 18

18 [33]. Finally, PGF2 causes smooth muscle contraction with in the ovary. Collectively, these coordinated events result in follicle rupture [33]. Luteal Development and Function Progesterone is the hormone responsible for preventing ovulation, pr eparing the uterus for pregnancy, and maintaining a pregnant state in cattle. Progesterone is produced by the CL, which is a mass of tissue created from differen tiated follicle cells around the time of ovulation. During ovulation, the follicle wa ll collapses and the basement membrane between the theca interna and granulosa cells breaks down, giving way to the development of an extensive vascular network where blood vessels invade the antral space of the follicle forming the corpus hemorrhagicum (bloody body) and eventually the co rpus luteum (CL; yellow body). In domestic animals the major luteotropin is LH, which stim ulates development of the CL and production of progesterone. The CL is comprised of two types of luteal cells: large and small. Unlike the small luteal cells, large luteal cells do not respond to LH stimulation, however, they are responsible for a majority of the basal secretion of progesterone [34]. In cattle, LH pulses are required for the development of a fully functional CL [35-39]. One of the important actions of progestero ne is to prevent ovulation. Progesterone acts on the hypothalamus to reduce GnRH pulse freque ncy and prevent the hypothalamic surge center from generating an LH surge. These activities reduce LH and FSH pulse frequency but increase pulse amplitude of LH and FSH; conditions that favor luteal functi on. Gonadotropin production is sufficient to allow follicular development to occur, however ovulation is prevented by the negative feedback actions of pr ogesterone on the hypothalamus [40]. Copious progesterone secretion by the CL is vital for the maintenance of pregnancy. Progesterone stimulates the endometrium to produce nutrients, growth factors,

PAGE 19

19 immunosuppressive agents, enzymes, and ions needed for conceptus development [41]. Among inseminated cows, lower systemic concentrations of progesterone are a ssociated with lower pregnancy rates [42, 43]. Progesteron e actions on the uterus are me diated by its interaction with progesterone receptors present in luminal and gla ndular epithelium, the primary site of uterine histotroph production. In several sp ecies, like the ewe, luminal a nd glandular epithelia show a loss of progesterone receptors ju st before conceptus implantation. This decrease in progesterone receptors seems to be needed for onset of differe ntiated functions of progesterone, in terms of secretory protein production, such as uterine milk proteins and osteopontin. The actions of progesterone on the endometrium throughout the majority of gest ation are mediated by the endometrial stroma, which remains progesterone receptor positive. Stromal cells are capable of producing several types of growth factors, including hepatocyte growth factor (HGF) and two forms of fibroblast growth factors, FGF-7 and -10. These factors are responsive to progesterone and mediate the interactions that ar e crucial for pregnancy support [44]. Luteolysis Luteolysis is the termed used to describe the structural a nd functional demise of the CL. This process is essential for the initiation of a new reproductive cycle and another mating opportunity if cows are not pregnant. Two main phase s of luteolysis include 1) a loss of ability to synthesize and secrete progesterone and 2) the stru ctural demise of the CL itself [38, 45]. During most of the estrous cycle, high levels of proge sterone create a block on th e uterus to prevent it from generating the luteolytic signal that prom pts CL regression. After a set period of time (12 days in the ewe; 16 days in the cow) this bloc k is diminished by the estrous cycle-dependent loss of progesterone receptors in luminal and gl andular epithelium [39], which enables the endometrium to become able to produce hormo nes that induce luteol ysis. Prostaglandin F2

PAGE 20

20 (PGF2 ) is a prostaglandin produced by the uterus whic h is capable of terminating a pregnancy at any time in the cow. Throughout the estrous cycle, progesterone stimulates the accumulation of phospholipids in the luminal and glandular epithelia of the endometrium, and these substrates are used to generate arachidonic acid, which is require d for synthesis and secretion of PGF2 [38, 46]. As progesterone receptor (PR) abundance decreases in luminal and glandular epithelium, estrogens acting through their receptors, termed estrogen receptors (ERs), stimulate the expression of oxytocin receptors (OTR) [46, 47]. In sheep, OTRs first appear on day 14 and reach a peak at estrus [48, 49], while in the cow OTRs appear between days 15 and 17, just prior to luteolysis [50-52]. There is some debate as to how this up-regulation of OTRs occurs. It has been hypothesized using ewes as the model species th at estradiol is acting through its receptor to cause the up-regulation on OTR. Ho wever if this were an estradiol-dependent event, then it would be plausible to assume then that ER recep tors would need to be up-regulated first. Two studies conducted by Robinson a nd colleagues would argue otherwise in the cow. In one experiment, luminal epithelium ER did not incr ease until between day 16 and 18 in the cow, slightly after, approximately 1-2 days later, th an the initial up-regulation of OTRs. Additionally, OTR mRNA was detectable on day 14, while ER mRNA was not detectable until day 16 [53]. In ewes, there is conflicting evidence as to whic h receptor is up-regulated first. Spencer and colleagues found that ER concentrations increas e before OTR [54] while Wathes and Hamilton observed the exact opposite [49]. It remains plausible that an up-regulation of ER is not required for estradiol to stimulate OTR expression in endometrium, or perhaps the OTR gene upregulation is mediated vi a estradiol acting in a paracrine manne r in other cells, such as the deep

PAGE 21

21 gland ERs [53]. Results from several studies i ndicate that estradiol is not required for the upregulation of OTRs, although it may enhance the process [55-58]. In the cow and other ruminant species, oxytocin is released by both th e posterior pituitary and the CL and can act on the endo metrial epithelium to produce PGF2 The synthesis and release of PGF2 from the uterus acts back on the CL to, amongst other things, induce further oxytocin release, and this oxytocin stim ulates further production of uterine PGF2 [59, 60]. This positive feedback loop that is crea ted continues until the CL is fully degraded and luteolysis is complete [61]. PGF2 is considered a luteolysin and causes luteolysis by both abating progesterone production and inducing the struct ural demise of the CL. While the exact mechanism remains unclear, it is hypothesized that functional luteolysis by PGF2 is mediated by the endothelial cellderived vasoconstrictive peptide known as e ndothelin-1 (ET-1), which can alter the normal pattern of progesteron e synthesis [62]. PGF2 is thought to activate the ET-1 gene in luteal endothelial cells, thus stimulati ng ET-1 production. ET-1 secretion th en further stimulates luteal PGF2 production, which will act in a paracrine manner on the CL to further enhance ET-1 synthesis and secretion [37, 62, 63]. ET-1 binds to a specific receptors found on both small and large luteal cells. This activation then d ecreases both basal and LH-induced production of progesterone, possibly by interrupting the cAMP mediated pathway leading to progesterone production [62]. This mechanism of action has been elucidated in the rat [64, 65] and pig [66], although it still remains unclear if this same pathway interruption occurs in the cow and ewe. At the time of structural luteolysis on the cow a nd ewe, it was shown that both ET-1 concentration and mRNA encoding for ET-1 were greatest in lut eal tissue at this time [62]. Further evidence

PAGE 22

22 shows that ET-1 blocks proge sterone production in bovine and ovine luteal ce ll culture and PGF2 treatment stimulates ET-1 expression and secretion in these lu teal cells [67-69]. A variety of agents have been im plicated as mediators in the PGF2 -induced structural demise of the CL in the bovine and ovine, but one of the major pl ayers is tumor necrosis factor(TNF). Several groups have shown that PGF2 elevates TNFproduction by macrophages in both the bovine and ovine CL [70, 71]. Th ere is evidence to suggest that TNFinduced apoptosis plays a role in struct ural luteolysis. Several reports indicate that apoptosis occurs during luteolysis in ruminants [72-74], while in rodents there is a strong correlation between apoptosis and maximal expression for PGF2 receptor mRNA [75]. PGF2 elevates TNFlevels in both the bovine and ovine CL and demonstrat ed that endothelial ce lls express high levels of TNFRI, a TNFreceptor type, and that th ey are sensitive to TNF-induced apoptosis in vitro [70, 71]. One group positively linked both PGF2 induced ET-1 and TNFin the resulting functional and structural demise of the corpus luteum. As previously discussed, ET-1 plays a key role in functional luteolysis through the inhib ition of progesterone production by luteal cells and TNFhas been linked to apoptosis in luteal endo thelial cells. It has been hypothesized that during early and mid luteal phase s, the high levels of progester one that still persist, in conjunction with low levels of TNFand a TNFR1, prevent apoptosis in endothelial cells. It seems the progesterone producing cell s are resistant to apoptosis and serves to protect luteal cells [76]. However, at the ti me of luteolysis, PGF2 stimulates ET-1 secretion by luteal cells and TNFproduction by local macrophages up-regulate one anothers production via a positive feedback loop, which synergize to inhibit pr ogesterone production. These low progesterone levels in conjunction with increased TNFR1 expression [70, 71] facilitate TNFapoptosis of

PAGE 23

23 the endothelial cells in the CL. The end result le ads to both functional and structural luteolysis [76]. Pregnancy If copulation takes place duri ng estrus, there is an opportunity for the sperm and egg to unite and form a new organism, which initially is termed a zygote or embryo. This embryo will develop first in the oviduct and then in the pr ogesterone-dominated uterine environment. By a particular period in development, the developi ng placenta must attach to the uterine wall to create a direct line of communi cation between mother and fetus and allow for nutrient, gas and waste exchange. The placenta will serve as this essential connection between the mother and fetus throughout the duration of gestation until a cri tical series of signals are initiated to induce parturition, or birth of the fetus. Fertilization Fertilization is the process by which sperm and egg unite and their haploid chromosomes form a new, genetically distinct diploid or ganism [77]. Sperm travels through the female reproductive tract in a regulated process that ensures those sp ermatozoa with normal morphology and vigorous motility will have the greatest chance of fertilization [78]. Sperm that remain viable through the arduous journey through th e cervix and vagina enter th e oviduct and travel to the uterotubal junction where a single sperm will fuse with the oocyte and contribute its genetic material to create a genetically distinct organism. Upon insemination, spermatozoa in the female reproductive tract are not capable of fertilizing an oocyte. Before fertilization can occur, three major phases of final spermatozoa maturation and development must take place. The first phase involves an alteration of the sperm membrane, an event termed capacitation. In the bovi ne, capacitation occurs in the oviduct of the female reproductive tract by interactions with glucosaminoglycans [79-82]. This interaction

PAGE 24

24 leads to altered flagellar motility and the developm ent of the capacity for the sperm to fuse with the oocyte [77]. The sperm then penetrates th e cumulus oophorus, which consists of cumulus cells and extracellular matrix. Once through the cumulus oophorus, the sperm comes in contact with and binds to the zona pellucida, at which point the acrosome reaction is initiated by ZP3. This constituent glycoprotein of the zone pell ucida, called ZP3, binds to the receptors on the anterior head of the acrosome-intact sperm [83, 84]. Once ZP3 binds, a chain of events occurs through the activation of phospholip ase C (PLC) and elevated cystolic calcium levels. This directly drives the acrosome reaction by continuously pushing calcium into the plasma membrane. Acrosome reacted sperm penetrate th e zona pellucida where fusion of the outer acrosome membrane to the plasma membrane take s place, resulting in sperm-egg adhesion and a release of acrosomal contents [77]. Finally, an increase in cytosolic calcium instigated by sperm egg fusion induces the oocyte to proceed through its final phases of meiosis. This activity also induces cortical granules to expel their contents This event alters the zona pellucida to limit polyspermy [85]. Early Embryonic and Trophectoderm Development Once fertilization has occurre d, the male and female pronucle i of the zygote will undergo synagmy within the cytoplasm. The newly formed zygote begins mitotic cellular divisions to generate multiple embryonic cells, or blastomeres. This newly formed entity generally is referred to as an embryo, but in reality this new entity is best referred to as a conceptus since it also contains the ability to form extraembryonic tissues (placenta). Both terms will be used in this narrative. During the first few mitotic divisions, each blastomere has the capability to develop into a completely formed individual [86], an event known as totipotency. As development progresses, totipotency is lost in some blasto meres but not others. The initial segmentation of

PAGE 25

25 embryonic cells is the first in many differe ntiation events that the embryo proper and extraembryonic membranes go through to ge nerate a viable fetus and placenta. It takes approximately 96 hours for the bovine embryo to travel through the oviduct and into the uterine body. Normally, bovine embryos have reached the 16-cell stage in development, which equates to developing through four cell divi sions. After this poin t, subsequent mitosis generates a mass of cells, termed a morula. Blas tomeres will continue to undergo division and will begin to compact. During compaction, tight j unctions form between blastomeres lining the outside of the embryo. Soon thereafter, the first si gns of blastocoele formation become evident as fluid begins to build up in the center of the morula [87, 88]. The timing of blastocoele formation varies among individual embryos and is dependent on the rate of fluid entry into the newly formed cavity and the extent of tight junc tion formation between blastomeres [87, 89, 90]. During the compaction phase of morula developmen t, two cells types may be distinguished from one another [88]. A population of flattened, outer cells will form the trophectoderm. This tissue will give rise to outer layer of the placenta. The remaining inner cells will gather at one pole of the embryo and form the inner cell mass, which al so is termed the embryonic disk, or epiblast [87]. These cells eventually will differentiate into various feta l and extraembryonic tissues. By approximately day 9-10 of development, a small slit forms in the zona pellucida, allowing the blastocyst to separa te from the zona pellucida in a process termed hatching [91, 92]. Once hatched, the inner cell mass will bulge to the outside of the sphere-shaped blastocyst and is clearly discernible as the embryonic dis k, which is still covere d by trophectoderm cells, termed the Raubers layer. As hatched blastocy st development progresses, trophectoderm cells within the Raubers layer will be shed.

PAGE 26

26 Over the next several days, the bovine con ceptus remains free floating in the uterine lumen as embryonic and extraembryonic developm ent continues [87]. Between days 8 and 10 of development, endoderm emerges from the inner cell mass and lines blasto coel cavity underneath the trophectoderm. This tissue eventually will fo rm the inner border of the yolk sac. Between days 12 to 14 of development, the outer cell s of the inner cell mass begin to polarize and differentiate into embryonic ectoderm. At this po int the inner cell mass appears to be composed of multiple cell layers [87]. Between days 14 to 16 of development, the mesoderm layer migrates from the inner cell mass between the trophectode rm and endoderm to separate the yolk and amniotic sacs, as well as forming the inner borde r of the chorion and eventually the umbilical cord [93]. Approximately the same time that embryonic cell lineages are emerging (days 12-16), the conceptus begins to elongate first into an ovoid shape and soon thereafter into tubular and finally a filamentous form [93]. After 17-18 days of de velopment, the embryo may occupy two-thirds of the uterine horn ipsilateral to the ovary bearing the ovulation and may be greater than 160 mm in length [94]. Within 1-3 days later (day 18-20), the embryo usually occupi es the entire gravid uterine horn and encroaches into the contralate ral horn. At this stage of the development the amnion forms through encroachment of mesoderm and inversion of the trophectoderm. Finally, a heartbeat is detectable between 20-22 days of development [95]. Implantation and Placental Development Implantation is best described as a tr ansitional stage during pregnancy where the conceptus assumes a fixed position on the endomet rium and begins its intimate physiological relationship with the uterus [96] The extent to which placenta l invasion into the uterus is dictated by the type of placenta utilized by that particular species. In the bovine and other ruminants, a synepitheliochorial placenta forms between mother and fetus. The placental cells

PAGE 27

27 responsible for uterine invasion in cattle and other ruminants are termed binucleate cells or multinucleated giant cells [97-100] These cells are first detect ed around day 16-17 of pregnancy and compose approximately 20% of the trophect oderm by day 25 of pregnancy in cows. They remain abundant until just a few days before part urition, at which time there is a significant decline in binucleate cell number [99, 101, 102]. The binucleate ce lls originate from mononucleated trophectoderm and undergo a pro cess known as acytokinesis, where nuclear division occurs but there is no cytoplasmic division. These binucleate cells contain large secretory granules which occupy about 50% of their volume [99, 100]. Once these cells have transformed to binucleate cells th ey migrate into the maternal ep ithelial layer and fuse with the maternal epithelial cells to form trinucleated hybrid cells. The lysis of these giant placentaluterine cells creates the syncytium during early pregnanc y, and their presence throughout pregnancy is thought to facil itate uterine-placental communica tions and potentially promote nutrient and gas exchange [97-99]. The trophoblast of the bovine conceptus posse sses areas of numerous microvilli that form into structures known as the cotyledons, which attach to specific ar eas on the endometrium known as caruncles. The finger-like projections on the fetal cotyledon fit into crypts of the maternal caruncle and together they make up the unit known as a placentome [93, 99]. The placentomes serve as the sites of nutrient, gas an d waste exchange between the mother and fetus. Additionally, they act as a barrier by preventing the migratio n of non-placental cells across the two entities [103]. These placentomes can grow to a size of 10-12 cm long and 2-3 cm thick towards the end of gestation in cattle. They are found throughout both uterine horns but are larger in the horn containing the pregnancy. The contact surface area between the fetus and

PAGE 28

28 mother can reach as much as 130 m2 due to this extensive interdigitation between the highly villus cotyledon and plentiful crypts of the caruncle [93, 99]. Grosser originally defined the ruminant placenta as syndesmochorial because the maternal epithelial layer degrad ed during early pregnancy [104]. Hi s initial conclusions were that the fetal epithelial layer lysed uterine epithelial ce lls so the placenta coul d be in direct contact with the maternal connective tissue. Future wo rk questioned this contention, notably because during midand late-gestation a majority of the uterine epithelium is reformed in the intercaruncular areas in cattle, sheep, deer and goats. In sheep and deer, caruncular areas remain free of uterine epithelium throughout pregnanc y, but epithelial re-growth is observed in caruncular regions of the cow and doe (femal e goat) [99, 105, 106]. In all ruminant species, limited placental cell migration and fusion persists in the in tercaruncular zones throughout pregnancy. Today, some researchers classify th e ruminant placenta as epitheliochorial (or desmochorial) under the misconception that the s yncytial state is transient [107, 108]. However, the synepitheliochorial or syndesmochorial cla ssification remains the more precise terminology for placentation in ruminants. Placental Hormones The placenta acts as a endocrine, paracrine, and autocrine organ and produces a wide range of steroid and peptide hormones that facil itate fetal development and aid in altering the maternal physiology to support pregnancy [103] Several hormones are commonly produced by mammalian placentae, including estrogens, proge sterone, and placental lactogen. However, ruminants also produce some unique secretory factors, including IFN the maternal recognition of pregnancy factor in ruminants. This unique hormone will be discussed later in the review. In ruminants and many other mammalian species, the placenta produces estrogens and progesterone. The main placenta-derived estrogen product is estrone-3-sulfate. Production of this

PAGE 29

29 variant may be detected locally as early as 33 days of gestation in cattle and is first detected in peripheral circulation around day 70-100 of gestation. Its peak concentrations reach 5-30 nmol/L from about day 265 of gestation through partur ition [109-111]. The biological role of this estrogen variant has yet to be discovered, however it is implicated in regulating myometrial activity, placental matura tion and softening of th e birth canal [109-112]. Placentae from various species produce copious quantities of progesterone. However, unlike the sheep and horse, where the placenta will take over as the dominant source of progesterone at a particular stage of gestati on, progesterone production by the bovine placenta is not sufficient to maintain pregnancy. Instead, lute al-derived progesterone mu st also be available throughout gestation to sustai n a pregnant state [110]. One of the main protein hormones produced by the ruminant placenta is placental lactogen (PL). PLs are produced by the binuc leate cells [113-115]. This non-glycosylated, single-chain, 23-kDa protein is st ructurally similar to prolactin and growth hormone. It can bind and activate prolactin and growth hormone r eceptors [103, 113-120]. The onset of PL production in the ewe occurs on day 16 of pregnancy [121, 122] In the cow, there is a slow rise of PL throughout pregnancy corresponding w ith a continuous fall in the fetal serum beginning at about day 75 of pregnancy [123]. Throughout gestation in the cow and greater than two thirds of gestation in the ewe, fetal PL c oncentrations remain consistently higher than those in maternal blood [123, 124]. The precise functions of PL in the rumi nant remains speculative, but recent work implicates this hormone in regulating endometrial gland development. In utero biological effects of ovine PL on endometrial differe ntiation and function has been de monstrated in ewes receiving ovarian steroid replacement therapy, where intraute rine injections affect ed endometrial function

PAGE 30

30 via uterine milk protein mRNA and osteopontin mRNA levels a nd uterine gland density were increased [125]. In addition to stimulating endom etrial gland morphogenesi s, placental lactogen has also been implicated in regulating water a nd electrolyte balance, mediating fetal growth and development, and potentially serving to regulate metabolism and behavior [126, 127]. Parturition Parturition is a hormonal process where the fetu s signals to the mother that it is ready to be expelled from the uterus. St ress signals initiated by the fetus must lead to enormous changes within the maternal environment to transition fr om a state conducive to maintaining a pregnancy to one that facilitates removal of the fetus fr om the uterus. Progesterone produced by the CL maintains its dominance as the main hormone of pregnancy in cattle, with a gestation of approximately 280 days. By approximately 250 days of gestation, plasma progesterone concentrations reach approximately 7-8 ng/mL an d drop steadily afterwards to about 3-4 ng/mL. The day before parturition progesterone drops to a nearly undetectable level [128]. Conversely, estradiol concentrations remain fairly low throughout mo st of gestation (approximately 300 pg/mL by mid-gestation) but increase just be fore parturition (4000-6000 pg/mL) [129]. Current dogma implicates fetal stress signals as the initiator of parturition in many mammals. In sheep and presumably cattle, fetal co rtisol appears to be the initial parturition signal. Cortisol, stimulated by corticotrophi n releasing hormone (CRH) and produced by the adrenal cortex, will act on the placenta to use progesterone as a substrate for estrogen production, thereby reducing progesterone secretion by th e placenta. This shift in steroid production increases myometrial contractions [130]. During th e cortisol surge at the onset of labor, a feedforward endocrine loop between cortisol, CRH, and ACTH is estab lished in the fetus, and this drives the endocrine events that produce active labor [131, 132]. It is hypothesized that increased uterine activity is, in pa rt, due to exogenous oxytocin producti on by the posterior pituitary of the

PAGE 31

31 mother, which is released in a pulsatile manner during pregnancy. Myometrial OTR concentrations in the cow will increase gradua lly throughout pregnancy, reaching its peak weeks before the onset of labor. Within caruncular endometrium, OTR levels remain low until just prior to parturition. A sudden surge of oxytocin into th e maternal blood system at the onset of labor along with the elevated concentrat ion of myometrial OTRs is th e likely cause of myometrial contractions during parturition [133]. Maternal Recognition of Pregnancy Once the newly created conceptus has begun to develop, an essential line of communication between the embryo and uterus must be established to maintain a pregnant state. It is particularly crucial that the embryo signal its presence to the mother to ensure continued CL function. This process is one component of what is commonly known as maternal recognition of pregnancy. In ruminant species, this key signa ling process is directed by a novel protein and member of the Type I interferon family, termed IFN Discovery of the Maternal Recognitio n of Pregnancy Factor in Ruminants Maternal recognition of pregna ncy in cattle and sheep was firs t investigated in the 1960s by Drs. Moor and Rowson. In a series of studies th ey determined that the presence of a viable embryo by day 12 and 16 post-estrus in ewes and cows, respectively, was required for the continuation of CL function beyond the time of a normal estrous cycle [134-138]. The nature of this factor remained unknown until Martal, et al. (1979) discovered the embryo-derived factor, initially termed trophoblastin, was proteinaceous in nature [139]. Intraute rine injections of homogenates from day 14-16 ovine embryos succ essfully maintained the CL lifespan by one month, while intrauterine injections of hom ogenates from day 21-23 ovine embryos did not extend the CL lifespan [139, 140]. Sh ortly thereafter, researchers at the University of Florida discovered that low molecular weight proteins isolated from ovine and bovine conceptus

PAGE 32

32 contained this antiluteolytic ac tivity. When injected into uterin e lumen of non-pregnant ewes or cows during diestrus, conceptus secretory prot eins from day 15-16 ovine conceptuses and 16-18 bovine conceptuses successfully extended luteal functi on in cyclic ewes and cows [140, 141]. These proteins were termed trophoblast protein1, or oTP-1 and bTP-1 for the ovine and bovine counterparts, respectively. Sequencing of oTP-1 cDNA isolated from a day 13 ovine conceptus cDNA expression library determined that the protein product was st ructurally similar to Type I interferons (IFN) [139, 142]. This newly disc overed trophoblast-derived interferon was then named interferon-tau (IFN ). The IFN Gene and its Expression Expression of IFN genes is distinct from many other Type I IFNs. Of pa rticular note is the lack of IFN expression in response to a virus or pathogen challenge, which is a hallmark feature of most other Type I and Type II (IFN ) IFNs [143, 144]. Also, IFN is not expressed by a variety of cell types, incl uding immune cells, as are mo st other IFNs. Instead, IFN genes are transcribed solely in the trophectoderm for a specific period during early pregnancy [145, 146]. These unique features of IFN expression likely were conc eived as the ancestral IFN gene was created in percoran ruminant ances tors soon after their divergence from other Artiodactyls, such as camels, llamas and pigs approximately 36 millions years ago. It is proposed that the ancestral IFN gene was created by duplication of the IFN gene. Coincident or soon after this duplication event, the promot er region of the new IFN gene was replaced by sequences that permitted placental-speci fic gene expression [16, 146, 147]. IFN genes have been identified in the Bovidae, Cervidae and Giraffidae families by Southern Blot analysis, but comparable genes could not be found in other mammalian species [16, 148].

PAGE 33

33 IFN mRNA is expressed for a brief peri od of time during ma ternal recognition, beginning early in bovine embryo development a nd reaching peak gene expression at day 14 coincident with the onset of concep tus elongation in cat tle. [145, 149-152]. IFN mRNA can be detected until approximately day 25 of devel opment but is not expr essed thereafter [145, 150, 151]. IFN protein can be detected in conditioned medium as early as the late morula or early blastocyst stage of development, approxima tely day 6-7 post-fertilization [153, 154]. Although IFN mRNA abundance reaches its peak at day 1314 of development, the overall production of IFN protein continues to increase between days 13 and 19 of pregnancy due to the considerable growth of trophectoderm over this period of development [149, 155-157]. Antiluteolytic Action of IFN The extension in CL function is consid ered as the primary activity of IFN during early pregnancy. IFN accomplishes this task by acti ng on the uterus to limit the pulsatile secretion of uterine-derived PGF2 normally occurring during late diestr us. Recombinant forms of both ovine and bovine IFN are able to mimic the biological activities of native IFN Intrauterine injections of recombinant bovine and ovine IFN in cattle extend luteal func tion and repress the pulsatile release of uterine PGF2 in response to oxytocin challenge [150, 158-160]. In a pregnant ewe and cow, IFN limits PGF2 pulsatility by limiting expression of oxytocin receptors within the e ndometrial epithelium. Flint and Sheldrick (1986) demonstrated that oxytocin receptor concentrations are decr eased in early pregnancy compared to the concentrations of a normal estr ous cycle in ewes [161]. This effect of pregnancy could be mimicked by administering IFN or ovine conceptus secretory pr oteins to cyclic ewes [139]. Studies in ewes also revealed that a decrease in estrogen rece ptor abundance corresponded with the decreased number and binding affinity of oxytocin receptors on the endometrium [162, 163].

PAGE 34

34 This activity is mediated within the endometr ium by two IFN-regulated transcription factors, Interferon Regulatory Factor-1 (IR F-1) and Interferon Regulatory Factor-2 (IRF-2). IRF-1 is a transcriptional activator of IRF-2, which is best known as a transcriptional repressor [164]. Luminal and glandular epithelial expression of both IRF-1 and IRF-2 increases in non-pregnant ewes supplemented with IFN [54]. The current working model of the antiluteolytic actions of IFN to limit oxytocin recepto r expression, and thus luteolysis, is that IFN stimulates IRF-1 gene transcription in endometrial epithelium, wh ich in turn increases IR F-2 gene transcription. IRF-2 represses ER gene expres sion by binding to an interferon re gulatory factor element, and this loss in ER expression prevents the up-regul ation of OTR expression. Collectively, this loss in OTR prevents oxytocin from inducing PGF2 synthesis [54, 164, 165]. The estrogen receptor-mediated theory of oxytocin receptor gene expression regulation appears to be well suited for the physiological events observed in ewes, but cattle are slightly different in their mechanism of action. Most notably, in a non-pregna nt state, increased abundance of OTRs precedes that of increased ER expression in bovine endometrium [52, 53]. Based on this physiological difference between ewes and cows, it is possible that cows and ewes differ in the mechanisms used to prevent oxytocin receptor gene expression. Perhaps IFN acts directly on oxytocin receptor expr ession in cattle and also acts indirectly by regulating estrogen receptor expression during early pregnancy in bo th species. As menti oned earlier, there is conflicting evidence in sheep as to which recepto r is up-regulated first. Spencer and colleagues found that ER concentrations increase before OTR [54] while observations by Wathes and Hamilton contradicted th ese findings [49]. Interferon-tau also modifies prostaglandi n metabolism in endometrium during early pregnancy. The production of endomet rial-derived prostaglandins is controlled by a series of

PAGE 35

35 enzymes. Phospholipase A2 (PLA2) is an OTR-responsive enzyme that cleaves membrane-bound phospholipids to generate arachidonic acid [166]. Arachidonic acid is then converted to PGH2 by cyclooxygenase -1 and -2 (COX-1, COX-2), which are also known as prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and PGHS-2). PGH2 is then converted to various other PGs by specific enzymes, including PG E and PGF synthases, which generate PGE2 and PGF2 respectively. In the presence of progesterone, recombinant bovine IFN can enhance the secretion of PGE2, thereby increasing the PGE2/PGF2 ratio [167]. A biphasic PG response to IFN treatment has been obser ved by several laboratori es. Lower levels of IFN similar to levels found during early conceptus de velop at the time of pregna ncy recognition suppress COX-2 expression in primary bovine endometrial cells or a bovine endometrial cell line (BEND cells) [160, 168, 169]. Conversely, exposure to high concentrations of IFN in these cell types, reminiscent of levels in the uterus after mate rnal recognition of pregna ncy, stimulates COX-2 expression [160, 169]. Collectively, IFN appears to shift the predominate prostaglandin production away from PGF2 and toward PGE2, thus permitting the putative luteotrophic actions of this prostaglandin to favor CL maintenance [59, 170-172]. Other Activities of IFN Interferon-tau possesses the classical ac tivities of Type I IFNs (antiviral, immunomodulatory), and it is possible that these activities facilitate pregnancy in cattle, sheep and other ruminants. IFN inhibits the proliferation of cultured lymphocytes [173, 174] and inhibits the proliferation of mitogen-stimulated or mixed population lymphocytes [173, 175178]. IFN also activates natural killer cells indire ctly by increasing granulocyte chemotactic protein-2 production in the endomet rium [132, 179]. Moreover, IFN limits the expression of major histocompatability complex (MHC) class I molecules in the luminal epithelium during

PAGE 36

36 placentation [180]. The physio logical importance of IFN s antiviral activity remains uncertain, but it seems intuitive to contend that IFN may act in a prophylactic capacity to limit viral infections early in pregnancy. IFN also is implicated in orchestrating events to create a favorable uterine environment for the maintenance of pregnancy. The expression of several uterine proteins is regulated by IFN One major IFN -induced uterine protein is ubiquitin cross reactive protein (URCP), which also is known as IFN stimulated gene-15 or 17 (ISG15 or ISG17) [ 181-183]. UCRP conjugates with cystolic proteins and prompts their degrad ation in cells. This activity of UCRP may be integral to establishing and maintaining pregnancy, although th is precise function of UCRP remains uncertain. Granulocyte ch emotactic protein-2 (GCP-2), wh ich was discussed earlier for its role in immunomodulatory activities, is induced by IFN Other IFN -induced uterine proteins include Mx proteins [184, 185], 2-oli goadenylate synthetase [186], and a member of the 1-8 family, termed Leu13 [187]. One well described function of Mx protein is in the antiviral response, and it is unclear if it has a critical activity during early pregnancy or merely is facilitative during early pre gnancy in ruminants [185, 188, 189]. 2-oligoadenylate synthetase activates a constitutively expressed latent endonuclease, ribon uclease L (RNAse L). This enzyme cleaves viral and cellular RNA and blocks viral replication and initiation of apoptosis in some cell types [186, 190, 191]. Its importance during ma ternal recognition of pregnancy remains uncertain. Leu13 was first described as a T-cell surface antigen implicated in inhibiting cell growth [192, 193]. Leu13 is locali zed primarily in the glandular epithelium, and it was hypothesized that this member of the 1-8 protei n family, which is known for its involvement in adhesion, wound healing, and inflammatory responses, plays a role in adhesion of the conceptus to the endometrium and in the development of the placentome [187, 192-194].

PAGE 37

37 Bovine Embryo Culture Todays bovine embryo culture systems are capable of generating blastocyst stage embryos that can be transferred to recipients with fair pre gnancy outcomes [195-199]. However, in vitro culture systems continue to remain inferior to the maternal envi ronment, and identifying components provided by the maternal uterine envir onment that are not curr ently present in these culture systems is a major focu s surrounding research regarding in vitro embryo culture. As the identification of more uterin e-derived factors affecting em bryonic development arise, our understanding of embryo development c ontinues to become more complete. Early attempts of bovine embryo culture met with little success as embryos would arrest at the 8to 16-cell stage due to a lack of adequate support embryonic genome activation [200202]. Several culture systems have been devised to overcome this obstacle. Traditionally, two types of culture systems were us ed: those utilizing somatic cells in the culture medium, or coculture systems, and those that do not depend on somatic cells or serum supplementation, or defined culture systems [203]. Co-culturing embryos with somatic cells was once the preferred method of bovine and ovine embryo culture [204] However, the disadvantage of co-culturing with somatic cells is that the identificati on of true embryotrophic compounds or other components of the physical environment are masked by the interaction that exists between the culture medium and somatic cells [205, 206]. This lead to the de velopment of several defined culture systems. Defined In vitro Culture Requirements There is much debate over an accurate de finition of defined embryo culture system. A definition given by J.G. Thompson (1996) is that a defined culture system is one implementing a culture medium and physical environment using id entifiable components prior to embryo culture. One of the primary reasons for such debate over the definition of a defined culture system is

PAGE 38

38 due to the common usage of serum, which c ontains uncharacterized compounds as well as defined compounds that vary in their concen tration among preparations [205]. Serum is a complex combination of both small and large molecules with growth-promoting and growthinhibiting activities, depending on the source of serum. The addition of serum is commonplace during final oocyte maturation in vitro and unknown agents in serum stimulate differentiation of cumulus cells [207-209]. Bovine serum albumin (BSA) is a common medium supplement and is the predominant protein in reproductive tract. So me of the other potential benefits of BSA in culture systems include its potential role in ch elation and blocking non-sp ecific binding sites on surfaces [203]. There are reports of serum-free bovi ne oocyte maturation protocols available, but others contend that these systems are not sufficien t to mature oocytes to the point where they can be fertilized in vitro and develop to the blas tocyst stage [210-212]. Presently, a variety of defined media exist for culturing bovine embryos after fertilization. Types of culture medium can be broken down into two main subtypes: complex and simple. Complex culture medium, such as Tissu e Culture Medium-199 (with Earles salts) and Menezos B2 medium, contain various compone nts including amino acids, vitamins, salts, nucleotides and purines. These t ypes of components typically refl ect the needs of somatic cell growth rather than for early embryo developm ent, although the bovine embryo will develop in these media [205, 213]. Simple medium formula tions, which include most of the so-called culture media designed specifically for embryos, typically are salt solutions supplemented with some specific substrates and a supplemental pr otein source, usually BSA. Examples include synthetic oviductal fluid (SOF; plus amino acids and BSA), which was formulated based on the make-up of luminal oviduct components, potassium simplex optimized medium (KSOM),

PAGE 39

39 chemically defined medium (CDM), Charles Rosenkrans 1 (CR-1), Chatot/Ziomek/Bavister medium (CZB), and Hamster embryo culture medium (HECM) [203, 214]. Carbohydrates are an important source of energy for the developing embryo. Prior to compaction and morula development, the early b ovine embryo does not readily utilize glucose as its primary energy source. In fact, glucose can be toxic to early developing embryos [215, 216]. Supplementation with pyruvate and/or lactate ar e excellent sources of energy for pre-compacted bovine embryos [203, 205, 217]. Work from Bavist er and colleagues found that supplementing culture medium with both essent ial and non-essential amino acids had an overall stimulatory effect on embryo development, although some specific amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited developm ent [218, 219]. Glutamine, in particular, is a vital component of embryo culture media based on work in mice where glutamine supports embryo development in mouse strains pr one to arresting at the 2-cell stage [220]. Bovine embryos undergo a change in their requ irements for essential and non-essential amino acids as development progresses [221], although most culture procedures do not alter medium formulations between the 1-cell and blastocyst stages. Optimal embryo development in vitro also relies on other impor tant considerations. The optimal temperature for bovine in vitro maturation, fertilization, and development is between 38.5 and 39 C [222, 223]. Maintenan ce of intracellular pH is cr itical for numerous cellular mechanisms [224]. In the oviduc t and uterine fluid, regulation of pH occurs through HCO3 and its equilibration with CO2, which varies throughout the estr ous cycle [225, 226]. Traditionally, embryo culture was conducted in an atmospheri c oxygen level (approximately 20%), which is considerably greater than that found in the maternal envir onment [227]. Recent work in bovine embryos note that reducing the oxygen levels during early deve lopment to between 5 and 7%

PAGE 40

40 improves proportion of embryos de veloping to the blastocyst stage [215, 228-231]. Presumably, the results from these studies in dicate that lowering the oxygen te nsion limits the production of reactive oxygen species, there by providing embryos with an environment that does not compromise membrane integrity a nd basic intracellular functions. Culture medium for early in vitro blastocyst development shoul d be tailored to the stage of development of the embryo. Earlier embryo development, up to day 5 post-fertilization, requires a simple base medium containing either no serum or a low concentration of serum, essential and non-essential amino acids (sometimes already in the base medium), pyruvate, lactate, glutamine, and preferably no glucose. Embryo development after the late morula/early blastocyst stage can be supported similarly by ei ther a simple or complex medium type with supplemented glucose. Throughout culture, it is pr eferable to utilize a 5% oxygen environment, as this more closely replicat es the oxygen environment an in vivo embryo is exposed to in utero. Differences between In vivo and In vitroDerived Embryos Although healthy looking blastocy sts are routinely obtained from in vitro maturation, fertilization, and culture me thods, these embryos remain somewhat inferior to their in vivo derived counterparts. Most notably, pr egnancy rates following transfer of in vitro -derived embryos is lower than transfer of in vivo -derived embryos to recipients [232-235]. Morphologically, in vitro -derived bovine embryos contain a darker cytoplasm, greater lipid content, and specifically more triglycerides, swollen blastomeres, and a more fragile zona pellucidae [236-239]. Also, in vitro -derived bovine embryos have a greater incidence of chromosomal abnormalities [236-242]. Blastocyst yield from in vitro produced embryos is affected by oocyte quality. While in the dominant follicle, the oocyte undergoes matura tion events that lead to developmental competence for fertilization and subsequent embr yo development. Several studies indicate that

PAGE 41

41 oocytes matured in vivo are more competent to become fertilized and develop to the blastocyst stage than those matured in vitro [243-248]. Interestingly, work by Lonergan and colleagues showed no differences in cleavage rate between in vivo and in vitro fertilized bovine oocytes. This suggests that although fertilization is re quired to create embryos, it probably is not a limiting factor in producing embryos in vitro The in vivo fertilized group had a greater proportion of oocytes reaching the blastocyst stage than in vitro fertilized oocytes, although there was no difference in quality, as determined by vitrification and thaw ing. However, a higher proportion of matured oocytes that devel oped to blastocysts after fertilization in vivo compared with in vitro fertilization. This result indicates that events surroundi ng the time of fertilization may play a role in developmental competen ce of oocytes, while th e actual location of fertilization has no effect on blastocyst quality [249]. Two related experiments conducted by Lonergan and colleagues exemplify how in vitro development is suboptimal [ 249]. In their studies, in vivo and in vitro matured and fertilized zygotes were placed in either an in vitro culture system or in the oviduct of ewes until day 7 post-fertilization. No differences in cleavage rates or the percentage of oocytes that developed to blastocysts were observed, but bl astocyst development and hatchi ng rates increased in embryos incubated in ovine oviducts regardless of wh ether oocytes were matured and fertilized in vivo or in vitro [249]. Similarly, other work i ndicates that viability of in vitro produced embryos is 1525% less than their in vivo produced counterparts [250-252]. The addition of serum to in vitro culture systems also is deleterious to post tran sfer development and promotes early blastulation, decreases cell number, increases apoptosis, and reduces rates of glycolysis [253-259]. A subset of genes also is expressed differentially between in vitro and in vivo derived bovine embryos. For example, mRNA for conne xin 43, a gap junction protein, is lower in

PAGE 42

42 abundance and Bax, a cell death factor, is elevated within in vitroderived bovine embryos compared with in vivo derived embryos [260, 261]. A study using cDNA microarrays evaluated the differential expression of 384 gene s or expressed sequence tags between in vitro and in vivo derived blastocysts. The results indicated that approximately 85% of these genes had reduced expression for in vitroderived blastocysts compared to their in vivo counterparts [262] In summary, the in vitro production of bovine embryos is a powerful tool to describe the events associated with normal embryo development in cattle. However, current in vitro systems are inadequate in several regard s and improvements in these system s or more stringent selection and/or grading of embryos produced by these sy stems is required to obtain higher quality embryos. Extended Blastocyst Culture The continuation of embryo culture during and beyond the blastocyst stage can be accomplished, at least to a certain degree, but most experiments conducted with in vitro -derived bovine embryos end on day 7 or 8 post-fertilizati on when they reach the blastocyst stage. One method of extending the traditional culture systems is by simply placing the embryo into a new medium drop. Using this type of conventional cultu re procedure, hatched bl astocysts are inclined to attach to the bottom of the culture dish and form trophectoderm outgrowths. Under these culture conditions, troph ectoderm cultures can be maintain ed for extended periods but the remainder of the embryo (i.e. the embryonic disk ) will degenerate on or after day 13-15 post-IVF [263, 264]. More recently, Alexopoul os and colleagues successfully extended embryo culture to day 27 post-fertilization when supplementing medi um with Fetal Bovine Serum (FBS). Embryo development reached a spherical stage and seemed to match up with typical in vivo embryonic development. This was determined by evaluation of trophectoderm proliferation and whether or not a hypoblast was established (D ay 11). More frequently, however, there was a divergence

PAGE 43

43 from normal in vivo embryo development when embryos attached down to collagen gel matrix and initiated (spread) putative tr ophectoderm cell growth [9]. Using agarose tunnels to promote elongati on is an intriguing concept for extended blastocyst culture. This method was first repo rted by Stringfellow and Thompson [265] and has been investigated more recently by others [13, 26 6]. These agar gel tunnels are created with 3% (w/v) agarose suspended in Dulbecco PBS with Ca2+ and Mg2+, poured into a frame for vertical gels with a comb containing glass capillaries. Once the agarose is set, the comb of capillaries is removed and cut into 3 pieces, each containing f our or five tunnels approximately 12-14 mm in length and 1 mm in diameter. Us ing this agarose scaffold, 52% of blastocysts elongated to a minimum of 1.6 mm by day 12 post-IVF. These embr yos completely filled the space between the tunnel walls, while blastocysts cu ltured without an agarose scaffo ld averaged 1.3mm in diameter and possessed no signs of elongation. By day 14 post-IVF, 24% of the blastocysts remained viable and continued to grow within the tunnels, reaching an average of 4.3 mm in length. However, by day 16 post-IVF, only 2 embryos con tinued to grow in the tunnels, one reached 12 mm in length and the other 8 mm in length. In summary, this system successfully induced elongation but few embryos remain ed viable by days 14 and 16. Another extended culture system utilizing ag arose gel tunnels that achieved success was developed by Brando and colleagues [12] This group developed the Post Hatching Development (PHD) system. In this system, a Petri dish was set up with two glass capillary combs were placed in opposite directions from on e another with the attached ends resting on the top edge of the dish and the closed ends on the bo ttom of the dish. The Petri dish was filled with 2.4% agarose in PBS, supplemented with either 4.5% or 9% FBS. When the gel was set, 2 mL of PHD medium (SOFaaci with 0.5% glucose and 10 % FBS) was poured over the surface and the

PAGE 44

44 combs were removed. Diagonal tunnels 20 mm in length and 1.2 mm wide were formed, filled with medium, and each tunnel contained one blastocyst. With this system, 56% of embryos initiated elongation and reached average leng th of 2.9-4.4 mm, depending on the quality score. Some embryos were not subjected to analysis and remained in the PHD system, reaching lengths of 10.1 mm by day 17 and 12.5 mm by day 19. Ho wever, only 23% showed continuous elongation by day 15 post-IVF. The development of this system allowed for a successful definition of a stable culture system for evaluating rapid growt h, elongation, and initial differentiation of bovine post-hatching embr yos that were produced exclusively by in vitro production [12]. In summary, while in vitro bovine embryo culture has met with some success in developing culture systems up to the hatching phase of development, there is still the issue of our inability to fully replicate the maternal uterine environment. Uterine endometrial glands secrete factors that are vital for proper conceptus deve lopment, elongation and placentation [4, 5, 267]. These more advanced stages of embryonic deve lopment create an even more problematic situation from what already exis ts in our culture systems. As the embryo grows and differentiates it requires a more complex array of energy, prot eins, growth factors and many other undefined components than our systems currently provide. N onetheless, attaining such a system will prove invaluable both commercially and experimentally. Uterine Factors Affecting Embryo Development This literature review has provided evidence arguing that the natural maternal environment provides factors that are vital to the production of healthy embryos, and scientists have been unable to precisely replicate embryo development using in vitro culture systems. Several uterine secretions are implicated as embr yotrophic factors, and an overview of some of these factors is provided below.

PAGE 45

45 One particularly well studied growth factor that plays a ro le in embryonic development is insulin-like growth factor-I (IGFI). Uterine and placental production of IGF-I occurs in several species, including the rat [268], human [268, 269], pig [270] and sheep [271, 272]. Several studies implicate IGF-I as an embryotrophic factor in porcine, murine, human and bovine embryo development [273-276]. More recently IGF-I has been proposed to function primarily as a survival factor, and decreases the rate of apoptosis during in vitro culture [277, 278]. Also, IGF-I limits the deleterious e ffects of heat shock during bovi ne embryo culture [279, 280], and supplementation with IGF-I in creases pregnancy rates of in vitro produced bovine embryos following transfer to recipients subjected to environmental conditions that could induce heat stress [281, 282]. Several other uterine-derived factors play vi tal roles in conceptus development. In the mouse, several factors, including transforming growth factor (TGFor TGF 1) plateletderived growth factor (PDGF), insulin-like growth factor (IGF-II), and ep ithelial growth factor (EGF) promote early embryo development in vitro [283-285]. Transcripts for TGF 2 and insulin-like growth factor (IGF-II) and the re ceptors for PDGF and IGF-II are detectable throughout preimplantation development in th e bovine, indicating that these mRNAs are products of both the maternal and the embryonic genome origin in the cow. Platelet derived growth factor (PDGF) is produced in myom etrial cells and pr omotes bovine embryo development beyond the 16-cell stage when treate d for 9 days after 24 hours post-fertilization, although it does not increase blastocyst rate [286]. In that same study, TGF was, in fact, able to increase the percentage of embryos devel oping to blastocysts wh ere PDGF could not.

PAGE 46

46 A few uterine-derived factors also are implicated in regulating IFN production during early pregnancy. The two that are particularly in teresting to this labora tory include fibroblast growth factor-2 (FGF-2) and granulocyte-macr ophage colony-stimulatin g-factor (GM-CSF). Fibroblast Growth Factor-2 (FGF-2) Fibroblast growth factors (F GF) represent a large family of paracrine and autocrine modulators consisting of 23 dis tinct polypeptides that contai n mitogenic, chemotactic, and angiogenic activities responsible for various physiological processe s, such as cell migration, cell differentiation, cell survival, and tumorgenesis [287-289]. These FGFs associate with a family of plasma membrane-spanning rece ptors containing intrinsic tyro sine kinase activity termed FGFRs. Four distinct genes encode these recepto rs and alternatively sp liced products generate receptor sub-types that contain two or thre e immunoglobulin-like extracellular domains [287, 289-291]. Alternative splic ing occurs primarily within th e third immunogl obulin-like domain. This alternative splicing creates receptor subtypes possessing dis tinct ligand-binding specificities [289]. Each of the four FGFR s are activated by a specific FGF subset, although FGF-1 and FGF2 have the ability to bind to numerous receptor subtypes and exert varying actions on target cells [289, 292]. Several members of the FGF family, such as FGF-4, and -7, have been implicated in influencing early embryo development. FGF-4 de termines the developmental fate of murine trophectoderm cells [293, 294]. FGF-7, typically know n to stimulate proliferation and migration of different epithelial cell types, was shown to increase the surface area of the trophoblast in the mouse [295]. Additionally, FGF-7 stimulates the proliferation and differentiation of porcine trophectoderm [296].

PAGE 47

47 The first FGF to be identified was FGF-2, or basic FGF. FGF-2 was first described as a mitogen for mouse 3T3 fibroblasts [290] and is now best known as a mitogen and angiogenic factor [297-300]. FGF-2 is produced by the uteri of several speci es during early pregnancy and has been implicated in regulating placental deve lopment in several species [297, 299]. In pigs, rodents and rabbits, FGF-2 is primarily localiz ed to the luminal epithelial layer of the endometrium during diestrus and early pregna ncy [8, 301-303]. The loss of FGF-2 function in mice does not have major impacts on embryonic and placental development although defects in wound healing and heart contractility have been observed [304, 305]. Supplementation of FGF-2 in mouse embryo culture medium increased trophectoderm outgrowths [295]. FGF-2 also promotes gastrulation in rabbits, limits differentiation in human embryonic stem cells in culture, and improves bovine blastocyst development wh en used in conjunction with transforming growth factor(TGF) [306-310]. Additionally, FGF-2 has b een implicated in the regulation of placental attachment and formation of the syncytium in sheep [311]. Most recently, FGF-2 has been im plicated in influencing IFN production in the bovine blastocyst. In ruminants, FGF-2 is produced by both the endometrium and the conceptus and in the endometrium is localized primarily in th e luminal and glandular epithelium throughout the estrous cycle and early pregnancy [8, 312]. FGF-2 supplementation increased IFN mRNA concentrations in a bovine trophectoderm cell line (CT-1 cells) and increased IFN protein secretion from both CT-1 cells and bovine blastocysts [8]. Granulocyte-Macrophage Colony-Stimulating-Factor (GM-CSF) This cytokine is best known for its abili ty to mediate inflammation and other immune responses in macrophages, granulocytes and eosinophils. GM-CSF was first identified in activated T cells for its role in proliferation and differentiation of myel oid haematopoietic cells

PAGE 48

48 [313, 314]. GM-CSF has been implicated in playi ng a role in embryo and placental development in several species. Mice exhibiting a disrupted GM-CSF gene produce smaller litter sizes and a reduction in offspring survival. Additionally, mice deficient in GM-CSF have exhibited reduced placental capacity, likely due to a reduction in the proliferation and differentiation of mononucleated trophoblast cells [313, 315]. GM-C SF injections in mice prone to abortion reversed their high absorption rate s and increased fetal and placen tal weights [316]. Furthermore, human and mouse embryos possess GM-CSF receptors beginning at the first cleavage stage and treatment with GM-CSF improves blastocy st development [317, 318]. GM-CSF mRNA is localized within the placenta of the mouse [317, 319-321]. GM-CSF is implicated in mediating embryo development and IFN production in sheep and cattle. GM-CSF mRNA and protein abundance is greatest in the lu minal and glandular epithelium of the bovine and ovine, while protein is detected in the ovid uct and uterine flushes [322, 323]. GM-CSF treatment improves blasto cyst development in cattle [317, 324, 325]. Several reports suggest that GM-C SF supplementation can increase IFN mRNA and/or protein production in bovine and ovine trophectoderm [ 314, 326-328], however others were not able to detect such affects [329]. There also is evidence that the conc eptus, and more precisely IFN regulate uterine production of GM-CSF. Based on several findings, the ability of IFN to stimulate uterine GM-CSF is indirect, and is caused by the IFN stimulating PGE2 production in uterine endometrium and PGE2 stimulating GM-CSF expression in uterine lymphocytes and endometrium [330-332]. Summary The estrous cycle consists of a series of predictable physiological events which occur throughout the females adult life and provides her with repeated opportunities to copulate and

PAGE 49

49 become pregnant. Follicles grow and develop in waves, eventually al lowing one to become dominant and ovulate, forming a corpus luteum (C L). If the female is pregnant, the conceptus will signal its presence, via th e maternal recognition of pregnancy signal Interferon-tau (IFN ), maintaining the pregnant state. Otherwise, a comp lex series of events will occur, leading to luteolysis and return to estrus for another breeding opportunity. Once the zygote has become fertilized, it develo ps first in the oviduct and later in the uterine lumen. At the blastocyst stage, trophectod erm is distinguishable from non-differentiated embryonic cells in the inner cell mass, and proliferation of troph ectoderm changes the size and shape of the developing conceptus into a filamentous structure th at will begin attaching to the uterine lining and forming the outer components of the placenta. Eventua lly, placentomes will be created by the coupling of pl acental-derived cotyledons with uterine-deri ved caruncles. Throughout the lifetime of the placenta, several placental hormones are produced and function in various capacities to drive placental and fetal de velopment and maintain the pregnant state until parturition. One vital product of the early placenta is IFN which is a type I interferon expressed solely by the trophectoderm. It is responsible for maintaining the uterus in a pregnancy-receptive state during the second and th ird weeks of pregnancy. Current bovine embryo culture systems are capab le of producing transferable embryos that result in fair pregnancy outcomes; however, they re main inferior to those created in the maternal environment. The maternal uterine environment contains a complex arra y of components which have yet to be fully characteri zed, and current culture systems ca nnot completely replicate that environment. Extension of embryo culture beyond th e hatched blastocyst stage of development is particularly challenging, yet a few systems have met with success in inducing elongation. Developing such extended embryo culture systems will certainly be a valuable tool for better

PAGE 50

50 understanding the physiological events associat ed with bovine embryo development and IFN production. The following research project was conceived to develop an in vitro culture system that extended bovine embryo culture past the initial formation of blas tocysts on days 6 to 8 post-IVF so that it could be used to improve our understa nding of embryo development during this crucial period of development. To follow is a culture scheme that was developed to sustain bovine blastocysts in culture up to at least day 11 post-IVF. Several experiments were completed to determine how medium formulation, atmospheri c conditions, and putative embryotrophic factors affect embryo quality, development, and IFN production in bovine blasto cysts. This work is expected to be valuable for future research describing how blastocyst development progresses and understanding how uterine-de rived factors influence bovine conceptus development.

PAGE 51

51 CHAPTER 3 MATERIALS AND METHODS In vitro Embryo Production In vitro maturation, fertilization and culture proc edures [333-335] were used to generate the bovine embryos used in these studies. Bovi ne ovaries from dairy and beef breeds were collected from Central Beef Pack ing Co. (Center Hill, FL) and st ored in sterile saline (or 0.9% [w/v] NaCl) with 100 units/mL penicillin and 100 g/mL streptomycin (Chemicon International) at room temperature during transport. Ovaries were rinsed in warmed (38C) sterile saline containing 100 units/mL penicillin and 100 g/mL streptomycin to remove blood and debris. Follicles (2-10 mm) were slashed with a scalpel blad e and rinsed in a beaker filled with 75 mL of Oocyte Collection Medium [Tissue Culture Me dium-199 (M-199) with Hanks salts, without phenol red; (Hyclone), 2% (v/v) bovine steer serum (BSS; Pel-Freez) containing 2 units/mL heparin, 100 units/mL penicillin, 0.1 g/mL streptomycin, 1 mM glutamine] to liberate and collect Cumulus Oocyte Complexes (COCs) contai ning one or more comple te layers of compact cumulus cells. COCs were transferred in groups of 10 to 50 L drops of Oocyte Maturation Medium [OMM; M-199 with Earles salts 10% (v/v) BSS, 2 g/mL estradiol 17, 20 g/mL bovine FSH (Folltropin-V; Bionich e), 22 g/mL sodium pyruvate, 50 g/mL gentamicin sulfate, 1 mM glutamine] equilibrated to 38.5C in 5% CO2 and 95% humidified air and covered with mineral oil. COCs were incubated for 22-24 hours at 38.5C in 5% CO2 and 95% humidified air. Following maturation, COCs were washed in 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid -Tyrodes Albumin Lactate Pyruvate solu tion (HEPES-TALP) and placed into 600 L of In vitro Fertilization-Tyrodes Albumin Lactate Pyruvate solution (IVFTALP) (Cell and Molecular Technologies). Froz en and thawed sperm from three different Holstein bulls (Genex Cooperative, Inc) were po oled and viable sperm were separated with a

PAGE 52

52 Percoll gradient [333]. COCs were fe rtilized with approximately 1 X 106 spermatozoa/well. Fertilization wells were spiked with 25 L of PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 M epinephrine) to promote fertiliz ation and incubated at 38.5C in 5% CO2/95% humidified air for 18-20 hours. Within each replicate, a different combination of three bulls was used for fertilization. After fertilization, prospec tive zygotes were vortexed in HEPES-TALP containing 1000 units/ml hyaluronidase (Sigma-Aldrich Co.) to re move cumulus layers. Denuded oocytes were cultured in 50 L drops (n=25-35 zygotes/drop) of m odified Potassium-Simplex-Optimized Medium (KSOM; Caisson Laborat ories) at 38.5C in 5% CO2 /5% O2/90% N2 [333]. This low oxygen environment was provided by a tri-gas chamber. The proportion of cleaved embryos assessed on day 3 postin vitro fertilization (IVF) averaged 73.0 1.7% among studies and the percentage of oocytes that developed to blastocysts on day 8 post-IVF was 16.1 1.2%. Individual Embryo Culture fr om Day 8 to 11 Post-IVF On day 8 post-IVF, all blastocysts were removed from culture in KSOM and washed in 0.01M Phosphate buffered saline containing 1 mg/mL of Polyvinyl Pyrrolidone (PBS/PVP; Fisher Scientific). The stage of blastocyst deve lopment was recorded (early, expanded, hatched) and embryos were evenly distribut ed across treatment groups accordi ng to the stage of blastocyst development and placed in medium drops (30 l) covered in mineral oil. Experiment 1: Determination of Whether Medium Type and Atmospheric Oxygen Concentration Affects Embryo De velopment to Day 11 Post-IVF Individual embryos were placed in 30 L drops of either M-199 containing 5% [v/v] fetal bovine serum (FBS; Invitrogen Corp.), 0.011 mM ge ntamicin (Sigma-Aldrich Co.), non-essential amino acids (Sigma-Aldrich Co.; 0.0001 mM of each L-alanine, L-asparagineH2O, L-aspartic

PAGE 53

53 acid, L-glutamic acid, glycine, L-proline, and Lserine), and 0.019 mM sodium pyruvate (SigmaAldrich Co.) or KSOM containing 5% [v/v ] FBS, 0.011 mM gentamicin, and non-essential amino acids. A comparison of M-199 and KSOM me dium components is pr ovided in Table 2-1 and 2-2. Embryos were incubate d at 38.5C in either a 5%CO2/5%O2/90% N2 (5% oxygen group) or a 5% CO2/95% air atmosphere (20% Oxygen group). On day 11 post-IVF the stage and quality of embryo development was record ed. Embryo quality was determined by visual observation for expansion and/or hatching and whether the embryo had degenerated. Degenerated embryos were classifi ed as having partial to complete collapse or deterioration of cell membranes, more specifically the trophectoderm, and/or rupt uring of the zona pellucida. Embryos were washed once in PBS/PVP, fixed in 4% [w/v] paraformaldehyde (Polysciences Inc.) for 10 minutes and stored in 500 L PBS/PVP with 0.02% [w/v] sodium azide (NaN3) until further use. Spent medium (25 L) was collected from each drop and stored at -20C. Four independent replicate studies we re completed with 5 to 12 blastocysts per treatment in each replicate (total n=20 to 46 blastocysts/treatment). Experiment 2: Determination of Whether S erum Type or Concentration Affects Embryo Dvelopment to Day 11 Post-IVF Blastocysts collected on day 8 post-IV F were cultured individually in 30 L drops of M199 with 0.011 mM Gentamicin, Non-Essential Am ino Acids (0.0001 mM of each L-alanine, LasparagineH2O, L-aspartic acid, L-glutamic acid, glyc ine, L-proline, and L-serine), and 0.019 mM sodium pyruvate was supplemented with e ither insulin-transferri n-selenium (670 g/L sodium selenite (anhydrous), 1.00 g/L insulin, and 0.55 g/L transferrin) (ITS; Invitrogen Corp.), 1% [w/v] bovine serum albumin (BSA; Sigma-Aldr ich Inc.), or 1%, 2.5%, or 5% [w/v] FBS at 38.5C in either a 5% or 20% oxygen atmosphere. On day 11 post-IVF, blastocyst development

PAGE 54

54 and quality was assessed and embryos and medi um samples were processed as described previously. Two independent rep licate studies were completed with 3 to 6 blastocysts per treatment in each replicate (total n=6 to 16 blastocysts/treatment). Experiment 3: Determination of Wh ether FGF-2 Supplementation and Oxygen Concentration Affects Embryo Development and IFN Concentration to Day 11 Post-IVF Day 8 blastocysts were cu ltured individually in 30 L drops of M-199 supplemented with 2.5% FBS, 0.011 mM gentamicin, non-essential am ino acids (0.0001 mM of each L-alanine, LasparagineH2O, L-aspartic acid, L-glutamic acid, glycin e, L-proline, and L-serine), 0.019 mM sodium pyruvate and either 0 or 100 ng/mL of r ecombinant bovine fibroblast growth factor-2 (boFGF-2; R&D Systems, Inc.) at 38.5C in ei ther a 5% or 20% oxyge n atmosphere. BoFGF-2 was purchased as a lyophilized powder and was reconstituted in M-199 containing 0.1% [w/v] BSA. Aliquots were stored at -20C. All treatme nts, including controls, were provided to cells with identical amounts of BSA carrier (20 g BS A/ml medium). On day 11 post-IVF, blastocyst development and quality was assesses and embryos and medium samples were processed as described previously Three independent replicate studies were completed with 8 to 10 blastocysts per treatment in each replicat e (total n=25 to 30 bl astocysts/treatment). Experiment 4: Determination of Wheth er FGF-2 and GM-CSF Co-supplementation Affects Embryo Development and IFN Concentration to Day 11 Post-IVF Day 8 blastocysts were cu ltured individually in 30 L drops of M-199 supplemented with 2.5% FBS, 0.011 mM Gentamicin, Non-Essential Amino Acids (0.0001 mM of each L-alanine, L-asparagineH2O, L-aspartic acid, L-glutamic acid, glyc ine, L-proline, and L-serine), 0.019 mM sodium pyruvate and either 100 ng/mL boFGF2, 100 ng/mL of recomb inant porcine GM-CSF (poGM-CSF; R&D Systems, Inc.), both FGF2 and GM-CSF (100 ng/ml of each), or no

PAGE 55

55 cytokine/growth factor treatment (control) at 38.5C in a 5% oxygen atmosphere. Both boFGF-2 and poGM-CSF were purchased as a lyophiliz ed powder and were reconstituted in M-199 containing 0.1 [w/v] BSA. Aliquot s were stored at -20C. All treatments, including controls, were provided to cells with iden tical amounts of BSA carrier (20 g BSA/ml medium). On day 11 post-IVF, blastocyst development and qua lity was assessed and embryos and medium samples were processed as described previously Three independent repl icate studies were completed with 8-10 blastocysts per treatment in each replicate (total n=24-28 blastocysts/treatment). Evaluating Apoptosis and Total Cell Number The Terminal dUTP Nick-End Labeli ng (TUNEL) procedure was performed on individual embryos fixed in 4% [w/v] paraformaldehyde using the In Situ Cell Death Detection Kit with a Fluorescein dye (Roc he Diagnostics) [334]. Individua l embryos were washed in 50 L drops of PBS/PVP and permeabilized in 50 L drops composed of PBS with 0.5% [v/v] Triton X-100 and 0.1% [w/v] sodium citrat e for 15 minutes at room temperature. Positive controls were incubated in a 50 L drop of RQ1 RNase-free DNase (50 U / ml; New England BioLabs) diluted in PBS/PVP at 37 C for 1 h. Embryos were washed in PBS/PVP and incubated with 25 L drop of TUNEL reaction mixture according to manufacture r instructions for 1 hour at 37C in a dark and humidified environment. Negative controls were incubated in the absence of terminal deoxynucleotidyltransferase. All embryos were then placed in 50 L drops of a 0.5 mg/mL Hoescht 33342 (Molecular Probes). Embr yos were washed 4 times in 500 L of PBS/PVP, placed on microscope slides, and mounted with 10-12 L of Prolong Gold Antifade mounting medium (Invitrogen Corp.). Epif luroescence microscopy was used to determine the total number

PAGE 56

56 of nuclei (blue fluorescent stain) and number of TUNEL-positive nuclei (green fluorescent stain) in each blastocyst. Interferon-tau Production The amount of biologically active IFN present in conditioned medium from each bovine blastocyst culture was determined by using antiv iral assays [178]. Briefly, 20 l of conditioned medium collected at day 11 post-IVF was diluted serially to determine when each sample was able to prevent vesicular stomatitis virus-induced cell lysis by 50%. The activity of each sample was compared with a bovine recombinant IFN developed in the laborato ry [151] that had been standardized with recombinant human IFN (EMD Biosciences Inc.; 3.84 x 108 IU/mg). The average activity of the bovine recombinant IFN standard was 8.03 x 108 IU/mg. Statistical Analysis All analyses were conducted using the general linear model of the Statistical Analysis System (SAS Institute Inc.; Cary, NC). Differences in percentage of degenerated embryos among treatments, total cell number per embryo, percentage of apoptotic cells per embryo, and antiviral activity of spent medium were determined us ing least-squares analysis of variance (LS-ANOVA). In most studies, the antiviral activity of IFN in medium samples was normalized for total cell number. Pair-wise comparisons were completed to further partition effects among treatment groups. In all experiments, the results are presen ted as arithmetic means + SEM.

PAGE 57

57 Table 2-1. A comparison of medium components for M-199 and KSOM. COMPONENTS M-199 MOLARITY (mM) KSOM MOLARITY (mM) AMINO ACIDS Glycine 0.667 0.050 L-Alanine 0.281 0.050 L-Arginine hydrochloride 0.332 0.299 L-Asparagine H2O 0.050 L-Aspartic acid 0.226 0.051 L-Cystine 0.050 L-Glutamic acid 0.455 0.045 L-Glutamine 0.685 1.001 L-Histidine hydrochloride-H2O 0.104 0.099 L-Isoleucine 0.305 0.200 L-Leucine 0.458 0.200 L-Lysine hydrochloride 0.383 0.199 L-Methionine 0.101 0.050 L-Phenylalanine 0.152 0.100 L-Proline 0.348 0.050 L-Serine 0.238 0.050 L-Threonine 0.252 0.200 L-Tryptophan 0.049 0.025 L-Tyrosine 0.100 L-Valine 0.214 0.200 INORGANIC SALTS Calcium chloride (CaCl2) (dihyd.) 1.701 Magnesium sulfate (MgSO4*7H2O) 0.200 Potassium chloride (KCl) 5.330 2.485 Monopotassium phosphate (KH2PO4) 0.353 Sodium bicarbonate (NaHCO3) 26.190 25.003 Sodium chloride (NaCl) 117.240 95.721 OTHER COMPONENTS D-Glucose (Dextrose) 5.560 0.200 EDTA 0.013 Sodium lactate syrup 10.003 Sodium pyruvate 0.200

PAGE 58

58 Table 2-2. Medium components th at are only included in M-199. AMINO ACIDS MOLARITY (mM) OTHER COMPONENTS MOLARITY (mM) L-Cysteine hydrochloride-H2O 0.000568 2-deoxy-D-ribose 0.00373 L-Cystine 2HCl 0.108 Adenine sulfate 0.0248 L-Hydroxyproline 0.0763 Adenosine 5'-phosphate 0.000576 L-Tyrosine disodium salt dihydrate 0.153 Adenosine 5'-triphosphate 0.00165 VITAMINS Cholesterol 0.000517 Alpha-tocopherol phosphate 0.0000142 Glutathione (reduced) 0.000163 Ascorbic Acid 0.000284 Guanine hydrochloride 0.0016 Biotin 0.000041 Hypoxanthine Na 0.00294 Choline chloride 0.00357 Phenol red 0.0531 D-Calcium pantothenate 0.000021 Ribose 0.00333 Folic Acid 0.0000227 Sodium acetate 0.61 i-Inositol 0.000278 Thymine 0.00238 Menadione (Vitamin K3) 0.0000581 Tween 80 20 mg/ml Niacinamide 0.000205 Uracil 0.00268 Nicotinic acid (Niacin) 0.000203 Xanthine-Na 0.00224 Para-Aminobenzoic Acid 0.000365 Pyridoxal hydrochloride 0.000123 Pyridoxine hydrochloride 0.000121 Riboflavin 0.0000266 Thiamine hydrochloride 0.0000297 Vitamin A (acetate) 0.000305 Vitamin D2 (Calciferol) 0.000252 INORGANIC SALTS Calcium chloride (CaCl2) (anhyd.) 1.8 Ferric nitrate (Fe(NO3)-9H2O) 0.00173 Magnesium sulfate (MgSO4) (anhyd.) 0.397 Sodium phosphate monobasic (NaH2PO4-H2O) 1.01

PAGE 59

59 CHAPTER 4 RESULTS Experiment 1: Effect of Medium Type and Oxygen Atmosphere on Blastocyst Survival, Development and IFN Production Bovine blastocysts were cultured individuall y in two medium types and under different oxygen tensions to assess if embryo developm ent could be extended to day 11 post-IVF in culture. On day 11 post-IVF, a portion of blas tocysts were consider ed degenerated. These embryos possessed a partial to complete collaps e or deterioration of cell membranes, more specifically the trophectoderm, a nd/or rupturing of the zona pellucida. Those embryos which appeared extremely unhealthy were small in size and did not seem to s how any signs of having an expanded trophectoderm. Embryos which app eared healthy upon visual observation were large, rotund, and expanded, some extensivel y so, and most were hatched from their zona pellucida by day 11 post-IVF ( 67.5 .8% overall mean and SEM) For a comparison of typical degenerated and healthy embryos under nuclear fluorescent staining (H oescht 33342), refer to Fig. 4-1 and 4-2. No main effects of medium type or oxygen concentration on rate of degeneration were detected but a medium type by oxygen interactio n was evident (P=0.05), where incubation in M199 in 5% oxygen prevented degeneration wherea s from 23 to 26.5% of embryos incubated in other medium and oxygen atmospheric conditions were degenerated at day 11 post-IVF (Fig. 41, 4-3). Accurate cell counts coul d not be retrieved from these embryos and conditioned medium from these embryos did not contain measurable an tiviral activity. Therefor e, only data collected from non-degenerated embryos were used for subsequent analyses. Total embryonic cell numbers on day 11 post-IV F were affected by the main effects of medium (P=0.04) and oxygen concentration (P =0.03) with M-199 and 5% oxygen treatments providing greater day 11 cell numbers (data no t shown). A medium by oxygen interaction was

PAGE 60

60 detected (P=0.006), and embryos cultured in TC M-199 in 5% oxygen contained greater (P<0.01) cell numbers at day 11 post-IVF than other culture treatments (Fig. 4-2, 4-4). No medium type or oxygen concentration effects, incl uding interactions, we re noted on the percentage of apoptotic nuclei on day 11 post-IVF (Fig. 4-5). In each gr oup, percent TUNEL positive nuclei ranged from 1.5-3%. Medium type did not affect the quantity of bioactive IFN as assessed with an antiviral assay, in conditioned medium from blastocysts cultured individually from day 8 to 11 post-IVF (Fig 4-6). However, embryos incubated in 5% oxygen secreted more (P=0.002) IFN into medium than embryos incubated in 20% oxygen (Fig. 4-6). This effect is evident in both medium types both before and after normalizing data according to embryonic cell numbers on day 11 post-IVF (Fig. 4-7). Experiment 2: Serum Requirements for Extended Bovine Blastocyst Culture In the previous study embryos were mainta ined in medium containing 5% [v/v] FBS based on the laboratorys previous observations for group-cultured bovine blastocysts (Michael, 2006). The objective of this study was to define th e minimum serum requirements for blastocyst survival in vitro to day 11 post-IVF. Treatments include d M-199 containing previously described additives and 1% BSA (Negative control), Insu lin-transferrin-selenium (ITS) containing 1% BSA, or 1%, 2.5% and 5 % FBS. Embryos were incubated in either 5% or 20% oxygen. During the experiment it became evident that embryos cultured with 1% BSA and ITS + 1% BSA had a high incidence of degeneration (approximately 75%) regardle ss of oxygen exposure and these treatments were not replicated. Rather, the rema inder of the experiment was completed only with 1%, 2.5% and 5% FBS treatment groups. The incide nce of degeneration wa s greater (P=0.04) for embryos cultured in 20% oxygen th an those cultured in 5% oxyge n (Data not shown). Moreover,

PAGE 61

61 serum concentration affected degeneration rate (P=0.04). This effect was most evident for embryos cultured in 1% FBS versus those cultur ed in 2.5 and 5% FBS in 5% oxygen (Fig. 4-8). Number of cells on day 11 post-IVF also was impacted by oxygen atmosphere (P=0.001) and serum supplementation to a lesser degree (P =0.09). As shown in Figure 4-9, cell numbers at day 11 post-IVF were increased (P<0.05) as serum concentrations in medium increased in embryos incubated in 5% oxygen. However, this effect was not observed in embryos incubated in 20% oxygen. There were no differences among serum and oxygen treatments in the proportion of apoptotic nuclei in blastocyst s on day 11 post-IVF (Fig. 4-10). Serum concentration and oxygen concentration influenced the quantity of bioactive IFN produced by the individual embryos (Fig. 4-11) Embryos cultured in 1% FBS produced less (P<0.05) IFN per cell at day 11 post-IVF than embryos either 2.5% or 5% FBS treatments, regardless of oxygen level. Moreover, IFN levels were below the detection limit for the assay (0.1 ng/ml) in a majority of the medium samp les derived from embryos cultured in medium containing 1 % FBS. A clear increase (P=0.02) in IFN concentrations was detected in embryos cultured in 20% oxygen than those cultured in 5% oxygen. The effects of serum concentrations in medium and oxygen atmosphere remained evident when individual treatment comparisons were made (Fig. 4-12). Experiment 3: FGF-2-Med iated Induction of IFN Production in Bovine Blastocysts Depends on the Level of Atmospheric Oxygen Based on the work completed in experiments 1 and 2, incubating embryos individually in drops of M-199 containing several additives, incl uding 2.5% FBS, permits continued blastocyst development and measurable IFN production from day 8 to 11 post -IVF. This extended culture system was used herein to determine if the le vel of atmospheric oxygen affects the ability of FGF-2 to influence IFN production. Bovine blastocysts were cultured individually from day 8

PAGE 62

62 to day 11 post-IVF with or wit hout 100 ng/mL FGF-2 in either 5% or 20% oxygen. No clearly degenerate embryos were observed in this study. Also, TUNEL analysis was not completed since previous studies did not detect any significant changes in percenta ge of apoptotic nuclei based on oxygen atmosphere (see experiments 1 and 2). As observed in previous work, embryos cultured in M-199 with 2.5% FBS in a 5% oxygen atmosphere contained greater cell numbe rs (P<0.001) on day 11 post-IVF than embryos cultured in 20% oxygen (Fig. 4-13). Supplementation with 100 ng/mL FGF-2 did not affect cell numbers for embryos regardless of the oxygen environment during culture. IFN concentration in conditioned medium at day 11 post-IVF was not affected by FGF-2 supplementation (P=0.1) and clearly increased by incubating embryos in 20% oxygen (P=0.01) (Fig. 4-14). When the effects of FGF-2 supplementation and oxygen levels were partitioned further, it became evident that FGF-2 supplementation was able to increase (P=0.06) IFN concentrations in conditioned medium when embryos were incubated in a 5% oxygen atmosphere but not when they were incubated in 20% oxygen (Fig. 4-15). Experiment 4: The Combined Effects of FG F-2 and GM-CSF on Blastocyst Development and IFNProduction Several reports implicate GM-CSF as a mediator of IFN production in bovine embryos and trophectoderm cell lines [314, 323, 328], although this observation could not be observed in some studies [329]. A final study was completed to determine if GM-CSF affects blastocyst development and/or IFN secretion in this newly deve loped embryo culture system and determine if GM-CSF and FGF-2 act cooperatively to mediate embryo development and/or IFN secretion. All embryos were cultu red individually in 30 l dr ops of M-199 containing 2.5% FBS under a 5% oxygen atmosphere. Either boFGF-2 (100 ng/mL), poGM-CSF (100 ng/mL), both FGF-2 and GM-CSF (100 ng/mL of each), or no growth factor treatment (Control) was added to

PAGE 63

63 medium immediately prior to incubation at da y 8 post-IVF. On day 11, embryos receiving GMCSF treatment had fewer (P=0.03) cells per em bryo regardless of whether FGF-2 was coadministered (data not shown). FGF-2 supplementa tion did not influence resulting cell numbers when compared with controls, but cell numbers were greater (P<0.04) in FGF-2-supplemented embryos on day 11 post-IVF than embryos s upplemented with poGM-CSF (Fig. 4-16). The incidence of apoptosis was not measured in this study. There were no main effects of FG F-2 or GM-CSF supplementation on IFN concentrations in conditioned medium at day 11 post-IVF when data were normalized to account for differences in day 11 cell counts. However, i ndividual treatment comparisons determined that IFN concentrations in conditione d medium were greater (P=0.02) in boFGF-2-supplemented cultures versus non-treated cont rols (Fig. 4-17). Interestingl y, poGM-CSF supplementation did not significantly affect IFN concentrations when compared to control values, but the mean concentrations of IFN in medium were increa sed slightly and were not significantly different from boFGF-2-supplemented samples. Co-supplementation with boFGF-2 and poGM-CSF did not increase IFN concentrations in conditioned medium when compared with controls.

PAGE 64

64 Figure 4-1. Examples of degenera ted embryos by day 11 post-IVF Degeneration on day 11 of development was determined by visual asse ssment of embryo integrity (see methods section for detail). Panel A and B depict degenerated embryos stained with Hoescht 33342 nuclear stain. Overall visual appearance is poor with inner cellular contents collapsed or absent, with presence of dege nerated cellular debris and in some cases the zona pellucida has ruptured.

PAGE 65

65 Figure 4-2. Examples of Hoescht 33342 and TUNEL staining for total cell counts and incidence of apoptosis. The total number of embryonic cell s and the percentage of embryonic cells that were TUNEL positive at day 11 post-IVF was determined for embryos cultured individually in KSOM or M-199 and 5% or 20% oxygen were recorded using epifluorescent microscopy. \

PAGE 66

66 Figure 4-3. Incubating blastocysts from day 8 to 11 post-IVF in M-199 and 5% O2 prevents degeneration. The incidence of embryo degeneration, as characterized by partial to complete collapse or deterioration of cell membranes, more specifically the trophectoderm, and/or rupturi ng of the zona pellucida, was determined for embryos cultured from day 8 to 11 post-IVF in KSOM or M-199 and 5% or 20% oxygen. Degeneration on day 11 was determined by visual assessment of embryo integrity (see methods section for detail). Four inde pendent replicate studies were completed with a total n=20 to 46 blastocysts/treatme nt. Different superscripts above bars represent differences (P<0.05) Error bars indicate mean standard error.

PAGE 67

67 Figure 4-4. Incubation in M199 under 5% oxygen conditions yields greater total cell numbers on day 11 post-IVF. Embryos were cultured individua lly in drops of KSOM or M-199 and 5% or 20% oxygen from day 8 to 11 pos t-IVF. Number of embryonic cells was determined at day 11 post-IVF was dete rmined by staining nuclear DNA (Hoescht 33342) and counting nuclei using epifluor escent microscopy. Four independent replicate studies were completed with a total n=20 to 37 blastocysts/treatment. Different superscripts above bars represent differences (P<0.05) Error bars indicate mean standard error.

PAGE 68

68 Figure 4-5. Incidence of apoptosis is not affected by medium t ype or oxygen tension in bovine embryos from day 8 to 11 post-IVF. The percentage of embryonic cells that were TUNEL positive at day 11 post-IVF was determined for embryos cultured individually in KSOM or M-199 and 5% or 20% oxygen were recorded using epifluorescent microscopy. Four independent replicate studies we re completed with a total n=20 to 37 blastocysts/treatment. Diffe rent superscripts above bars represent differences (P<0.05) Error bars indicate mean standard error.

PAGE 69

69 A. Figure 4-6. Incubation in atmo spheric oxygen increases IFN secretion from bovine blastocysts. The quantity of bioactive IFN present in conditioned medium on day 11 post-IVF was determined for embryos cultured from day 8 to 11 post-IVF in KSOM or M-199 and 5% or 20% oxygen. Results depict the ma in effects of medium type (A.) and oxygen concentration (B.) on IFN concentrations in conditioned medium. Four independent replicate studies were completed with a total n=20 to 37 blastocysts/treatment. Different superscr ipts above bars represent differences (P=0.02) Error bars indicate mean standard error. B.

PAGE 70

70 Figure 4-7. Interaction of medium type and oxygen concentration on IFN secretion on day 11 post-IVF. Concentrations of IFN in conditioned medium are depicted for embryos cultured in KSOM or M-199and 5% or 20% oxygen from day 8 to 11 post-IVF. Data presented include IFN concentrations in medium prior to (A.) or after (B.) data were normalized to account for variation in embryo cell number. Four independent replicate studies were completed with a total n=20 to 37 blastocysts/treatment. Different superscripts above bars represent differences (P<0.05) Error bars indicate mean standard error A. B.

PAGE 71

71 Figure 4-8. Incubating embryos in M-199 containing 2.5 or 5% FBS with 5% oxygen limits degeneration from day 8 to 11 post-IVF. The incidence of embryo degeneration was determined for embryos cultured individually from day 8 to 11 post-IVF in M-199 containing 1%, 2.5% or 5% FB S. Degeneration on day 11 was determined by visual assessment of embryo integrity (see methods section for detail). Two independent replicate studies were completed with 6 to 12 blastocysts/treatment. Different superscripts above bars re present differences (P<0.05) Error bars indicate mean standard error.

PAGE 72

72 Figure 4-9. Supplementation with FBS improves embryonic cell number after incubation in 5% oxygen. Embryos were cultured in M-199 suppl emented with 1%, 2.5% or 5% FBS and incubated in 5% or 20% oxygen from da y 8 to 11 post-IVF. Number of cells at day 11 post-IVF was counted by epifluores cent microscopy after staining nuclear DNA (Hoescht 33342 staining). Two independe nt replicate studies were completed with a total of 6 to 12 blas tocysts/treatment. Different superscripts above bars represent differences (P<0.05) Error bars indicate mean standard error.

PAGE 73

73 Figure 4-10. Effect of serum supplementation on percentage of TUN EL positive cells during individual embryo culture from day 8 to 11 post-IVF. Embryos were cultured individually from day 8 to 11 post-IVF in M-199 containing 1% 2.5% or 5% FBS. The percentage of TUNEL positive cells in each embryo was determined using a fluorescent stain for DNA nicking follo wed by nuclear DNA counter-staining. TUNEL positive and total nuclei were count ed by epifluorescent microscopy. The number of TUNEL positive cells was compared to the total cell numbers. Two independent replicate studies were completed with a total of 6 to 12 blastocysts/treatment. Different superscr ipts above bars represent differences (P<0.05) Error bars indicate mean standard error.

PAGE 74

74 Figure 4-11. Degree of serum supplementation a nd oxygen concentrations during culture impact IFN secretion. Blastocysts were cultured in dividually in M-199 supplemented with 1%, 2.5% or 5% FBS in either 5% or 20% oxygen from day 8 to 11 post-IVF. Concentration of bioactive IFN in conditioned medium at day 11 post-IVF was normalized by cell number. Data represent the main effects of level of serum supplementation (A.) and atmospheri c oxygen concentration (B.) on IFN secretion. Two independent replicate studies were completed with a total of 6 to 12 blastocysts/treatment. Different superscr ipts above bars represent differences (P<0.05) Error bars indicate mean standard error. A. B.

PAGE 75

75 Figure 4-12. Serum supplementation and atmos pheric oxygen conditions act independently to mediate IFN production Blastocysts were cultured individually in M-199 supplemented with 1%, 2.5% or 5% FBS in either 5% or 20% oxygen from day 8 to 11 post-IVF. Concentration of bioactive IFN in conditioned medium at day 11 postIVF was normalized by cell number. Two independent replicate studies were completed with a total of 6 to 12 blastocyst s/treatment. Different superscripts above bars represent differences (P<0.05) Error bars indicate mean standard error.

PAGE 76

76 Figure 4-13. Effect of FGF-2 supplementation on cell numbers in embryos on day 11 post-IVF. Blastocysts were cultured individu ally from day 8 to 11 post-IVF in M-199 supplemented with 0 or 100 ng/mL boFGF-2 under 5% or 20% atmospheric oxygen conditions. Cell number was determined at day 11 post-IVF by staining nuclear DNA (Hoescht 33342) and coun ting nuclei using epifluor escent microscopy. Three independent replicate studies were completed with a total of 25-30 blastocysts/treatment. Different superscr ipts above bars represent differences (P<0.05) Error bars indicate mean standard error.

PAGE 77

77 Figure 4-14. Main effects of FGF-2 supplem entation and oxygen concentrations on IFN secretion from bovine blastocysts Blastocysts were cultured individually from day 8 to 11 post-IVF in M-199 supplemented with 0 or 100 ng/mL boFGF-2 under 5% or 20% atmospheric oxygen conditions. The quantity of bioactive IFN in conditioned medium on day 11 post-IVF was determined and normalized for embryo cell number. Data represent the main effect of FG F-2 supplementation (A.) and oxygen condition (B.) on IFN concentrations in conditioned me dium. Three independent replicate studies were completed with a total of 25-30 blastocysts/treatment. Different superscripts above bars re present differences (P<0.05) Error bars indicate mean standard error. A. B.

PAGE 78

78 Figure 4-15. Treatment with FGF2 and 20% oxygen increases IFN production from bovine blastocysts. Embryos were cultured individua lly from day 8 to 11 post-IVF in M199 supplemented with 0 or 100 ng/mL boF GF-2 under 5% or 20% atmospheric oxygen conditions. The quantity of bioactive IFN in conditioned medium on day 11 post-IVF was determined and normalized fo r embryo cell number. Three independent replicate studies were completed with a tota l of 25-30 blastocysts/treatment. Different superscripts above bars re present differences (P<0.06) Error bars indicate mean standard error.

PAGE 79

79 Figure 4-16. Effect of FGF-2 and GM-C SF supplementation on embryo cell number. Blastocysts were cultured individually in drops of M-199 containing 2.5% FBS and 100 ng/ml boFGF-2, 100 ng/ml poGM-CSF, both FGF-2 and GM-CSF (100 ng/ml of each), or no growth factor treatment (contro l) from day 8 to 11 post-IVF. Cell number was determined at day 11 post-IVF by staining nuclear DNA (H oescht 33342) and counting nuclei using epifluor escent microscopy. Three i ndependent replicate studies were completed with a total of 24-28 blas tocysts/treatment. Different superscripts above bars represent differences (P<0.05) Error bars indicate mean standard error.

PAGE 80

80 Figure 4-17. Supplementation with FG F-2 but not GM-CSF increases IFN production from bovine embryos. Blastocysts were cultured individua lly in drops of M-199 containing 2.5% FBS and 100 ng/ml boFGF-2, 100 ng/ml poGM-CSF, both FGF-2 and GM-CSF (100 ng/ml of each), or no growth factor treatment (control) from day 8 to 11 postIVF. Quantity of bioactive IFN in conditioned medium at day 11 post-IVF was determined and normalized for embryo ce ll number. Three independent replicate studies were completed with a total of 24-28 blastocysts/treatment. Different superscripts above bars re present differences (P<0.05) Error bars indicate mean standard error.

PAGE 81

81 CHAPTER 5 DISCUSSION Reproductive failure is a considerable issue in the beef and dairy i ndustries, with early embryonic mortality being responsible for a substantial proportion of reproductive insufficiencies. A more articu late understanding of the seri es of events of embryonic development may lead to possible resolutions to these issues. The most valuable method of studying embryonic development is through in vitro production and culture of embryos. Conventional bovine embryo culture systems are not designed to adequately support blastocyst development. This is because the full extent of components provided by the natural uterine environment has yet to be defined, which proves to be problematic in developing embryo culture systems, particularly during post-hatching developm ent. The work presented here establishes that blastocysts can be sustained in culture until at least day 11 post-IVF with the proper medium formulation, by supplementing medium with se rum, and by exposing embryos to a low oxygen environment. The medium formulation used in these expe riments had a major impact on blastocyst viability and development between days 8 and 11 post-IVF. A majority of the components in the modified KSOM (with non-essential amino acids) and M-199 were fairly well balanced except for their glucose levels (5.5 mM for M-199; 0.2 mM for KSOM). Gl ucose can be toxic to bovine embryos, and especially female embryos, during initial cleavage stages [336-338]. As embryos become more advanced, rate of glycolysis a nd glucose uptake increase s [336, 337], and medium containing glucose is required for propagating bovine embryos during and beyond the blastocyst stage [6, 7, 10, 12, 13]. However, si nce glucose withdrawal and/or replacement studies were not completed during the course of this work, it re mains possible that minor differences between media formulations also contributed to the superi ority of M-199 as a blas tocyst culture medium.

PAGE 82

82 Serum supplementation, previously shown to be a required supplement in culture medium [210, 211], limited the degeneration of bovine bl astocysts between days 8 and 11 post-IVF in these experiments. Attempts to supply M-199 with defined agents known to substitute for serum in several somatic cell lines ( i.e. insulin, transferrin, selenium, BS A) failed to provide adequate conditions for blastocyst development and survival Others have noted th e same requirement for serum in extended bovine embryo cultures [9, 12, 13]. In one study [9], FBS was superior to mature cow serum as a medium supplement and both serum types were better than media formulations lacking serum at sustaining bovine embryonic outgrowths formed on collagencoated plates. In the present work, 2.5% FB S limited degeneration and sustained a modest amount of embryonic growth during culture. An i ndividual blastocyst culture system was used throughout our studies to remove the confounding effects of group embryo culture on outcomes and to permit using blastocyst cell numbers for normalizing IFN production data. The minimum serum requirement for blastocysts cultur ed in groups remains unknown, but serum supplementation is routine during group culture [6-8]. Taken together, serum, and preferably FBS supplementation is required du ring bovine blastocyst culture wh en using M-199 or a related medium formulation. Traditional embryo culture systems incubate d embryos at an atmospheric oxygen level (approximately 20%) is greater th an that found in the uterus a nd oviducts (approximately 2-9%) [227]. Recent work from several groups investig ating bovine embryo development indicate that reducing the oxygen levels during in vitro culture to between 5 and 7% improves proportion of embryos developing to the blastocyst stage and in several species [215, 228-231, 339-342]. In this work, a 5% oxygen atmosphere prevented degeneration and sustai ned cell proliferation between days 8 and 11 post-IVF. It is generally accepted that reducing the oxygen tension during

PAGE 83

83 embryo culture limits the production of reactive oxygen species that cause oxidative damage in embryos [229, 230, 343]. Level of oxidative stress was not measured in th ese studies, but day 8 to 11 bovine blastocysts were sensitive to tert -butylhydroperoxide (tBH), an agent that oxidizes various intracellular molecules, including gl utathione, in mammalian embryos [344, 345]. This oxidant produced a dose responsiv e increase in the proportion of degenerated blastocysts and decrease in cell numbers but did not affect IFN levels in conditioned medium [Cooke et al., Unpublished observations]. In related work, addi tion of hydrogen peroxide to culture medium negatively affected developmental competence of bovine embryos but did not affect IFN production [346]. The agent tBH mimicked some but not all of the effects of 20% oxygen on blastocysts, thereby suggesting that oxidative stress probabl y is at least partial responsible for the inferior development of blas tocysts cultured in 20% oxygen. It was surprising that incuba tion in 20% oxygen increased IFN concentrations in conditioned medium. This oxygen te nsion compromised the overall fitness of blastocysts, as evidenced by reduced cell numbers and increase d the incidence of degeneration, but increased the amount of IFN produced. Work by others did not detect differences in IFN mRNA or protein content in bovine blasto cysts cultured in 5% versus 20% oxygen [215, 346]. In previous studies, exposure to different oxygen environments began on the day following fertilization (1cell stage) [215] or on day 3 postIVF (8-cell stage) [346] and IFN levels were evaluated either on day 7 post-IVF by measuring IFN mRNA abundance [215] or after 24 or 48 h culture of individual day 8 blastocysts by measur ing the quantity of bioactive IFN in conditioned medium [346]. Early exposure to high oxygen also compro mised the developmental potential of bovine embryos in these studies. One explanation for why outcomes differ between this and previous studies is that the early expos ure to a high oxygen environment may provide embryos with an

PAGE 84

84 opportunity to adapt to high oxygen conditions prior to the blastocyst stage, and embryos that reached the blastocyst stage represented the subs et of embryos that overcame the limitations of this environment. In the present study, embryos were not given an opportunity to adapt to high oxygen before the blastocyst stag e. Alternatively, various geneti c and environmental factors, including gender, paternal ge notype, medium com position, and time of blastocyst formation, influence IFN production in blastocysts [6, 7, 338, 347] and any one of these factors or variables that remain undefined could have contributed to this novel response to the high oxygen environment. The 20% oxygen environment also precluded blastocysts from responding to FGF-2 supplementation. FGF-2 stimulates IFN mRNA and protein production in bovine trophectoderm and IVF-derived blastocysts, a nd its presence in the uterine lumen throughout the estrous cycle and early pregnancy implicate it as an uterine-derived mediator of IFN production during early pregnancy in cows and ew es [8, 312]. Detecting IFN responses to FGF-2 treatment in blastocysts cultured individuall y in 5% oxygen supports previous findings, where groups of embryos (n=8-11 blastocysts/50 l drop of medium) produced more IFN in response to FGF-2 supplementation than non-supplemented controls [8]. FGF-2 did not contain detectable mitogenic activity in bovine blastocysts in a pr evious study [8] and likewise this activity could not be detected in present work when embryos were cultured indivi dually. The biochemical processes that induce basal IFN levels but prevent FGF-2-induced IFN production in blastocysts exposed to 20% oxygen remain unclear, but the inducti on of oxidative stress is not directly involved with this event. The failure of GM-CSF to increase IFN production and cell numbers from in vitro derived bovine embryos is in direct disagreement with previous reports indicating that GM-CSF

PAGE 85

85 supplementation increases IFN mRNA and/or protein pr oduction in bovine and ovine trophectoderm [314, 326-328] and stimulates embr yo development [329]. Reasons for these unexpected outcomes remain uncertain. The activity of the poGM-CSF may have been compromised within the particular lot used in the experiment. Additionally, perhaps the dose of GM-CSF tested was inappropriate. In an ear lier study, 150-300 pM (3.3-6.6 ng/mL) stimulated IFN production whereas 600 pM did not [326]. GM-CSF was not test ed at different doses in this experiment and the greater concentra tion utilized (100 ng/m L poGM-CSF) may have prevented a beneficial effect to be detected. Conversely, this dose was effective when used on a bovine trophectoderm cell line (CT-1 cells) [328]. It was interesting to observe that GM-CSF prevented FGF-2 from stimulating IFN production. This study is the first to combine FGF2 and GM-CSF treatments, and the presumptive block in FGF-2 effects by GM-CSF suggest that 1) the GM-CSF preparation contained bi ological activity and 2) perhap s these two uterine-derived factors do not cooperate with one another but rather conflict with each other in mediating conceptus development and IFN production. Further study is need ed to resolve these issues before definitive roles of GM-CSF on blastocyst development and IFN production can be described. It remains unclear if the effect of high oxygen on IFN production has any physiological relevance or if it merely is an artifact of the cu lture system. Even so, this aberrant production of IFN may reflect the inferiority of these embryos for producing viable pregnancies. Bovine blastocysts producing lower amounts of IFN prior to transfer generate more pregnancies than blastocysts producing greater amounts of IFN before transfer [348]. Also, bovine blastocysts forming early in culture, which ar e superior at producing pregnanc ies after transfer than embryos lagging in development [197], produce lower amounts of IFN than blastocysts forming two to

PAGE 86

86 three days later [6]. However, although early forming blastocysts produce less IFN initially, they produce much more IFN after three to four days than blastocysts forming later in culture [6]. Taken together, several genetic a nd environmental factors influence IFN production in bovine embryos, and these variables co mplicate the potential use of IFN as a predictor of pregnancy success in cattle. But, the contention that producing more IFN could predict embryos will be less able to produce pregnancies is a provocative concept that may explain why blastocysts cultured in 20% oxygen produce more IFN but have a grea ter incidence of degeneration and fewer cell numbers by day 11 post-IVF. In summary, studies presented here pr ovide evidence that M-199 containing 2.5% FBS and a 5% oxygen environment is able to sustain bovine blastocyst viabilit y until at least day 11 post-IVF. This system also permits blastocyst s to respond to putative embryotrophic factors, such as FGF-2. It is anticipated th at this system will be used by ot hers to resolve some of the key issues relating to conceptus wastage in cattle. Implications: The extension of this research must continue and further expand upon recent discoveries. This group continues to investig ate this research area a nd limits of the culture system developed. Utilizing slight modifications to this culture system resulted in successfully lengthening its boundaries and obtaining health y bovine blastocysts until day 14 post-IVF (Cooke et al., Unpublished observations). Extensi on beyond this point met with little success. Further expansion of this system will include utilizing these methods in conjunction with treatment of other curre ntly known uterine-derived factors and those that have yet to be discovered, as well as developing an agaros e tunnel mold, which will induce embryonic elongation in culture, under treatment with FGF2 and GM-CSF to evaluate for further progress upon previous work.

PAGE 87

87 Future work examining embryotrophic factor s FGF-2 and GM-CSF involve the transfer of embryos treated with these gr owth factors into r ecipients to evaluate filamentous embryonic development, placentation and pregnancy rates compared to in vivo embryos. The analysis of growth factor treatment effects on in vivo derived embryos flushed from donors on day 8 postinsemination and comparisons betwee n their growth factor treated in vitro counterparts for development and IFN secretion will provide further insight on the research presented here. Further interests include the e ffects of these growth factors at various stages of embryonic development between in vitro and in vivo derived embryos beginning at the time of in vitro fertilization to day 8 post-IVF, day 8-11 pos t-IVF and then day 11-14 post-IVF. Finally, evaluating the effects of intrauterine infusion/in jection of these growth factors post-insemination will bring this research full circle, completely evaluating FGF-2 and GM-CSF effects from a strictly in vitro environment to an entirely in vivo one. A more complete understanding of the events associated with embryonic development is a vital component in the complex series of events surrounding the fe tus and mother, which results in either the gain or loss of a pregnancy. The work presented in this thesis is but one piece to the puzzle and must continue so that methods for the ultimate resolution of embryonic loss and reproductive failure may be devised.

PAGE 88

88 LIST OF REFERENCES [1] Inskeep EK, Dailey RA. Embryonic death in cattle. Vet. Clin. Food Anim. 2005; 21: 437-461. [2] Roche JF, Bolandl MP, McGeady TA Reproductive wastage following artificial insemination of heifers. Vet. Rec. 1981; 109: 401-404. [3] De Vreis A. Economic value of pregnanc y in dairy cattle. J. of Dairy Sci. 2006; 89: 3876-3885. [4] Gray CA, Taylor KM, Ramsey WS, Hill JR, Bazer FW, Bartol FF, Spencer TE. Endometrial glands are required for preimp lantation conceptus el ongation and survival. Biol. Reprod. 2001; 64: 1608-1613. [5] Gray CA, Burghardt RC, Johnson GA, Bazer FW, Spencer TE. Evidence that absence of endometrial gland secretions in uterin e gland knockout ewes compromises conceptus survival and elongation. Reproduction 2002; 124: 289-300. [6] Kubisch HM, Larson MA, Roberts RM. Relationship between age of blastocyst formation and interferon-tau secretion by in vitro-derived bovine embryos. Mol. Reprod. Dev. 1998; 49: 254-260. [7] Kubisch HM, Larson MA, Kiesling DO. Cont rol of interferon-tau secretion by in vitroderived bovine blastocysts during extended culture and outgrowth formation. Mol. Reprod. Dev. 2001; 58: 390-397. [8] Michael DD, Alvarez IM, Ocon OM, Po well AM, Talbot N, Johnson SE, Ealy AD. Fibroblast Growth Factor-2 Is Expresse d by the Bovine Uterus and Stimulates Interferon-tau Production in Bovine Trophectoderm. Endocrinology 2006; 147: 35713579. [9] Alexopoulos NI, Vajta G, Ma ddox-Hyttel P, French AJ, Trounson AO. Stereomicroscope and histochemical ex amination of bovine embryos following extended in vitro culture. Repr od. Fertil. Dev. 2005; 17: 799-808. [10] Talbot NC, Caperna TJ, Edwards JL Garrett W, Wells KD, Ealy AD. Bovine blastocyst-derived trophect oderm and endoderm cell cultures: interferon tau and transferrin expression as re spective in vitro markers. Biol. Reprod. 2000; 62: 235-247. [11] Talbot NC, Powell AM, Camp M, Ealy AD. Establishment of a bovine blastocystderived cell line collec tion for the comparative analysis of embryos created in vivo and by in vitro fertilization, somatic cell nuclear transfer, or parthenoge netic activation. In Vitro Cell Dev. Biol. 2007; 43: 59-71. [12] Brandao DO, Maddox-Hyttel P, Lovendahl P, Rumpf R, Stringfellow D, Callesen H. Post hatching development: a novel system for extended in vitr o culture of bovine embryos. Biol. Reprod. 2004; 71: 2048-2055.

PAGE 89

89 [13] Vajta G, Alexopoulos NI, Callesen H. Rapid growth and elongation of bovine blastocysts in vitro in a three-dimensi onal gel system. Theriogenology 2004; 62: 12531263. [14] Vejlsted M, Du Y, Vajta G, MaddoxHyttel P. Post-hatching development of the porcine and bovine embryo: Defi ning criteria for expected development in vivo and in vitro. Theriogenology 2006; 65: 153-165. [15] Roberts RM. Embryonic loss and conceptu s interferon production. St rauss, J. F., III and Lyttle, C. R. Uterine and Embryonic Fact ors in Early Pregnancy. 21-31. 1991. New York, NY, Plenum Press. [16] Roberts RM, Ezashi T, Rosenfeld CS, Ealy AD, K ubisch HM. Evolution of the interferon-tau genes and their promoters, and maternal-trophoblas t interactions in control of their expression. Reprod. Suppl. 61, 239-251. 2003. 1.1.2. [17] Bilby TR, Sozzi A, Lopez MM, Silvestre FT, Ealy AD, Staples CR, Thatcher WW. Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows: I. Ovarian, conceptus, and growth hormone-insu lin-like growth factor system responses. J. Dairy Sci. 2006; 89: 3360-3374. [18] Mann GE, Fray MD, Lamming GE. Effect s of time of progesterone supplementation on embryo development and interferon-tau produc tion in the cow. Vet. J. 2006; 171: 500503. [19] Robinson RS, Fray MD, Wathes DC Lamming GE, Mann GE. In vivo expression of interferon tau mRNA by the embryonic tr ophoblast and uterine concentrations of interferon tau protein during early pregnancy in the cow. Mol. Reprod. Dev. 2006; 73: 470-474. [20] Spicer LJ, Echternkamp SE. Ovarian follic ular growth, function and turnover in cattle: a review. J. of Ani. Sci. 1986; 62: 428-451. [21] Findlay JK. An update on the roles of inhibin, activin, and follstatin as local regulators of folliculogenesis. Biol. Reprod. 1993; 48: 15-23. [22] Hodgen G. The dominant ovarian follicle. Fertil. Steril. 1982; 38: 281-300. [23] Hsueh AJW, Billig H, Tsafriri A. Ovar ian follicle atresia: A hormonally controlled apoptotic process. Endocr. Rev. 1994; 15: 707-724. [24] Bleach EC, Peiris ID, Gr ewal TS, Shepherd DA, Savva D. Effect of administration of a novel recombinant bovine in terferon on length of oestrous cycl e in cattle. Res. Vet. Sci. 1998; 64: 73-77. [25] Kaneko H, Kishi H, Watanabe G, Taya K, Sasamoto S, Hasegawa Y. Changes in plasma concentrations of immunoreactive inhi bin, estradiol and FSH associated with

PAGE 90

90 follicular waves during the estrous cycle of the cow. J. of Reprod. and Dev. 1995; 41: 311-320. [26] Fortune JE. Ovarian follicular grow th and development if mammals. Biol. Reprod. 1994; 50: 225-232. [27] Badinga L, Driancourt MA, Savio JD Wolfenson D, Drost M, De La Sota RL, Thatcher WW. Endocrine and ovarian res ponses associated with the first-wave dominant follicle. Biol Reprod. 1992; 47: 871-883. [28] Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Selection of the dominant follicle in cattle. Biol. Reprod. 1996; 55: 1187-1194. [29] Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the estrous cycle of heifers. J. Reprod. Fertil. 1988; 83: 663-671. [30] Sirois J, Fortune JE. Ovarian follicul ar dynamics during the es trous cycle in heifers monitored by real-time ultrasonogr aphy. Biol. Reprod. 1988; 39: 308-317. [31] Quirk SM, Cowman RG, Harman R.M., Hu C-L, Porter DA. Ovarian follicular growth and atresia: The relationship between cell prol iferation and survival. J. of Ani. Sci. 2007; 82: E40-E52. [32] Webb R, Garnsworthy PC, Gong J-G, Armstrong DG. Control of follicular growth: Local interactions and nutr itional influences. J. of Ani. Sci. 2004; 82: E63-E74. [33] Espey LL. Ovulation as an inflamma tory reaction A hypothesis. Biol. Reprod. 1980; 22: 73-106. [34] Fields MJ, Fields PA. Morphological char acteristics of the bovine corpus luteum during the estrous cycle and pregnanc y. Theriogenology 1996; 45: 1295-1325. [35] Barnes MA, Bierley ST, Halman RD, Henricks DM. Follicle stimulating hormone, luteinizing hormone and estr adiol-17beta response in GnRH treated prepubertal Holstein heifers. Biol. Reprod. 1980; 22: 459-465. [36] Kaltenbach CC, Dunn TG, Kiser TE, Co rah LR, Akbar AM, Niswender GD. Release of FSH and LH in beef heifers by synthetic gon adotropin releasing hormone. J. of Ani. Sci. 1974; 38: 357-362. [37] McCracken JA, Custer EE, Lamsa JC. Luteolysis: A neuroendocrine-mediated event. Physiol Rev. 1999; 79: 263-323. [38] Niswender GD, Juengel JL, McGuire WJ Belfiore CJ, Wiltbank MC. Luteal function: the estrous cycle and early pre gnancy. Biol. Reprod. 1994; 50: 239-247.

PAGE 91

91 [39] Niswender GD, Juengel JL, Silva PJ, Rollyson MK, McIntush EW. Mechanisms controlling the function and lif e span of the corpus luteum. Physiol Rev. 2000; 80: 129. [40] Evans ACO, Komar CM, Wandji S-A, Fo rtune JE. Changes in androgen secretion and luteinizing hormone pulse amplitude are asso ciated with recruitment and growth of ovarian follicles during the luteal phase of the bovine estrous cy cle. Biol. Reprod. 1997; 57: 394-401. [41] Geisert RD, Morgan GL, Short EC, Zavy MT. Endocrine events associated with endometrial function and conceptus developm ent in cattle. Reprod. Fertil. Dev. 1992; 4: 301-305. [42] Lamming GE, Darwash AO, Back HL. Co rpus luteum function in dairy cows and embryo mortality. J. Reprod. Fertil. Suppl 1989; 37: 245-252. [43] Lukaszewska JH, Hansel W. Corpus luteum maintenan ce during early pregnancy in the cow. J. Reprod. Fertil. 1980; 59: 485-493. [44] Spencer TE, Bazer FW. Biology of pr ogesterone action during pregnancy recognition and maintenance of pregnancy. Front. Biosci. 2002; 7: d1879-d1898. [45] Demmers KJ, Derecka K, Flint A. Trophoblast interferon and pregnancy. Reproduction 2001; 121: 41-49. [46] Spencer TE, Johnson GA, Burghardt RC, Bazer FW. Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biol. Reprod. 2004; 71: 2-10. [47] Goff AK. Steroid hormone modulation of prostaglandin secre tion in the ruminant endometrium during the estrous cy cle. Biol. Reprod. 2004; 71: 11-16. [48] Stevenson KR, Riley PR, Stewart HJ, Flint AP, Wathes DC. Localization of oxytocin receptor mRNA in the ovine uterus during th e oestrous cycle and early pregnancy. J. Mol. Endocrinol. 1994; 12: 93-105. [49] Wathes DC, Hamon M. Localization of oestradiol, proge sterone and oxytocin receptors in the uterus during the oestrous cycle and early pregnancy of the ewe. J. Endocrinol. 1993; 138: 479-492. [50] Jenner LJ, Parkinson TJ, Lamming GE. Uterine oxytocin receptors in cyclic and pregnant cows. J. Reprod. Fertil. 1991; 91: 49-58. [51] Mann GE, Lamming GE. Use of repeated biopsies to monitor endometrial oxytocin receptors in cows. Vet. Rec. 1994; 135: 405.

PAGE 92

92 [52] Robinson RS, Mann GE, Lamming GE, Wa thes DC. The effect of pregnancy on the expression of uterine oxytocin, oestrogen and progesterone receptors during early pregnancy in the cow. J. Endocrinol. 1999; 160: 21-33. [53] Robinson RS, Mann GE, Lamming GE, Wa thes DC. Expression of oxytocin, oestrogen and progesterone receptors in uterine biopsy samples througho ut the oestrous cycle and early pregnancy in cows. Reproduction 2001; 122: 965-979. [54] Spencer TE, Ott TL, Bazer FW. Expressi on of interferon regulatory factors one and two in the ovine endometrium: effects of pregna ncy and ovine interfer on tau. Biol. Reprod. 1998; 58: 1154-1162. [55] Horn S, Bathgate R, Lioutas C, Bracke n K, Ivell R. Bovine endometrial epithelial cells as a model system to study oxytocin recepto r regulation. Hum. Reprod. Update 1998; 4: 605-614. [56] Leung ST, Wathes DC. Es tradiol regulation of oxytocin r eceptor expression in cyclic bovine endometrium. J. Reprod. Fertil. 2000; 119: 287-292. [57] Sheldrick EL, Flick-Smith HC. Eff ect of ovarian hormones on oxytocin receptor concentrations in explants of uterus fro m ovarectomised ewes. J. Reprod. Fertil. 1993; 97: 241-245. [58] Wathes DC, Flick-Smith HC, Leung ST, Stevenson KR, Meier S, Jenkin G. Oxytocin receptor development in the ovine uterus and cervix throughout pregnancy and at parturition as determined by in situ hybridi zation analysis. J. Reprod. Fertil. 1996; 106: 25-31. [59] Asselin E, Bazer FW, Fortier MA. R ecombinant ovine and bovine interferons tau regulate prostaglandin production and oxytocin response in cultured bovine endometrial cells. Biol. Reprod. 1997; 56: 402-408. [60] Bazer FW, Spencer TE, Ott TL, Ing NH. Regulation of endometrial responsiveness to estrogen and progesterone by pr egnancy recognition signals during the periimplantation period. Dey, S. K. Molecular and cellular asp ects of periimplantati on processes. 27-47. 2000. Norwell, MA, Springer. [61] Flint APF, Sheldrick EL, McCann TJ, J ones DSC. Luteal oxytocin: characteristics and control of synchronous episodes of oxytocin a nd PGF2alpha secretion at luteolysis in ruminants. Domest. Anim Endocrinol. 1990; 7: 111-124. [62] Milvae RA. Inter-rela tionships between endothelium and prostaglandin f2alpha in corpus luteum function. Rev. of Reprod. 2000; 5: 1-5. [63] Ohtani M, Kobayashi S, Miyamoto A, Hayashi K, Fukui Y. Real-time relationships between intraluteal and plasma concen trations of endot helin, oxytocin, and progesterone during prostaglandi n f2alpha-induced luteolysis in the cow. Biol. Reprod. 1998; 58: 103-108.

PAGE 93

93 [64] Tedeschi C, Hazum E, Kokia E, Ricciarelli E, Adas hi EY, Payne DW. Endothelin-1 and a luteinization inhibitor: Inhibition of rat granulosa cell progesterone accumulation via selective modulation of key steroidoge nic steps affecting both progesterone formation and degradation. Endocrinology 1992; 131: 2476-2478. [65] Tedeschi C, Loham C, Hazum E, I ttop O, Ben-shlomo I, Resnick CE, Payne DW, Adashi EY. Rat ovarian granulosa cell as a site of ET-1 re ception and action: attenuation of gonadotropin-stimulated steroido genesis via perturbati on of the A-kinase signaling pathway. Biol. Reprod. 1994; 51: 1058-1065. [66] Iwai M, Hasegawa M, Taii S, Sagawa M, Nakao K, Imura H, Nakanishi S, Mori T. Endothelin inhibits luteinization of cultu red porcine granulosa cells. Endocrinology 1991; 129: 1909-1914. [67] Girsh E, Milvae RA, Wang W, Mcidan R. Effect of endothelin-1 on bovine luteal cell funtion: role in prostagla ndin F2alpha-induced antistero idogenic action. Endocrinology 1996; 137: 1306-1312. [68] Girsh E, Wang W, Arditi F, Friedm an A, Milvae RA, Meidan R. Regulations of endothelin-1 expression in the bovine cor pus luteum: elevation by prostaglandin F2alpha. Endocrinology 1996; 137: 5191-5196. [69] Miyamoto A, Kobayashi S, Arata S, Ohtani M, Fukui Y, Schams D. Prostaglandin F2alpha promotes the inhibito ry action of endothelin-1 on th e bovine luteal function in vitro. J. of Endocrinol. 1997; 152: R7-R11. [70] Ji I, Slaughter RG, Ellis JA, Ji TH, Murdoch WJ. Analysis of ovine corpora lutea for tumor necrosis factoor mRNA and bioactivit y during prostaglandin-induced luteolysis. Mol. Cell. Endocrinol. 1991; 81: 77-80. [71] Shaw DW, Britt JH. Concentrations of tumor necrosis factor alpha and progesterone within the bovine corpus luteum samp led by continuous-flow microdialysis during luteolysis in vitro. Bi ol. Reprod. 1995; 53: 847-854. [72] Juengel JL, Garverick HA, Johnson AL Young-Quist RS, Smith MF. Apoptosis during luteal regression in cattle Endocrinology 1993; 132: 249-254. [73] Sawyer HR, Niswender KD, Niswender GD. Nuclear changes in ovi ne luteal cells in response to PGF2alpha. Domest. Anim. Endocrinol. 1990; 7: 229-238. [74] Zheng J, Fricke PM, Reynolds LP Redmer DA. Evaluation of growth, cell proliferation, and cell death in bovine corpora lutea thr oughout the estrous cycle. 51 1994; 632. [75] Hasumoto K, Sugimoto Y, Yamasaki A, Morimoto K, Negishi M, Ichikawa A. Association of expression of mRNA encoding PGF2alpha receptor with luteal apoptosis in ovaries of pseudopregnant mice. J. Reprod. Fertil. 1997; 109: 45-51.

PAGE 94

94 [76] Friedman A, Weiss S, Levy N, Meidan R. Role of tumor necrosis factor alpha and its type I receptor in luteal regression: I nduction of programmed cell death in bovine corpus luteum-derived endothelial cells. Biol. Reprod. 2000; 63: 1905-1912. [77] Evans JP, Floyd JG. The state of the union: the cell biol ogy of fertilization. Nat. Cell. Biol. 2002; 4: s57-s63. [78] Suarez SS, Pacey AA. Sperm transport in the female reproductive tract. Hum. Reprod. Update 2006; 12: 23-27. [79] Florman HM, First NL. The regulation of acrosomal exocytosis. I. Sperm capacitation is required for the induction of acrosome r eactions by the bovine zona pellucidae in vitro. Dev. Biol. 1988; 128: 453-463. [80] Handrow R., Boehm SK Lenz RW, Robinson JA, Ax RL. Specific binding of the glycosaminoglycan 3H-heparin to bull, m onkey, and rabbit spermatozoa in vitro. J. Androl. 1984; 5: 51-63. [81] Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol. Reprod. 1988; 35: 608-617. [82] Parrish JJ, Susko-Parrish JL, Handrow R.H., Sims MM, First NL. Capacitation of bovine spermatozoa by oviduct fluid. Biol. Reprod. 1989; 40: 1020-1025. [83] Florman HM, Wassarman PM. O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. Cell 1985; 41: 313-324. [84] Wassarman PM, Litscher ES. Towards th e molecular basis of sperm and egg interaction during mammalian fertilization. Cell s Tissues Organs 2001; 168: 36-45. [85] Schultz RM, Kopf GS Molecular basis of of ma mmalian egg activation. Curr. Top. Dev. Biol. 1995; 30: 21-62. [86] Rossant J. Postimplantation developmen t of blastomeres isolated from 4and 8-cell mouse eggs. J. of Embryol. Exp. Morphol. 1976; 36: 283-290. [87] Betteridge KJ, Flechon J-E. The anat omy and physiology of pre-attachment bovine embryos. Theriogeno logy 1988; 29: 155-187. [88] Van Soom A, Boerjan M, Ysebaert M-T, de Kruif A. Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media. Mol. Reprod. Dev. 1996; 45: 171-182. [89] Garbutt CL, Chisholm JC, Johnson MH. The establishment of the embryonicabembryonic axis in the mouse em bryo. Development 1987; 100: 125-134. [90] Kidder GM, McLachlin JR. Timing of tr anscription and protei n synthesis underlying morphogenesis in preimplantation mous e embryos. Dev. Biol. 1985; 112: 265-275.

PAGE 95

95 [91] Flechon JE, Renard JP. A scanning electron microscope st udy of the hatching of bovine blastocysts in vitro. J. Reprod. Fertil. 1978; 53: 9-12. [92] Perry JS. The mammalian fetal memb ranes. J. Reprod. Fertil. 1981; 62: 321-335. [93] Schlafer DH, Fisher PJ, Davies CJ The bovine placenta before and after birth: placental development and function in health and disease. Anim Reprod. Sci. 2000; 6061: 145-160. [94] Chang MC. Development fo the bovine blastocyst with a note on implantation. Anat. Rec. 1952; 113: 143-161. [95] Curran S, Pierson RA, Ginther OJ Ultrasonographic appear ance of the bovine conceptus from days 20 thr ough 60. JAVMA 1986; 189: 1295-1302. [96] Schlafke S, Enders A.C. Cellular basi s of interaction between trophoblast and uterus at implantation. Biol. Reprod. 1975; 12: 41-65. [97] Wango EO, Wooding FB, Heap RB. The role of trophobl astic binucleate cells in implantation in the goat: a morphologi cal study. J. Anat. 1990; 171: 241-257. [98] Wooding FB. Role of binuc leate cells in fetomaternal cel l fusion at implantation in the sheep. Am. J. Anat. 1984; 170: 233-250. [99] Wooding FB. Current topi c: the synepitheliochorial plac enta of ruminants: binucleate cell fusions and hormone producti on. Placenta 1992; 13: 101-113. [100] Wooding FBP, Chambers SG, Perry JS, George M, Heap RB. Migration of binucleate cells in the sheep placenta during nor mal pregnancy. Anat. Embryol. 1980; 158: 361370. [101] Wooding FB, Flint AP Heap RB, Morgan G, Buttle HL, Young IR. Control of binucleate cell migration in the placenta of sheep and goats. J. Reprod. Fertil. 1986; 76: 499-512. [102] Wooding FBP. Frequency and localisation of binucleate cells in the placentomes of ruminants. Placenta 1983; 4: 527-540. [103] Gootwine E. Placental hormones and fe tal-placental development. Anim Reprod. Sci. 2004; 82-83: 551-566. [104] Grosser O. Fruhentwicklung, Eiha utbildung und Placentation des Menschen und der Saugetiere. Munich: 1927. [105] Wooding FB, Morgan G, Brandon MR, Camous S. Membrane dynamics during migration of placental cells through trophectod ermal tight junctions in sheep and goats. Cell Tissue Res. 1994; 276: 387-397.

PAGE 96

96 [106] Wooding FB, Morgan G, Adam CL. Structure and function in the ruminant synepitheliochorial placenta: central role of the trophoblas t binucleate cell in deer. Microsc. Res. Tech. 1997; 38: 88-99. [107] Lang CY, Hallack S, Leiser R, Pfarrer C. Cytoskeletal f ilaments and associated proteins during resricted trophoblast invasion in bovi ne placentomes: light and transmission electron microscopy and RT-PCR. Cell Tissue Res. 2004; 315: 339-348. [108] Pfarrer C, Wirth C, Schuler G, Kl isch K, Leiser R, Hoffman B. Frequency, ultrastructural features, and relevance of a poptosis in the bovine placenta. Biol. Reprod. 1999; 60: 126. [109] Eley RM, Thatcher WW, Bazer FW. Ho rmonal and physical changes associated with conceptus development. J. Reprod. Fertil. 1979; 55: 181-190. [110] Hoffman B, Schuler G. The bovine placen ta; a source and target of steroid hormones: observations during the second half of ge station. Domest. Anim Endocrinol. 2003; 23: 309-320. [111] Hoffmann B, Goes de Pinho T, Schul er G. Determination of free and conjugated oestrogens in peripheral blood plasma, feces and urine of cattle throughout pregnancy. Exp. Clin. Endocrinol. Di abetes 1997; 105: 296-303. [112] Birgel EH, Zerbe H, Grunert E. Untersuchugen uber Zusammenhange zwischen Anzeichen der nahenden Abkalbung und Steroi dhormonprofilen. Prakt Tierarzt 1996; 77: 627-630. [113] Anthony RV, Liang R, Kayl EP, Pratt SL. The growth of hormone/prolactin gene family in ruminant placentae. J. Reprod. Fertil. Suppl 1995; 49: 83-95. [114] Duello TM, Byatt JC, Bremel RD. Immunohistochemical localization of placental lactogen in binucleate cells of bovine placentomes. Endocrinology 1986; 119: 13511355. [115] Wooding FBP, Beckers JF Trinucleate cells and the ul trastructural localization of bovine placental lactogen. Ce ll Tissue Res. 1987; 247: 667-673. [116] Anthony RV, Pratt SL, Liang R, Holla nd MD. Placental-fetal hormonal interactions: impact on fetal growth. J. Anim Sci. 1995; 73: 1861-1871. [117] Forsyth I.A., Wallis M. Growth hor mone and prolactin-molecular and functional evolution. J. Mammary Gland Bi ol. Neoplasia 2002; 7: 291-312. [118] Gluckman PD, Pinal CS. Regulation of fetal growth by the somatotrophic axis. J. Nutr. 2003; 133: 1741S-1746S. [119] Soares MJ, Muller H, Orwig KE, Peters TJ, Dai G. The uteroplacental prolactin family and pregnancy. Biol. Re prod. 1998; 58: 273-284.

PAGE 97

97 [120] Gertler A, Djiane J. Mechanism of ruminant placental la ctogen action: molecular and in vivo studies. Mol. Genet. Metab. 2002; 75: 189-201. [121] Johnson GA, Burghardt RC, Spencer TE, Newton GR, Ott TL, Bazer FW. Ovine osteopontin: II. Osteopontin and alpha(v)beta(3 ) integrin expression in the uterus and conceptus during the periimplantation period. Biol. Reprod. 1999; 61: 892-899. [122] Johnson GA, Spencer TE, Burghardt RC, Bazer FW. Ovine osteopontin: I. Cloning and expression of messenger ribonucleic acid in the uterus during the periimplantation period. Biol. Reprod. 1999; 61: 884-891. [123] Beckers JF, Decoster R, Wouters-Ballman P, Fromont -Lienard CH, Van der Zwalmen P, Ectors F. Dosage radioimmunologique de l'hormone placentaire somatotrope et mammotrope bovine. Ann. Vet. Med. 1982; 126: 9-21. [124] Chan JSD, Robertson HA, Friesen HG. Maternal and fetal con centrations of ovine placental lactogen measured by radi oimmunoassay. Endocrinology 1978; 102: 16061613. [125] Noel S, Herman A, Johnson GA, Gray CA, Stewart MD, Bazer FW, Gertler A, Spencer TE. Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus. Biol. Reprod. 2003; 68: 772-780. [126] Bole-Feysot C, Goffin V, Edery M, Ke lly PA. Prolactin (PRL) a nd its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr. Rev. 1998; 19: 225-268. [127] Igwebuike UM. Trophoblas t cells of ruminant placentas-A minireview. Anim Reprod. Sci. 2005. [128] Goff JP, Horst RL. P hysiological changes at parturition and their relationship to metabolic disorders. J. of Dairy Sci. 1997; 80: 1260-1268. [129] Chew BP, Keller HF, Er b RE, Malvern PV. Periparturient concentrations of prolactin, progesterone, and estrogens in blood plasma of cows retaining a nd not retaining fetal membranes. J. of Ani. Sci. 1977; 44: 1055. [130] Challis JRG, Sloboda D, Matthews SG, Holloway A, Alfaidy N, Patel FA, Whittle W, Fraser M, Moss TJM, Newnham J. The fe tal hypothalamic-placental-adrenal (HPA) axis, parturition and post natal health Mol. Cell. Endocrinol. 2001; 185: 135-144. [131] Bloomfield FH, Oliver MH, Hawkin s P, Holloway AC, Campbell M, Gluckman PD, Harding JE, Challis JRG. Periconcepti onal undernutrition in sheep accelerates maturation of the fetal hypothalamic-pituitary-adrenal axis in late gestation. Endocrinology 2004; 145: 4278-4285. [132] Tuo W, Ott TL, Bazer FW Natural killer cell activity of lymphocytes exposed to ovine, type I, trophoblast interferon. Am J Reprod Immunol 1993; 29: 26-34.

PAGE 98

98 [133] Fuchs AR, Helmer H, Behrens O, Li u H-C, Antonian L, Chang SM, Fields MJ. Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors precedes onset of labor. Biol. Reprod. 1992; 47: 937-944. [134] Moor RM, Rowson LE. The corpus lute um of the sheep: effect of the removal of embryos on luteal function. J. Endocrinol. 1966; 34: 497-502. [135] Moor RM, Rowson LE. The corpus lu teum of the sheep: functional relationship between the embryo and the corpus lu teum. J. Endocrinol. 1966; 34: 233-239. [136] Moor RM, Rowson LE, Hay MF, Caldwell BV The corpus luteum of the sheep: effect of the conceptus on luteal function at seve ral stages during pregnancy. J. Endocrinol. 1969; 43: 301-307. [137] Rowson LE, Moor RM, La wson RA. Fertility following egg transfer in the cow; effect of method, medium and synchronization of oe strus. J. Reprod. Fertil. 1969; 18: 517523. [138] Rowson LE, Lawson RA, Moor RM, Baker AA. Egg transfer in the cow: synchronization requirements. J. Reprod. Fertil. 1972; 28: 427-431. [139] Martal J, Lacroix MC, Loudes C, Sauni er M, Wintenberger-Torres S. Trophoblastin, an antiluteolytic protein present in early pr egnancy in sheep. J. Reprod. Fertil. 1979; 56: 63-73. [140] Godkin JD, Bazer FW, Th atcher WW, Roberts RM. Proteins released by cultured Day 15-16 conceptuses prolong luteal maintenan ce when introduced into the uterine lumen of cyclic ewes. J. Reprod. Fertil. 1984; 71: 57-64. [141] Knickerbocker JJ, Thatcher WW, Bazer FW, Drost M, Barron DH, Fincher KB, Roberts RM. Proteins secret ed by day-16 to -18 bovine conceptuses extend corpus luteum function in cows. J. Reprod. Fertil. 1986; 77: 381-391. [142] Imakawa K, Anthony RV, Kazemi M, Marotti KR, Polites HG, Roberts RM. Interferon-like sequence of ovine tro phoblast protein secreted by embryonic trophectoderm. Nature 1987; 330: 377-379. [143] Cross JC, Roberts RM. Constitutive and trophoblast-specific expression of a class of bovine interferon genes. Proc. Natl. Acad. Sci. U. S. A 1991; 88: 3817-3821. [144] Leaman DW, Cross JC, Roberts RM. Mu ltiple regulatory elements are required to direct trophoblast interfer on gene expression in choriocarcinoma cells and trophectoderm. Mol. Endocrinol. 1994; 8: 456-468. [145] Farin CE, Imakawa K, Hansen TR McDonnell JJ, Murphy CN, Farin PW, Roberts RM. Expression of trophoblasti c interferon genes in sheep and cattle. Biol. Reprod. 1990; 43: 210-218.

PAGE 99

99 [146] Roberts RM, Liu L, Alexenko A. New a nd atypical families of type I interferons in mammals: comparative functions, structures and evolutionary relationships. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 287-325. [147] Roberts RM, Liu L, Guo Q, Leaman D, Bixby J. The evolution of the type I interferons [published erratum appears in J Interfe ron Cytokine Res 1999 Apr;19(4):427]. J. Interferon Cytokine Res. 1998; 18: 805-816. [148] Leaman DW, Roberts RM. Genes for th e trophoblast interferons in sheep, goat, and musk ox and distribution of related genes among mammals. J. Interferon Res. 1992; 12: 1-11. [149] Bartol FF, Roberts RM, Bazer FW, Lewis GS, Godkin JD, Thatcher WW. Characterization of proteins produced in vi tro by periattachment bovine conceptuses. Biol. Reprod. 1985; 32: 681-693. [150] Ealy AD, Green JA, Alexenko AP, Keisler DH, Roberts RM. Different ovine interferon-tau genes are not expressed identically and th eir protein products display different activities. Biol. Reprod. 1998; 58: 566-573. [151] Ealy AD, Larson SF, Liu L, Alex enko AP, Winkelman GL, Kubisch HM, Bixby JA, Roberts RM. Polymorphic forms of expresse d bovine interferon-tau genes: relative transcript abundance during early placental development, pr omoter sequences of genes and biological activity of protein pr oducts. Endocrinology 2001; 142: 2906-2915. [152] Stewart HJ, McCann SH, Northrop AJ, Lamming GE, Flint AP. Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels. J. Mol. Endocrinol. 1989; 2: 65-70. [153] Hernandez-Ledezma JJ, Sikes JD, Mu rphy CN, Watson AJ, Schultz GA, Roberts RM. Expression of bovine trophoblas t interferon in conceptu ses derived by in vitro techniques. Biol. Re prod. 1992; 47: 374-380. [154] Hernandez-Ledezma JJ, Mathialaga n N, Villanueva C, Sikes JD, Roberts RM. Expression of bovine trophoblas t interferons by in vitroderived blastocysts is correlated with their morphological quality and stage of development. Mol. Reprod. Dev. 1993; 36: 1-6. [155] Godkin JD, Bazer FW, Moffatt J, Sessions F, Roberts RM. Purification and properties of a major, low molecular weight prot ein released by the trophoblast of sheep blastocysts at day 13-21. J. Reprod. Fertil. 1982; 65: 141-150. [156] Roberts RM. Minirevi ew: Conceptus interferons a nd maternal recognition of pregnancy. Biol. Repr od. 1989; 40: -449. [157] Roberts RM, Cross JC, Leaman DW. Unique features of the trophoblast interferons. Pharmacol Ther 1992; 51: 329-345.

PAGE 100

100 [158] Dannet-Desnoyers, ., Wetzels C, Th atcher WW. Natural and recombinant bovine interferon tau regulate basal and oxytocin-induc ed secretion of prostaglandins F2 alpha and E2 by epithelial cells a nd stromal cells in the endom etrium. Reprod. Fertil. Dev. 1994; 6: 193-202. [159] Meyer MD, Hansen PJ, Thatcher WW, Dr ost M, Badinga L, Roberts RM, Li J, Ott TL, Bazer FW. Extension of corpus luteum lifes pan and reduction of uterine secretion of prostaglandin F2 alpha of cows in response to recombinant interferontau. J. Dairy Sci. 1995; 78: 1921-1931. [160] Parent J, Villeneuve C, Alexenko AP Ealy AD, Fortier MA. Influence of different isoforms of recombinant trophoblastic inte rferons on prostaglandin production in cultured bovine endometrial cells Biol. Reprod. 2003; 68: 1035-1043. [161] Flint APF, Sheldrick EL. Ovarian oxyt ocin and maternal recognition of pregnancy. J. Reprod. Fertil. 1986; 76: 831-839. [162] Mirando MA, Harney JP, Zhou Y, Ogle TF, Ott TL, Moffatt RJ, Bazer FW. Changes in progesterone and oestrogen receptor mRNA and protein and oxytocin receptors in endometrium of ewes after intrauterine inj ection of ovine trophoblas t interferon. J. Mol. Endocrinol. 1993; 10: 185-192. [163] Spencer TE, Bazer FW. Ovine interferon-tau suppresses transcription of the estrogen receptor and oxytocin receptor genes in the ovine endometrium. Endocrinology 1996; 137: 1144-1147. [164] Harada H, Takahashi E, Itoh S, Hara da K, Hori TA, Taniguchi T. Structure and regulation of the human inte rferon regulatory factor-1 (Irf-1) and Irf-2 genes Implications for a gene network in the interferon system. Mol. Cell Biol. 1994; 14: 1500-1509. [165] Bazer FW, Spencer TE, Ott TL. Interferon tau: a novel pregnancy recognition signal. Am. J. Reprod. Immunol. 1997; 37: 412-420. [166] Clark JD, Lin L, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, Milona N, Knopf JL. A novel arachidonic acid-selective cytoso lic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell 1991; 65: 1043-1051. [167] Xiao CW, Liu JM, Sirois J, Goff AK. Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-tau in bovine endometrial cells. Endocrinology 1998; 139: 2293-2299. [168] Binelli M, Subramaniam P, Diaz T, Johnson GA, Hansen TR, Badinga L, Thatcher WW. Bovine Interferon-tau Stimulates the Janus Kinase-Signal Transducer and Activator of Transcription Pathway in B ovine Endometrial Epithelial Cells. Biol. Reprod. 2001; 64: 654-665.

PAGE 101

101 [169] Guzeloglu A, Michel F, Thatcher WW Differential effects of interferon-tau on the prostaglandin synthetic pathway in bovine e ndometrial cells treate d with phorbol ester. J. of Dairy Sci. 2004; 87: 2032-2041. [170] Asselin E, Lacroix D, Fortier MA IFN-tau increases PGE2 production and COX-2 gene expression in the bovine endometriu m in vitro. Mol. Cell Endocrinol. 1997; 132: 117-126. [171] Asselin E, Drolet P, Fortier MA. In vitro response to oxytocin and interferon-tau in bovine endometrial cells from caruncular and intercaruncular areas. Biol. Reprod. 1998; 59: 241-247. [172] Smith WL, Marnett LJ DeWitt DL. Prostaglandin and thromboxane biosynthesis. Pharmacol Ther 1991; 49: 153-179. [173] Skopets B, Li J, Thatcher WW, Ro berts RM, Hansen PJ. Inhibition of lymphocyte proliferation by bovine trophoblast protein-1 (type I trophoblast in terferon) and bovine interferon-alpha I1. Vet. Imm unol. Immunopathol. 1992; 34: 81-96. [174] Thatcher WW. Antiluteolytic signa ls between the conceptus and endometrium. Theriogenology 47, 131-140. 1997. [175] Fillion C, Chaouat G, Reinaud P, Char pigny JC, Martal J. Imm unoregulatory effects of ovine trophoblastin protein (o TP): all five isoforms s uppress PHA-induced lymphocyte proliferation. J. Reprod. Immunol. 1991; 19: 237-249. [176] Newton CR, Vallet JL, Hansen PJ, Bazer FW. Inhibition of lymphocyte proliferation by ovine trophoblast protein-1 a nd a high molecular weight glycoprotein produced by the peri-implantation sheep conceptus. Am. J. Reprod. Immunol. 1989; 19: 99. [177] Niwano Y, Hansen TR, Kazemi M, Malathy PV, Johnson HD, Roberts RM, Imakawa K. Suppression of T-lymphocyte blastogene sis by ovine trophoblast protein-1 and human interferon-alpha may be independent of interleuki n-2 production. Am J Reprod Immunol. 1989; 20: 21-26. [178] Roberts RM, Imakawa K, Niwano Y, Kazemi M, Malathy PV, Hansen TR, Glass AA, Kronenberg LH. Interferon production by the preimplantation sheep embryo. J. Interferon Res. 1989; 9: 175-187. [179] Proost P, Wuyts A, Conings R, Lenaer ts JP, Billiau A, Opdenakker G, Van Damme J. Human and bovine granulocyte chemotactic protein-2: co mplete amino acid sequence and functional characteriza tion as chemkines. Bioc hemistry 1993; 32: 10170-10177. [180] Choi Y, Johnson GA, Spencer TE, Bazer FW. Pregnancy and interferon tau regulate major histocompatibility complex class I and B2-microglobulin expression in the ovine uterus. Biol. Reprod. 2003; 68: 1703-1710.

PAGE 102

102 [181] Naivar KA, Ward SK, Austin KJ, Moor e DW, Hansen TR. Secretion of bovine uterine proteins in response to type I in terferons. Biol. Reprod. 1995; 52: 848-854. [182] Staggs KL, Austin KJ, Johnson GA, Te ixeira MG, Talbott CT, Dooley VA, Hansen TR. Complex induction of bovine uterine proteins by interfer on-tau. Biol. Reprod. 1998; 59: 293-297. [183] Teixeira MG, Austin KJ, Perry DJ, Dooley VD, Johnson GA, Francis BR, Hansen TR. Bovine granulocyte chemotactic protein-2 is secreted by the endometrium in response to interferon-tau (IFN-tau). Endocrine. 1997; 6: 31-37. [184] Johnson GA, Joyce MM, Yankey SJ, Hans en TR, Ott TL. The Interferon Stimulated Genes (ISG) 17 and Mx have different tem poral and spatial expression in the ovine uterus suggesting more complex regulation of the Mx gene. J. Endocrinol. 2002; 174: R7-R11. [185] Ott TL, Yin J, Wiley AA, Kim HT, Gera mi-Naini B, Spencer TE, Bartol FF, Burghardt RC, Bazer FW. Effects of the estrous cycle and early pregnancy on uterine expression of Mx protein in sheep (Ovis ar ies). Biol. Repr od. 1998; 59: 784-794. [186] Johnson GA, Stewart MD, Gray CA, C hoi Y, Burghardt RC, Yu-Lee LY, Bazer FW, Spencer TE. Effects of the estrous cycle, pregnancy, and interferon tau on 2',5'oligoadenylate synthetase expression in the ovine uterus. Biol. Reprod. 2001; 64: 13921399. [187] Pru JK, Austin KJ, Haas AL, Hansen TR. Pregnancy and interferon-tau upregulate gene expression of members of the 1-8 family in the bovine uterus. Biol. Reprod. 2001; 65: 1471-1480. [188] Charleston B, Stewart HJ. An inte rferon-induced Mx protein: cDNA sequence and high-level expression in the endometrium of pregnant sheep. Gene 1993; 137: 327-331. [189] Hicks BA, Etter SJ, Carnahan KG, Joyce MM, Assiri AM, Carling SJ, Kodali K, Johnson GA, Hansen TR, Mirando MA, Woods GL, Vanderwall DK, Ott TL. Expression of the uterine Mx protein in cyclic and pregnant cows, gilts and mares. J. of Ani. Sci. 2003; 81: 1561. [190] Horisberger MA, Staehel i P, Haller O. Interferon induces a unique protein in mouse cells bearing a gene for resistance to influen za. Proc. Natl. Acad. Sci. U. S. A 1983; 80: 1914. [191] Horisberger MA, Gunst MC. Interferon-induced proteins: identification of Mx proteins in various mammalian species. Virology 1991; 180: 185-190. [192] Deblandre GA, Marinx OP, Evans SS, Ma jjaj S, Leo O, Caput D, Huez GA, Wathelet MG. Expression cloning of an interfer on-inducible 17-kDa membrane protein implicated in the control of cell growth. J. Biol. Chem. 1995; 270: 23860-23866.

PAGE 103

103 [193] Evans SS, Collea RP, Leasure JA, Lee DB. IFN-alpha induc es homotypic Leu-13 expression in human B lymphoid cells. J Immunol 1993; 150: 736-747. [194] Chen YX, Welte K, Gebhard DH, Evans RL. Induction of T cell aggregation by antibody to a 16kD human leukocyte surf ace antigen. J Immunol 1984; 133: 24962501. [195] Al-Katanani YM, Drost M, Monson RL, Rutledge JJ, Krininger CE, Block J, Thatcher WW, Hansen PJ. Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lacta ting dairy cows under heat stress conditions. Theriogenology 2002; 58: 171-182. [196] Franco M, Block J, Jousan FD, de Cast ro e Paula LA, Brad AM, Franco JM, Grisel F, Monson RL, Rutledge JJ, Hansen PJ. Pregna ncy rates in heat-stressed dairy cattle receiving one or two on vitr o-produced embryos in a timed embryo transfer program. Reprod.Fertil.Dev. 18[2], 202-203. 2006. [197] Hasler JF, Henderson WB, Hurtgen PJ Jin ZQ, McCauley AD, Neely B, Shuey LS, Stokes JE, Trimmer SA. Production, freezing an d transfer of bovine IVF embryos and subsequent calving results. Theriogenology 1995; 43: 141-152. [198] Putney DJ, Drost M, Thatcher WW. Influence of Summer He at-Stress on Pregnancy Rates of Lactating Dairy-Cattle Following Embryo Transfer Or Artificial-Insemination. Theriogenology 1989; 31: 765-778. [199] Reichenbach HD, Liebrich J, Berg U, Brem G. Pregnancy rates and births after unilateral or bilatera l transfer of bovine embryos produ ced in vitro. J. Reprod. Fertil. 1992; 95: 363-370. [200] Thibault C. In vitro culture of cow egg. Ann. Biol. Anim. Biochem. Biophys. 1966; 6: 159-164. [201] Barnes FL, Eyestone WH. Early cleavage and the maternal zygo tic transition in bovine embryos. Theriogeno logy 1990; 33: 141-152. [202] Frei RE, Schultz GA, Church RB. Qu alitative and quantitativ e changes in protein synthesis occur at the 8-16-cel l stage of embryogenesis in the cow. J. Reprod. Fertil. 1989; 86: 637-641. [203] Thompson JG. Defining the requirem ents for bovine embryo culture. Theriogenology 1996; 45: 27-40. [204] Gandolfi F, Moor RM. Stimulation of early embryonic development in the sheep by coculture with oviduct cells. J. Reprod. Fertil. 1987; 81: 23-28. [205] Bavister BD. Culture of preimplant ation embryos: facts and artifacts. Hum. Reprod. Update 1995; 1: 91-148.

PAGE 104

104 [206] Leese HJ, Alexiou M, Comer MT, Lamb VK, Thompson JG. Assessment of embryo nutritional requirements and role of co-cul ture techniques. Assisted Reproduction[7]. 1995. Carnforth, Parthenon. Advances in Repr oductive Endocrinology Series. Shaw, R. W. [207] Brackett BG. In vitro culture of the zygote and embryo. In: Mast roianni L, Jr., Biggers J (eds.), Fertilization and Embryonic Developm ent In Vitro. New York: Plenum Press; 1981: 61-79. [208] Maurer HR. Towards chemically-d efined, serum-free media for mammalian cell culture. In: Freshney RI (ed.). Oxford: IRL press; 1986: 13-31. [209] Merten OW. Safety issues of animal products used in serum-free media. Dev Biol Stand 1999; 99: 167-180. [210] Gutierrez CG, Ralph JH, Telfer EE, Wilm ut I, Webb R. Growth and antrum formation of bovine preantral follicle s in long-term culture in vitro. Biol. Reprod. 2000; 62: 1322-1328. [211] Newton H, Picton H, Gosden RG. In v itro growth of oocyte-granulosa cell complexes isolated from cryopreserve d ovine tissue. J. Reprod. Fertil. 1999; 115: 141-150. [212] Senbon S, Fukumi Y, Hamawaki A, Yo shikawa M, Miyano T. Bovine oocytes grown in serum-free medium acquire fertilizati on competence. J. of Reprod. and Dev. 2004; 50: 541-547. [213] Bavister BD, Rose-Hellekant TA Pinyopumminter T. Development of in-vitro matured/in-vitro fertilized bovine embryos into morulae and blastocysts in defined culture media. Theri ogenology 1992; 37: 127-146. [214] Mastromonaco GF, Semple E, Robe rt C, Rho GJ, Betts DH, King WA. Different culture media requirements of IVF and nuclear transfer bovine embryos. Reprod. Domest. Anim. 2004; 39: 462-467. [215] Correa GA, Rumpf R, Mundim TCD, Fr anco MM, Dode MAN. Oxygen tension during in vitro culture of bovine embryos: Effect in production and expression of genes related to oxidative stress. Anim. Reprod. Sci. 2007. [216] Kim JH, Niwa K, Lim JM, Okuda K. Effects of Phosphate, energy substrates, and amino acids on development of in vitro-mature d, in vitro-fertilized bovine oocytes in a chemically defined, protein-free cultur e medium. Biol. Reprod. 1993; 48: 1320-1325. [217] Gardner DK, Lane M. Embryo Culture Systems. In: Trounson A, Gardner DK (eds.), Handbook of In Vitro Fertilization. Bo ca Raton: CRC Press Inc.; 1993: 85-114. [218] Bavister BD, Arlotto T. Influence of single amino acids on the development of hamster one-cell embryo in vitro. Mol. Reprod. Dev. 1990; 25: 45-51.

PAGE 105

105 [219] Carney EW, Bavister BD. Stimulator y and inhibiting effects of amino acids on the development of hamster eight-cell embryos in vitro. J. In Vitro Fert. Embryo Transf. 1987; 4: 162-167. [220] Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mo use embryos in vitro. J. Reprod. Fertil. 1989; 86: 679-688. [221] Steeves TE, Gardner DK. Temporal a nd differential effects of amino acids on bovine embryo development in culture. Biol. Reprod. 1999; 61: 731-740. [222] Ball GD, Liebfried ML, Lenz RW, Ax RL, Bavister BD, First NL. Factors effecting successful in vitro fertilisation of bovine follicular oocytes. Biol. Reprod. 1983; 28: 717-725. [223] Lenz RW, Ball GD, Liebfried ML, Ax RL, First NL. In-vitro maturation and fertilisation of bovine oocytes are temper ature dependent processes. Biol. Reprod. 1987; 29: 173-179. [224] Busa WB, Nuccitelli R. Metabolic re gulation via intracellular pH. Am J Physiol 1984; 246: R409-R438. [225] Hunter RHF. "The Fallo pian Tubes. Their role in fer tility and in fertility". Berlin: Springer-Verlag; 1988. [226] Olds D, VanDemark NL. Luminal fluids of bovine female genitalia. J. Am. Vet. Med. Assoc. 1957; 31: 555-556. [227] Fischer B, Bavister BD Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J. Reprod. Fertil. 1993; 99: 673-679. [228] Guerin P, Mouatassim S, Menezo ElY. Oxidative stress and protection against reactive oxygen species in the pre-implantation em bryo and its surroundings. Hum. Reprod. Update 2001; 7: 175-189. [229] Kitagawa Y, Suzuki K, Yoneda A, Wa tanabe T. Effects of oxygen concentration and antioxidants on the in vitro developmen tal ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology 2004; 62: 1186-1197. [230] Takahashi M, Keicho K, Takahashi H, Ogawa H, Schultz RM, Okano A. Effect of oxidative stress on development and DNA chan ge in in-vitro cu ltured bovine embryos by comet assay. Theriogenology 2000; 54: 137-145. [231] Yuan YQ, Van Soom A, Coopman FOJ, Mintiens K, Bo erjan ML, Van Zeveren A, de Kruif A, Peelman L.J. Influence of oxyge n tension on apoptosis and hatching bovine embryos cultured in vitro. Th eriogenology 2003; 59: 1585-1596.

PAGE 106

106 [232] Holm P, Walker SK, Seamark RF. Embr yo viability, duration of gestation and birth weight in sheep after transfer of in vitro matured and in vi tro fertilized zygotes cultured in vitro or in vivo. J. Re prod. Fertil. 1996; 107: 175-181. [233] Papadopoulous S, Rizos D, Duffy P, Wade M, Quinn K, Boland MP, Lonergan P. Embryo survival and recipient pregnancy rates after transfer of fresh or vitrified, in vivo or in vitro produced ovine blastocy sts. Anim. Reprod. Sci. 2002; 74: 35-44. [234] Hasler JF. Factors aff ecting frozen and fresh embryo tran sfer pregnancy rates in cattle. Theriogenology 2001; 56: 1401-1415. [235] Peterson AJ, Lee RSF. Improving su ccessful pregnancies af ter embryo transfer. Theriogenology 2003; 59: 687-697. [236] Abd El Razek IM, Charpigny G, Kodj a S, Marquant-Le Guienne B, Mermillod P, Guyader-Joly C, Humblot P. Differences in lipid composition betwee n in vivoand in vitro-produced bovine embryos. Ther iogenology 2000; 53: 346 abstract. [237] Duby RT, Hill JL, O'Callaghan D, Over strom EW, Boland MP. Changes induced in the bovine zona pellucida by ovine and bovi ne oviducts. Theriogenology 1997; 47: 332 abstract. [238] Pollard JW, Leibo SP. Chilling se nstivity of mammalian embryos. Theriogenology 1994; 41: 101-106. [239] Van Soom A, Vlaenderen IV, Mahm oudzadeh AR, Deluyker H, de Kruif A. Compaction rate of in vitro fertilized bovi ne embryos related to the interval from insemination to first cleavage Theriogenology 1992; 38: 905-919. [240] Boni R, Tosti E, Roviello S, Dale B. Intracellular communicati on in in vivoand in vitroproduced bovine embryos. Biol. Reprod. 1999; 61: 1050-1055. [241] Slimane W, Heyman Y, Lavergne Y, Humblot P, Renard JP. Assessing chromosomal abnormalities in two-cell bovine in vitro fertilized embryos by using in situ hybridization with thr ee cloned probes. Biol. Reprod. 2000; 62: 628-635. [242] Viuff D, Rickords L, Offenberg H, Hytte l P, Avery B, Greve T, Olsaker I, Williams JL, Callesen H, Thomsen PD. High proportion of b ovine blastocysts produced in vitro are mixoploid. Biol. Reprod. 1999; 60: 1273-1278. [243] Bordignon V, Morin N, Durocher J, Bousquet D, Smith LC. GnRH improves the recovery rate and the in vitro developmental competen ce of oocytes obtained by transvaginal follicular aspiration from s uperstimulated heifers. Theriogenology 1997; 48: -291. [244] Greve T, Xu KP, Callesen H, Hyttel P. In vivo development of in vitro fertilized bovine oocytes matured in vivo versus in vitro. J. In Vitro Fert. Embryo Transf. 1987; 4: 281285.

PAGE 107

107 [245] Liebfried-Rutledge ML, Crister ES, Eyestone WH, Northey DL, First NL. Development potential of bovine oocytes matu red in vitro or in vivo. Biol. Reprod. 1987; 36: 376-383. [246] Marquant-Le Guienne B, Gerard M, Solari A, Thibault C. In v itro culture of bovine egg fertilized either in vivo or in vitro. Reprod. Nutr. Dev. 1989; 29: 559-568. [247] Rizos D, Ward F, Duffy P, Boland MP. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: Implications for blastocyst yield and blastocyst quality. Mol. Reprod. Dev. 2002; 61: 234-248. [248] van de Leemput EE, Vos PLAM, Zein stra EC, Bevers MM, van der Weijden GC, Dieleman SJ. Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a cont rolled pre-ovulatory surge. Theriogenology 1999; 52: 335-349. [249] Lonergan P, Rizos D, Ward F, Bo land MP. Factors influencing oocyte and embryo quality in cattle. Reprod. Nutr. Dev. 2001; 41: 427-437. [250] Enright BP, Dinnyes A, Fair T, Ward F, Yang X, Boland MP. Culture of in vitro produced bovine zygotes in vitro vs. in vivo: Implications for early blastocyst development and quality. Theriogenology 2000; 54: 659-673. [251] Galli C, Lazzari G. Practical aspects of IVM/IVF in cattle. Anim Reprod. Sci. 1996; 42: 371-379. [252] Holm P, Walker SK, Petersen BA, As hman RJ, Seamark RF. In vitro versus in vivo culture of ovine IVM-IVF ova: Effect on lambing. Theriogenology 1994; 41: 217 abstract. [253] Carolan C, Longeran P, Van Langendonc kt A, Mermillod P. Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro. Th eriogenology 1995; 43: 1115-1128. [254] Dorland M, Gardner DK, Trounson AO. Serum in synthetic oviduct fluid causes mitochondrial degeneration in ovine embryos. J. Reprod. Fertil. 1994; Abstract series: 70. [255] Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: Amino acids, vitamins and culturing embryos in groups stimulate development. Biol. Reprod. 1994; 50: 390-400. [256] Thompson JG. Comparison between in vivo-derived and in vitro-produced preelongation embryos from domestic ruminant s. Reprod. Fertil. Dev. 1997; 9: 341-354.

PAGE 108

108 [257] Thompson JG, Allen NW, McGowan LT, Bell ACS, Lambert MG, Tervit HR. Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following tr ansfer. Theriogenology 1998; 49: 1239-1249. [258] Van Langendonckt A, Donnay I, Schuur biers N, Auquier P, Carolan C, Massip A, Dessy F. Effects of supplementation with fetal calf serum on development of bovine embryos in synthetic oviduct fluid medi um. J. Reprod. Fertil. 1997; 109: 87-93. [259] Walker SK, Hartwich KM, Seamark RF The production of unusually large offspring following embryo manipulation: Concepts and challenges. Ther iogenology 1996; 45: 111-120. [260] Wrenzycki C, Herrmann D, Neimann H. Expression of the gap junction gene connexin43 (C X 43) in preimplantation bovine embryos derived in vitro or in vivo. J. Reprod. Fertil. 1996; 108: 17-24. [261] Lonergan P, Fair T, Corcoran D, Ev ans ACO. Effect of culture environment on gene expression and developmental chatactersitic s in IVF-derived embryos. Theriogenology 2006; 65: 137-152. [262] Corcoran D, Fair T, Park S, Rizos D, Patel OV, Smith GV, Coussens PM, Ireland JJ, Boland MP, Evans ACO, Lonergan P. Suppr essed expression of genes involved in transcription and translation in in vitro compared to in vivo cultured bovine embryos. Reproduction 2006; 131: 651-660. [263] Alexopoulos NI, Maddox-Hyttel P, Vajta G. Effect of protein supplementation on establishment of a hypoblast layer in IV P bovine embryos. Theriogenology 2002; 57: 213. [264] Brackett BG, Zuelke KA. Analysis of f actors involved in the in vitro production of bovine embryos. Theriogenology 1993; 39: 43-64. [265] Stringfellow DA, Thom pson MS. Maintenance and devel opment of bovine embryos in vitro. Alabama Agricultural Experiment Sta tion: Highlights of Agricultural Research 1986; 33: 11. [266] Vajta G, Hyttel P, Trounson AO. Post -hatching development of in vitro produced bovine embryos on agar and collagen gels. Anim Reprod. Sci. 2000; 60-61. [267] Gray CA, Bartol FF, Tarleton BJ, Wiley AA, Johnson GA, Bazer FW, Spencer TE. Dev. Biol. of uterine glands Biol. Reprod. 2001; 65: 1311-1323. [268] Zhou J, Bondy C. Insulin-like growth f actor-II and its binding proteins in placental development. Endocrinology 1992; 131: 1230-1240. [269] Han VKM, Bassett N, Wa lton J, Challis JRG. The expr ession of Insulin -like growth factor (IGF) and IGF-binding protein (IG FBP) genes in the human placenta and

PAGE 109

109 membranes: evidence for IGF-IGFBP interactions at the feto-maternal interface. J. Clin. Endocrinol. Metab 1996; 81: 2680-2693. [270] Simmen RCM, Ko Y, Simmen FA. In sulin-like growths factors and blastocyst development. Theriogenology 1993; 39: 163-175. [271] Reynolds TS, Stevenso n KR, Wathes DC. Pregnancy-sp ecific alterations in the expression of the insulin-like growth factor system during early placental development in the ewe. Endocrinology 1997; 138: 886-897. [272] Stevenson KR, Gilmour RS, Wathes DC. Localization of insulinlike growth factor-I and -II messenger ribonucleic acid and type 1 IG F receptors in the ovine uterus during the estrous cycle and early pre gnancy. Endocrinology 1994; 134: 1655-1664. [273] Herrler A, Krusche CA, Beier HM. Insu lin and insulin-like grow th factor-1 promote rabbit blastocyst development and preven t apoptosis. Biol. Reprod. 1998; 59: 13021310. [274] Matsui M, Takahashi Y, Hishinuma M, Kanagawa H. Insulin and insulin-like growth factor-I (IGF-I) stimulate the de velopment of bovine embryos fertilized in vitro. J. Vet. Med. Sci. 1995; 57: 1109-1111. [275] Mihalik M, Rehak P, Koppel J. The infl uence of insulin on the in vitro development of mouse and bovine embryos. Physiol. Res. 2000; 49: 347-354. [276] Xia P, Tekpetey FR, Armstrong DT. Effect of IGF-I on pig oocyte maturation, fertilization, and early em bryonic development in vitro, and on granulosa and cumulus cell biosynthetic activity. Mol. Reprod. Dev. 1994; 38: 373-379. [277] Makarevich AV, Markkula M. Apoptosis and cell proliferatio n potential of bovine embryos stimulated with insu lin-like growth factor I du ring in vitro maturation and culture. Biol. Reprod. 2002; 66: 386-392. [278] Spanos S, Becker DL, Winston RML, Hardy R. Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development. Biol. Reprod. 2000; 63: 1413-1420. [279] Jousan FD, Hansen PJ. Anti-apoptoti c and growth-promoting actions of insulin-like growth factor-1 in bovine preimplantation embryos are mediated through distinct signaling pathways. [38]. 2005. The Society for the Study of Reproduction. [280] Jousan FD, Hansen PJ. Insulin-like gr owth factor-I as a surv ival factor for bovine embryos subjected to heat shock. [38]. 2005. The Society for the Study of Reproduction. [281] Block J, Hansen PJ. Interaction betw een season and culture w ith insulin-like growth factor-1 on survival of in vitro produced em bryos following transfer to lactating dairy cows. Theriogenology 2007; 67: 1518-1529.

PAGE 110

110 [282] Block J, Drost M, Monson RL, Rutle dge JJ, Rivera RM, Paula-Lopes FF, Ocon OM, Krininger CEI, Liu J, Hansen PJ. Use of insulin-like growth f actor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed, lactating cows. J.Anim Sci. 81, 1590-1602. 2003. [283] Paria BC, Dey SK. Preimplantation embryo development in vitro: cooperative interactions among embryos and ro le of growth factors. Proc. Natl. Acad. Sci. U. S. A 1990; 87: 4756-4760. [284] Rappolee DA, Sturm KS, Behrendtsen O, Schulz GA, Pede rsen RA, Werb Z. Insulinlike growth factor II acts through an endogenous growth pathway regulated by imprinting in early mouse embryos. Genes Dev. 1992; 6: 952. [285] Watson AJ, Hogan A, Hahnel A, Schultz GA, Weirner KE. Expression of growth factor ligand and receptor genes in the preimp lantation bovine embryo. Mol. Reprod. Dev. 1992; 31: 87-95. [286] Larson RC, Ignotz GG, Currie WB. Platel et derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle. Development 1992; 115: 821-826. [287] Bottcher RT, Niehrs C. Fibroblast gr owth factor signaling during early vertebrate development. Endocr. Rev. 2005; 26: 63-77. [288] Burgess WH, Maciag T. The heparin-bi nding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem. 1989; 58: 575-606. [289] Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr. Relat Cancer 2000; 7: 165-197. [290] Armelin HA. Pituitary extracts and st eroid hormones in control of 3T3 cell growth. Proc. Natl. Acad. Sci. U. S. A 1973; 70: 2702-2706. [291] Ornitz DM, Itoh N. Fibroblast grow th factors. Genome Biol. 2001; 2: Reviews3005. [292] Itoh N, Ornitz DM. E volution of the Fgf and the Fgfr gene families. Trends Genet 2004; 20: 563-569. [293] Feldman B, Poueymirou W, Papaioannou VE, DeChiara TM, Goldfarb M. Requirement of FGF-4 for postimplantation mouse de velopment. Science 1995; 267: 246-249. [294] Leunda-Casi A, de Hertogh R, Pampfer S. Control of trophect oderm differentiation by inner cell mass-derived fibroblas t growth factor-4 in mous e blastocysts and corrective effect of fgf-4 on high gluc ose-induced trophoblast disr uption. Mol. Reprod. Dev. 2001; 60: 38-46.

PAGE 111

111 [295] Taniguchi F, Harada T, Yoshida S, Iw abe T, Onohara Y, Tanikawa M, Terakawa N. Paracrine effects of bFGF and KGF on the pr ocess of mouse blasto cyst implantation. Mol. Reprod. Dev. 1998; 50: 54-62. [296] Ka H, Jaeger LA, Johnson GA, Spencer TE, Bazer FW. Keratinocyte growth factor is up-regulated by estrogen in the porcine uterine endometrium and functions in trophectoderm cell proliferation and differentiation. Endocrinology 2001; 142: 23032310. [297] Baird A, Walicke PA Fibroblast growth-factors. Br it. Med. Bulletin 1989; 45: 438-452. [298] Folkman J, Klagsbrun M. Angi ogenic factors. Science 1987; 235: 442-447. [299] Gospodarowicz D, Ferrara N, Schweigere r L, Neufeld G. Structural characterization and biological functions of fibroblast growth-factor Endocr. Rev. 1987; 8: 95-114. [300] Sato Y, Rifkin DB. Autocrine activities of basic fibroblast growth factor regulation of endothelial cell movement, plasminogen-activ ator synthesis, a nd DNA-synthesis. J. Cell Biol. 1988; 107: 1199-1205. [301] Carlone DL, Rider V. Embryonic modulation of basic fibroblast grow th factor in the rat uterus. Biol. Reprod. 1993; 49: 653-665. [302] Grundker C, Kirchner C. Uterine fibrobl ast growth factor-2 and embryonic fibroblast growth factor receptor-1 at the beginning of gastrulation in the rabbit. Anat. Embryol. (Berl) 1996; 194: 169-175. [303] Gupta A, Bazer FW, Jaeger LA. Imm unolocalization of acidic and basic fibroblast growth factors in porcine uterine and conceptus tissues. Biol. Reprod. 1997; 56: 15271536. [304] Ortega S, Ittmann M, Tsang SH, Ehr lich M, Basilico C. Neuronal defects and delayed wound healing in mice lacing fibroblast growth factor 2. Proc. Natl. Acad. Sci. U. S. A 1998; 95: 5672-5677. [305] Zhou M, Sutliff RL, Paul RJ, Lorenz JN, Hoying JB, Haudenschild CC. Fibroblast growth factor 2 control of vasc ular tone. Nat Med 1998; 4: 201-207. [306] Ludwig TE, Levenstein ME, Jone s JM, Berggren WT, Mitchen ER, Frane JL. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 2006; 24: 185-187. [307] Xu RH, Peck RM, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated prol iferation of human ES cells. Nat Meth 2005; 2: 185-190.

PAGE 112

112 [308] Seli E, Zeyneloglu HB, Senturk LM, Bah tiyar OM, Olive DL, Arici A. Basic fibroblast growth factor: peritoneal and follicular fl uid levels and its effect on early embryonic development. Fertil. Steril. 1998; 69: 1145-1148. [309] Larson RC, Ignotz GG, Currie WB. Tran sforming growth factor beta and basic fibroblast growth factor s ynergistically promote early bovine embryo development during the fourth cell cycle. Mol. Reprod. Dev. 1992; 33: 432-435. [310] Lim JM, Hansel W. Roles of growth factors in the development of bovine embryos fertilized in vitro and cult ured singly in a defined me dium. Reprod. Fertil. Dev. 1996; 8: 1199-1205. [311] Reynolds LP, Redmer DA. Angiogenes is in the placenta. Biol. Reprod. 2001; 64: 10331040. [312] Ocon-Grove OM, Alvarez IM, Johnson SE, Ott TL, Ealy AD. Ovine endometrial expression of fibroblast grow th factor (FGF) 2 and conceptus expression of FGF receptors during early pregnancy. Domest. Anim. Endocrinol. 2007. [313] Guthridge MA, Stomski FC, Thomas D, Woodcock JM, Bagley CJ, Berndt MC, Lopez AF. Mechanism of activation of the GM-CSF, IL-3, and IL-5 family of receptors. Stem Cells 1998; 16: 301-313. [314] Rooke J, Ewen M, McEv oy T, Entrican G, Ashworth C. Effect of inclusion of serum and granulocyte-macrophage colony stimula ting factor on secreti on of interferon-tau during the in vitro culture of ovine embryos. Reprod. Fertil. Dev. 2005; 17: 513-521. [315] Robertson SA, Roberts CT, Farr KL, D unn AR, Seamark RF. Fertility impairment in granulocyte-macrophage colony-stimulating factor-deficient mice. Biol. Reprod. 1999; 60: 251-261. [316] Chaouat G, Menu E, Clark DA, Dy M, Minkowski M, Wegmann TG. Control of fetal survival in CBA x DBA/2 mice by lymphoki ne therapy. J. Reprod. Fertil. 1990; 89: 447-458. [317] Robertson SA, Sjoblom C, Jasper MJ, Norman RJ, Seamark RF. Granulocytemacrophage colony-stimulating factor prom otes glucose transport and blastomere viability in murine preimplantati on embryos. Biol. Reprod. 2001; 64: 1206-1215. [318] Sjoblom C, Wikland M, Robertson SA. Granulocyte-macrophage colony-stimulatingfactor (GM-CSF) acts inde pendently of the beta co mmon subunit of the GM-CSF receptor to prevent inner cell mass apoptos is in human embryos. Biol. Reprod. 2002; 67: 1817-1823. [319] Armstrong DT, Chaouat G. Effects of lymphokines and immune complexes on murine placental cell growth in vitr o. Biol. Reprod. 1989; 40: 466-474.

PAGE 113

113 [320] Crainie M, Guilbert L, Wegmann TG. Expression of novel cytokine transcripts in the murine placenta. Biol. Reprod. 1990; 43: 999-1005. [321] Lea RG, Riley SC, Antipatis C, Hann ah L, Ashworth CJ, Clark DA, Critchley HO. Cytokines and the regulation of apoptosis in reproductive tissues: a review. Am. J. Reprod. Immunol. 1999; 42: 100-109. [322] de Moraes AA, Paula-Lopes FF, Chegin i N, Hansen PJ. Localization of granulocytemacrophage colony-stimulating factor in the bovine reproductiv e tract. J. Reprod. Immunol. 1999; 42: 135-145. [323] McGuire WJ, Imakawa K, Tamura K, Meka CS, Christenson RK. Regulation of endometrial granulocyte macrophage-colony stim ulating factor (GM-CSF) in the ewe. Domest. Anim Endocrinol. 2002; 23: 383-396. [324] de Moraes AA, Hansen PJ. Granul ocyte-macrophage colony-stimulating factor promotes development of in vitro produ ced bovine embryos. Bi ol. Reprod. 1997; 57: 1060-1065. [325] Sjoblom C, Wikland M, Robertson SA. Granulocyte-macrophage colony-stimulatingfactor promotes human blastocyst devel opment in vitro. Hum. Reprod. 1999; 14: 30693076. [326] Imakawa K, Helmer SD, Nephew KP, Meka CS, Christenson RK. A novel role for GM-CSF: enhancement of pregnancy specifi c interferon producti on, ovine trophoblast protein-1. Endocrinology 1993; 132: 1869-1871. [327] Imakawa K, Carlson KD, McGuire WJ, Christenson RK, Taylor A. Enhancement of ovine trophoblast interferon by granulocyte macrophageco lony stimulating factor: possible involvement of protein kinase C. J. Mol. Endocrinol. 1997; 19: 121-130. [328] Michael DD, Wagner SK, Ocon OM, Talbot NC, Rooke JA, Ealy AD. Granulocytemacrophage colony stimulating factor increas es interferon-tau protein secretion in bovine trophectoderm cells. Am.J.R eprod.Immunol. in press. 2006. [329] de Moraes AA, Davidson JA, Fleming JG, Bazer FW, Edwards JL, Betts JG, Hansen PJ. Lack of effect of granulocyte-macrophage colony-stimulating factor on secretion of interferon-tau, other proteins, and prostagla ndin E2 by the bovine and ovine conceptus. Domest. Anim Endocrinol. 1997; 193-197. [330] Emond V, Fortier MA, Murphy BD, La mbert RD. Prostaglandin E-2 regulates both interleukin-2 and granulocyte-macrophage colo ny-stimulating-factor gene expression in bovine lymphocytes. Biol. Reprod. 1998; 58: 143-151. [331] Fortin M, Ouellette MJ, Lambert RD. TGFbeta2 and PGE2 in rabbit blastocoelic fluid can modulate GM-CSF production by human lymphocytes. Am J Reprod Immunol 1997; 38: 129-139.

PAGE 114

114 [332] Emond V, Asselin E, Fo rtier MA, Murphy BD, Lambert RD Interferon-tau stimulates granulocyte-macrophage colony-stimulati ng factor gene expression in bovine lymphocytes and endometrial stroma l cells. Biol. Reprod. 2000; 62: 1728-1737. [333] Moreira F, Paula-Lopes FF, Hansen PJ Badinga L, Thatcher WW. Effects of growth hormone and insulin-like growth factor-1 on development of in vitro derived bovine embryos. Theriogeno logy 2002; 57: 895-907. [334] Paula-Lopes FF, Hansen PJ. Heat shoc k-induced apoptosis in preimplantation bovine embryos is a developmentally regulate d phenomenon. Biol. Reprod. 2002; 66: 11691177. [335] Rivera RM, Hansen PJ. Development of cultured bovine embryos after exposure to high temperatures in the physiologica l range. Reproduction 2001; 121: 107-115. [336] Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development a nd their significance in embryo culture. Theriogenology 1998; 49: 83-102. [337] Khurana NK, Niemann H. Energy me tabolism in preimplantation bovine embryos derived in vitro or in viv o. Biol. Reprod. 2000; 62: 847-856. [338] Larson MA, Kimura K, Kubisch HM Roberts RM. Sexual dimorphism among bovine embryos in their ability to make the tran sition to expanded blastocyst and in the expression of the signaling molecule IFN-tau. Proc. Natl. Acad. Sci. U. S. A 2001; 98: 9677-9682. [339] Farrell PB, Foote RH. Bene ficial effects of culturing rabb it zygotes to blastocysts in 5% oxygen and 10% carbon dioxide. J. Reprod. Fertil. 1995; 103: 127-130. [340] Petersen A, Mikkelsen AL, Linde nberg S. The impact of oxygen tension on developmental competence of post-thaw human embryos. Acta. Obstet. Gynecol. Scand. 2005; 84: 1181-1184. [341] Thomson JG, SImpson AC, Pugh PA Donnelly PE, Tervit HR. Effect of oxygen concentration on in-vitro development of pr eimplantation sheep and cattle embryos. J. Reprod. Fertil. 1990; 89: 573-578. [342] Umaoka Y, Noda Y, Narimoto K, Mori T. Effects of oxygen toxicity on early development of mouse embryos. Mol. Reprod. Dev. 1992; 31: 28-33. [343] Takahashi M, Nagai T, Hamano S, Ku wayama M, Okamura N, Okano A. Effect of thiol compounds on in vitro development and intracellular glutathione content of bovine embryos. Biol. Reprod. 1993; 49: 228-232. [344] Gardiner CS, Reed DJ. Status of gl utathione during oxidant-i nduced oxidative stress in the preimplantation mouse embr yo. Biol. Reprod 1994; 51: 1307-1314.

PAGE 115

115 [345] Stover SK, Gushansky GA, Salmen JJ, Gardiner CS. Regulation of gamma-glutamatecysteine ligase expression by oxidative st ress in the mouse preimplantation embryo. Toxicol. Appl. Pharmacol. 2000; 168: 153-159. [346] Kimura K, Spate LD, Green MP, Roberts RM. Effects of oxidative stress and inhibitors of the pentose phosphate pathway on sexua lly dimorphic production of IFN-tau by bovine blastocysts. Mol. Reprod. Dev. 2004; 68: 88-95. [347] Kimura K, Spate LD, Green MP, Murphy CN, Seidel GE, Roberts RM. Sexual dimorphism in interferon-t au production by in vivo-derived bovine embryos. Mol. Reprod. Dev. 2004; 67: 193-199. [348] Kubisch HM, Sirisathien S, Bosch P, Hernandez-Fonseca HJ, Clements G, Liukkonen JR, Brackett BG. Effects of developmental stage, embryonic interferon-tau secretion and recipient synchrony on pre gnancy rate after transfer of in vitro produced bovine blastocysts. Reprod. Domest. Anim. 2004; 39: 120-124.

PAGE 116

116 BIOGRAPHICAL SKETCH Teresa Marie Rodina was born and raised in Ridge, New York on Long Island where she grew up with her twin sister Kr istin, older brother James and her parents, Barbara and Robert. At an early age she developed a passionate interest in animals and sought to one day find a career in the field. She spent her undergra duate career in Kingston, Rhode Is land at the quaint campus of the University of Rhode Island as an animal science and technology major. After graduating Cum Laude in 2005, she accepted a position with Dr. Al an Ealy as a graduate research assistant at the University of Florida. For the next two years she worked unde r Dr. Ealy pursuing her Master of Science in Animal Sciences. She hope s to one day attend the College of Veterinary Medicine at the University of Florida and conquer the final cha llenge in her ultimate goal of developing a career as a veterinarian.