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Consequences of Post-Ejaculatory Stress on the Bovine Spermatozoa for the Resultant Embryo

Permanent Link: http://ufdc.ufl.edu/UFE0023958/00001

Material Information

Title: Consequences of Post-Ejaculatory Stress on the Bovine Spermatozoa for the Resultant Embryo
Physical Description: 1 online resource (132 p.)
Language: english
Creator: Hendricks, Katherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: butyl, damage, development, dna, ejaculatory, embryo, heat, hyfroperoxide, irradiation, menadione, oxidative, post, sperm, stress, tert, tunel
Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Animal Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The ability of the preimplantation embryo to complete its developmental program is determined in large part by its acquisition of genetic and non-genetic components from the sperm and oocyte. While the role of damage to the oocyte on embryo competence has been well established, it is not clear whether damage to the sperm after ejaculation results in formation of an embryo with reduced competence for development. Accordingly, a series of experiments were conducted to determine whether this was the case. Stresses examined were aging, heat shock, irradiation, and oxidative stress. The experimental approach involved evaluation of oocytes inseminated with sperm exposed to stress to cleave and develop to the blastocyst stage of development. Aging of sperm by incubation for 4 h at a temperature characteristic of normal body temperature (38.5 degree Celsius) or hyperthermia (40 degree Celsius) reduced cleavage, but did not alter embryo competence as measured by the percent of cleaved embryos that became blastocysts. Similarly, exposure of sperm to X-irradiation characteristic of airport screening devices for checked or carry-on luggage had no effect on embryo competence. Cleavage rate was reduced by X-irradiation after multiple exposures at the checked luggage dose but there was no reduction in the percent of cleaved embryos becoming blastocysts at any exposure. In contrast, oxidative stress did affect embryo competence to develop to the blastocyst stage. Oocytes inseminated with sperm treated with menadione or tert-butyl hydroperoxide had reduced cleavage and a reduced percentage of cleaved embryos that became blastocysts. One potential mechanism for sperm damage affecting the resultant embryo is the initiation of DNA damage during sperm cell apoptosis. To test whether apoptosis could be induced in ejaculated spermatozoa, bull and stallion sperm were subjected to heat shock and aging and features of apoptosis evaluated. Endpoints included terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), mitochondrial membrane potential, and presence and activation of procaspase-9 and procaspase-3. There was only a slight increase in TUNEL labeling after 24 h aging and the increase was not blocked with a caspase inhibitor. Moreover, procaspase-9 was detected in bovine sperm but not activated by 4 h aging at 38.5, 40 and 41 degree Celsius and procaspase-3 was not detected. Taken together, these results indicate that specific types of stress occurring after ejaculation can negatively impact the ability of the subsequent embryo to develop. Understanding these effects on early embryonic development may lead to new approaches for reducing early embryonic loss.
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.
Statement of Responsibility: by Katherine Hendricks.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Hansen, Peter J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023958:00001

Permanent Link: http://ufdc.ufl.edu/UFE0023958/00001

Material Information

Title: Consequences of Post-Ejaculatory Stress on the Bovine Spermatozoa for the Resultant Embryo
Physical Description: 1 online resource (132 p.)
Language: english
Creator: Hendricks, Katherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: butyl, damage, development, dna, ejaculatory, embryo, heat, hyfroperoxide, irradiation, menadione, oxidative, post, sperm, stress, tert, tunel
Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Animal Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The ability of the preimplantation embryo to complete its developmental program is determined in large part by its acquisition of genetic and non-genetic components from the sperm and oocyte. While the role of damage to the oocyte on embryo competence has been well established, it is not clear whether damage to the sperm after ejaculation results in formation of an embryo with reduced competence for development. Accordingly, a series of experiments were conducted to determine whether this was the case. Stresses examined were aging, heat shock, irradiation, and oxidative stress. The experimental approach involved evaluation of oocytes inseminated with sperm exposed to stress to cleave and develop to the blastocyst stage of development. Aging of sperm by incubation for 4 h at a temperature characteristic of normal body temperature (38.5 degree Celsius) or hyperthermia (40 degree Celsius) reduced cleavage, but did not alter embryo competence as measured by the percent of cleaved embryos that became blastocysts. Similarly, exposure of sperm to X-irradiation characteristic of airport screening devices for checked or carry-on luggage had no effect on embryo competence. Cleavage rate was reduced by X-irradiation after multiple exposures at the checked luggage dose but there was no reduction in the percent of cleaved embryos becoming blastocysts at any exposure. In contrast, oxidative stress did affect embryo competence to develop to the blastocyst stage. Oocytes inseminated with sperm treated with menadione or tert-butyl hydroperoxide had reduced cleavage and a reduced percentage of cleaved embryos that became blastocysts. One potential mechanism for sperm damage affecting the resultant embryo is the initiation of DNA damage during sperm cell apoptosis. To test whether apoptosis could be induced in ejaculated spermatozoa, bull and stallion sperm were subjected to heat shock and aging and features of apoptosis evaluated. Endpoints included terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), mitochondrial membrane potential, and presence and activation of procaspase-9 and procaspase-3. There was only a slight increase in TUNEL labeling after 24 h aging and the increase was not blocked with a caspase inhibitor. Moreover, procaspase-9 was detected in bovine sperm but not activated by 4 h aging at 38.5, 40 and 41 degree Celsius and procaspase-3 was not detected. Taken together, these results indicate that specific types of stress occurring after ejaculation can negatively impact the ability of the subsequent embryo to develop. Understanding these effects on early embryonic development may lead to new approaches for reducing early embryonic loss.
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.
Statement of Responsibility: by Katherine Hendricks.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Hansen, Peter J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023958:00001


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1 CONSEQUENCES OF POST-EJACULATORY STRESS ON THE BOVINE SPERMATOZOA FOR THE RESULTANT EMBRYO By KATHERINE E. M. HENDRICKS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Katherine Elizabeth May Hendricks

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3 To my husband, Gregory Hendricks, for all the l ove, laughter and tears th at we have shared together through this adventure and to my mother, Elsa-May, who believed in me and taught me that you can do anything you put your mind to.

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4 ACKNOWLEDGMENTS This dissertation would not have been co m pleted without the knowledge, guidance and dedication of Dr. Peter J. Hansen, chair of my supervisory committee. It was Dr. Hansen who invited me to join the Animal Molecular and Cell Biology group and continue my academic career, for this I am deeply indebted. I woul d like to thank the memb ers of my supervisory committee: Dr. William C. Buhi, Dr. Alan Ealy, Dr. Charles Guy, and Dr.Chad Chase. It has been a pleasure working with such a talented gr oup of people. I would lik e to thank each member for their knowledge and support and their willingness to help in completion of this dissertation. I would be remiss in not thanking the individual s in the Hansen laboratory for their help in gaining the requisite techniques used throughout this dissertation. Special thanks to Amber M. Brad, Maria B. Padua and visiti ng scientist Leydson Martins. I would like to extend my sin cere thanks to the management and personnel at Central Packing Co., Center Hill, FL for providing the ovaries used in the experiments of this dissertation and to William Rembert for his assist ance in collecting ovaries. Special thanks to Dr. Linda Penfold, White Oaks Conservation, Yulee, FL for providing x-irradiated bull semen and to Andrea Desvouges and Dr Mats Troedsson for providing help in acquiring stallion semen used in experiments of this dissertation. I am thankful to the faculty, staff and student s of the Department of Animal Sciences and the Animal Molecular and Cell Biology Program for a ll their support. I am especially grateful for the camaraderie of Lilian Olivera, Kathleen Pennington, Justin Fear and James Moss thanks for being my sounding boards, for your friendship and support. Finally, I would like to express my deepest gratitude to my parents, Elsa Wilmot and Lloyd Binns, my grandmother, Mu riel Binns, my godparents, Junior and Merle Bingham, my

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5 sister, Sabrena Hardware and extended family for their encouragement throughout my academic career.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 ABSTRACT...................................................................................................................................11 CHAP TER 1 LITERATURE REVIEW.......................................................................................................13 Sire Effects on Embryonic Development............................................................................... 14 Fertilization.................................................................................................................. ...........16 Paternal Contributions to the Embryo....................................................................................18 Post-ejaculatory Sperm Damage.............................................................................................22 Heat Shock.......................................................................................................................22 Cryopreservation............................................................................................................. 23 Hydrogen Peroxide (H2O2)..............................................................................................26 Menadione.......................................................................................................................27 Gossypol..........................................................................................................................28 X-irradiation....................................................................................................................29 Pathways of Apoptosis...........................................................................................................31 Synopsis..................................................................................................................................33 2 CONSEQUENCES FOR THE BOVINE EM B RYO OF BEING DERIVED FROM A SPERMATOZOAN SUBJECTED TO POST -EJACULATORY AGING AND HEAT SHOCK: DEVELOPMENT TO THE BLAS TOCYST STAGE AND SEX RATIO............ 36 Introduction................................................................................................................... ..........36 Materials and Methods...........................................................................................................37 Materials..........................................................................................................................37 Sperm Preparation........................................................................................................... 38 Sperm Motility................................................................................................................. 39 In Vitro Production of Embryos......................................................................................39 Determination of Fertilization......................................................................................... 40 Embryo Sex Determination............................................................................................. 40 Experiments.................................................................................................................... .41 Statistical Analyses.......................................................................................................... 42 Results.....................................................................................................................................43 Sperm Motility................................................................................................................. 43 Cleavage and Development to the Blastocyst Stage....................................................... 43

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7 Fertilization.................................................................................................................. ....43 Sex of Blastocysts............................................................................................................44 Discussion...............................................................................................................................44 3 CONSEQUENCES FOR THE BOVINE EM B RYO OF BEING DERIVED FROM A SPERMATOZOAN SUBJECTED TO X-I RRADIATION CHARACTERISTIC OF AIRPORT SCREENING DEVICES......................................................................................53 Introduction................................................................................................................... ..........53 Materials and Methods...........................................................................................................54 Materials..........................................................................................................................54 Sperm Treatment and Preparation................................................................................... 54 In Vitro Production of Embryos......................................................................................55 Statistical Analyses.......................................................................................................... 56 Results.....................................................................................................................................56 Discussion...............................................................................................................................57 4 CONSEQUENCES FOR THE BOVINE EM B RYO OF BEING DERIVED FROM A SPERMATOZOAN SUBJECTED TO POST -EJACULATORY STRESS: OXIDATVE STRESS......................................................................................................................... .........63 Introduction................................................................................................................... ..........63 Materials and Methods...........................................................................................................64 Materials..........................................................................................................................64 Sperm Preparation........................................................................................................... 65 In Vitro Production of Embryos......................................................................................66 Experiments.................................................................................................................... .66 Statistical Analyses.......................................................................................................... 67 Results.....................................................................................................................................68 Menadione.......................................................................................................................68 Tert -Butyl Hydroperoxide ............................................................................................... 68 Discussion...............................................................................................................................69 5 CAN PROGRAMMED CELL DEATH BE I NDUCED IN POST-EJACULATORY BULL AND STALLION SPERM ATOZOA?....................................................................... 75 Introduction................................................................................................................... ..........75 Materials and Methods...........................................................................................................77 Chemicals and Reagents.................................................................................................. 77 Sperm Preparation........................................................................................................... 78 BEND Cells..................................................................................................................... 78 Evaluation of TUNEL Labeling......................................................................................79 Evaluation of Mitochondrial Membrane Potential.......................................................... 81 Western Blotting for Caspase-9 and Caspase-3.............................................................. 81 Statistical Analysis.......................................................................................................... 82 Results.....................................................................................................................................83 Term inal Deoxynucleotidyl Transferas e dUTP Nick End Labeling (TUNEL) ..............83

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8 Bull Sperm................................................................................................................ 83 Equine Sperm...........................................................................................................83 Evaluation of Mitochondrial Membrane Potential.......................................................... 84 Western Blotting.............................................................................................................. 84 Discussion...............................................................................................................................85 6 GENERAL DISUSSION........................................................................................................ 92 LIST OF REFERENCES.............................................................................................................104 BIOGRAPHICAL SKETCH.......................................................................................................132

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9 LIST OF TABLES Table page 2-1. Effect of sperm aging on cleavage ra te and blastocyst developm ent following insemination of matured oocytes with sperm for 8 h......................................................... 49 2-2. Fertilization rate at 18 h post-insemina tion as affected by aging of sperm at 38.5 or 40C for 4 h........................................................................................................................50 2-3. Sex of blastocysts at Day 7 and Day 8 af ter insem ination as affected by sperm aging at 38.5 or 40C for 4 h........................................................................................................51 3-1. Effect of sperm irradiation with a carry-on X-ray m achine on cleavage rate, blastocyst development and embryo competence (blastocyst/cleaved) following insemination of matured oocytes....................................................................................... 61 3-2. Effect of sperm irradiation with a ch ecked luggage x-ray m achine on cleavage rate, blastocyst development and embryo competence (blastocyst/cleaved) following insemination of matured oocytes....................................................................................... 62 4-1. Effect of treatment of sp erm with menadione (5 M) or tert -butyl hydroperoxide (TBHP; 10 nM) on cleavage and blastocyst formation following insemination of matured oocytes................................................................................................................ .72 4-2. Effect of treatment of sperm with m enadione (0, 15 and 30 M) on cleavage and blastocyst formation following in semination of matured oocytes..................................... 73 4-3. Effect of treatment of sperm with tert -butyl hydroperoxide (TBHP; 0, 150 and 300 M) on cleavage and blastocyst form ation following insemination of matured oocytes...............................................................................................................................74 5-1. Effect of aging for 4 h at temperatures characteristic of norm othermia (38.5C) or heat stress (40 and 41C) and of mitochondr ial depolarization w ith carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the per cent of frozen-thawed, ejaculated bull spermatozoa positive for the TUNEL reaction.................................................................. 87 5-2. Effect of aging for 24 h at temperatures characteristic of norm othermia (38.5C) or heat stress (40 and 41C) on the percen t of frozen-thawed, ejaculated bull spermatozoa positive for the TUNEL reacti on as affected by the group II caspase inhibitor, z-DEVD-fmk...................................................................................................... 88

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10 LIST OF FIGURES Figure page 1-1. Simplification of induction of apopt osis by heat shock with emphasis on the m itochondrial or intrinsic apoptotic pathwa y. Other pathways controlling apoptosis are not shown here............................................................................................................. 35 2-1. Representative results for analysis of embryo sex by PCR............................................... 52 5-1. Effect of aging for 4 h at temperatures characteristic of norm othermia (38.5C) or heat stress (41C) and of mitochondria l depolarization with carbonyl cyanide 3chlorophenylhydrazone (CCCP) on the per cent of freshly-ejaculated equine spermatozoa positive for the TUNEL reaction.................................................................. 89 5-2. Effect of aging for 4 h at temperatures characteristic of norm othermia (38.5C) and heat stress (42C) on mitochondrial memb rane potential of frozen-thawed bull spermatozoa as measured by the cationic fluoroprobe JC-1.............................................. 90 5-3. Representative western blots for the de tection of caspase-9 (A) and caspase-3 (B). ........ 91 6-1. Model describing points in the mitochondrial/in trinsic pathway for apoptosis that are blocked in bovine ejaculated spermatozoa...................................................................... 102 6-2. Possible scenario for the effect of post-ejaculatory oxidative stress on fertilization and subsequent em bryo development.............................................................................. 103

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CONSEQUENCES OF POST-EJACU LATORY STRESS ON THE BOVINE SPERMATOZOA FOR THE RESULTANT EMBRYO By Katherine E. M. Hendricks December 2008 Chair: Peter J. Hansen Major: Animal Molecula r and Cellular Biology The ability of the preimplantation embryo to complete its developmental program is determined in large part by its acquisition of ge netic and non-genetic components from the sperm and oocyte. While the role of damage to the oocyte on embryo competence has been well established, it is not clear whether damage to the sperm after ejaculation results in formation of an embryo with reduced competence for developmen t. Accordingly, a series of experiments were conducted to determine whether th is was the case. Stresses examined were aging, heat shock, irradiation, and oxidative stress The experimental approach involved evaluation of oocytes inseminated with sperm exposed to stress to cl eave and develop to the blastocyst stage of development. Aging of sperm by incubation for 4 h at a temperature characteristic of normal body temperature (38.5 degree Celsius) or hyperthermia (40 degree Celsius) reduced cleavage, but did not alter embryo competence as measured by the percent of cleaved embryos that became blastocysts. Similarly, exposure of sperm to X-i rradiation characteristic of airport screening devices for checked or carry-on luggage had no effect on embryo competence. Cleavage rate was reduced by X-irradiation after multiple exposures at the checked luggage dose but there was no reduction in the percent of cleaved embr yos becoming blastocysts at any exposure.

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12 In contrast, oxidative stress did affect embryo competence to develop to the blastocyst stage. Oocytes inseminated with sperm treated with menadione or tert-butyl hydroperoxide had reduced cleavage and a reduced percentage of cleaved embryos that became blastocysts. One potential mechanism for sperm damage aff ecting the resultant embryo is the initiation of DNA damage during sperm cell apoptosis. To test whether apoptosis could be induced in ejaculated spermatozoa, bull and stallion sperm were subjected to heat shock and aging and features of apoptosis evaluated. Endpoints included terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), mitochondrial membrane potential, and presence and activation of procaspase-9 and procaspase-3. There was only a slight increase in TUNEL labeling after 24 h aging and the increase was not blocked with a ca spase inhibitor. Moreover, procaspase-9 was detected in bovine sperm but not activated by 4 h aging at 38.5, 40 and 41 degree Celsius and procaspase-3 was not detected. Taken together, these results indicate that specific types of st ress occurring after ejaculation can negatively impact the abil ity of the subsequent embryo to develop. Understanding these effects on early embryonic de velopment may lead to new approaches for reducing early embryonic loss.

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13 CHAPTER 1 LITERATURE REVIEW It is conceivable, and indeed probable, that every part of the adult contains m olecules derived from the male and from the female parent; and that, re garded as a mass of molecules, the entire organism may be compar ed to a web of which the warp is derived from the female and the woof from the male.--Huxley and Sully, 1888 The ability of the preimplantation embryo to complete its developmental program is determined in large part by its acquisition of ge netic and non-genetic components from the sperm and oocyte. While the role of damage to the oocyte on embryo competence has been well established (Sirard et al., 2006), it is not clear whether damage to the sperm after ejaculation results in formation of an embryo with reduced competence for development. Spermatozoal abnormalities which lead to failure in embryogenesis prior to maternal recognition are defined as uncompensable sinc e these cannot be minimized or eliminated by increasing sperm dosage alone. Several examples in the literature illustrate the importance of the sperm for embryo quality. For example, fertilization with sperm produced from bulls with thermally-insulated testis leads to a delay in pronuclear formation (Walters et al., 2006), reduction in embryo cleavage rates, blastocyst development, production of a higher frequency of low quality embryos, and incr eased early embryo losses in vivo (Walters et al., 2005ab) and in vitro (Saacke et al, 1994). Similarly, fertilization with sperm produced by low fertility bulls results in a reduction in early cl eavage rates and pronuclear formati on (Eid et al., 1994; Saacke et al., 1994) and reduction in embryo development (Saacke et al., 1994). Most research on contributions of the sp erm to embryonic competence has focused on effects of stress on the male gamete that occu rred during spermatogenesis. Little is known regarding whether stress of sp erm occurring after ejaculation (while sperm are in the female reproductive tract or cryopreserve d) leads to decreased embryonic potential for development. In the past 50 years the interest in post-ejaculatory sperm damage has increased, especially in light

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14 of improvements in artificial reproductive t echnology (ART). Today, a single sperm can be selected based on morphological qualities to be used for intra-cytoplasmic sperm injection (ICSI), yet a morphologically norm al sperm does not always give rise to a competent embryo (Gmez et al., 1997). The view that sperm were si mply a DNA bullet is changing as more focus is being placed on the paternal contributions to the embryo. Sire Effects on Embryonic Development Differences in em bryonic development can be attributed in part to differences in spermatozoal contribution to the embryo. Desp ite a group of bulls pr oducing embryos having similar cleavage rates, the ability of the embryos produced by individual bulls to develop can be drastically different (Shi et al., 1990; 1991; Saacke et al., 1994) This phenomenon, the bull or sire effect is due to differences in sperm quality rather than quantity and has been termed uncompensable. These uncompe nsable differences reflect spermatozoal abnormalities which lead to failure of fertilization or sustained em bryogenesis (Saacke et al., 1994). This paternal effect has also been noted in other species, including the ram (Morri s et al., 2003) and men (Tesarik et al., 2002). There are genes that control embryonic deve lopment. One of the best known is the Ped gene in the mouse. The product of the Ped gene is Qa-2, a cell surface protein and mice are defined as either Ped fast (expressing Qa-2 protein) or Ped slow (Qa-2 is not expressed). When exposed to anti-Qa-2 monoclonal Ab at the 2-cell stage cross-linking of the protein occurs and results in a significant increase in blastocyst development in the embryos expressing Qa-2 (McElhinny and Warner, 2000). There appears to be a bovine homo log (bovine MHC I 4221.1/Ped gene; Fair et al., 2004), however it is not known whether this gene is maternally imprinted and hence paternally e xpressed. Fibroblast growth factor 2 (FGF2) gene has also been associated with embryonic survival and fertilizat ion rate in cattle; however, FGF2 seems to be

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15 maternally governed (Khatib et al., 2008a). However, there is evidence to suggest that the paternal inherited signal trans ducer and activator of transcript ion 5A (STAT5A) gene plays a role in fertilization rates and embryonic survival in cattle (Khatib et al., 2008b). The sire effect extends to the ability of male s to fertilize the oocyte and may be reflected for example, in a group of bulls by their non-return rates. This parameter for the evaluation of reproductive performance of bulls gives the percent of cows/heife rs that did not return for breeding within a specific time after insemination. In one study where bulls were divided into two groups based on their lifetime non-return rates as either low fertility (66) or high fertility bulls (781), sperm from low fertility bulls were less able to penetrate the oocyte and to sustain embryonic development to the morula and blastocy st stage (Hillery et al., 1990). Further, bulls differ in their ability to form the sperm as ter following successful penetration and sperm incorporation into the oocyte (Navara et al., 1996). In this study sire effects on sperm aster size and microtubule organization during bovine fertilization was inves tigated using three bulls of known field and/or in vitro fertility high, medium and low. It was demonstrated that the bull with the highest field fertility (79.5% nonreturn rate) and development after in vitro fertilization had the largest and most organized sperm aster, while the bull with the poorest development after in vitro fertilization (IVF) had the smallest and least organized aster (Navara et al., 1996). Earlier cleaving embryos are more likely to de velop to the blastocyst stage (Longerhan et al., 1999; Dinnys et al., 1999; Ward et al., 2001) and sperm could affect an embryos competence for development if the timing of fi rst cleavage is delayed. The timing of first cleavage following insemination differ between bulls (Ward et al., 2001) and embryos produced by high-fertility bulls entered S-phase of the firs t cell cycle earlier and ha d a longer S-phase than

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16 those produced by low-fertility bulls (Eid et al., 1994; Comi zzoli et al., 2000) which may influence the timing of first cleavage a nd hence embryo competence for development. Sire effects extend to differences in an embr yos potential to devel op under heat stress conditions. In one study, lactating Holstein co ws inseminated with Gyr semen during the Brazilian summer were more likely to be diagnosed pregnant 80 days after artificial insemination (AI) when compared to cows inseminated with Ho lstein semen (Pegorer et al., 2007). Moreover, Pegorer et al. (2007) demonstrated that even within breed (Gyr) sires can affect embryonic loss, where one Gyr bull had lower embryo/fetal loss between the first and second pregnancy diagnosis (30 to 40 days and 60 to 80 days af ter AI) than other Gyr bulls and even between breeds (Holstein bulls). It has b een suggested that the oocyte ha s a more crucial role in the genetic ability of an embryo to resist effect s of heat shock than the contribution of the spermatozoa (Block et al., 2002). However, other studi es have indicated that the breed of the sire plays just as much a crucial role in embryo thermotolerance both in in vivo derived (Pegorer et al., 2007) and in vitro derived embryos (Barros et al., 2006). Eberhardt et al. (2005) demonstrated that both the oocyte and the sper m contribute to thermotolerance where embryos that had a predominant Bos indicus genotype were more likely to develop to the blastocyst stage following heat shock at 41C for 12 h at 48 h post-insemination. Fertilization Following capacitation within the fem ale repr oductive tract, spermato zoa utilize enzymatic and mechanical (forward propulsion -hyperactivation) action to pene trate the cumulus cells of the cumulus oocyte complex. Once in contact with the zona pellucida the spermatozoon binds to the zona by receptor-ligand interaction. In the bovine this is achieved by interactions between galactosyltransferase (GalT) on the sperm membrane and N-ac etylglucosamine residues on the ZP3 (Tengowski et al., 2001). Interaction with the ZP3 receptor stimulates the acrosome

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17 reaction plasma membrane and outer acrosomal membrane fusi on, vesiculation and release of the acrosomal content. Forward motion and acrosom al enzymes help the spermatozoon to create a tract through the zona pellucida. Once through the zona, sperm bind and fuse to the plasma membrane of the oocyte. Spermoocyte fusion begi ns from the equatorial segment (between the inner acrosomal and plasma membranes overlying the nucleus in the posterior region of the sperm head (Yanagimachi and Noda, 1970, Bedford et al., 1979) and is th ought to be mediated by a group of proteins which include the dimeri c sperm glycoprotein fe rtilin (Kaji and Kudo, 2004). Upon fusion of the sperm with the plasma membrane of the oocyte, the oocyte becomes activated (intracellular calcium [Ca2+] oscillations, completion of th e second meiotic division and the oocyte undergoes the cortical reaction exoc ytosis of cortical granules; Sutovsky et al., 2003b). The cortical reaction leads to hardening of the zona pellu cida, loss of sperm receptors and constitutes the major bl ock to polyspermy (Sun, 2003). Fusion of the sperm membrane with the ooc yte membrane and inte raction with oocyte microvilli leads to engulfment of the entire sperm by the ooplasm (Sutovsky et al., 1996). Once within the oocyte the nuclear envelope of the sperm disperses and nuclear decondensation occurs; mitochondria within the connecting piece are displaced, the sperm proximal centriole is exposed to the ooplasm and formation of the sp erm aster occurs (Sutov sky et al., 1996). The paternal mitochondria are targ eted for destruction by ubiquitin (Sutovsky et al., 1999) and the remains of the sperm principal sheath ar e destroyed (Sutovsky et al., 1996; 2003a). One of the first steps to male pronucleus formation involves desolution of the sperm nuclear envelope and the reduction of interand intraprotamine disulphide bonds formed during sperm maturation between cystein residues by oocyte derived glutathione. These events normally

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18 occur as the fertilized oocyte tr ansits from metaphase II to tel ophase II (Adenot et al., 1991). As telophase II advances sperm chromatin re-c ondenses and decondense once more concomitant with female pronucleus development (Adenot et al., 1991). The final steps of fertilization involve apposition of the male and female pronuclei and syngamy, mediated by the sperm aster and associated cytoskeletal structures. Paternal Contributions to the Embryo Paternal investm ents in development of th e embryo consist of genetic and non-genetic factors. The genetic factors incorporated into the oocyte include the paternal DNA and mitochondrial DNA (mtDNA). However, paternal mitochondria are selectively ubiquitinated (Sutovsky et al., 1996) and shunted to the lysoso mal apparatus for destruction in the bovine oocyte (Sutovsky et al., 2000). Nevertheless, pa ternally derived mitoc hondria are found in the bovine embryo as late as 2 to 4 cell stage (Sutovsky et al., 1996). It is possible that the paternal mtDNA might contribute to early embryonic developmen t, but this is unlike ly in the pig since complete degradation of sperm mitochondria in the cytoplasm of fertilized porcine oocytes occurs within 20 to 30 h after insemination prio r to the first cleavage event (Sutovsky et al., 2003c). Others would argue that paternal mitoch ondria inheritance does occur. It has been demonstrated that a small amount of paternal mtDNA can survive in the mouse embryos of interspecific mitochondrial congenic mice and that 0.1 to 0.01% mtDNA in all tissues is of paternal origin (Gyllensten et al., 1991). However, Shitara and colleagues demonstrated that in interspecific hybrids, paternal mtDNA was not di stributed to all tissues and was not transmitted from the females to the following generation (Sh itara et al., 1998) and that sperm mitochondria were uniquely selected for elimination from mouse embryos when compared to liver mitochondria (Shitara et al., 2000).

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19 The inheritance of paternal mtDNA has been associated with disease. The New England Journal of Medicine reported the case of a 28-year-old man with mitochondrial myopathy due to a 2-bp mtDNA deletion in the ND2 gene. It was determined that the mutated mtDNA was paternal in origin and account ed for 90% of the patients musc le mtDNA (Schwartz and Vissing, 2002). How does one account for the finding of patern al mtDNA in the offspring? Perhaps, the ubiqitination process of sperm is a species -specific phenomenon. Cross-breed embryos from interspecific mice; hybrid embryos formed from domestic cow eggs and sperm of wild cattle (gaur; Sutovsky et al., 2000) and hybrid birds fo rmed from major and minor subspecies of the Great Tit ( Parus major; Kvist et al., 2003) have been show n to contain paternally inherited mtDNA, and this may reflect the inability of the maternal ubiqitin-a ssociated degradation machinery to recognize and eliminate paternal m itochondria with an inter-species ubiquitin tag. The most important contribution of the sp erm to the embryo is its DNA. Paternal DNA must undergo morphological and biochemical cha nges such that the chromatin structure is compatible with that of the oocyte for DNA replication, transcription and mitosis. Mammalian sperm chromatin structure and composition differs from that of somatic cell chromatin and that of the oocyte (Clarke, 1992). Th e basic unit is the to roid consisting of 50 kb of DNA wrapped around highly basic proteins protamines (Hud et al., 1993). Protamines consist largely of arginine and cysteine residue s which allow for strong DNA bindi ng and for disulphide crosslinkage (Brewer et al., 2003) dur ing sperm nuclear maturation in the epididymis. There are two types of protamines, P1 and P2. Bull sperm c ontains P1 only (Lee and Cho, 1999) and may have residual histones. Human sperm contain some of th eir histones (Zalensky et al., 1997; Gineitis et al., 2000) usually associated with the nuclear periphery and telo meric regions (Zalensky et al.,

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20 1997; Gineitis et al., 2000) Since the DNA associated with hi stones is less tightly wound than DNA associated with protamines it is postulated th at these areas may be the first structures to respond to oocyte signals for pronucleus formation (Gineitis et al., 2000). The non-genetic factors incor porated into the oocyte upon fe rtilization include the oocyteactivating factor, centriole, sperm nuclear ma trix and RNAs. During fe rtilization the sperm perinuclear theca is solubilized in the oocyte cyt oplasm and releases a number of sperm factors into the oocyte (Sutovsky et al., 1997; 2003b). On e such factor is the sperm oocyte-activating factor (SOAF), which triggers Ca2+ oscillations and a block to polyspermy (Sutovsky et al., 2003b). Several candidates for SOAF have been extracted from sperm. However, phospholipase C-zeta (PLC ) seems to be the likely candidate for SOAF (Swann et al., 2006; Saunders et al., 2007). Studies using oocytes and oocyte extracts established that microinjection of PLC complementary RNA (Saunders et al., 2002; Y oneda et al., 2006) and recombinant PLC (Kouchi et al., 2004) trigger Ca2+ oscillations similar to those seen at fertilization. Phospholipase C-zeta-immunodepleted soluble sperm extr acts are incapable of triggering Ca2+ oscillations (Saunder et al., 2002) and RNAi knockdown of PLC reduces the number of Ca2+ oscillations and the activation rates in ooc ytes fertilized by transgen ic sperm (Knott et al., 2005). Phospholipase C-zeta has been identified in the soluble sperm extracts of the mouse, pig, hamster and man (Saunders et al., 2002; Fujimoto et al ., 2004; Kurokawa et al., 2005; Young et al., 2008; Grasa et al., 2008) and localized to the equatorial area of bull sperm and to the post-acrosomal region of mouse sperm (Yoon and Fissore, 2007). Importantly, the quantity of PLC required to activate the oocyte is in the range of that carried by a single sperm (Saunders et al., 2002). Boveri first recognized (in 1901) that the egg typically losses the centrosome during oogenesis and that the sperm introduces this stru cture at fertilization (S chatten, 1994). In most

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21 mammals with exception of some rodents, the centrioles are lost during oogenesis and partially degraded during spermatogenesis (Schatten, 1994 ; Manandhar et al., 2005). Concurrently, the pericentriolar matrix is retained in the oocyte but lost in the sperm. During fertilization mammalian embryos inherit the proximal centriole fr om the sperm, which is responsible for aster formation during the first cell-cy cle (Navara et al., 1994; 1995; Palermo et al., 1997). Proper sperm aster formation is required for successful fertilization as sperm aster microtubules provide the tracks for pronuclear migration and apposition of the male and female pronuclei facilitating syngamy (Navara et al., 1996; Suto vsky et al., 1996). The paternally derived centriole duplicates during interphase concurrent wi th the pronuclear stag e and the centrosome in itially organizes a sperm aster concurrent with male and female pronuclei breakdown (prometaphase). The sperm aster now containing two centrio les split and moves to opposite poles of a bipolar spindle to establish bipolarization (anaphase), and patern al and maternal chromosomes organize on the equator of a metaphase spindle, at syngamy (Schatten, 1994; Sutovsky et al., 1996; Palermo et al., 1997). During cytokinesis the centrioles are at opposite ends and will form the centrosome of the daughter blastomeres (Schatte n, 1994; Navara et al., 1995). The sperm nuclear matrix is also inherited by the fertilized oocyte and there is some evidence that the sperm nuclear matrix is essent ial for events during fert ilization (Ward et al., 1999; Shaman et al., 2007). In mice, removal of the sperm nuclear matrix from the DNA prior to injection into mouse oocytes results in failu re of paternal DNA rep lication and pronuclear formation (Shaman et al., 2007) without derailmen t of maternal DNA replication and pronuclear formation. Injection of isolated sperm nuclear ma trix and isolated matrix associated DNA does not reconstitute the ability for paternal DNA replication (Shaman et al., 2007). While injection of matrix-associated regions (areas where the DNA l oop domain attach to the sperm nuclear matrix)

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22 into mouse oocytes without large portions of the DNA loop initiate DNA replication (Shaman et al., 2007). Ribonucleic acids (RNAs; messenger RNAs, microRNAs and antisense RNAs) have been localized within ejaculated sperm (Ostermeier et al., 2002; Miller and Ostermeier, 2006; Amanai et al., 2006) and unique paternal mRNA have been found to persist in the embryo up to 3 h post fertilization (Ostermeier et al., 2004). However, the role of pa ternal RNAs in the developing embryo remains a mystery (Lalancette et al., 2008). Post-ejaculatory Sperm Damage Investigations into post-ejaculatory sperm da mage have been limited to studies designed to evaluate the effect of cryopr eservation, short term storage, h eat stress or the female tract on sperm. While much emphasis has been placed on effects of stress on the fertilizing capability of sperm, few studies have suggest ed that damage to sperm afte r ejaculation may compromise the resultant embryo. Heat Shock Evidence to support the idea that sperm da ma ge after ejaculation can affect embryo competence of an embryo formed from post-ejaculatory damaged sperm includes work done utilizing heat shock as a model. Burfen ing and Ulberg (1968) demonstrated that in vitro heat shock of ejaculated sperm decrease embryo survival as determined by the number of implantation sites noted on day 9 or 12 post inse mination per number of cleaved ova 30 h post coitus in rabbits. Moreover, pos t-ejaculatory sperm damage in utero can result in the formation of an embryo with reduced competence for development (Howarth et al., 1965). This phenomenon has also been confirmed by work done more recently by Cozzi et al. (2001) where epididymal sperm harvested from male mice and heat shocked (56C for 30 min) prior to ICSI

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23 resulted in embryos produced that failed to develop to the blastocyst stage compared to controls (16% vs 62%, respectively). Cryopreservation Since the advent of artificial reproductive technologies (ARTs) in the late 1940s by the acciden tal freezing and recovery of live motile sperm cells in the presence of glycerol (Polge et al., 1949; Lovelock and Polge, 1954) the practice of freezing sperm for later use in artificial insemination (AI) programs in the livestock industry has be come common practice. This technique has been adopted in companion anim al and human reproductive medicine and in wildlife conservation and preservation program s (Wildt, 2000; Pukazhenthi et al., 2006; Swanson et al., 2007). Frozen-thawed mammalian spermatozoa how ever differ from freshly ejaculated spermatozoa in many ways. These differences incl ude a shorter lifespan (Gillian and Maxwell, 1999; Sankai et al., 1994; Rodrguez-Martnez et al., 2008), decreased motil ity (Gandini et al., 2006; Jin et al., 2008; Rodr guez-Martnez et al., 2008), a highe r degree of membrane damage or membrane alterations (Hammerstedt et al., 1990 ; De Leeuw et al., 1990; Gillian et al., 1997; Gillian and Maxwell, 1999; Pegg, 2002; Nishizono et al., 2004), increased incidence of acrosome reacted sperm (Gillian et al., 1997; Gillian and Maxwell, 1999; Gill ian et al., 1999), increase in chromatin abnormalities and DNA fragmentation in some studies (Horse: Baumber et al., 2003; Ram: Peris et al., 2004, 2007; Hu man: Gandini et al., 2006; Boar : Fraser and Strzezek, 2007; Mouse: Yildiz et al., 2007, 2008) but not in others (Humans Ch ernos and Martin, 1989; Martin et al., 1991; Okada et al., 1995) and decreased fertility followi ng intrauterine or cervical insemination (Dogs: Gill et al., 1970. Ewes: Ja bbour and Evans, 1991; Maxwell et al., 1993; Gillian et al., 1997; Gmez et al., 1997; Donovan et al., 2004; Niza ski, 2006. Mouse: Nishizono et al., 2004.) and even after in tracytoplasmic sperm injection (ICSI; Gmez et al., 1997).

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24 Cryopreservation results in formation of intr acellular ice crystals (Pegg, 2002), and an increase in osmotic pressure, that is, the remaining solutes become more concentrated as water is removed from solution (Watson and Duncan, 1988; Pegg, 2002) consequently leading to mechanical and osmotic damage of membranes (plasma and nuclear) and cellular organelles (Hammerstedt et al., 1990; Pegg, 2002; Nishiz ono et al., 2004). Cryopreservation induces oxidative damage to sperm membrane lipids (m id-piece) and reduces the ability of frozenthawed sperm to withstand further oxidative stre ss (Neild et al., 2005), a nd induces lateral phase separation in plasma membranes of bovine a nd porcine sperm (De Leeuw et al., 1990). Cryopreservation also results in the production of reactive oxygen species (Bilodeau et al., 2000; Ball et al., 2001; Chatterjee et al ., 2001), redistribution of the an tioxidant defense system (Marti et al., 2008) and a reduction in glutathione in sp erm (Bilodeau et al., 2000; Gadea et al., 2004). Cryopreservation results in sperm damage a nd embryos formed from cryo-damaged sperm have reduced developmental competence. For exam ple, mouse oocytes fertilized with frozenthawed epididymal sperm have reduced fertilization (as assessed by synchronous pronuclei at 7.5 hours post insemination), reduced cleavage rates a nd hatching rates when compared to oocytes produced from fresh epididymal sperm (Songsasen et al., 1997). This obse rvation is not unique to frozen-thawed mouse sperm, as ewes undergoing cervical insemination of frozen-thawed semen have reduced pregnancy rates and produced smaller litter size (Donovan et al., 2004) when compared to fresh semen. Furthermore, the use of frozen-thawed semen for vaginal insemination in the dog results in reduced preg nancy rates, whelping ra tes and litter size (Nizaski, 2006). Moreover, deposition of frozen-t hawed dog semen into the uterus does not improve pregnancy rates over deposition of fresh semen into the cranial vagina (Linde-Forsberg and Forsberg, 1989). In horses, pregnancy rate s following hysteroscopic insemination with

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25 frozen-thawed sperm tends to be lower than with fresh semen (Linds ey et al., 2002). Even following ICSI embryo quality, pregnancy and de livery rates of human embryos produced with frozen-thawed testicular sperm is lower than in embryos produced with fresh testicular sperm (Aoki et al., 2004) and spontaneous abortion rates are higher wh en embryos are produced from frozen-thawed sperm (Aoki et al., 2004). Add itionally, human embryos produced by ICSI from frozen-thawed testicular sperm have lower im plantation rates (measured as the number of gestational sacs observed by ultrasound at 6 weeks of pregna ncy divided by the number of embryos transferred) when compared to embryos produced with fresh testicular sperm (De Croo et al., 1998). Even in domestic animals, such as the horse, ICSI performed with frozen-thawed ejaculated spermatozoa yielded zygotes with re duced pronuclear formation, and embryos with reduced cleavage rate and a re duction in the average number of nuclei at 96 h when cultured either in vitro or in vivo (Choi et al., 2002). However, these reductions in embryo development parameters were not statistically significant when compared to ICSI performed with fresh ejaculated equine spermatozoa (C hoi et al., 2002). However, it is possible that developmental differences may have become significant had the em bryos been allowed to develop further as in the human studies. Interestingly, frozen-thawed epididymal boar sperm as opposed to frozen-thawed ejaculated sperm retains motility similar to fresh ejaculated boar sperm and frozen-thawed epididymal sperm show enhanced fertilizing capacity as measured by pronuclear formation (Rath and Niemann, 1997). Furthermore oocytes fertilized with frozen-thawed epididymal boar sperm have increased cleavage rates, with significantly more embryos developing to the 2and 4-cell stages compared with oocytes fertilized with fr esh or with frozen-thawe d ejaculated semen (Rath and Niemann, 1997).

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26 Hydrogen Peroxide (H2O2) The effect of hydrogen peroxide on ejacula ted sperm is two fold. At physiological concentrations H2O2 facilitates sperm hyperac tivation and acrosome reaction (Griveau et al., 1994), capacitation (Rivlin et al., 2004; de Lamirande and OFlah erty, 2008), zona pellucida binding and penetration thus improving in vitro fertilization (Ford, 2004). The deleterious effects of H2O2 typically occur at high concentrations (100 M to 1.5 mM H2O2) but may also depend on the species involved. In human ejaculated sperm, concentrations ranging from 230 M to 1.5 mM reduce motility (Chen et al., 1997; Armstrong et al., 1999; Kemal Duru et al., 2000) and are associated with decreased mitoc hondrial transmembrane potential ( m) and reduced ATP sperm content (Armstrong et al., 19 99). Furthermore, 100 to 230 M H2O2 induces the formation of toxic lipid peroxide and DNA damage in hu man ejaculated sperm (Chen et al., 1997; Kemal Duru et al., 2000) and 200 M H2O2 reduces sperm-zona pellucid a binding (Oehninger et al., 1995). Ram sperm however exhibit a re duction in motility when exposed to lower concentrations (50 to 300 M H2O2) and exhibit DNA damage at H2O2 concentration similar to human sperm (150 to 300 M H2O2; Peris et al., 2007). Interestingly, th e reduction in motility has not been associated with increased lipid peroxidation in ram sperm (50 to 300 M; Peris et al., 2007). Similarly, ejaculated ram sperm exposed to 0.0375 and 0.375% H2O2 exhibit no increase in lipid peroxidation as measured by fluorescence recovery after photobleaching (FRAP) to monitor 5(N-octadecanoyl)-aminofluorescein (ODAF; lipid reporter probe) diffusion (Christova et al., 2004). In addition exposure to 150 and 300 M H2O2 prevents the acrosome reaction in ram sperm (Peris et al., 2007). In the boar, incubation with 300 M H2O2 reduces motility, but is not associated with decreased m or sperm ATP content (Guthrie et al ., 2008) in contrast to human ejaculated

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27 sperm (Armstrong et al., 1999). Interestingly, even lower concentration of H2O2 reduced motility (25 M) and 10 to 250 M H2O2 reduced the capacity of bovine frozen-thawed ejaculated spermatozoa to undergo capacitation, however an increase in acrosome reacted sperm was noted at 25 M H2O2 as detected by the chlortetracycline assay (O'Flaherty et al., 1999). Hydrogen peroxide results in sperm damage and embryos formed from damaged sperm have reduced developmental competence. For ex ample, bovine frozen-thawed ejaculated sperm exposed to 500 M H2O2 or a cocktail (100 mM ascorbic acid, 20 mM FeSO4 and 500 mM H2O2) produced embryos with reduced capacity to cleave by Day 5 post insemination and to develop to the blastocyst stage by Day 9 (Silv a et al., 2007). The proportion of Day 5 cleaved embryos that had 8 blastomeres was reduced in embryos produced by bovine sperm exposed to 500 M H2O2 and no 8-cell or greater embryos were form ed from sperm treated with the cocktail (Silva et al., 2007). Human ejaculated sperm exposed to media containing 0.23 mM H2O2, 1.8 mM ADP and 2.7 mM FeSO4 have increased levels of 8-hydroxydeoxyguanosine (8-OH-dG; Chen et al., 1997) and embryos formed fr om sperm containing 8-OH-dG demonstrate fragmentation and overall reduced em bryo quality (Meseguer et al., 2008). Menadione Menadione (2-m ethyl-1,4-naphthoqu inone) is a polycyclic aromatic ketone precursor to vitamin K2. Menadione is metabolized by flavoprotein reductase to a semiquinone, which can be oxidized back to a quinone in the presence of molecular oxygen. In this redox cycle, the superoxide anion radical (O 2), hydrogen peroxide (H2O2) and other reactiv e oxygen species are generated (Monks et al., 1992). As such menadione has been utilized to generate reactive oxygen species (ROS) in order to study oxidative damage in vitro on fresh and frozen-thawed ejaculated

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28 sperm (Guthrie and Welch, 2006, 2007; Guthrie et al., 2008). Menadione reduces sperm motility, depresses m within 30 min and will eventually re duce ATP content (Guthrie et al., 2008). Gossypol Gossypol, a polyphenolic yellow pigm ent f ound in cotton plants of the genus, Gossypium has been implicated in reducing or obliterating male reproductive function in a number of species (reviewed in Randel et al., 1992). Uptake of goss ypol by sperm may be due to interactions of gossypol with membrane phospholipids (Ueno et al., 1988). Work with human and hamster ejaculated sperm indicate that gossypol preven ts capacitation and penetration of zona-free oocytes (Kennedy et al., 1983; Aitken et al., 1983) as a result of a reduction in acrosin activity by preventing the conversion of proacrosin to ac rosin (Kennedy et al., 1983; Yuan and Shi, 2000) and in the inactivation of other acrosomal en zymes (Yuan et al., 1995). Gossypol also induces uncoupling of the respiratory chai n and oxidative phosphorylation in boar ejaculated sperm (Tso and Lee, 1982). Other mechanisms of action of gossypol include inhibition of anion exchange (Haspel et al., 1985). In isolated plasma membranes of human ejaculated sperm, gossypol inhibits Ca2+ transport and Ca2+-activated ATPase activity (Kanwar et al., 1989). It also inhibits the membrane bound Mg2+and Na+-K+ dependent ATPases, 5'-nucleotidas e and alkaline phosphatase systems (Kanwar et al., 1989). Similar findings have been noted in mouse, ram and bull ejaculated sperm (Shi et al., 2003; Breitbart et al., 1984). Further, gossypol in hibits glucose uptake in human ejaculated sperm, increases thiobarbituric acid reaction products and decreases total phospholipids establishing the lipi d peroxidative effect of goss ypol on sperm plasma membranes (Kanwar et al., 1990). Kanwar et al. (1990) speculated that inhi bition of glucose uptake was linked to lipid peroxidation of the plasma membranes and subsequent membrane damage.

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29 The mechanism of action of gossypol and their effects on cells are varied and complicated, because gossypol can be metabolized to yield se veral derivatives aldehyde, enol, hemiacetal forms and gossypolone. Gossypol has the ability to generate reactive oxygen species (Kovacic, 2003) and has been investigated for its an ti-oxidant (Dodou et al., 2005), contraceptive (Coutinho, 2002), anticancer (Balakrishnanet al., 2008), antiprotozoan, antiparasitic and carcinogenic properties (Kovacic, 2003). Gossypol induced sperm damage reduces fert ilizing ability and could have a negative impact on embryo competence. For example, treatm ent of bovine ejaculated sperm with gossypol has been reported to reduce cleavage rate when sperm are used for in vitro fertilization and lead to the formation of embryos with reduced proba bility of developing to the blastocyst stage (Brocas et al., 1997). Precoital in trauterine administration of gossypol reduces the number of ejaculated spermatozoa reaching the ampullae assessed the morning after mating and the number of penetrated oocytes assessed on Day 2 and 3 postcoitus in rats (Moore et al., 1988). Furthermore, precoital intraute rine administration of gossypol inhibited implantation, however it is difficult to ascertain if this was a direct result of gossypol-i nduced sperm damage alone, or in combination with gossypol-induced oocyte or zy gote and uterine damage (Moore et al., 1988). Hernndez-Cern et al. (2005) de monstrated that gossypol reduces the ability of one-cell bovine embryos to develop to the blastocyst stage but has no effect on later stag es of development. X-irradiation X-irradiation causes DNA strand breaks and ch romoso mal aberrations in somatic cells (Haines et al., 2001, 2002; Cordelli et al., 2003), ooc ytes (Matsuda et al., 1985b; Griffin et al., 1990), ejaculated sperm (Kami guchi et al., 1990) and embryo s (Matsuda et al., 1985b). Mechanism through which this damage occur are either the direct effect of ionizing radiation (Cadet et al., 2003) or through the indirect e ffects generation of fr ee radicals from surrounding

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30 water molecules (Cadet et al., 2003). The generation of OH via radiolysis of water account for 60-70% of cellular DNA damaged produced by ionizing radiation (Ward, 1988). Damage to DNA by OH include oxidation of bases, abas ic sites, DNA-DNA interstrand adducts, DNA single and double strand breaks and oxidation of DNA-proteins cr oss-links (Cadet et al., 2004). Oxidative damage to the sugar-phosphate backb one of DNA leads to singleand double-strand breaks (Cadet et al., 2004). The dose at which X-irradiation causes DNA dama ge in ejaculated sperm varies from one species to another. McKelvey-M artin et al. (1997) exposed huma n ejaculated sperm to increasing doses to X-rays (5, 10 and 30 Gy) and found that only when sperm were exposed to the highest dose of radiation (30 Gy) increased DNA degrada tion as measured by the alkaline comet assay. However, Fatehi et al. (2006) de monstrated that there was a si gnificant increase in DNA damage at much lower doses of X-irradiation in bovi ne sperm (0.6 Gy) using the TUNEL assay in conjunction with flow cytometry and this increased with increasing radiation dose (1.25, 2.5, 5.0 and 10 Gy). X-irradiation results in sperm damage (DNA damage) and embryos formed from damaged sperm have reduced developmental competence. For example, exposure of bovine ejaculated sperm to X-irradiation (1.25, 2.5 and 5.0 Gy) reduces the proportion of oocy tes that cleave after insemination and the proportion of cl eaved embryos that became blas tocysts (Fatehi et al., 2006). Furthermore, exposure of caudal epididymal mous e sperm to X-irradiation (50 to 400 cGy) does not affect fertilization (Matsuda et al., 1985a; Pampfer et al., 1989) and there is a dose-dependent increase in the proportion 2-cell embryos cont aining fragmented DNA (Matsuda et al., 1985a; Pampfer et al., 1989) despite the ability of the oocyte to repair DNA dama ge (Matsuda et al., 1989). Additionally, the consequences of DNA damage in the male gamete are low fertilization

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31 rates (Sakkas et al., 1996; Hst et al., 2000a,b; Bakos et al., 20 07), reduced embryo development (Morris et al., 2002; Tomsu et al., 2002; Seli et al., 2004; Fatehi et al., 2006; Paul et al., 2008) and increased embryo and fetal loss (Tomlinson et al., 2001; Tomsu et al ., 2002; Virro et la., 2004; Waterhouse et al., 2006; Paul et al., 2008). Sperm DNA damage has also been associated with an increased incidence of cancer in offs pring produced from such damaged sperm (Lord, 1999; Lewis and Aitken, 2005). Pathways of Apoptosis The typical response to DNA da mage in somatic cells is arrest of the cell cycle and activation of DNA repair mechanisms (Friedberg, 2003). In some cases the damage is tolerated (Goodman, 2002) or repaired (Friedberg, 2003). Ho wever, in the event that the DNA cannot be repaired the cell undergoes progr ammed cell death apoptosis (F riedberg, 2003). It is possible that one mechanism by which sperm damage compromises the competence of the resulting embryo to develop is activation of a poptosis responses in the sperm cell. Apoptosis may be an important mechanism for sperm damage and experimental elevation in testicular temperature causes germ cell apoptosis vi a the intrinsic pathway (Hikim et al., 2003; Vera et al., 2004; 2005). Furthe rmore, apoptosis or programmed cell death appears to be a normal phenomenon during spermatogenesis and has b een proposed to maintain homeostasis in the testes (Blanco-Rodriguez, 1998). There are two well-known pa thways involved in apoptosis in mammalian cells; the intrinsi c or mitochondrial pathway and the extrinsic or receptormediated pathway. The intrinsic pathway is depicted in Figure 1-1. It involves the release of cytochrome c from the mitochondrial intermembrane space into th e cytosol. The molecular events which lead to the formation of pores in the mitochondrial out er membrane and release of cytochrome c in response to stress are not well unde rstood. It is believed that stre sses such as, heat shock (Chung

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32 et al., 2003), infections with S. aureus (Esen et al., 2001) N. gonorrhoeae (Grassme et al., 1997), or treatments with gamma-irradiation (Santa na et al., 1996) and UV li ght (Zhang et al., 2001) can induce the activation of sphinogomyelinase (SMase) whic h cleaves plasma membrane sphinogomyelin (SM) to ceramide. It has been shown that approximately 13% of bovine sperm plasma membrane is made up of sphinogomyelin which is concentrat ed in the acrosomal membrane (Parks et al., 1987). Ceramide then triggers the activation of signal transduction pathways which activate pro-apopt otic proteins of the Bcl-2 fa mily, Bax and Bad. Activated Bax translocates from the cytosol to the mitoc hondria where they form pores in the outer mitochondrial membrane and cytochrome c is rel eased into the cytosol (Zamzami and Kroemer, 2001; Fumarola and Guidotti, 2004). Cytosolic cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1), i nducing oligomerization and exposure of the caspase recruitment domain (CARD; Riedl and Salvesen, 2007) resulti ng in the recruitment of procaspase 9 and the formation of the apoptosome (Fumarola and Guidotti, 2004; Riedl and Salvensen, 2007). Procaspase 9 undergoes activation via proteolyti c autoactivation (Fumarola and Guidotti, 2004). The active caspase 9 then activates the down st ream executioner caspases (caspase 3 and 7) (Fumarola and Guidotti, 2004). Along with cyto chrome c, Smac/Diablo, apoptosis-inducing factor (AIF) and endonuclease G are released from the intermembrane space (Fumarola and Guidotti, 2004). The pro-apoptotic factor Smac/Diablo binds to IAPs (inhibitors of apoptosis proteins) promoting caspase activity and hen ce apoptosis (Fumarola and Guidotti, 2004). Inhibitors of apoptosis proteins bind to procaspases and activated ca spases inhibiting there activation and activity respectively (Riedl and Shi, 2004) Apoptosis-inducing factor and endonuclease G translocate to the nucleus wher e they induce caspase-independent DNA damage (Fumarola and Guidotti, 2004).

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33 The receptor mediated pathway, involves the in teraction of death re ceptor and its ligand, such as FasFasL interaction (Waring and Mllbacher, 1999; Sharma et al., 2000). In this case, binding of FasL to Fas induces tr imerization of the Fas receptor and recruitment of the adapter molecule Fas-associated death domain (FADD) to the cytoplasmic tail of Fas to form the deathinducing signaling complex (DISC; Sharma et al., 2000). Fas-associated death domain binds and activates procaspase 8 (Sharma et al., 2000). Active caspase 8 either goes on to activate the executioner caspase, caspase 3 or it may cleave Bid, a proapoptotic Bcl-2 family member, to form a truncated form of Bid, tBid (Li et al., 1998). The truncated form of Bid, tBid, facilitates the cross-talk between the receptor mediated and mitochondrial pathways, by stimulating the release of cytochrome c via Bax and/or Bak olig omerization and inserti on into the mitochondrial membrane with the resultant release of cytochrome c (Korsmeyer et al., 2000). The overall importance of exposure of ejacu lated sperm to hydrogen peroxide, menadione, and X-irradiation is that once the sperm DNA is damaged, unlike in somatic cell or even the oocyte, the DNA damage is not repaired as sperm lack DNA repair mechanisms. Secondly, failure to undergo complete apoptosis implies that these defective sperm remain in the fertilizing population increasing the possibility that oocytes will become fertilized with damaged sperm. This is especially true in a ssisted-reproductive technology (ART), where the mechanisms which have evolved to ensure selection of high qual ity sperm for fertilization have been bypassed. Synopsis It is possible that post-ejacula tory sperm damage can compromise the ability of embryos formed from damaged sperm to fulfill their de velopmental potential, and this could involve damage to paternally inherited macromolecules. Moreover, damage to paternally inherited macromolecules may occur by apoptosis. Using ejacu lated bull sperm as a model this thesis will examine the effects of aging and heat shock, airpor t security X-irradiation and oxidative stress on

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34 embryo competence. These will be addressed in or der and Chapter 5 will investigate the role of apoptosis in aging and heat s hock as a possible mechanism of sperm damage using ejaculated bull and stallion sperm as a model. Finally, the General Discussion in Chapter 6 will provide an overview of the findings and possible mechanis ms for the reduction in the developmental competence of embryos formed fr om damaged ejaculated sperm.

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35 Apoptosome Procaspase 3 Active caspase 3 Active caspase 9 DNA fragmentation and cellular death Cytochrome c Apaf-1 Pro-caspase 9 Caspase activated DNaseAIF Endonuclease G Smac/Diablo IAPs SMCeramide HEAT SHOCK Figure 1-1. Simplification of induction of apoptosis by heat shock with emphasis on the mitochondrial or intrinsic apoptotic pathwa y. Other pathways controlling apoptosis are not shown here.

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36 CHAPTER 2 CONSEQUENCES FOR THE BOVINE EM B RYO OF BEING DERIVED FROM A SPERMATOZOAN SUBJECTED TO POST-EJA CULATORY AGING AND HEAT SHOCK: DEVELOPMENT TO THE BLASTOCYST STAGE AND SEX RATIO Introduction Developm ental competence of the mammalia n embryo is dependent on genetic and nongenetic contributions from its parents (Warner et al., 1998; Sirard et al., 2006; Mnzo, 2006; Baumann et al., 2007; Khatib et al., 2008ab). Sp erm could affect an embryos competence for development if the timing of fertilization or ear ly cleavage is delayed. For example, embryos produced by high-fertility bulls entered S-phase of the first cell cycle ear lier and had a longer Sphase than those produced by lowfertility bulls (Eid et al., 19 94). In another study, spermatozoa from 50% of bulls identified as being of low fertility in artificial insemination studs experienced premature capacitation (Kuroda et al., 2007). Da mage to the macromolecular portions of the sperm that are incorporated by the embryo could also result in formation of embryos with reduced developmental competence. Among thes e sperm contributions are DNA, the centriole (Sutovsky and Schatten, 2000), and RNA (Ostermeier et al., 2004). Embryos fertilized with semen containing a high proporti on of sperm with extensive DNA damage have reduced competence for development (Virro et al., 2004; Se li et al., 2004; Muriel et al., 2006), but the importance of damage to the centr iole or sperm RNA is not known. Damage to sperm can occur in the male repr oductive tract or after de position of sperm in the female. In bulls, for example, thermal stress of the scrotum leads to pr oduction of sperm that produce embryos with delayed or reduced pronuclea r formation (Walters et al., 2006), a reduced ability of cleaved embryos to become blastocy sts for some bulls (Walters et al., 2004; 2005a), and increased embryo apoptosis (Walters et al., 2005b). Sperm from diabetic mice have reduced capacity to fertilize oocytes and for the resultant embryos to give rise to blastocysts (Kim and

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37 Moley, 2008). There is also evid ence that sperm can be damage d after ejaculation by stresses that lead to reduced embryo competence after fe rtilization. Irradiation of mouse sperm did not affect fertilizing abilit y but did reduce rates of blastocyst development and implantation (Ahmadi and Ng, 1999ab). Exposure of ejacu lated frozen-thawed bull spermatozoa to gossypol (Brocas et al., 1997) or reactive oxygen species reduced the percent of cleaved embryos that developed to the blastocyst stage (Silva et al., 2007). For the current study, we used bull spermatozo a to test the hypothesis that aging of ejaculated sperm for 4 h after freeze-thawing woul d damage sperm and lead to embryos with reduced developmental competence after fertilizati on. The term aging was used to represent incubation in vitro since this treatment can cause a reduction in sperm motility after 3 h (Monterroso et al., 1995). A second hypothesis was that sperm damage would be enhanced if aging occurred at elevated temperatures. The heat -stress temperature used, 40C, is characteristic of rectal temperatures of lactating cows exposed to heat stress (Elvinger et al., 1992; de Castro e Paula et al., 2008) and effects of aging sperm at this temperature could be relevant to understanding causes of reduced fertility of da iry cows during heat stress (Hansen, 2007). Exposure of human ejaculated sperm to mild heat shock caused DNA damage (Mann et al., 2002) and studies in the rabbit i ndicate that fertilization with sperm incubated at elevated temperature in vitro or in the female rabbit results in embryos with reduced implantation rates (Burfening and Ulberg, 1968; Howarth et al., 1965). Materials and Methods Materials The m edia HEPES-Tyrodes Lactate (HEPES-T L), IVF-TL, and Sperm-TL were purchased from Caisson (Sugar City, ID, USA) and used to prepare HEPES-Tyrodes albumin lactate pyruvate (TALP), IVF-TALP, and Sperm-TALP as previously described (Parrish et al., 1986).

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38 Oocyte collection medium (OCM) was Tissue Culture Medium-199 (TCM-199) with Hanks salts without phenol red (Atlanta Biologicals, No rcross, GA, USA) supplemented with 2% (v/v) bovine steer serum (Pel-Freez, R ogers, AR) containing 2 U/mL he parin, 100 U/mL penicillin-G, 0.1 mg/mL streptomycin, and 1 mM glutamine. Oocyte maturation medium (OMM) was Tissue Culture Medium 199 (Gibco, Invitrogen, Carlsbad, CA, USA) w ith Earles salts supplemented with 10% (v/v) bovine steer serum, 2 g/mL estradiol 17, 20 g/mL bovine FSH (FolltropinV; Bioniche Animal Health, London, ON, Canada), 22 g/mL sodium pyruvate, 50 g/mL gentamicin sulfate, and 1 mM glutamine. Perc oll was from GE Healthca re (Uppsala, Sweden). Potassium simplex optimized medium (KSOM) containing 1 mg/mL BS A was obtained from Caisson. Essentially fatty-acid fr ee (EFAF) BSA was from Sigma (St. Louis, MO). On the day of use, KSOM was modified for bovine embryos to produce KSOM-BE2 as described elsewhere (Soto et al., 2003). Hoechst 33342 was from Calbiochem (San Diego, CA, USA). PCR oligonucleotide primers were obtained from Inte grated DNA Technology (C oralville, IA, USA). Taq DNA polymerase and 100 mM dNTPs were fr om Invitrogen. All other reagents were purchased from Sigma or Fisher Scientific (P ittsburgh, PA, USA) unle ss otherwise stated. Sperm Preparation Extended and frozen sem en from Holstein bulls was obtained from Select Sires Inc. (Plain City, OH, USA) and ABS Global (Deforest, WI, USA). Semen was thawed, subjected to Percoll gradient purification to obtain motile spermatozo a (Parrish et al., 1986), diluted in Sperm-TALP medium to 20 x 106 spermatozoa/mL, and aged by incubation at 38.5 C or 40 C in air for 4 h using a water bath. Additional semen was th awed to prepare a non-incubated spermatozoa control. In this case, semen wa s thawed, subjected to Percoll purification, and diluted to 20 x 106 spermatozoa/mL at a time to coincide with the end of the incubation period for aged sperm.

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39 Sperm Motility The percent of sperm exhibiting motility wa s assessed by visual examination. Briefly, 20 L of sperm suspension were placed on a glass slide pre-warmed at 37C and examined under 200x magnification. Motility was estimated for 100 sp ermatozoa located in 10 different fields. In Vitro Production of Embryos Em bryo production was performed as previously described (Soto et al., 2003) except that sperm were subjected to incuba tion prior to fertilization as de scribed above and oocytes in a single replicate were inseminated with seme n from a single bull. Briefly, cumulus-oocyte complexes (COCs) were obtained by slicing 2 to 10 mm follicles on the surface of ovaries (a mixture of beef and dairy cattle) obtained from a local abattoir. Cumulus-oocyte complexes containing at least one layer of compact cumu lus cells were selected for maturation and fertilization. They were washed twice in OC M and placed in groups of 10 in 50 L drops of OMM overlaid with mineral oil and matured for 22 h at 38.5 C, 5% CO2 in humidified air. Matured oocytes were then washed once in HEPE S-TALP and transferred in groups of 30 to 4well-plates containing 600 L IVF-TALP per well and 25 L PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 M epinephrine in 0.9% [w /v] NaCl) per well and fertilized with 25 L (5 x 105) Percoll-purified spermatozoa from a single Ho lstein bull. After 8 h of co-incubation at 38.5C, 5% CO2 in humidified air, putative zygotes were removed from fertilization wells and denuded of cumulus cells by vortexing in 100 L hyaluronidase (1000 U/mL in approximately 0.5 mL HEPES-TALP). Denuded putative zygotes were cultured in groups of 25 to 30 in 50-L drops of KSOM-BE2 overlaid with mineral oil at 38.5oC in a humidified atmosphere of 5% CO2, 5% O2 and the balance nitrogen. Fertilization was assessed at 18 h post-insemination (hpi), cleavage was assessed on Day 3 after insemination and presence of blastocysts was determined on Day 7 and/or Day 8 after insemination.

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40 Determination of Fertilization Insem inated oocytes were transferred at 18 hpi from KSOM-BE2, washed in 10 mM KPO4 (pH 7.4) containing 0.9% (w/v) NaCl (PBS) and 1 mg/mL polyvinylpyr ollidone (PVP) (PBSPVP) and transferred onto poly-L-lysine coated s lides. Slides were allowed to air dry and fixed overnight in 100% ethanol and then stained with Hoechst 33342 (1 g/mL in PBS-PVP) for 10 min in the dark at room temperature. Slides were washed three to four times with PBS-PVP, and cover slips mounted using 5 L mounting medium containing ProLong Gold antifade reagent (Invitrogen). Pronuclei of inseminated oocytes were identified by fl uorescence using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Inc., Gttingen, Germany) with an ultraviolet excitation filter. Oocytes were classified in four groups: X, unknown, unable to assess presence of pronuclei; M, unfertilized oocyte in Metaphase II with one polar body vi sible; 1PN, presence of a single decondensed pronucleus; 2PN, presence of two pronuclei, indica tive of fertilization; and PPN; presence of 3 or more pronuclei. Oocyte s with two pronuclei (2PN) were considered as fertilized and those with more than 2PN we re considered as fertilized but polyspermic. Embryo Sex Determination Blastocys ts were removed from culture drops, washed in PBS-PVP and transferred into a solution of 0.1% (wt/vol) protease from Streptomyces griseus (Sigma) in PBS for 1.5 min Embryos were then washed 3 times in 150 l PBS-PVP, collected individually in 10 l drops of 0.1% (wt/vol) diethylpyrocarbonate in water, transf erred into 0.2 mL PCR tubes and stored at 20C until analysis. To prepare samples for PCR, tubes were thawed at room temperature and centrifuged at 2000 x g for 5 s, heated to 98 C for 10 min and centrifuged at 2000 x g for 5 s prior to addition of PCR reagents. Two sets of PCR primers were used to de termine embryo sex: Y-chromosome specific primers that amplify a 141 bp pr oduct and auotosomal bovine-specifi c satellite sequence primers

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41 that amplify a 216 bp product (Park et al., 2001). The amplification reacti ons were conducted in a total volume of 20 l. The first amplification consiste d of 10x PCR buffer, 2.5 mM dNTPs, 50 mM MgCl2, 1 units of Taq DNA polymerase and 10 M Y-specific forward primer (5GATCACTATACATACACCACT-3) and 10 M Y-specific reverse primer (5GCTATGCTAACACAAATTCTG-3). The first PCR was programmed for an initial denaturation at 95C for 7 min followed by 10 cycl es of 95C for 30 s, 55C for 30 s and 72C for 30 s; after the 10 cycles the r eaction mixtures were kept at 72C for 7 min. Tubes were centrifuged at 2000 x g for 5 s prior to addition of the second PCR mix for autosomal primers containing 10 M forward (5-TGGAAGCAAAGAACCCCGCT-3) and 10 M reverse primers (5-TCGTCAGAAACCGCACACTG-3). The s econd PCR was programmed for initial denaturation at 95C for 7 min, 30 cycles of 95 C for 30 s, 55C for 30 s and 72C for 30 s, and a final step at 72C for 7 min. P CR amplification products were separated by electrophoresis on 3% (w/v) agarose gels in a 1 x TBE buffer ( 89 mM Tris, 88.9 mM boric acid, 2.2 mM EDTA, pH 8.3) containing 10 g/mL ethidium bromide. Experiments The f irst experiment tested the effects of aging on sperm motility. Sperm motility was assessed for non-incubated sperm immediately fo llowing Percoll purification and for sperm at the end of the incubation period at 38.5 C or 40 C in air for 4 h using a water bath. The experiment was replicated a total of 21 times with sperm from 17 bulls. The second experiment tested effects of ag ing on cleavage rate and development to the blastocyst stage when oocytes were fertilized fo r 8 h. Oocytes were fertilized with unincubated control sperm, sperm aged at 38.5C or sperm aged at 40C. Another gro up of oocytes remained unfertilized (i.e., incubation of oocytes in fertilization medium without sperm) to determine

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42 parthenogenesis. After fertilizat ion for 8 h, oocytes (fertilized i.e., putative zygotes, and unfertilized, possible parthenotes) were pl aced in groups of 25 to 30 in 50 l microdrops of KSOM-BE2 at 38.5C in a humid ified atmosphere of 5% CO2, 5% O2 and the balance nitrogen. Cleavage was accessed on Day 3 after insemination and blastocyst development on Day 8 after insemination. The experiment was replicated a to tal of 11 times with a different bull for each replicate and with a total of 226 to 853 oocytes/g roup. For 10 of these replicates, blastocysts were harvested at Day 7 and again at Day 8 for determination of embryo sex. A third experiment was designed to determine the effect of aging on fertilization. Oocytes were fertilized with unincubated control sperm, sperm aged at 38.5C or sperm aged at 40C. After fertilization for 8 h, oocytes (i.e., putativ e zygotes) were placed in groups of 25 to 30 in drops of KSOM-BE2 medium until processing for fertilization determination at 18 hpi at 38.5oC in a humidified atmosphere of 5% CO2, 5% O2 and the balance nitrogen (vol/vol). The experiment was replicated three times using a different bull for each replicat e and with a total of 59 to 72 inseminated oocytes/group COCs. Statistical Analyses For each rep licate, percent sperm that were motil e, percent of oocytes that were fertilized, cleaved or developed to the blastocyst stag e, percent of cleaved embryos that became blastocysts, and percent of blastocysts that were male were calculated fo r all oocytes or embryos within the same treatment. Thus, the group of embryos treated alike within each replicate was the experimental unit. Data were subjected to le ast squares analysis of variance using the GLM procedure of the Statistical An alysis System (SAS for Windows Release 9.0, SAS Institute, Inc., Cary, NC). Data were analyzed without transfor mation and again after arcsin transformation to correct for any non-normality associated with per centage data. The mathematical model included

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43 effects of bull, sperm treatment and treatment x bull (i.e., error). Data are reported as leastsquares means SEM from the analysis of the untransformed data while probability values are derived from analyses of transf ormed data. The CONTRAST stat ement of SAS was utilized to compare individual treatments. Treatment effects on sex ratio were determined by logistic regression using the logistic procedure of SAS. Two comparisons were made : between control sperm and sperm aged at 38.5C and between control sper m and sperm aged at 40C. Results Sperm Motility As compared to nonincubated control sperm (79.3 1.8%), a smaller percent of sperm exhibited motility after aging for 4 h at either 38.5C or 40C (P<0.001). Moreover, motility was lower (P<0.01) for sperm aged at 40C than for sperm aged at 38.5 C (38.3 1.8% vs 46.6 1.8%). Cleavage and Development to the Blastocyst Stage As com pared to oocytes inseminated with c ontrol sperm, cleavage rate was lower for oocytes inseminated with sperm aged at 40 C (P<0.05) and tended to be lower (P=0.08) for oocytes inseminated with sperm aged at 38.5 C (Table 2-1). However, there was no significant difference in cleavage rates between oocytes produced from sperm pre-incubated at 38.5 C vs 40C. There was no effect of agi ng at either temperature on the percent of oocytes that became blastocysts or on the percent of cleaved embr yos that became blastocysts (Table 2-1). Fertilization The effects of aging on the proportion of ooc ytes fertilized af ter 8 h was evaluated by counting the number of pronuclei at 18 hpi (Table 2-2). Overall fer tilization rate, as determined

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44 by the proportion of embryos with at least two pronuclei, was not affected by aging of sperm at 38.5C or at 40 C. Similarly, the percent of oocytes that were fertilized with a single spermatozoon (i.e., those with two pronuclei) was si milar for all three groups and not affected by treatment. The percent of oocytes undergoing po lyspermy (i.e., those with more than two pronuclei) tended (P=0.08) to be lower for oocytes fertili zed with sperm aged at 40 C as compared to the controls. Aging at 38.5C did not affect ra te of polyspermy. Sex of Blastocysts A total of 375 blastocysts were produced and 367 of these we re successfully sex ed. PCR reactions in which there were two amplicons (for Y-specific primers and for autosomal primers) were classified as males while those exhibiting an autosomal am plicon only were classified as female (Figure 2-1). The effect of sperm treatme nt on the proportion of bl astocysts at Day 7 and 8 that were male is presented in Table 2-3. For embryos produced from oocytes inseminated with non-incubated sperm, there was a prepondera nce of male blastocysts at both Day 7 (57.5%) and Day 8 (59.6%) after insemination. The percent of blastocysts that were male was reduced for embryos produced with sperm aged at 38.5 C (P=0.08) but not for embryos produced with sperm aged at 40 C. Discussion Aging of sperm after freeze-thawing reduced motility but had no effect on the fertilizing ability of bovine spermatozoa, a slight effect only on the pro portion of oocytes cleaving after insemination, and no effect on the competence of the resultant embryo to develop to the blastocyst stage. Lack of effect of aging on embryo competence was true even when spermatozoa were incubated at a temperature of 40 C that is characteristic of heat-stressed cows.

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45 Aging did affect sperm function, however, sin ce sperm motility and cleavage rate was reduced and the blastocyst sex ratio tended to be altered by aging of sperm at 38.5 C. Sperm survival after ejaculat ion is transient unless sperm are maintained with oviductal cells (Pollard et al., 1991), cryopreserved or suspended in diluents designed to prolong viability while stored cold. Bull sperm incubated for 24 h at 39 C had reduced competence for fertilizing oocytes as determined by subsequent cleavage (Po llard et al., 1991; Lechni ak et al., 2003). Even short-term aging can compromise sperm func tion. In this study, motility of bull sperm was decreased by 4 h incubation at 38.5C and slightly more so by incubation at 40C. Previous work indicates bull sperm motility was decreased by as little as 3 h incubation at 39C (Monterroso et al., 1995). The reduction in cleavage rate when oo cytes were fertilized with sperm aged at 38.5C or 40 C in the current study could reflect d ecreased sperm motility and fertilizing capacity. However, examination of pronuclear fo rmation after fertilizati on failed to indicate a decline in fertilization rate in oocytes inseminated with aged sperm. It may be, therefore, that the reduction in cleavage rate in oocyt es inseminated with aged sperm reflects a delay in fertilization and aging of the oocyte (Agung et al., 2006). It is also possible that aging damaged the sperm centriole so that syngamy was compromised. The lack of effects of aging on the proportion of inseminated oocytes or cleaved embryos that became blastocysts agrees with other studies finding no effect of aging sperm for 3 to 6 h at 39C on cleavage rate or on the proportion of oo cytes becoming blastocysts (Kochhar et al., 2003; Lechniak et al., 2003). Em bryos produced by sperm aged for 24 h did exhibit reduced competence for development however (Lechniak et al., 2003). Aging in vivo is likely to result in less steep decline in sperm function than seen here because oviducta l epithelial cells can maintain fertilizing capacity of bull sperma tozoa for up to 30 h (Pollard et al. 1991).

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46 Lactating dairy cows exposed to heat stress of ten have rectal temperatures that reach or exceed 40C (Elvinger et al., 1992; de Castro e Paula et al., 2008). There is the potential, therefore, for sperm in the reprodu ctive tract to be damaged by exposure to elevated temperature. Most of the data presented here are not supportive of such a hypothesis. As compared to aging at 38.5C, there was no effect of 40C on the fe rtilizing capacity of sperm as measured by pronuclear formation or on cleavage rate of oocytes at Day 3 afte r insemination. As compared to sperm at 38.5C, aging at 40C did not reduce the proportion of oocytes and cleaved embryos becoming blastocysts. Aging of sperm at 40C also reduced the rate of polyspermy. This effect might reflect a reduction in motility or ability of sperm to attach to and penetrate the zona pellucida of the oocyte. Developmental competence in the present study wa s evaluated to the blastocyst stage. One cannot rule out effects of sperm aging on embryo competence for development to later stages of embryogenesis. Studies in the rabbit using sperm exposed to heat shock in vitro (Burfening and Ulberg, 1968) or in vivo (Howarth et al., 1965) indicate incr eased embryonic loss at Day 9 or 12 after insemination. One effect of aging sperm was on blastocyst sex ratio. In the absence of sperm aging, the sex ratio of blastocysts was skewed to males and aging at 38.5C resulted in a sex ratio close to an equal number of males and female embryos. A similar effect of sperm aging on the sex ratio of the resultant blastocysts has been seen else where (Kochhar et al., 2003 ; Lechniak et al., 2003; Iwata et al., 2008). A preponderance of male blastocysts is a characteristic of the in vitro embryo production system in our laboratory (Block et al., 2003; Block and Hanse n, 2007; Franco et al., 2006) and other laboratories (Lechniak et al., 2003; Agung et al., 2006; Koc hhar et al., 2003; Iwata et al.,

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47 2008, King et al., 1991, Hasler et al., 2002). The reason for this bias in sex gender is not clearly understood. It has been reported that male embr yos develop faster in KSOM than female embryos (Nedambale et al., 2004), making it more likely that the first emerging blastocysts would be male. However, the increased proporti on of males for embryos produced with control sperm in the present study was seen for embryos becoming blastocysts by Day 7 and between Day 7 and 8. Kimura et al. (2005) demonstrated that glucose in excess of 1 mM is toxic to female bovine embryos but the concentrations of glucose in embryo culture medium in the present experiment (0.2 mM) was too low to be t oxic (Soto et al., 2003). It seems mostly likely that the gender bias is due to differential fertilizing ability of Y-bearing vs X-bearing spermatozoa. In support of this are the findings that the sex bias occu rs as early as the 4 to 8 cell stage (Kochhar et al., 2003) and that lengthening fertilization tim e beyond 5 to 6 h eliminated the male bias in the sex ratio of embryos (Kochhar et al., 2003; Iwata et al ., 2008). Iwata et al. (2008) speculates that the more rapid fertiliza tion achieved with Y-bearing sperm reflects earlier capacitation for Y-bearing sperm. Thus, it is li kely that the reduction in the proportion of blastocysts that were male caused by aging of sperm at 38.5C reflects differential effects of aging on fertilizing ability of Ybearing and X-bearing sperm. Energy store depletion, free radical damage, membrane changes or other aging-associated changes (Vishwanath and Shannon, 1997; Krzyzosiak et al., 2000, 2001) could occur more rapidly for Y-bearing sperm, particularly if they are more activ e because of earlier capacitation. The reduction in male bias in sex ratio cau sed by aging at 38.5C wa s not significant when sperm were aged at 40C. Possibly, aging at 38.5 C affects Y-bearing sperm preferentially while aging at a higher temperature results in agingassociated changes in both Yand X-bearing sperm.

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48 The observation that aging of sperm can lead to changes in sex ratio of the resultant embryo points out the potential fo r changes in sperm function to effect the embryo formed by fertilization with that sperm. Nonetheless, de spite nuclear and non-nuclear contributions of the sperm to the embryo (Sutovsky and Schatten, 2000) there was no evidence that the competence of the embryo to develop to the blastocyst stage was determined by aging at temperatures characteristic of normothermia or hyperthermia Thus, at least under the conditions tested, damage to the sperm is more likely to lead to a reduction in fertilizing abi lity than to the cellular characteristics of the resultant embryo that determine its developmental potential.

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49 Table 2-1. Effect of sperm aging on cleavage rate and blastocyst development following insemination of matured oocytes with sperm for 8 h Treatment n a Cleavage (%) Blastocysts/oocyte (%) Blastocyst/cleaved embryo (%) Control 367 65.7 4.7 30.4 3.0 46.6 3.6 38.5C 845 56.6 2.7 28.9 1.7 50.9 2.1 40C 853 52.2 2.7* 25.1 1.7 46.9 2.1 Parthenogenesis 226 3.5 2.7*** -0.3 1.7*** -0.3 2.1*** Data are least-squares means + SEM of the percent cleaved or percent blastocysts for each of 11 replicates using a separate bull fo r each replicate. Means that di ffer from controls are indicated by superscripts: P=0.08; P<0.05; *** P<0.001. There were no differences between 38.5 and 40C. a n: total number of embryos evaluated per treatment.

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50 Table 2-2. Fertilization rate at 18 h post-insemin ation as affected by aging of sperm at 38.5 or 40C for 4 h Percent Fertilized Percent Polyspermy Treatment n a 2PN 2PN >2PN Control 64 79.8 8.8 63.0 8.6 16.8 3.5 38.5C 72 74.0 8.8 60.1 8.6 13.9 3.5 40C 59 66.3 8.8 61.3 8.6 5.0 3.5 Data are least-squares means + SEM of the percent fertilized or percent polyspermy for each of three replicates using a separa te bull for each replicate. Ther e were no differences between 38.5 and 40C. a n: total number of oocytes evaluated per treatment; 2PN : embryos having 2 or more pronuclei; 2PN : embryos having 2 pronuclei. Differs from control (P=0.08).

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51 Table 2-3. Sex of blastocysts at Day 7 and Day 8 after insemination as affected by sperm aging at 38.5 or 40C for 4 h Blastocysts at Day 7 Blastocysts at Day 8b Day 7 and 8 combined Sperm treatment n M % Male N M % Male n M % Male Control 78 45 57.7 47 28 59.6 125 73 58.4 38.5C 77 35 45.5 48 23 47.9 125 58 46.4 40C 80 42 52.5 37 19 51.4 117 61 52.1 n: total number of blastocysts ev aluated per treatment; M: total number of embryos that were males. Blastocysts at Day 8 represent embryos that were not blastocy sts at Day 7 but which became blastocysts by Day 8. Differs from control (P=0.08).

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52 216 bpauto 141 bpsexEmbryos F M M F BDNA Ladder 216 bpauto 141 bpsexEmbryos F M M F BDNA Ladder Figure 2-1. Representative results fo r analysis of embryo sex by PCR. ( )=female bovine DNA isolated from whole blood. ()=male bovine DNA isolated from whole blood. B= blank-PCR reaction mixture without embryo. Amplificons for the Y-specific primer (141 bp) and autosomal primer (216 bp) are indicated by arrows. Note that embryos that produced both the 216 and 141 bp amplicons were classified as male (M), while those with only the 216 bp product we re classified as female (F).

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53 CHAPTER 3 CONSEQUENCES FOR THE BOVINE EM B RYO OF BEING DERIVED FROM A SPERMATOZOAN SUBJECTED TO X-IRRADI ATION CHARACTERISTIC OF AIRPORT SCREENING DEVICES Introduction X-irradiation has been shown to cause DNA strand breaks and chromosomal aberrations in somatic cells (Dobrzy ska, 2007; Liang et al., 2007), germ cell s (Griffin et al., 1990; Haines et al., 2002; Cordelli et al., 2003), ejaculated sperm (Matsuda et al., 1985; Kamiguchi et al., 1990) and embryos (Molls and Streffer, 1984; Streffer et al., 1993). DNA damage in the male gamete has been attributed to low fertilization rates (Sakkas et al., 1996; Hst et al., 2000ab; Waterhouse et al., 2006), reduced embryo deve lopment (Seli et al., 2004; Fatehi et al., 2006) and increased embryo and fetal loss (Virro et la., 2004; Paul et al., 2008). Sperm DNA damage has also been associated with an increased incidence of cancer in offspring produced from such damaged sperm (Lord, 1999; Lewis and Aitken, 2005). Worldwide and inter-continental transportation of biological material such as sperm, ova and embryos has become more common with the advent in artificial reproductive technologies. Transportation of these biological materials may re quire that they be exposed to X-irradiation due to airport security at ports of entry and exit. Currently, it is not known whether the levels of X-irradiation employed at airpor t security checkpoints adversely affect sperm or the embryos produced from sperm. It was hypot hesized that exposure of fro zen bovine sperm to x-rays generated by airport security x-ray machines for a) checked luggage and b) carry-on luggage would induce changes in sperm DNA and cause a reduction in competence of the resultant embryos to develop to the blastocyst stage.

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54 Materials and Methods Materials The m edia HEPES-Tyrodes Lactate (HEPES -TL) and IVF-TL were purchased from Caisson (Sugar City, ID, USA) and used to prepare HEPES-Tyrodes al bumin lactate pyruvate (TALP) and IVF-TALP as previously described (P arrish et al., 1986). Oocyte collection medium (OCM) was Tissue Culture Medium-199 (TCM199) with Hanks salts without phenol red (Atlanta Biologicals, Norcross, GA, USA) supplemented with 2% (v /v) bovine steer serum (PelFreez, Rogers, AR) containing 2 U/mL he parin, 100 U/mL penicillin-G, 0.1 mg/mL streptomycin, and 1 mM glutamine. Oocyte maturation medium (OMM) was Tissue Culture Medium 199 (Gibco, Invitrogen, Carlsbad, CA, USA) w ith Earles salts supplemented with 10% (v/v) bovine steer serum, 2 g/mL estradiol 17, 20 g/mL bovine FSH (Folltropin-V; Bioniche Animal Health, London, ON, Canada), 22 g/mL sodium pyruvate, 50 g/mL gentamicin sulfate, and 1 mM glutamine. Perc oll was from GE Healthca re (Uppsala, Sweden). Potassium simplex optimized medium (KSOM) containing 1 mg/mL BS A was obtained from Caisson. Essentially fatty-acid fr ee (EFAF) BSA was from Sigma (St. Louis, MO). On the day of use, KSOM was modified for bovine embryos to produce KSOM-BE2 as described elsewhere (Soto et al., 2003). Extended and frozen semen from nine bulls was obtained from Genex Cooperative, Ithaca, NY and exposed to x-irradia tion under the supervision of Dr. Linda Penfold, White Oak Conservation Center, Yulee, FL. Sperm Treatment and Preparation X-irradiated bovine sem en straws were donated by Dr. Linda Penfold, White Oak Conservation Center, Yulee, FL. Straws of se men extened with a milk-based extender from known fertile bulls (n= 9) were stored in a dry shipper at -196C and processed through airport security x-ray machines. Straws were x-rayed 0, 1, 2, or 3 times using a checked luggage x-ray

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55 machine or a carry-on luggage x-ray machine. The zero group was passed through the x-ray machine without exposure to X-irradiation. In Vitro Production of Embryos Em bryo production was performed as previously described (Soto et al., 2003) except that oocytes in a single replicate were inseminated with semen from a single bull. Briefly, cumulusoocyte complexes (COCs) were obtained by slicing 2 to 10 mm follicles on the surface of ovaries (a mixture of beef and dairy cattle) obtained fr om a local abattoir. COCs containing at least one layer of compact cumulus cells were selected for maturation and fertilization. They were washed twice in OCM and placed in groups of 10 in 50 L drops of OMM overlaid with mineral oil and matured for 20 to 22 h at 38.5C, 5% CO2 in humidified air. Matured oocytes where then washed once in HEPES-TALP and transferred in groups of 30 or 50 to 4-well-plates containing 425 L IVF-TALP per well and 20 L PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 M epinephrine in 0.9% [w/v] NaCl) per well and fertilized with 30 L (5 x 105) Percollpurified spermatozoa from a single bull. Extern al controls were used to monitor the IVF procedure and consisted of a pool of three bulls (a different pool of bulls was used for each replicate). Sperm used for the external control wa s extended in an egg yo lk based extender prior to cryopreservation. After 8 to 9 h of co-incubation at 38.5C, 5% CO2 in humidified air, putative zygotes were removed from fertilization wells and denuded of cumulus cells by vortexing in 100 L hyaluronida se (1000 U/mL in approximately 0.5 mL HEPES-TALP). Denuded putative zygotes were cultured in grou ps of 25 to 30 in 50-L drops of KSOM-BE2 overlaid with mineral oil at 38.5C in a humidified atmosphere of 5% CO2, 5% O2 and the balance nitrogen. Cleavage was assessed on Day 3 af ter insemination and presence of blastocysts was determined on Day 7 and/or Day 8 after insemination. The experiment for sperm exposed

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56 to checked luggage x-ray machine was replicated a total of 9 time s with 9 bulls and with a total of 652 to 821 oocytes/group. The experiment fo r sperm exposed to carry-on luggage x-ray machine was replicated a total of 5 times with 5 bulls and with a total of 421 to 510 oocytes/group. Statistical Analyses For each rep licate, percent of presumptive z ygotes that cleaved, percent of embryos that developed to the blastocyst stag e and percent of cleaved embryos that became blastocysts were calculated for all embryos within the same treatment. Thus, the group of embryos treated alike within each replicate was the expe rimental unit. Data were subjected to least squares analysis of variance using the GLM procedure of the Stat istical Analysis System (SAS for Windows, Release 9.0, SAS Institute, Inc., Cary, NC). Percentage data were analyzed without transformation and again after ar csin transformation to correct for any non-normality associated with percentage data. All main effects and intera ctions were included in the mathematical models for ANOVA. Replicate was considered random for analysis of the developmental parameters (cleavage rate, blastocyst/total and blastocyst/cle aved) and other main effects were considered fixed. Hence, treatment replicate was the erro r term for treatment. Th e CONTRAST statement of SAS was utilized to compare each irradia tion dose to the zero dose and to compare all treatments (0, 1, 2 and 3 exposures) to the external control. Results Results for carry-on luggage doses are shown in Table 3-1 an d results for checked luggage doses are shown in Table 3-2. As compared to the 0 dose, there was no effect of 1, 2 or 3 exposures to the carry-on luggage dose on the proportion of oocytes that cleave after fertilization or on blastocyst development rates (expressed as percent of oocytes becoming blastocysts or percent of cleaved embryos becoming blastocysts) For the checked luggage dose, exposure of

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57 sperm 1 or 2 times did not affect cleavage or bl astocyst development. There was, however, a tendence towards a reduction in the proportion of oocytes that cl eaved (P=0.07) and the percent of oocytes becoming blastocysts (P=0.06) when sperm received 3 doses of X-irradiation using checked x-ray machine. The percent of cleaved embryos becoming blastocysts (a measure of embryo competence for development) was not si gnificantly affected by any checked luggage exposure. Overall, cleavage rates (P<0.001) and the per cent of oocytes becoming blastocysts (P<0.001) were low compared to the external control. The percent of oocytes that cleaved for the external control was 77.7 4.7% for the carry-on luggage experiment and 82.7 3.7% for the checked luggage experiment. The percent of oocytes that became blastocysts was 37.7 3.5% and 32.6 2.0% for the carry-on and checked luggage experi ments, respectively. The proportion of cleaved embryos that developed to the blastocyst stage was similar for the external control and treated sperm (P>0.10). The percent of cleaved embryos that became blastocysts was 48.4 6.5% and 37.6 6.7% for the carry-on and checked luggage experiments, respectively. Discussion Except at the highest cumulative dose (exposure 3 tim es to checked luggage X-irradiation), exposure of frozen semen straws in dry shippers to airport security x-ray machines did not induce changes in sperm that were reflected as a reduction in oocyte cl eavage rate or embryo competence to develop to the blastocyst stag e. Exposure to 3 doses of checked luggage irradiation induced changes in sperm that te nded to cause a reduction in cleavage rate and resulted in reduced blastocyst development. Ev en at this high cumulative dose, however, embryo competence for development as measured by th e proportion of cleaved embryos that became blastocysts was not different from results for cont rol sperm exposed to the 0 dose or for external control sperm. Taken together, re sults indicate that fertilizing ab ility of sperm could be damaged

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58 by repeated exposure to checked luggage doses of irradiation employed in airport security x-ray machines. There is no evidence to suggest that competence of the resultant embryos to develop to the blastocyst stage is reduced by exposure to X-irradiation doses char acteristic of airport aray machines. The tendency for the reduction in cleavage rate for oocytes inseminated with sperm exposed three-times to checked luggage X-irradia tion is most likely a reflection of a reduction in sperm fertilizing capacity. One cannot rule out, howe ver, that fertilizing ability was not affected by irradiation but that some zygotes formed from X-irradiated sper m had defects, such as in the spindle apparatus, that precluded completion of the first cleavage division. The fact that embryos formed from X-irradiated sperm did not have a reduced capacity for development to the blastocyst stage is consistent with an earlier study (Fahet i et al., 2006) where there was no adverse effect of exposure of bovine sperm to a single low dose of Xirradiation (0.6 Gy). In that study, exposure to higher doses (1.25, 2.5 and 5.0 Gy) reduced the proportion of oocytes that cleaved after insemination and the proportion of cleaved embryos that became blastocysts (Fatehi et al., 2006). The resiliency of ejaculated sperm DNA to low dose irradiation can be attributed to sperm chromatin structure and compos ition. Unlike somatic cells and sp ermatogonia, the majority of the histones in spermatozoa have been replaced by protamines (Poccia 1986) and the DNA is organized into toroids (Ward and Coffey 1991; Ward 1993). In addition, protamines are crosslinked by disulphide bonds and together these changes cause sperm DNA to become compacted into one-sixth the volume of a somatic cell nuc leus (Ward and Coffey 1991). The resilience of sperm DNA is reflected by the high levels of irradiation re quired to damage sperm DNA compared with somatic cells. McKelvey-Martin et al. (1997) exposed human ejaculated sperm to

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59 increasing doses of X-irradi ation (5, 10 and 30 Gy) and found that DNA degradation as measured by the alkaline comet assay occurred on ly when sperm were exposed to the highest dose of radiation. In contrast, exposure of mice to 0.1 Gy whole-body X-irradiation caused DNA damage in peripheral blood cells as determin ed by the comet assay (Giovanetti et al., 2008). It is possible that radiation damage su stained by sperm DNA wa s repaired following fertilization by the oocyte DNA repair machinery since the oocyte cont ains the requisite DNA repair machinery and is capable of initiating and carrying out repair of damaged paternal DNA prior to syngamy (Brandriff and Pederse n, 1981; Ashwood-Smith and Edwards, 1996). The DNA damage caused to spermiogenic cells by X-irra diation that persist in the spermatozoon can be repaired by the oocyte in the mouse (Matsuda et al., 1989). The sperm used for the X-irradiation expe riments resulted in significantly reduced proportions of oocytes that cleaved and that became blastocysts wh en compared to sperm used as external controls. This may be a reflection of the extender used the experimental sperm were in a milk extender while the external control sperm were in an egg yolk extender. Lonergan et al. (1994) demonstrated that performing IVF with bovi ne sperm extended in a milk-based extender results in lower cleavage rates than when IVF is performed with sperm in an egg-yolk-based extender. Despite the possibility that X-ray induced DNA damage may have been limited or repaired, one must take into consideration the pot ential for epigenetic and genetic changes to the original DNA in the embryo formed from X-i rradiated sperm. These changes may not be exhibited as a reduction in embryo competence to the blastocyst stage but as a reduced implantation rate, or increased rates of mid and late gestational abnormalities, death or increased

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60 susceptibility to disease in the offspring (Brinkworth, 2000; Anderson, 2005; Dobrzyska and Czajka, 2005; Cordier, 2008).

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61 Table 3-1. Effect of sperm irradiation with a ca rry-on X-ray machine on cl eavage rate, blastocyst development and embryo competence (blastoc yst/cleaved) following insemination of matured oocytes Treatment Cleavage (%) Blastocysts/ooc ytes (%) Blastocysts/cleaved (%) 0 exposure 24.5 4.7 13.0 3.5 40.3 6.5 1X exposure 31.3 4.7 12.8 3.5 34.0 6.5 2X exposure 31.1 4.7 14.0 3.5 34.4 6.5 3X exposure 30.6 4.7 13.5 3.5 34.7 6.5 Data are least-squares means SEM for five repli cates involving five bulls The total number of oocytes inseminated varied from 412 to 510 pe r treatment. There was no difference between 0 exposure and all other doses (P>0.10).

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62 Table 3-2. Effect of sperm irradiation with a checked luggage x-ray machine on cleavage rate, blastocyst development and embryo competence (blastocyst/cleaved) following insemination of matured oocytes Treatment Cleavage (%) Blastocysts/ oocytes (%) Blastocysts/cleaved (%) 0 exposure 29.4 3.1 13.8 1.7 45.4 5.7 1X exposure 24.9 3.1 11.5 1.7 40.4 5.7 2X exposure 25.7 3.3 12.6 1.9 46.4 6.1 3X exposure 21.6 3.1a 9.0 1.7 b 41.8 5.7 Data are least-squares means SEM for nine rep licates involving nine bulls The total number of oocytes inseminated varied fr om 652 to 821 per treatment. a Different from 0 at P=0.07. b Different from 0 at P=0.06.

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63 CHAPTER 4 CONSEQUENCES FOR THE BOVINE EM B RYO OF BEING DERIVED FROM A SPERMATOZOAN SUBJECTED TO POST -EJACULATORY STRESS: OXIDATVE STRESS Introduction Developm ental competence of the mammalia n embryo is dependent on genetic and nongenetic contributions from its parents (Warner et al., 1998; Sirard et al., 2006; Mnzo, 2006; Baumann et al., 2007; Khatib et al., 2008ab). Individual males having similar in vitro cleavage rates can have different abilities to produce em bryos competent for continued development (Shi et al., 1990; Saacke et al., 1994; Tesarik et al., 2002; Morris et al., 2003). The sperm contribution to the embryo includes the paternal DNA, plas ma membrane, centriole (Sutovsky and Schatten, 2000) and RNAs (Ostermeier et al., 2004). Damage to these components could theoretically compromise the developing embryo. Sperm DNA dama ge has been associated with failure of spermatozoal pronuclear decondensation, reduc tion in embryo developmental potential and reduced implantation rates (Tomsu et al 2002; Seli et al., 2004). The degree to which damage to sperm af ter ejaculation actually reduces embryo competence is not clear. In the previous chapters (Chapter 2 and 3) aging, heat shock and Xirradiation from airport x-ray machines did not damage post-ejaculated sperm in a manner that reduced embryo competence to develop to the blastocyst stage. There is some evidence, however, that molecules that increase sperm oxidative stress can reduce embryo competence. The proportion of cleaved bovine embryos that deve loped to the blastocyst stage was reduced for embryos treated with the pro-oxidants: goss ypol (Brocas et al., 1997) and hydrogen peroxide (Silva et al., 2007). Spermatozoa are extremely sensitive to oxidative damage owing to their high polyunsaturated fatty acid content (Poulos et al., 1986; Alvarez and St orey, 1995) and exposure

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64 to pro-oxidants have been shown to increase oxidation of molecular components of the sperms plasma membrane, mitochondria, cytosol and DNA (Silva et al., 2007; Peris et al., 2007). For the current study, we used bull spermatozoa to te st the hypothesis that oxidative stress of ejaculated sperm after freeze-thawing would dama ge sperm in a manner that leads to the formation of embryos with reduced developmen tal competence. Two pro-oxidant chemicals were examined menadione and tert -butyl hydroperoxide. Me nadione is a vitamin K2 precursor that generates superoxide anion radical (O 2), hydrogen peroxide (H2O2) and other reactive oxygen species in its conversion from a quinone to a semiquinone and back to a quinone in the presence of molecular oxygen (Monks et al., 1992). Menadione reduces sperm motility, depresses mitochondrial transm embrane potential and reduces AT P content (Guthrie et al., 2008). Finally, tert-butyl hydroperoxide is an organic pe roxide, which has been reported to decrease sperm count, sperm motility and reduce litte r size following intra-pe ritoneal injection in male mice (Kaur et al., 2006) and mid-piece lipi d peroxidation in fresh and frozen-thawed stallion sperm (Neild et al., 2005). Materials and Methods Materials The m edia HEPES-Tyrodes Lactate (HEPES-T L), IVF-TL, and Sperm-TL were purchased from Caisson (Sugar City, ID, USA) and used to prepare HEPES-Tyrodes albumin lactate pyruvate (TALP), IVF-TALP, and Sperm-TALP as previously described (Parrish et al., 1986). Oocyte collection medium (OCM) was Tissue Culture Medium-199 (TCM-199) with Hanks salts without phenol red (Atlanta Biologicals, No rcross, GA, USA) supplemented with 2% (v/v) bovine steer serum (Pel-Freez, R ogers, AR) containing 2 U/mL he parin, 100 U/mL penicillin-G, 0.1 mg/mL streptomycin, and 1 mM glutamine. Oocyte maturation medium (OMM) was Tissue

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65 Culture Medium 199 (Gibco, Invitrogen, Carlsbad, CA, USA) w ith Earles salts supplemented with 10% (v/v) bovine steer serum, 2 g/mL estradiol 17, 20 g/mL bovine FSH (FolltropinV; Bioniche Animal Health, London, ON, Canada), 22 g/mL sodium pyruvate, 50 g/mL gentamicin sulfate, and 1 mM glutamine. Perc oll was from GE Healthca re (Uppsala, Sweden). Potassium simplex optimized medium (KSOM) containing 1 mg/mL BS A was obtained from Caisson. Essentially fatty-acid fr ee (EFAF) BSA was from Sigma (St. Louis, MO). On the day of use, KSOM was modified for bovine embryos to produce KSOM-BE2 as described elsewhere (Soto et al., 2003). Menadione (2-methyl-1,4-naphthoquinine) was purchased from Sigma (St. Louis, MO) and dissolved in absolute etha nol to make a 10 mM stock solu tion. Fresh stock solutions of menadione were made every two weeks. Tert -butyl hydroperoxide solution (TBHP) was purchased from Sigma (St. Louis, MO) and dilu ted to 20 nM [0.1% (v/v) ethanol] where the effect of low concentrations of menadione and TBHP were assessed in the same experiment or 300 and 600 M [0 % (v/v) ethanol] in Sp-TALP on the day of use from the original solution. Hoechst 33342 was from Calbiochem (San Diego, CA, USA). All other reagents were purchased from Sigma or Fisher Scientific (P ittsburgh, PA, USA) unle ss otherwise stated. Sperm Preparation Extended and frozen sem en from bulls was obtai ned from Select Sires Inc. (Plain City, OH, USA), Southeast Semen (Wellborn, FL, US A) and ABS Global (Deforest, WI, USA). Semen was thawed, subjected to Percoll gradie nt purification to obtain motile spermatozoa (Parrish et al., 1986), diluted in Sperm-TALP to 40 x 106 spermatozoa/mL, and mixed 1:1 with Sp-TALP containing treatment chem icals prior to incubation at 38.5 C in air for 3 h using a water bath. Treatments were design ed so that the final concentra tion of ethanol was the same for

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66 all treatments (including the vehicle control). Depending on the experiment, the final concentration of ethanol varied from 0 to 0.3% At the end of the incubation period, treated sperm were washed and resuspend in Sp-TALP. In Vitro Production of Embryos Em bryo production was performed as previously described (Soto et al., 2003). Briefly, cumulus-oocyte complexes (COCs) were obtained by slicing 2 to 10 mm follicles on the surface of ovaries (a mixture of beef and dairy cattle) obtained from a lo cal abattoir. COCs containing at least one layer of compact cumulus cells were selected for maturation and fertilization. They were washed twice in OCM and placed in groups of 10 in 50 L drops of OMM overlaid with mineral oil and matured for 20 to 22 h at 38.5 C, 5% CO2 in humidified air. Matured oocytes where then washed once in HEPES-TALP and tran sferred in groups of 30 or 60 to 4-well-plates containing 425 L IVF-TALP per well and 20 L PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 M epinephrine in 0.9% [w/v] Na Cl) per well and fertilized with 30 L (6 x 105) Percoll-purified spermatozoa from a single bull. After 8 to 9 h of co-incubation at 38.5C, 5% CO2 in humidified air, putative zygotes were removed from fertilization wells and denuded of cumulus cells by vortexing in 100 L hyaluronidase (1000 U/mL in approximately 0.5 mL HEPES-TALP). Denuded putative zygotes were cultured in groups of 25 to 35 in 50-L drops of KSOM-BE2 overlaid with mineral oil at 38.5oC in a humidified atmosphere of 5% CO2, 5% O2 and the balance nitrogen. Cleavage wa s assessed on Day 3 after insemination and presence of blastocysts was determined on Day 7 and Day 8 after insemination. Experiments Effects of menadione and TBHP we re evaluated in three experim ents. In the first, oocytes were fertilized with sperm that had been incuba ted in vehicle [0.05% (v/v ) ethanol in Sp-TALP),

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67 sperm that had been incubated in Sp-TALP cont aining 5 M menadione or sperm that had been incubated in Sp-TALP containing 10 nM TBHP. The experiment was replicated seven times with a total of five bulls (i .e., two bulls were used twice) and with a to tal of 445 to 682 oocytes/group. Subsequently, experiments were conducted to evaluate effects of higher concentrations of menadione and TBHP. In one e xperiment, oocytes were fertilized with sperm that had been incubated in vehicle [0.3% (v/v) ethanol in Sp-TALP], or sperm that had been incubated with either 15 or 30 M menadione. Th e experiment was replic ated a total of four times with a separate bull for each replicate and with a total of 278 to 347 oocytes/group. In the other experiment, oocytes were fertilized with sp erm that had been incubated with vehicle (Sp-TALP) or sperm that had been incubated in Sp-TALP containing 150 or 300 M TBHP. The experiment was replicated a total of five times w ith a different bull for each replicate and with a total of 266 to 464 oocytes/group. Statistical Analyses For each rep licate, the percent of oocytes th at cleaved, percent of oocytes that became blastocysts and the percent of cleaved embryos that became blastocysts were calculated for all embryos within the same treatment. Thus, th e group of embryos treated alike within each replicate was the experimental unit. Data were s ubjected to least squares analysis of variance using the GLM procedure of the Statistical An alysis System (SAS for Windows, Release 9.0, SAS Institute, Inc., Cary, NC). Data were an alyzed without transfor mation and again after arcsin transformation to correct for any non-normality associated with percentage data. All main effects and interactions were included in the mathematical models. Replicate was considered random and other main effects were considered fi xed. Hence, treatment replicate was the error term for treatment. The CONTRAST statement of SAS was utilized to compare treatments (TBHP and menadione) against their respective vehi cle controls. Data reported for P values are

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68 from the analysis of transformed data while le ast-squares means are from the analysis of nontransformed data. Results Menadione In a prelim inary experiment, incubation of sperm with 5 M menadione had no effect on the cleavage rate or blastocyst development rate as compared to sperm incubated with vehicle (Table 4-1). Subsequently, highe r concentrations of menadione were evaluated (Table 4-2). Cleavage of oocytes following fertilization with sperm preincubated with either 15 or 30 M menadione was reduced (P<0.001) as compared to cl eavage of oocytes fertil ized with sperm with 0 M dose. At 15 M, there was no significant reduction in the proportion of cleaved embryos that became blastocysts although va lues were numerically lower than for oocytes inseminated with control sperm. At 30 M, the percent of cleaved embryos that became blastocyts was lower (P<0.01) than for controls; in fact, no cleaved embryo derived from sperm treated with 30 M menadione became a blastocyst. Tert-Butyl Hydroperoxide When tested at a concentration of 10 nM, tert -butyl hydroperoxide treatm ent of sperm had a positive effect on the percentage of oocytes that were blastocy sts at Day 7 and 8 (P<0.05) after insemination (Table 4-1). In addition, compet ence of embryos formed from treated sperm tended to be increased as determined by a gr eater proportion of cleaved embryos that became blastocysts at Day 8 after insemination (P=0.07). At higher concentrations, however, treatment of sperm with TBHP had deleterious effects on oocyte cleavage and development (Table 4-3). In particular, cleavage of oocytes following insemination with sperm preincuba ted with 300 M TBHP was redu ced (P<0.05) as compared to cleavage of oocytes fertilized with sperm preincubated with 0 M dose. Moreover, the

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69 proportion of oocytes that were bl astocysts at Day 7 or 8 after insemination was less for oocytes inseminated with sperm treated with 150 or 300 M TBHP (P<0.05 or less) than for oocytes inseminated with sperm incubated with 0 M dos e. The proportion of cleaved embryos that were blastocysts at Day 7 or 8 after inseminati on was also less for oocytes inseminated with sperm treated with 150 M (P<0.05 or less) or 300 M TBHP (P<0.01). Discussion Exposure of frozen-thaw ed ejaculated sperm to the pro-oxidants used in this study menadione and tert -butyl hydroperoxide reduced cleavage rate Such an effect, which is most likely due to damage to the fertilizing ability of sperm, is not surprising because similar effects have been seen for other molecules causing oxidative stress such as hypoxanthine-xanthine oxidase system (Blondin et al., 1997), hydrogen pe roxide (Hsu et al., 1999; Silva et al., 2007) and gossypol (Kennedy et al., 1983; Aitken et al., 1983). Menadione reduces sperm motility, mitochondrial membrane potential and AT P content (Guthrie et al., 2008) while tert-butyl hydroperoxide causes mid-piece lip id peroxidation in fresh and frozen-thawed stallion sperm (Neild et al., 2005). The present results extend previous observations found with hydrogen peroxide treatment of bull sperm (Silva et al., 20 07) that high concentrations of oxidative stress to ejaculated sperm also can cause the form ation of embryos with reduced competence for development to the blastocyst stage. Thus, damage to the sperm not only reduces sperm fertilizing ability but also causes changes in the resultan t embryo that reduces embryonic function. An unexpected result was the observation that low concentrations of tert -butyl hydroperoxide tended to improve embryo competence. This result implies that alterations in the sperm can also result in beneficial changes to the resultant embryo. There are several potential causes for reduced developmental competence of embryos derived from sperm exposed to oxidative stress Perhaps, oxidative stress caused damage to

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70 paternal DNA. There is some evidence to suppor t the idea of limited capacity for the bovine oocyte to repair damaged paternal DNA (Generoso et al., 1979; Brandr iff and Pedersen, 1981; Fatehi et al., 2006) and DNA damage introduced in to the embryo by sperm can lead to genomic instability affecting the matern ally derived genome in zygotes and in embryos (Niwa and Kominami, 2001) and may reduce embryo comp etence. X-irradiation induced sperm DNA damage does not cause arrest at the G1/S border in mouse zygote, but retards S-phase by 2 h (Shimura et al., 2002) and such a delay in cell cycle could conceivable disrupt embryonic development. Bovine zygotes that de monstrate a delayed cleavage following in vitro fertilization have a reduced competence for development to blastocyst stage (Lone rgan et al., 1999). Tert butyl hydroperoxide causes mid-piece lipid pero xidation in fresh and frozen-thawed stallion sperm (Neild et al., 2005) and it is possible that lipid peroxidation in this region of the sperm cell may result in damage to the centriole which is inherited by the zygote and is essential for pronuclear apposition and syngamy (Sutovsky et al., 1996). The role of paternal RNAs in early embryo development is not known. However oxidative damage to these may play some role in reducing embryo competence as oxidized base s in mRNAs cause ribosome to stall on the transcripts and leads to a decrease in protein expression (Shan et al., 2007). Another possibility is that da mage to sperm reduces fertilizing ability and could lead to aging of the oocyte and reduced developmenta l competence. That aging can compromise oocytes is indicated by the obser vation that inseminated bovine oocytes matured for 28 or 34 h are less likely to produce cleaved embryos that develop to the blas tocyst stage than inseminated oocytes aged for 22 h (Agung et al., 2006). Aged oocytes have been linked to asynchrony in pronuclear formation (Goud et al., 1999) and in alte rations in methylation patterns of maternally imprinted genes (Liang et al., 2008).

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71 It is possible that the beneficial effects of low concentrations of tert -butyl hydroperoxide on embryo competence also results from a change in oocyte aging before fe rtilization in this case, accelerated fertilization and reduced oocyte aging. A low level of reactive oxygen species can facilitate sperm functions directly involved in fertilization (Sengoku et al., 1998; Aitken et al., 1989; de Lamirande et al., 1997; Ford, 2004). Sengoku et al. (1998) demonstrated that low concentrations of nitric oxide enhance sperm capacitation and zona pellucida binding while de Lamirande et al. (1997) reviewed the enhancem ent in spermatozoa hyperactivation, capacitation and acrosomal reaction in response to low levels of superoxide anion, hydrogen peroxide and nitric oxide in human sperm. Similarly, hydrogen peroxide and nitric oxide are essential for sperm capacitation and the acrosome reaction in cattle (OFlaherty et al., 1999, 2003; Rodriguez et al., 2005ab). The observation that oxidative stre ss can lead to a reduction in fertilization and in embryo competence demonstrates that change in sperm function can impact the embryo formed from that sperm. Such effects are likely to be physiolo gically relevant because sperm may encounter oxidative stress in the female re productive tract (Agarwal et al., 2005; Agarwal and Prabakaran, 2005), male reproductive tract (Tremellen, 2008) or during processing (Aga rwal et al., 2005; Agarwal and Prabakaran, 2005).

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72Table 4-1. Effect of treatment of sperm with menadione (5 M) or tert -butyl hydroperoxide (TBHP; 10 nM) on cleavage and blastocyst formation following in semination of matured oocytes Day 7 Day 8 Treatment n a Cleavage (%) Blastocysts/oocyte (%) Blastocysts/cleaved (%) Blastocysts/oocyte (%) Blastocysts/cleaved (%) Vehicle control 7 (483) 48.3 3.9 13.4 1.3 30.0 3.9 20.4 1.9 43.6 3.0 Menadione, 5 M 7 (668) 43.6 3.9 12.7 1.3 27.1 3.9 16.9 1.9 38.6 3.0 TBHP, 10 nM 7 (682) 53.9 3.9 18.4 1.3 34.7 3.9 27.4 1.9 51.6 3.0 Data are least-squares means SEM. Means that diffe r from vehicle control are indicated by superscripts: P=0.07; P<0.05. a number of replicates (total number of embryos evaluated per treatment).

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73Table 4-2. Effect of treatment of sperm with menadione (0, 15 and 30 M) on cleavage and blastocyst formation following insemination of matured oocytes Day 7 Day 8 Menadione (M) n a Cleavage (%) Blastocysts/oocyte (%) Blastocysts/cleaved (%) Blastocysts/oocyte (%) Blastocysts/cleaved (%) 0 4 (301) 77.4 3.1 26.0 1.9 34.2 8.4 34.7 1.3 45.4 9.6 15 4 (347) 7.4 3.1*** 2.6 1.9*** 22.8 8.4 3.2 1.3*** 29.5 9.6 30 4 (285) 3.9 3.1*** 0.0 1.9*** 0.0 8.4* 0.0 1.3*** 0.0 9.6* Data are least-squares means SEM. Means that differ from 0 M are in dicated by superscripts:* P<0.05, ** P<0.01; *** P<0.001. a number of replicates (total numbe r of embryos evaluated per treatment)

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74Table 4-3. Effect of treatment of sperm with tert -butyl hydroperoxide (TBH P; 0, 150 and 300 M) on cleavage and blastocyst formation following insemination of matured oocytes Day 7 Day 8 TBHP (M) n c Cleavage (%) Blastocysts/oocyte (%) Blastocysts/cleaved (%) Blastocysts/oocyte (%) Blastocysts/cleaved (%) 0 5 (323) 66.6 5.3 19.6 1.7 30.3 2.0 28.2 2.9 43.5 4.4 150 5 (464) 60.6 5.3 10.7 1.7** 17.6 2.0** 17.2 2.9* 28.2 4.4* 300 5 (315) 47.4 5.3* 4.9 1.7*** 10.2 2.0** 8.6 2.9** 18.8 4.4** Data are least-squares means SEM. Means that differ from 0 M are in dicated by superscripts:* P<0.05, **P<0.01; ***P<0.001. a number of replicates (total number of embryos evaluated per treatment)

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75 CHAPTER 5 CAN PROGRAMMED CELL DEATH BE INDU CED IN POST-EJACULATORY BULL AND STAL LION SPERMATOZOA? Introduction Apoptosis is a common characteristic of ga metes undergoing spermatogenesis and is required to maintain homeostasis in the testes (Blanco-Rodriguez, 1998; Huckins, 1978; Allan et al., 1992; Sinha Hikim and Swerdloff, 1999). Ejaculates of healthy males contain two populations of sperm; a non-apopt otic fraction containing mor phologically superior quality sperm and an apoptotic fraction associated w ith increased abnormal sperm morphology (Bull Anzar et al., 2002; Boar Pea et al., 2003; Man Aziz et al., 2007). Insults to the testis can lead to an increase in the proporti on of tubular spermatocytes or ejaculated spermatozoa with characteristics of apoptosis and abnormal morphology. Among these insults are heat stress (Paul et al., 2008), scro tal heating (Sinha Hikim et al., 2003; Vera et al., 2004) and varicocele (Chen et al., 2004; Wu et al., 2008). There are two major pathways for induction of apoptosis in mammalia n cells. The intrinsic pathway involves the release of cytochrome c from the mitoc hondrial intermembrane space into the cytosol and sequential activat ion of caspase-9 and caspase-3 (Ravagnan et al., 2002; Riedl and Shi, 2004). Heat shock is one stress that activates the intrinsic pathway (Milleron and Bratton, 2007) as do agents such as carbonyl cyanide 3-chlorophenylhydrazone (CCCP) that depolarize mitochondria (Brad et al., 2007; Chaudha ri et al., 2007). The extrinsic pathway is activated by ligands such as tumor necrosis factorthat bind to receptors that activate caspase-8 and downstream caspases (Jin and El-Deir y, 2005). Given the profound morphological and molecular changes in male germ cells during sper matogenesis, it is possible that the signaling or effector pathways for induction of apoptosis be come dysfunctional in th e mature ejaculated sperm. Whether ejaculated sper matozoa can be induced to un dergo apoptosis is not clear.

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76 Cellular changes characteristic of apoptosis have been report ed in ejaculated spermatozoa subjected to cryopreservation for the bull (Martin et al., 2004), stallion (Orteg a-Ferrusola et al., 2008) and man (Weng et al, 2002; Paasch et al, 2004ab). Whil e hydrogen peroxide has been reported to activate caspase-3 and -9 in man (Bejarano et al., 2007). Induction of apoptosis after ej aculation by exposure of sperm to a stressful environment could compromise fertilizati on rate and developmental comp etence of the resulting embryo. Several studies indicate reduced fertilizing abil ity of sperm populations having a high percentage of apoptotic sperm (Paul et al., 2008; Tomsu et al., 2002; Benchaib et al., 2003; Seli et al., 2004). Moreover, an embryo formed from fertilization by an apoptotic sperm could conceivably have reduced developmental potential if DNA damage wa s not repaired or the sperm centriole which contributes to first cleavage in the em bryo (Sutovsky et al., 1996) was damaged. For the current study, bull and stallion spermato zoa were used to test the hypothesis that apoptosis can be triggered in ejac ulated sperm. The pro-apoptotic signals were aging of sperm at 38.5C, which leads to a decline in fertilizing ability of sperm (Monterrosso et al., 1995; Lechniak et al., 2003; Hendric ks et al., 2008), heat shock, and treatment with CCCP. The temperatures used for heat shock, 40 and 41C, are characteristic of lactating dairy cows exposed to heat stress (Elvinger et al., 1992; deCastro e Paula et al., 2008). Heat shock at 41C can cause apoptosis in bovine oocytes a nd preimplantation embryos (Rot h and Hansen, 2004; Paula-Lopes et al., 2002; Brad et al., 2007) a nd CCCP has been demonstrated to cause apoptosis in bovine embryos (Brad et al., 2007). A second object ive was to examine the mechanism by which apoptosis is blocked in bull spermatozoa by ev aluating stress-induced ch anges in mitochondrial depolarization and activation of the caspase cascade.

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77 Materials and Methods Chemicals and Reagents The m edium Sperm-TL was purchased from Ca isson (Sugar City, ID, USA) and used to prepare three media. Sp-TALP was made as previously described (Parrish et al., 1986), SpTALP-PVP was made similarly to Sp-TALP ex cept bovine serum albumin was replaced with 6.3 mg/mL polyvinylpyrrolidone (PVP) (Eastman Kodak, Rochester, NY) and Sp-TALP-MOPS was prepared like Sp-TALP except the HEPES was re placed with MOPS (10 mM) and 1 mM sodium pyruvate and 0.1 mg/mL gentamicin were also added. Percoll was fr om GE Healthcare (Uppsala, Sweden). Carbonyl cyanide 3-chlorophenylhydr azone (CCCP) was from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in dimet hyl sulfoxide (DMSO) to make a 100 mM stock solution. Aliquots were stored at C until th e day of use. The stock solution of CCCP was diluted to 100 M CCCP in 0.1% (v/v) DMSO. An equivalent amount of DMSO was added to Sp-TALP for control media. Tissue Culture Medium-199 (TCM-199) Eagles Minimum Essential Medium (MEM), MEM with D-valine, Hams F12, Dulbeccos phosphate buffered saline (DPBS), and penicillinstreptomycin were purchased fr om Sigma-Aldrich (St Louis, MO, USA). Horse serum was obtained fr om Hyclone (Logan, UT, USA), Frozen semen from bulls of various beef breeds was donated by Southeastern Semen Services (Wellborn, FL, USA) and frozen Hols tein semen was purchased from Select Sires (Rocky Mount, VA, USA) and ABS Global (Deforest, WI, USA). Mini-Brahman semen (Liu et al., 1999) was obtained from USDA-ARS Subt ropical Agricultural Research Station, Brooksville, FL courtesy of Chad Chase Jr. B ovine endometrial (BEND) cells were obtained from ATCC (Rockvill e, MD, USA). The In Situ Cell Death Detect ion Kit (fluorescein or TMR red) was obtained from Roche Diagnostics Corporation (Indiana polis, IN). An 8% (w/v) paraformaldehyde stock solution was

PAGE 78

78 from Electron Microscopy Sciences (Hatfiel d, PA, USA). RQ1 RNase-free DNase was from Promega (Madison, WI, USA). Hoechst 33342 was from Sigma-Aldrich. Rabbit polyclonal antibody recognizing synthetic pep tide derived from the sequence of human caspase-9 and rabbit polyclonal antibody against recombinant human cas pase-3 were from Stressgen Bioreagents (Ann Arbor, MI, USA). Horseradis h peroxidase-conjugated goat an ti-rabbit IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The mitochondrial polarity probe JC-1 (Molecu lar Probes, Invitrogen Eugene, Oregon) was prepared as a 7.67 mM stock in DMSO at C and diluted in Sp-TALP-PVP to 4 M on the day of use. Caspase-3 inhibitor, z-DEVD -fmk, was from R & D Systems (Minneapolis, MN) and diluted with DMSO to make a 20 mM stock solution. All other reagents were purchased from Sigma-Aldrich or Fisher Scientific (P ittsburgh, PA, USA) unless otherwise stated. Sperm Preparation Extended bull sem en was thawed, subjected to Percoll gradient pur ification to obtain motile spermatozoa (Parrish et al., 1986) and diluted in Sp-TALP medium to 10 to 20 x 106 spermatozoa/mL for experiments. Semen was co llected from two fertile stallions using an artificial vagina, and the gel por tion was removed with a mesh f ilter. The semen was diluted 1:2 with Sp-TALP, centrifuged (300 g; 10 min) and resuspended in 2 mL Sp-TALP. One milliliter was layered over a 45% 90% Percoll gradient and centrifuged (300 g; 10 min). The sperm pellet was collected, resuspended in 5 mL HE PES-TALP, washed by centrifugation (300 g; 10 min), and sperm pellet resuspended in 2 mL Sp-TALP and then adjusted to 20 106 sperm/mL. BEND Cells BEND cells, a bovine endometrial cell line (Stagg s et al., 1998) were grown in a 1:1 (v:v) mixture of Hams F12 and the D-valine modifi cation of Eagles MEM supplemented with 200 U/L insulin, 10% (v/v) heat in activated fetal bovine serum, 10% horse serum, 200 U/mL

PAGE 79

79 penicillin and 2 mg/mL streptomycin. At confluence, cells were trypsinized, mixed with an equal amount of culture medium, centrifuged for 5 mi n at 110 g, resuspended in culture medium and split into two equal aliquots. At approximate ly 80% confluence, medium was removed and replaced with fresh medium and used for experiments. Evaluation of TUNEL Labeling DNA frag mentation was determined by the terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) pr ocedure, whereby free 3OH ends of DNA are labeled with TMR redor fluorescein-c onjugated dUTP by the enzyme terminal deoxynucleotidyl transferase. Three experiments were performed. In the fi rst, bull sperm were incubated at 20 x 106 spermatozoa/mL in Sp-TALP at 38.5C, 40C, 41C or 38.5C in Sp-TALP containing 100 M CCCP in air for 4 h using a water bath. Additio nally an aliquot of sperm was used as a nonincubated spermatozoa control. All treatment s contained a similar c oncentration of DMSO [0.1% (v/v)]. At the end of the incubation period, sperm were fixed and subjected to the TUNEL labeling procedure. The experiment was replicat ed 4 to 7 times using a separate Holstein bull for each replicate. The second experiment was pe rformed similarly except that incubation was for 24 h and sperm were incubated with a nd without 100 M z-DEVD-fmk. All treatments contained the same amount of DMSO [0.1% (v /v)] with the excepti on of the non-incubated controls. The experiment was replicated 3 ti mes using a separate Holstein bull for each replicate. The third experiment was performe d using freshly ejaculated and Percoll-purified equine sperm. Sperm were incubated at 38.5C with or without 100 M CCCP or at 41C in air for 4 h using a water bath. The experiment was replicated twice using a separate stallion for each replicate.

PAGE 80

80 After incubation as described above, sperm were washed once by centrifugation (600 x g, 10 min) in 1mL PBS-PVP [10 mM KPO4, pH 7.4, containing 0.9% (w/v ) NaCl (PBS) and 1 mg/mL PVP] and fixed. In some cases, sperm we re fixed in 4% (w/v) paraformaldehyde in PBS for 15 min on ice, washed, and stored in PBS-PVP at 4C for up to 2 to 3 wk before assay. Slides were prepared using 20-50 L of fixed sperm on the day of assay. Alte rnatively, sperm smears were prepared immediately following treatment, air dried and sperm fixed by covering slides with 4% (w/v) paraformaldehyde in PBS for 15 mi n on ice. Slides were washed gently three times by flooding with PBS-PVP, air dried and stored in the dark at 4C for up to 2 wk until assayed. The TUNEL assay was initiated by permeabilizing fixed sperm with 0.5 or 0.1% (v/v) Triton X-100 containing 0.1% (w/v) sodium citr ate in PBS for 30 min at room temperature. Samples were then incubated in 50-100 L of TUNEL reaction mixt ure (containing TMR-red conjugated dUTP or fluorescei n-conjugated dUTP and terminal deoxynucleotidyl transferase) for 1 h at 37C in the dark. Sperm were washed in PBSPVP and incubated with 50 L of 1 mg/mL Hoechst 33342 in PBS-PVP for 30 min at r oom temperature. Slides were washed three to four times with PBS-PVP, and cover slips mounted using 5 L mounting medium containing ProLong Gold antifade reagent (Invitrogen, Molecular Probes). Each TUNEL procedure contained sperm treated with RQ1 RNase-free DNase (50 U/mL) at 37C for 1 h as positive controls and sperm incubated with the T UNEL reagent in the absence of terminal deoxynucleotidyl transferase as a negative control. TUNEL labeli ng was observed using a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Inc., Gttingen, Germany). Images were acquired using AxioVision software and an Axio Cam MRm digital camera (Zeiss).

PAGE 81

81 Evaluation of Mitochondrial Membrane Potential Percoll-purified sperm were diluted to 10 106 sperm/mL in pre-equilibrated Sp-TALPMOPS maintained in an incuba tor at 38.5C and 5% (v/v) CO2 or 42C and 7% (v/v) CO2. The CO2 concentration was varied with temperature to account for temperature-associated changes in gas solubility and to maintain an equal pH at both temperatures. Sperm were incubated at 38.5C and 5% CO2 or 42C and 7% CO2 for 4 h. A third aliquot of sperm was used as a killed control by freeze-thawing two to three times. Fo llowing incubation, mitochondrial polarity was measured by determining fluorescence emission of the polarity-dependent dye, JC-1. A total of 1 106 spermatozoa were incubated in triplicate with 2 M JC-1 in a volume of 200 L and Sp-TLP-MOPS medium at 37C in air for 15 min. Incubations were carried out in 96-well plates (black, tissue-culture treated, Corning, Acton, MA). Red (exc itation, 550 nm; emission, 600 nm) and green fluorescence (excitation, 485 nm; em ission, 535 nm) was measured using a BioTek FLx 800 fluorometer (BioTek, Winooski VT) and th e ratio of red : green used to determine mitochondrial membrane potential. The experiment was replicated 4 times with a separate Angus, Brahman and Holstein bull in each replicate. Experiment 2 was conducted similarly to Experiment 1 except that sperm from individual bulls of 4 breeds were used in each replicate: Angus, Brahman, Holstein and Mini-Brahman. The experiment was replicated a total of 6 times w ith 22 bulls, so that 3-4 bulls per breed were examined. Western Blotting for Caspase-9 and Caspase-3 Sperm cells were subjected to the following treatments: control no incubation, incubation at 38.5, 40 and 41C in Sp-TALP for 4 h and incubation at 38.5C in Sp-TALP containing 100 M CCCP for 4 h. In addition, BEND ce lls were used to verify that the caspase-9 and -3 antibodies cross-reacted with bovine protei ns. Cells were incubated at 37C in medium

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82 with and without 100 M CCCP for 24 h. Followi ng treatments, sperm aliquots containing 106 cells were washed once by centrifugation in cold DPBS (1,800 g for 10 min), resuspended in 15 L DPBS and stored at -80C until analysis Following thawing, cells were solubilized by boiling for 5 min in an equal volume of ly sis buffer [125 mM Tris-HC1 buffer pH 6.8, containing 10% (w/v) sodium dodecyl sulfate (S DS), 20% (w/v) sucrose, and 5% (v/v) 2mercaptoethanol]. Proteins in lysed sperm (106 cell equivalents per lane) and lysed BEND cells (105 cell equivalents per lane) were separated under reducing c onditions using one-dimensional, discontinuous SDSpolyacrylamide gel elect rophoresis (SDSPAGE) with 4% (w/v) gradient polyacrylamide gels and Tris-HCl buffer, pH 6.8. Proteins were transferred electrophoretically to H ybond ECL 0.2 mm nitrocellulose membranes. Membranes were washed for 10 min in TTBS [10 mM Tris pH 7.6, 0.9% (w/v) NaCl, and 0.3% (v/v) Tween-20] and blocked for 2 h or overnight in blocking buffer [TTBS containing 1% (w/v) gelatin]. Membranes were incubated for 2 h or overnight at room temperature with a rabbit polyclonal antibody recognizing caspase-9 (2 g/mL in blocking buffer) or caspase-3 ( 0.5 g/mL in blocking buffer), washed, and then incubated for 2 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution in blocking buffer). After additional washing, blots were developed using the ECL Plus Western blotti ng detection reagents as per manufacturer recommendations (GE Healthcare). Statistical Analysis Data were s ubjected to least-squares anal ysis of variance using the GLM and MIXED procedures of the Statistical Analysis System (SAS for Win dows, Release 9.0, SAS Institute, Inc., Cary, NC). The p-diff procedure with Tuke y adjustment was used as a means separation test. Data on percent TUNEL la beling were analyzed without transformation and again after arcsin transformation to correct for any non-normality associated wi th percentage data. Bull (i.e.

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83 replicate) was considered random and other main effects were considered fixed. Data are reported as least-squares means SEM from th e analysis of the untransformed data while probability values are derived from analyses of transformed data. Data on mitochondrial membrane potential were analyzed without transformation. Results Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Bull Sperm In the first experim ent, the TUNEL assay wa s used to assess differences in DNA damage among bull sperm aged for 4 h in Sp-TALP at 38.5 C and under heat shock conditions of 40 and 41C. Results are summarized in Table 5-1. When compared to non-incubated controls, percent of sperm positive for TUNEL labeling was not incr eased by aging at 38.5, 40 or 41C. Similarly the mitochondrial depolarizing agent, CCCP, did not increase the percent of cells positive for TUNEL. A second experiment was conducted where sperm were incubated for 24 h instead of 4 h. In addition, some sperm were treated with z-DEVD-fmk to block group II caspases. Sperm incubated for 24 h had a higher percentage that we re positive for TUNEL as compared to control sperm that were not incubated (P<0.01), but th ere was no difference in the degree of TUNEL labeling between sperm incubated at 38.5, 40 and 41 C (Table 5-2). In addition, the increase in TUNEL labeling caused by incubation fo r 24 h was not blocked by z-DEVD-fmk. Equine Sperm To investigate if TUNEL responses in stalli on sperm were similar to those seen for bull sperm, equine sperm were evaluated for TUNE L labeling following aging for 4 h at 38.5C, 38.5C with 100 M CCCP, and 41C (Figure 5-1). Two stallions were examined. For stallion 1, the proportion of sperm positive for the TUNEL r eaction ranged from 5.7 to 9.2% and was not

PAGE 84

84 affected by treatment. For stallion 2, it was noted that a large fraction of sperm (39.5%) were without tails. Among those with tails, the per cent of cells positive for TUNEL was less than 3% and was unaffected by treatment (2.7%, 1.5% an d 0.7% for sperm aged at 38.5C, with CCCP and at 41C respectively). However, ther e was a high incidence of TUNEL labeling among sperm without tails at all temperatures (94 to 97%). Evaluation of Mitochondrial Membrane Potential Results are given in Figure 5-2. Overall, ther e was no breed effect or interactions with breed and data are pooled across this classifi cation. There was no difference in m itochondrial membrane potential between sperm incubated at 38.5 vs 42C in either experiment (P>0.05). In contrast, the ratio of red to green fluorescence was lower for sperm killed by freeze-thawing when compared to sperm incubated at 38.5 and 42C (P<0.001). Western Blotting Procaspase-9 (~50-54 kDa) and -3 (~36 kDa) were observed by W ester n blotting in bovine endometrial cells (BEND cells, Figu re 5-3). There was also a band of ~30 kDa dete cted with the antibody to caspase-3. The manufact urer states that this protei n is not caspase-3 because the band appears in cells lacking caspase-3. After addition of 100 M CCCP, cleaved fragments of procaspase-9 (~30 kDa) and procaspase-3 (~20 kD a and 18 kDa) were observed; these represent the active caspases (Figure 5-3). In bovine spermatozoa, procaspase-9 was evid ent in frozen-thawed sperm (control) and in sperm aged at 38.5, 40 and 41C. However, aging and exposure to 100 M CCCP did not lead to the appearance of caspase-9. Moreover, neithe r procaspase-3 nor caspase-3 was observed (Figure 5-3).

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85 Discussion Short term aging of bull or equine sperm ha d no effect on DNA integrity as determined by the TUNEL assay. This was true even when sperma tozoa were incubated at temperatures of 40C and 41C that are characteristic of heat-stressed co ws (Elvinger et al., 1992; de Castro e Paula et al., 2008). Long-term aging for 24 h caused DNA da mage to bovine spermatozoa but this was a caspase-independent phenomenon. The failure of aging to induce caspase-dependent apoptosis is due, at least in part, to failure of activation of procaspase-9 a nd a lack of procaspase-3. These latter results are similar to thos e of Martin et al. (2007) where procaspase-9 was present in bull sperm but procaspase-3 and procaspase-8 were absent. One reason why procapase-9 was not activated by aging was that there was no change in mitochondrial potential caused by aging or heat s hock. Other aspects of the activation pathway for procaspase-9 must be dysfunctional also sin ce artificial depolarization of mitochondria with CCCP did not result in cleavage of procaspase -9. Cryopreservation, in contrast, has been reported to activate caspase-9 in bull sperm (Martin et al., 2007). The lack of a functioning intrinsic pathway for activation of apoptosis may be a speciesspecific phenomenon. Cryopreservation has been repor ted to activate caspase-3 and -9 in man (Paasch et al., 2004ab; Bejarano et al., 2008) and activated caspase-3 has been found in ejaculated sperm in stallion (O rtega-Ferrusola et al., 2008), ma n (Kotwicka et al., 2008), and boar (Choi et al., 2008). There was an increase in TUNEL labeling caused by aging of sperm for 24 h. However, this phenonomen was independent of the intr insic activation pathway because the group II caspase inhibitor z-DEVD-fmk di d not reduce the proportion of sperm cells that were positive for TUNEL. Mitochondria can induce DNA damage in a caspase-independent manner that involves apoptosis inducing factor (AIF) and endonuclease G (EndoG; van Gurp et al., 2003).

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86 Sperm cells are particularly susceptible to ox idative stress because of their high content of polyunsaturated fatty acids in th e plasma membrane (Parks et al., 1987; Aitken and Fishel, 1994). Perhaps, incubation for 24 h causes oxidative damage leading to mitochondrial membrane damage and the subsequent release of AIF and E ndoG into the cytosol. Other nucleases also exist within the sperm (Sotolongo et al., 2005; Sh aman et al., 2006) and these could also be activated by long-term incubation. The fact that bull and stallion sperm are re sistant to the induction of apoptosis after ejaculation by aging does not mean that apoptotic sperm are unlikely to be present in the ejaculate. Insults to the testis can increase the inci dence of apoptosis of ma le germ cells (Paul et al., 2008; Sinha Hikim et al., 2003; Vera et al., 2004; Chen et al., 2004; Wu et al., 2008). The high incidence of apoptosis in the tailless sper m of one stallion in the present study probably represents the elimination of defective sperm by induction of apoptosis during spermatogenesis. While it is improbable that tailless sperm participat e in fertilization, it is conceivable that other types of sperm with apoptotic changes in th e nucleus could. Perhaps embryos formed from apoptotic sperm do not develop normally. Sper m with severe DNA damage decondense when injected into mouse oocytes but paternal DNA is not replicated during the first round of DNA synthesis and becomes digested (Yamauchi et al., 2007). One hypothesis of the current st udy was that induction of apopt osis in ejaculated sperm by elevated temperatures, such as occur in the re productive tract of heat-s tressed females, could result in formation of defective embryos and be one source of the redu ced fertility of heatstressed females (Hansen, 2007). However, the failure of heat shock to induce apoptosis does not support this hypothesis.

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87 Table 5-1. Effect of aging for 4 h at temperatures characteristic of normothermia (38.5C) or heat stress (40 and 41C) and of mitochondr ial depolarization w ith carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the per cent of frozen-thawed, ejaculated bull spermatozoa positive for the TUNEL reaction Treatment n a Percent of spermatozoa positive for TUNEL Control (no incubation) 4 1.2 0.7 38.5C, 4 h 7 1.6 0.6 40C, 4 h 7 2.7 0.5 41C, 4 h 4 1.5 0.8 100 M CCCP 7 1.8 0.5 DMSO (control for CCCP) 7 2.1 0.5 a Number of bulls examined per treatment. Data are least-squares means + SEM. There was no effect of treatment.

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88 Table 5-2. Effect of aging for 24 h at temperatur es characteristic of normothermia (38.5C) or heat stress (40 and 41C) on the percen t of frozen-thawed, ejaculated bull spermatozoa positive for the TUNEL reacti on as affected by the group II caspase inhibitor, z-DEVD-fmk Percent of spermatozoa positive for TUNEL Incubation temperature na inhibitor + inhibitor Control 3 1.0 1.4 38.5C 3 4.0 1.4 4.2 1.4 40C 3 6.2 1.4 4.4 1.4 41C 3 7.0 1.4 8.5 1.4 a Number of bulls examined per treatment. Data represents least-squares means SEM. The percent of cells positive for TUNEL was greater for cells incuba ted for 24 h than for control cells (P<0.01). When analyzed as a 2 x 3 factorial design (i.e., excluding the control), there was no effect of incubation temperature, inhibito r or temperature x in hibitor (P>0.05).

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89 0 10 20 30 40 50 60 70 80 90 100 Stallion 1 Stallion 2 No Tail IntactPercent TUNEL Positive Sperm38 5 C 100 M CCCP 41 C 38 .5 C 10 0 M CC CP 4 1 C 38. 5 C 100 M CCC P 41 C Figure 5-1. Effect of aging for 4 h at temperatures characteristic of normothermia (38.5C) or heat stress (41C) and of mitochondria l depolarization with carbonyl cyanide 3chlorophenylhydrazone (CCCP) on the per cent of freshly-ejaculated equine spermatozoa positive for the TUNEL reacti on. Data on TUNEL labeling for stallion 2 were determined separately for sp erm with and without tails because 39.5% of sperm were without tails. Few sperm (<1%) for stallion 1 were tailless.

PAGE 90

90 0.0 0.5 1.0 1.5 2.0 2.5 3.0123Ratio of red to green fluorescence Experiment 1bExperiment 2c** ** C o n t r o l H e a t S h o c k K i l l e d C o n t r o l H e a t S h o c k K i l l e d Figure 5-2. Effect of aging for 4 h at temperatures characteristic of normothermia (38.5C) and heat stress (42C) on mitochondrial memb rane potential of frozen-thawed bull spermatozoa as measured by the cationic fluoroprobe JC-1. Data are least-squares means SEM of data from 16 bulls (Experi ment 1) or 22 bulls (Experiment 2). There was no effect of temperature on m itochondrial membrane potential in either experiment (P>0.05) but killing of sper m reduced potential in both experiments (P<0.01;**).

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91 Figure 5-3. Representative wester n blots for the detection of ca spase-9 (A) and caspase-3 (B). Bovine sperm: Control a non-incubated sperm contro l; -CCCP sperm incubated at 38.5C for 4 h without CCCP; +CCCP sperm incubated at 38.5C for 4 h in 100 M CCCP; 40C sperm incubated at 40C for 4 h and 41C sperm incubated at 41C for 4 h; BEND cells: -CCCP cells incubated at 37C for 4 h without CCCP; +CCCP cells incubated at 37C for 4 h in 100 M CCCP. Note that the 30 kDa protein in the BEND cell lanes (panel B) is not caspase-3.

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92 CHAPTER 6 GENERAL DISUSSION The ability o f the preimplantation embryo to complete its developmental program is determined in part by its genetic inheritan ce and non-genetic acquisition from the sperm and oocyte. The role of the oocyte in determinati on of embryo competence has been well established (Sirard et al., 2006). The thesis of this dissert ation is that the sper m cell, too, contributes components to the embryo that affect its deve lopmental competence and that damage to the sperm after ejaculation can result in the forma tion of an embryo with reduced competence for development. Sperm may be exposed to post-ejaculatory st ress while in the female reproductive tract following natural mating and/or AI or under condi tions such as sperm processing for AI and IVF. Sperm must traverse the female reproductive tr act to reach the site of fertilization. In cattle, sperm may remain in the female reproductive trac t for up to 18 h or more prior to ovulation (Hawk, 1987) and up to 36 h prior to ovulation in swine (Hawk, 1987) Furthermore, intercourse in humans is not usually associated with ovula tion, and sperm can remain in the reproductive tract for several days in women (Suarez a nd Pacey, 2006). Hence the time from deposition of sperm into the female reproductive tract and fert ilization may be quite long with ample time for sperm to be exposed to stressors in the female reproductive tract. Sperm within the female reproductive tract ar e foreign bodies and as such generate a robust inflammatory response that exposes th em to immune cells. Within minutes of insemination, there is an influx of neutrophils into the uterus which peaks between 1 and 12 h after insemination (Schuberth et al., 2008). In th e pig, seminal plasma induces the expression of pro-inflammatory cytokines in the uterus within 34 h of insemination (OLeary et al., 2004).

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93 Under normal physiological conditi ons it would be expected that the secretions of the female reproductive system would protect, ma intain and/or enhance sperm function. For example, spermatozoa spend a large portion of time in the oviduct in sperm reservoirs waiting for ovulation to occur, and it has been demonstrat ed in vitro that the ov iductal secretions may play a role in protecting sperm DNA structure (Robert et al., 2008), and in maintaining sperm survival and function (Pollard et al., 1991; Zhu et al., 1994; Quintero et al., 2005). Pathological conditions which lead to a change in the componen ts of oviductal secretions may lead to loss of this protective effect, such as ch anges in pH (Rizvi et al., 2008). Under both physiological and pathological c onditions oxidative stress due to elevated concentrations of ROS has been associated with change in the reproductive and peritoneal microenvironments that can have negative effect on spermatozoa and hence on fertilization and early embryonic development (A garwal et al., 2003). Surprisingly, in a study to examine the presence of ROS in the follicular fluid of women undergoing IVF, Atta ran et al. (2000) found that women who became pregnant had higher leve ls of ROS than those who did not. However under pathological conditions where nitric oxi de is generated in excessive amount over prolonged periods, such as urogeni tal tract infection in males and females it is possible that reduced fertility in these individuals is due to NO-induced sperm toxicity (Rosselli et al., 1995). Furthermore, it has been suggested that pa thological changes due to oxidative stress and autoimmunity in infertile women could lead to changes in protein function and hence inhibit post-ejaculatory sperm functions such as sperm capacitation and oocyte fert ilization, in addition to embryo implantation (Iborra et al., 2005). Post-ejaculatory sperm processing for AI and IVF may entail staining sperm with Hoechst followed by flow cytometry sorting to produce sex-sorted semen and/or cryopreservation for

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94 long term storage (Levinson et al., 1995; J ohnson, 2000; Garner, 2008). Removal of seminal plasma with its associated anti-oxidant during sperm processing results in a pro-oxidant state, while repetitive washing and cen trifugation increases the produc tion of ROS and impairs sperm function (Aitken and Clarkson, 1988). Furthermore, sex-sorted sperm have lower viability and fertility than their unsorted c ounterparts (Maxwell et al., 1998; de Graaf et al., 2008). This reduction in viability and fertility may be associated to exposure to stressors, prior to, during and after passage through th e flow cytometer and may incl ude staining with Hoechst 33342, exposure to UV light, high temperat ures during incubation, pressure changes, physical stress and shear forces as sperm are being sorted (Seide l and Garner, 2002; de Graaf et al., 2008). The process of cryopreservation can lead to degenerative changes that include shortened lifespan (Gillian and Maxwell, 1999; Sankai et al., 1994; Rodr guez-Martnez et al., 2008), decreased motility (Gandini et al., 2006; Jin et al., 2008; Rodrguez-Ma rtnez et al., 2008), a higher degree of membrane damage or membra ne alterations (Hammerstedt et al., 1990; De Leeuw et al., 1990; Gillian et al., 1997; Gillian and Maxwell, 1999; Pegg, 2002; Nishizono et al., 2004), increased incidence of acrosome reacted sp erm (Gillian et al., 1997; Gillian and Maxwell, 1999; Gillian et al., 1999), incr ease in chromatin abnormalities and DNA fragmentation (Horse: Baumber et al., 2003; Ram: Peris et al., 2004, 2007; Human: Gandini et al., 2006; Boar: Fraser and Strzezek, 2007; Mouse: Yildiz et al., 2007, 2008) and decreased fertility following intrauterine or cervical inse mination (Dogs: Gill et al., 1970. Ewes: Jabbour and Evans, 1991; Maxwell et al., 1993; Gillian et al., 1997; Gmez et al., 1997; Donovan et al., 2004; Niza ski, 2006; Mouse: Nishizono et al., 2004) and after in tracytoplasmic sperm injection (ICSI; Gmez et al., 1997).

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95 Most of the stresses that we re applied to sperm did not a ffect the competence of the resulting embryos to become blastocy sts. These stresses include aging in vitro heat shock, and exposure to low doses of X-irradia tion (Chapters 2 and 3). Failure of these stresses to affect the resultant embryo probably reflects the resistance of sperm DNA to damage as well as the limited contributions of other components of the sper m to the embryo. The highly compact nature of spermatozoal DNA due to the preponderance of pr otamines (Poccia, 1986), its organization into toroids (Ward and Coffey 1991; Ward 1993) and further disulphide-cross-linkage between protamines may protect spermatozoal DNA from da mage due to mild stress. Perhaps, a greater magnitude of stress than applied here would compromise embryonic surv ival. Cozzi et al. (2001) found that exposure of epid idymal mouse sperm to heat s hock prior to ICSI resulted in embryos that had a lower ability to develop to th e blastocyst stage compared to controls. More intense radiation than given here caused DNA damage in bovine ejaculated spermatozoa as measured by TUNEL labeling and embryos formed from X-irradiated spermatozoa had a lower ability to develop to the blas tocyst stage compared to c ontrols (Fatehi et al., 2006). Tert -butyl hydroperoxide and menadione did reduce the competence of the resulting embryos to become blastocysts (measured as th e proportion of cleaved em bryos that developed to the blastocyst stage; Chapter 4). One of the possible mechanisms by which sperm damage affected embryo competence may be by delaying fer tilization. A delay in fertilization leads to fertilization of an aged oocyt e and this may account for the reduction in embryo competence seen in Chapter 4. Fertilization of aged oocytes results in asynchrony in pronuclear formation (Goud et al., 1999) and pronuclear asynchrony has b een linked to developmental arrest in human zygotes (Zenezes et al., 1985; Schmiady and Kentenich, 1993). Another possibility for the reduction in the embryos competence may have been due to the inhe ritance of damaged

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96 macromolecules DNA, centriole, membranes and/or RNAs. Pro-oxidants have been shown to cause oxidative damage to sperm plasma memb rane, mitochondria, cytosol and DNA (Silva et al., 2007; Peris et al., 2007). Paternal DNA damage causes failure of spermatozoal pronuclear decondensation, reduction in embryo developmenta l potential and implan tation rates following transfer in humans (Tomsu et al 2002; Seli et al., 2004) and in duces developmental block after first cleavage in bovine embryos (Khalifa et al., 2007). The oocyte has a limited capacity to repair paternal DNA damage (Generoso et al., 197 9; Fatehi et al., 2006) and thus some embryos will continue to develop. The mammalian zygote, with exception to some rodents inherits the proximal centriole (which double and together forms the centrosom e becoming the organizing center of the mitotic spindle) from sperm. Damage to the paternally inherited centriole may result in failure of pronuclear apposition and syngamy, in addition to the possibility of aberrant segregation of chromosomes following first cleavage, which has been linked to embryos with reduced competence for development (Chatzimeletiou et al., 2005). Finally, the role of paternal RNAs in em bryo development is unknown. In the event that these are essential to preimplantation embryo de velopment, damage to mRNA, such as oxidation of bases will lead to failure in ribosomal tran slation and reduced protei n expression (Shan et al., 2007). If vital to embryo survival and continued development, damage in this manner will certainly reduce the embryos ability to develop. Apoptosis is the typical route utilized to eliminate damaged sperm cells within the testes, both under physiological conditions, during norma l spermatogenesis (Bla nco-Rodriguez, 1998) or under conditions such as testicular hypertherm ia (Hikim et al., 2003; Vera et al., 2004; 2005). There is also evidence in the l iterature that ejaculated sperm ma y have the capacity to undergo

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97 cellular changes charact eristic of apoptosis in re sponse to stress (cryopreservation Martin et al., 2004; Ortega-Ferrusola et al 2008 : Weng et al., 2002; hydrogen peroxide Bejarano et al., 2007). However, the results presented in Chapter 5 make it clear that the ejaculated bull spermatozoa are not capable of caspase-mediated apoptosis (Figure 6-1). This is because mitochondrial membrane depolarization due to aging, heat sh ock and CCCP (a known depolarizing agent) does not occur in ejaculated frozen-thawed sperm. Se condly, activation of procaspase 9 is blocked and this may be due to the resist ance of mid-piece mitoc hondria to depolarize in response to the stresses used. Thirdly, procaspa se 3 was not found in ejaculated bovine sperm; hence caspaseactivated DNase activation is not likely. The fact that apoptosis is not possible in ejaculated spermatozoa increases the probabi lity that a damaged spermato zoon fertilizes the oocyte. Apoptosis is a mechanism utilized by most cell types to initiate removal of damaged cells, without damaging the surrounding tissu e or cells. What biological function could the block to apoptosis in ejaculated spermatozoa serve? Pe rhaps the signaling mechanisms employed during apoptosis are utilized during sperm capaci tation. One of the early events in the mitochondrial/intrinsic pathway of apoptosis is the release of cytochrome c into the cytosol (Figure 6-1). In ejaculated boar sperm, cytoch rome c is up regulated during capacitation without activation of caspase-3 and apopt osis (Choi et al., 2008). This early apoptotic event can be detected using annexin V (Vermes et al., 1995). Incubation of human ejaculated sperm with A23187 a calcium ionophore induces capacitated sperm to undergo the acrosome reaction and results in plasma membrane scrambling as measur ed by flow cytometric analysis of annexin V binding (Martin et al., 2005). Furthermore, the externalization of PS due to A23187 is not associated with other key hallmarks of apoptosis in human sperm (atte nuation of mitochondrial membrane potential,

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98 caspase activation, increased plasma membrane permeability or increas ed DNA fragmentation; Martin et al., 2005). Activation of the apoptotic pathway while in the process of capacitation would be detrimental to sperm survival and blockade of the apoptosis pathway ensures fertilization by a competent spermatozoa. It is possible that there were changes in em bryo competence caused by stress of sperm that were not evident in the simple measure of embr yo competence used in this study (blastocyst development). Perhaps, transfer of embryos to recipients would have revealed errors in embryonic or fetal development later in gestatio n. Burfening and Ulberg (1968) demonstrated that in vitro heat shock of ejaculated sperm decreased embryo survival as determined by the number of implantation sites note d on day 9 or 12 post insemination per number of cleaved ova 30 h post coitus in rabbits. Moreove r, post-ejaculatory sperm damage in utero can result in the formation of an embryo with reduced competen ce for development (Howarth et al., 1965). It was not until the end of this research that agents causing sperm damage that carried over into the embryo were identified. As a result, it was not possible to perform experiments to examine the mechanism for actions of tert -butyl hydroperoxide and mendaione. Figure 6-2 illustrates the possible outcomes of post-ejaculatory oxidative stress on fertilization and subsequent embryo developmen t. Post-ejaculatory oxidative damage could cause free radical lipid peroxida tion (Chen et al., 1997) there by destabilizing sperm membranes including those of mitochondria located in the mid-piece. Damage to sperm mitochondria can lead to a reduction in mitochondrial transmem brane potential and reduce the ATP content of sperm (Armstrong et al., 1999). The disturbance to the mitochondria and depletion in ATP can lead to a reduction in sperm mo tility. Fertilization failure occurs when the sperm fail to reach the oocyte or when sperm fail to bind to and penetrat e the zona pellucida of the oocyte (Oehninger et

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99 al., 1995), both of which may be due to either a reduction in sperm motilit y and/or damage to sperm plasma membrane. Fertilization delay may also result from a re duction in sperm motility, as sperm take longer to reach the oocyte; at the same time the oocyte continues to age and fertilization of an aged oocyte can lead to pronuclear asynchrony and developmental arrest (Agung et al., 2006). However, in some cases the asynchrony in pronuclear development may not be severe enough to disrupt further development and the embryo develops normally. Post-ejaculatory oxidative stress can lead to damage to the macromolecules inherited by the embryo. These marcomolecules include the pa ternal DNA and associated nuclear matrix, centriole and RNAs (especially mRNAs). Dependi ng on the extent of paternal DNA damage, the damage may either be overlooked or repaired. However due to the limited ability of the oocyte to repair damaged DNA (Generoso et al., 1979; Bra ndriff and Pedersen, 1981), damage to the paternal genome may persist in the developing embryo. DNA damage leading to errors in the cell cycle, gene expression and/or metabolism may trigger cell cycle arrest at anytime during embryo development leading to apoptosis and em bryo death. However, in the event that DNA damage does not compromise the embryo, development will continue unperturbed. Oxidative damage to the paternally inherited centriole can lead to failure of pronuclear apposition and syngamy. Furthermore, centrio lar damage may preven t duplication of the centriole and lead to aberrant chromosomal segregation prior to first cleavage. Failure of blastomeres to inherit the normal complement of chromosomes can lead to activation of cell cycle check points and cell cy cle arrest, triggering apopt osis and embryo death. There is currently a lack of knowledge on the role of pate rnal RNAs in early embryo development but there is one study that has id entified unique paternal mRNA in the embryo

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100 (Ostermeier et al., 2004). However, in the event that paternally inherited RNAs are essential for normal embryo development, oxidative damage mR NAs may lead to reduction in essential proteins (Shan et al., 2007) lead ing to developmental arrest, most likely prior to the maternalzygotic transition. Future research evaluating the effect of post-ejaculatory sperm damage on embryo competence should focus on the timing of fertiliza tion and developmental arrest as a means of identifying which paternally inherited sperm components negatively affects embryo competence. Post-ejaculatory oxidative stress could affect fertilizing ability through sperm plasma membrane damage to either prohibit or delay fertilizati on (Figure 6-2). A time course study evaluating the timing of fertilization and the proportion of oocytes with sperm incorporated into the ooplasm would answer the question of delayed fertilization and concomitantly the possibility of fertilization of aged oocytes. In addition, de fining the stage at which developmental arrest occurs could identify which paternally inher ited sperm components negatively affect embryo competence. For example, failure of paternal pronuclei formation could point to DNA and nuclear matrix oxidative damage, while arrest at the 8to 16cell stage could point to inheritance of paternal damaged DNA which the oocyte was unable to repair. Examination of the zygote prior to syngamy may lead to evidence of inheritance of a damaged centriole by examining aster formation or in the 2-cell em bryo examination of chromosome numbers may point to defective chromosome segregation associat ed with inheritance of a defective centriole. A more difficult proposal is to examine the e ffect of inheritance of oxidative damaged mRNA, due to the current lack of knowledge on the role patern al mRNA play in the embryo and how these affect embryo competence. Gene e xpression profiles of 2, 8 and 16-cell embryo produced from sperm exposed to oxidative stre ss could give some insights into markers

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101 associated with a damaged embryo. These markers may be used later to make selection of embryos that are more competent to complete its developmental program. The fertility of lactating dairy cows has decl ined over the last 40 to 50 years (Lucy, 2001; Lopez-Gatius, 2003; Dobson et al., 2 007). Some of this decline in fe rtility has been attributed to increased milk production and associated intens ive management practices leading to disease such as mastitis, lameness, retained fetal me mbranes and endometritis (Dobson et al., 2007). Endocrine disrupters including environmental contamin ants of either natural or synthetic origin, have been implicated as potential causes of reduced fertility in animals a nd humans (Brevini et al., 2005). Mouse embryos produced from fertilizat ion of oocytes with spermatozoa from male mice fed the plant growth regulator, 2-chloroet hyltrimethyl ammonium chloride, have reduced embryo survival (Torner et al., 1999) despite no alterati on in spermatogenesis. The results of this dissertation clearly indicat e that post-ejaculatory sperm damage can reduce the resultant embryos competence for preimplantation development. Thus, the sperm is an important determinant in determining embryonic survival and should be considered when identifying causes of embryonic failure in dairy cows and other species.

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102 Apoptosome Procaspase 3 Active caspase 3 Active caspase 9 DNA fragmentation and cellular death Cytochrome c Apaf-1 Procaspase 9 Caspase activated DNase m Figure 6-1. Model describing points in the mitoc hondrial/intrinsic pathway for apoptosis that are blocked in bovine ejaculated spermatozoa. Induction of apoptosis by post-ejaculatory stress triggers depolarization of the mitochondria (reduction in m) within the midpiece and release of various pro-apoptotic f actors. Of these factors, Cytochrome c, APAF-1 and procaspase-9 form the apopt osome, resulting in the cleavage of procaspase-9 to its active form and subseque nt activation of caspa se 3. In addition to activation of other executione r caspases, caspase 3 cleaves caspase-activated DNase and DNA fragmentation and cell death occurs This pathway is blocked at three points in the ejaculated spermatozoa. First, depolarization due to aging, heat shock and CCCP (a known depolarizing agent) does not occur in sperm. Activation of procaspase 9 is blocked, probably as a result of the resistance of mid-piece mitochondria to depolarization. Thirdly, pro caspase 3 is absent in bovine ejaculated sperm so caspase-activated DNase activation via this enzyme is not possible.

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103 Post-ejaculatory oxidative stressFree radicals, Energy depletion, Membrane destabilization Fertilization failure Fertilization delay Cytoplasmic and nuclear aging Damage to molecules inherited from spermDNA CentrioleSperm mRNA Errors in cell cycle, gene expression and/or metabolism Cell cycle arrest Error in cell cycle Role unclear Activate cell cycle checkpoint Possible decrease in critical mRNA ? REPAIR Normal developmentAPOPTOSIS Figure 6-2. Possible scenario for th e effect of post-ejaculatory oxidative stress on fertilization and subsequent embryo development

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104 LIST OF REFERENCES Adenot, P.G., Szllsi, M.S., Geze, M., Renard, J.P., Debey, P., 1991. Dynam ics of paternal chromatin changes in live one-cell mouse embryo after natural fertilization. Mol. Reprod. Dev. 28, 23-34. Agarwal, A., Saleh, R.A., Bedaiwy, M.A., 2003. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil. Steril. 79, 829-843. Agarwal, A., Gupta, S., Sharma, R., 2005. Oxida tive stress and its implications in female infertility a clinician's perspective. Reprod. Biomed. Online. 11, 641-650. Agarwal, A., Prabakaran, S.A., 2005. Mech anism, measurement, and prevention of oxidative stress in male reproductive physiology. Indian J. Exp. Biol. 43, 963-974. Agung, B., Otoi, T., Wongsrikeao, P., Taniguchi M., Shimizu, R., Watari, H., Nagai, T., 2006. Effect of maturation culture peri od of oocytes on the sex ratio of in vitro fertilized bovine embryos. J. Reprod. Dev. 52, 123-127. Ahmadi, A., Ng, S.C., 1999a. Fertilizing ability of DNA-damaged spermatozoa. J. Exp. Zool. 284, 696-704. Ahmadi, A., Ng, S.C., 1999b. Developmental ca pacity of damaged spermatozoa. Hum. Reprod. 14, 2279-2285. Aitken, R.J., Liu, J., Best, F.S., Richardson, D. W., 1983. An analysis of the direct effects of gossypol on human spermatozoa. Int. J. Androl. 6, 157-167. Aitken, R.J., Clarkson, J.S., 1988. Significance of reactive oxygen species and antioxidants in defining the efficacy of sp erm preparation techniques. J. Androl. 9, 367-376. Aitken, R.J., Clarkson, J.S., Fishel, S., 1989. Generation of reactive oxygen species, lipid peroxidation, and human sper m function. Biol. Reprod. 41, 183-197. Aitken, R.J., Fisher, H., 1994. Reactiv e oxygen species generation and human spermatozoa, the balance of benefit and risk. Bioessays. 16, 259-266. Allan, D.J., Harmon, B.V., Roberts, S.A ., 1992. Spermatogonial apoptosis has three morphologically recognizable phases and s hows no circadian rhythm during normal spermatogenesis in the ra t. Cell. Prolif. 25, 241-250. Alvarez, J.G., Storey, B.T., 1995. Differentia l incorporation of fa tty acids into and peroxidative loss of fatty acids from phos pholipids of human spermatozoa. Mol. Reprod. Dev. 42, 334-346. Amanai, M., Brahmajosyula, M., Perry, A.C ., 2006. A restricted role for sperm-borne microRNAs in mammalian ferti lization. Biol. Reprod. 75, 877-884.

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105 Anderson, D., 2005. Male-mediated developmenta l toxicity. Toxicol. Appl. Pharmacol. 207, 506-513. (supp) Anzar, M., He, L., Buhr, M.M., Kroetsch, T. G., Pauls, K.P., 2002. Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility. Biol, Reprod. 66, 354-360. Aoki, V.W., Wilcox, A.L., Thorp, C., Ha milton, B.D., Carrell, D.T., 2004. Improved in vitro fertilization embryo quality and pregna ncy rates with intracytoplasmic sperm injection of sperm from fresh testicular biopsy samples vs. frozen biopsy samples. Fertil. Steril. 82, 1532-1535. Armstrong, J.S., Rajasekaran, M., Chamulitrat W., Gatti, P., Hellstrom, W.J., Sikka, S.C., 1999. Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy meta bolism. Free Radic. Biol. Med. 26, 869880 Ashwood-Smith, M.J., Edwards, R.G., 1996. DNA repair by oocytes. Mol. Hum. Reprod. 2, 46-51. Attaran, M., Pasqualotto, E., Falcone, T., Go ldberg, J.M., Miller, K.F., Agarwal, A., Sharma, R.K., 2000. The effect of follicu lar fluid reactive oxygen species on the outcome of in vitro fertilization. In t. J. Fertil. Womens Med. 45, 314-320. Aziz, N., Said, T., Paasch, U., Agarwal, A., 2007. The relationship between human sperm apoptosis, morphology and the sperm deformity index. Hum. Reprod. 22, 14131419. Bakos, H.W., Thompson, J.G., Feil, D ., Lane, M., 2007. Sperm DNA damage is associated with assisted reproductive t echnology pregnancy. Int. J. Adrolo. 30, 1-9. Balakrishnan, K., Wierda, W.G., Keating, M.J., Gandhi, V., 2008. Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells. Blood. 112, 1971-1980. Ball, B.A., Vo, A.T., Baumber, J., 2001. Gene ration of reactive oxygen species by equine spermatozoa. Am. J. Vet. Res. 62, 508-515. Barros, C.M., Pegorer, M.F., Vasconcelos, J. L., Eberhardt, B.G., Monteiro, F.M., 2006. Importance of sperm genotype (indicus vers us taurus) for fertility and embryonic development at elevated temp eratures. Theriogenology. 65, 210-218. Baumann, C.G., Morris, D.G., Sreenan, J.M., Leese, H.J., 2007. The quiet embryo hypothesis, molecular characteristics favoring viability. Mol. Reprod. Dev. 74, 1345-1353.

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106 Baumber, J., Ball, B.A., Linfor, J.J., Meyers, S.A., 2003. Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. J. Androl. 24, 621-628. Bedford, J.M., Moore, H.D., Franklin, L.E ., 1979. Significance of the equatorial segment of the acrosome of the spermatozoon in eutherian mammals. Exp. Cell. Res. 119, 119-126. Bejarano, I., Lozano, G.M., Ortiz, A., Garca, J.F., Paredes, S.D., Rodrguez, A.B., Pariente, J.A., 2007. Caspase-3 activation in human spermatozoa in response to hydrogen peroxide and progesterone Fertil. Steril. (in press). Benchaib, M., Braun, V., Lornage, J., Hadj, S., Salle, B., Lejeune, H., Gurin, J.F., 2003. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum. Reprod. 18, 1023-1028. Bilodeau, J.F., Chatterjee, S., Sirard, M.A., Gagnon, C., 2000. Levels of antioxidant defenses are decreased in bovine spermato zoa after a cycle of freezing and thawing. Mol. Reprod. Dev. 55, 282-288. Blanco-Rodriguez J., 1998. A matter of death an d life, the significance of germ cell death during spermatogenesis. Int. J. Androl. 21, 236-248. Block, J., Chase, C.C. Jr., Hansen, P.J ., 2002. Inheritance of resistance of bovine preimplantation embryos to heat shock, re lative importance of the maternal versus paternal contribution. Mol. Reprod. Dev. 63, 32-37. Block, J., Drost, M., Monson, R.L., Rutledge, J.J., Rivera, R.M., Paula-Lopes, F.F., Ocon, O.M., Krininger, C.E. 3rd, Liu, J., Hansen, P.J., 2003. 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. Block, J., Hansen, P.J., 2007. Interaction betw een season and culture with insulin-like growth factor-1 on survival of in vitro produced embryos following transfer to lactating dairy cows. Theriogenology. 67, 1518-1529. Blondin, P., Coenen, K., Sirard, M.A., 1997. Th e impact of reactive oxygen species on bovine sperm fertilizing ability and ooc yte maturation. J. Androl. 18, 454-460. Brad, A.M., Hendricks, K.E., Hansen, P.J., 2007. The block to apoptosis in bovine twocell embryos involves inhibi tion of caspase-9 activat ion and caspase-mediated DNA damage. Reproduction. 134, 789-797. Brandriff, B., Pedersen, R.A., 1981. Repair of the ultraviolet-irradiated male genome in fertilized mouse eggs. Science. 211, 1431-1433.

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BIOGRAPHICAL SKETCH Katherine Elizabeth May Hendric ks was born in 1972, the first of two daughters, to E lsa May Binns and Lloyd Ivanhoe Binns, in St. Andr ew, Jamaica. In 2002, she received her degree in veterinary medicine from the University of the West Indies, St. Augustine campus, located in the Republic of Trinidad and Tobago. In January 2003 she started her masters program in the College of Veterinary Medicine at the University of Florida unde r the supervision of Dr. Louis Archbald. Her masters research focused on repro ductive strategies in the post partum cow with emphasis on anovulation and postpartum uterine health. In August 2004 Katherine was awarded a University of Florida Gra duate Alumni Fellowship and be gan working on a Doctor of Philosophy degree in the Animal Molecular an d Cell Biology Graduate Program under the supervision of Dr. Peter J. Hansen. After comple tion of her program, Katherine will continue her career in academia.