Change in dehydrodolichyl diphosphate synthase during spermatogenesis in the rat

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Material Information

Title:
Change in dehydrodolichyl diphosphate synthase during spermatogenesis in the rat
Physical Description:
xii, 146 leaves : ill. ; 29 cm.
Language:
English
Creator:
Chen, Zhong, 1944-
Publication Date:

Subjects

Subjects / Keywords:
Transferases -- metabolism   ( mesh )
Spermatogenesis   ( mesh )
Testis -- physiology   ( mesh )
Muridae   ( mesh )
Sertoli Cells -- physiology   ( mesh )
Biochemistry and Molecular Biology thesis Ph.D   ( mesh )
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references (leaves 137-145).
Additional Physical Form:
Also available online.
Statement of Responsibility:
by Zhong Chen.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 030567875
oclc - 18721388
System ID:
AA00025838:00001

Full Text















LCOHDURAI NG SPRAOEEI VN TH RATa# i &40um'R










ML~ZH N CHENMt M WN 4f




















ACKNWEGMN




Sincere appreciationi4xrse om red D r C Aharles M. Alln Jr1 fo hinndrn guidne and support. Ia epygaeu o
proofrednyEgih i ble ntefna

















TAB



ACKNOWLEDGEMENT S 0 * LIST OF TABLES..........

LIST OF FIGURES.........














APPENDICES

A SUMMARY OF EXPEIMNA DTAPENEDI
FIGURE 2-w12. DEHYR1O PSNHS CII
IN SONICATES OF TBLSFO ASO
D IFFERENT AGES.....*...*.*.*.*.............

B TYPICAL NUMBEROFRTTTL UBEWIGT
AND NUMBER OF ASSYUElFRECiEPRMN
AS A FUNCTION OFRA

C SUMMARY O XE
IGUrE^1T1" 3-5c AG DEPNDN VAITINI




















Table,

2-1w Incorporation of A'C-~jIoeney ihs
and (afia32P]-I*sopentnlDpopaeit
Dehydro DOL PP andDeyr DO

2-aw2 Formation of EnzymatcPouc tDffrn r


3-1 AA010 f Dehdr Doichy Dihopht Syths Activity 40

















LISTO IUE

Fig1 ure

1-1 The Cellular Composiino te1 tae fL
Cycle of the Seminifru Epteim nRa..

1-2 Schematic DrawingofHmnS iieru Eptl

13* DOL. Cycle for GlyrcopoenFraini










2-am10 A Double Reciprocal Po fteSmo eyr
DOL PP and Dehydro DOI omtonv.Frey
Diphosphate Concentraio

2-11 Time Course of Incorprto:f(4]loetn
Diphosphnate into DehyrlO Pan eyr O

2-12 Dehydro DOL PP SynthaeiletclrHmgnt
0of D iffe rent Age od Rats......*.*..

2-13 Dehydro DOL PP SynthaeAtvt]nSnctso
Tubules from Rats of ifrn ge......

2-14 Comparison of ChangesiO ocnrto
and Dehydro, DOL PP Snhs ciiya
F~~ -wrot io of Ra Age .f o% .: D ^ .o A .r ^

















KEY I




BSA bovi
C 0 1
I curi

Cm cent












Man manr


MEM, Eagl


micl

0
pc i mict

0
Ag MICI


JAl mici


MM, micl

0
jAmo 1 e micl




















Abstract of Di*ssertation
th Univers*t-- of FloAr
Requirements for the


CHANGE IN DEHYDROE
TNYYDTMr. QPVIDI










The a ct iv it y i n hooeasof ptae
seminferous tubulesancelfctosnr


spermatogenic c ellIs or Setlcls asohng

function of age in the ratTehges nyai

occurred in each case a g 3dy.Cl

enriched i*n pachytene spemtctsspraiso

cells were shown to havehgeytaeatvt whole testicular homogenate oramxue fclspe



















I



SR













preleptotene. primary speraoyewhcunrg replication of nuclear DNAbfr nern eoi

Thepre leptotene pr

c om p ar t ment to t headu nlcoprm t

spermatogenesis Is comltd Thscnd p

spermatogenesis, melo s is, ocrwhlte spm

remain on the adluminalsieo th inrclua












camera, a series of diffrn elascain

seen, until the cycle wa opltd Te tm

between the appearanceoftesmcllaoiti given point of the tuble s cle h yl

seminifru epithelium.Tenm rofsasint

is constant for a given speis a a ,muea

have 12,I the rat, 14 (Figur -)




























































#

#
#

#




to

It














-dO 0'
16












Exocrine and Endocrine u

The testis has both xciead norn u

The exocrine function ofth te isr idsnteC

the semini*ferous epitheliu hc rduetsiua

and spermatozoa. The enor e fucinf th
resides prmril in theLyi elpplto


synthesizes and secretes tepicplcruaiga












expressed during meiosisan sprigeisThe

b a sic c hrom os omal prten tha unegru

trans itions during speraoei cel dfee

(Meistrich et al., 1981), svrlsemtgncc1isozymes (Goldberg,, 197) ad saeseii glycoprote ins identifiedwt bohmca Hl




Millettei n 198 It ha bee sugete thtmn1












remainder of spermatogenest Fokta.,18)

is thought to be an essetartae rqie

proteolys is of the of thoou

f ertil ization (Har tree, 17) n prfe

proacrosin, glucosamine,maosglcsend il

were found in the ratio o ,,, e oeo r

which is consistent withth raiexcedo tes












described in 1865 by Sertol ohv usn uc

provide mechanical support o h eeoigsem

cells as well to be phaoyi't i eiv

circulating hormones, whic c n semtgfe

their effects mediated via h etl el(aso

1976)*

Sertoli cells are baialcounrels















under the inf luence of vrosfcosbtte divide any more.

Adjacent Sertoll cell r ondb h et

junctional complex, whichisauqestcur no f

other epithelium tissues.Thsfntoa bried

in the rat at about 16to1 das fag(Val

1973).* The Sertoli-mSertolucinlcmlxdv












]Physilolog ical and morhlgclstde aei

that the Sertol i cell undrg cylc cags i

metabolic activity which aerltdt pcfcs

the cycle of the s em inifru pteimyo

studies showed that severl ezm ciiis i

cells vary depending on te sae o h yl

s emi niferous epithelium. Fo intceth pak ci












have been studied, suchasadoebi inprti
(Fritz& et al,1976), plasmngnatvtr(ari


1977), test icular transferrn(kne trsod 9

sulfated glycoprotein 1 (S1I n S0I Ci

al., 1986).*

The stage specif icnaueothelbr in

glycoprotein, androgen bindnrrtihsbe at












characterized this compoundfo i ivr(enc

1960).o Mammal ian polyprenl oti agrnx

isoprene residues C85-mC115(123xC)ta ths pr

plants and bacteria C50-mC60(01 5.Plpeo

in bacteria have an oa-unsatuae speeui. Hw

is known that liver continsmlaoutof -a

shorter polyprenols (MankosieIl,17) n












liver, kidney, testis lugadhat) (ulra

1984)*

DOL may have someefetonmbresrct

fluidity (Valters son etal18)Otews, m

apparently has no direcieainhpt lc

synthesis siLnce the bulk o h O speetmn

other than the endoplasmineiuu Wn ta.












live r, and 65mw90% in moue tse n rpta

(Malvar et al. 1985). Th means wic dtrme


di*stribut ions isno ow


DOL P in Glycoprotein Synthei

In the early 1970s, BhesadLli eosr

invo lvement gflyco






























1 AuPGLcNAc

r%& aftLIA&ftfl













The f irst s te p i h sebyoii
oligosaccha ride involves teadto fGe~- r


GleNAc, to DOL P to gender OPGc~.Te molecule reacts with an addiinlUPGc~ ofr

(ClcNAc) 2. Five mannose resde r de etfo

mannose to form DOL PP-w(lcc)4a5Ithdbe oligos-Accha ^ riftd chaint In the olgoachaid lipi ,












D)OL P plays a majorrlinteboyhss linked sic DO P no onlyo ei


oligosaccharide unit carrie u as s natiao

reacts with certain nucleoiesgr n aiia

sugar transfer to the cor lgschrd his

involvement of DOL P inIh rdcino h

ol igosaccharide and t he usqettase












was inhibited by compacia ptn nii

hydroxymethyl glutaryl Goreutsancne

polyisopreno id b iosyntheipoin gyslai

impaired and the oligosacchrd cais yneizdw

negatively charged (Carson&Lnazc91.I report inhibition of DOLPboyteiinuda gastrulation in sea urchin mro Crsn&Lnaz












since DOL P is animotn prcsrofbh

mono saccharide, a nd DOPolgschrd

understandable that a shortgfDLPcudhv effects on the biosynthesiso"iidoioachrd cause the production of defciegyortin. Ca

shown that a mouse lymphomacl uat akn O

not synthesize DOL Pm*Man Camneil, 18)












controm-%Llling DOL P level I* the only de nov'o,, 'Di*osyntl "bridge" connecting the CoA, with the lar.cre DOL
0
pathway is deh--dro DOL p Y

synthesis of dehydro DOL
0
isopentenyl d'phosphate.


























































































"am 4m
VP laftL 0" Amp&

isopentenyl Diphubpl











Farnesyl D*phosphat(















40IS
EMEWL or m
DeL--Lvdro DOL PP





Synthase
-Imw,- -












present in the whole ratadithlve24r injec tO:ion. Furthermore, itwssilprsn0dy almost entirely as 4CDL The haflf ofDOAPi

liver has been estimate.,,d to e71 aso h ai

size of the DOL P pool.,h aaoi rdcsdn

labeled DOL have not yet bee on Rpe l* 95












condensation o f I s op e ntey ihshtis dime thylal lyl d Iph osphaepie ad te

c ons equential1 allyl ic dpopae ale is

experiments conf irmed ta h spee rsd

polyisoprenoid alcoholsaradeinasrece specific manner (Hemming,17) h i di







moeuftrans, trans-farnesyl diphsat (Fgr















Therefore, it is likely tha th ptwyofDLsne

h ave i1t s own regulatory ponwhcisnded cholesterol biosynthesis, hscudmk eyr

synthase a rate-limiting stpiDObosnhisT coupled with the findingthtDLsneisndv s yst e ms is greatly enhacdrltv o co

biosynthesis, makes it likely htlreicessi












cle a rly demons trate d hih DL snessn spermatogenic cell populatosHwve h n

enzymes that might be reonsbefrtinra synthesis were not. identifid

In order to understand ho4lcpoti se

coordinated wi1.t h di*f f eretain t iees

understand how the indiviul sesilh eu











































































Ammmm













d hj


qmwm












Adair and Keller (1982)hae sod tathen

products of the li.ver enzmweeagopfdhy monophosphates ranging insiefo C7to 95(5

Recent data f rom thi laoatr hae son t

testicular homogenates and termmrae fato

catalyze the synthesis of 7-8teyr O n

DOL PP f rom t Itmef arnesldpohaend ip













in spermatogenic cells. Tse rm tee mc

compared to normal control eosrtdmrel

ratios of1 C acetate incoprt nitoDLa cmp

cholesterol. These resultssgetdthttehg

DOL synthesis ina mouse teseia eatrbtdt

more types of spermatogenic el lhuhSroic

not be excluded. It wassusqetyhon ha pr












DOL P levels (day 15), it wssgetdta O i

function primarily in mainann, dqae eeso

for glycoprotein biosynthesi fe h nta us

P biosynthesis. Theref ore DO kiaewsp tu td

little or no effect in regltn(h iei O

during the ini1Lti#al phasso ifrnito

spermatogenesils. It was sgetdisedta ey












anxLd ro ge nic %.and tropic hormoeato nsemtgn

mediated by the Sertoli celsTheclshaebt androgen receptors (Sanboreta197Snb ne

1979; Means & Vaitukaitis17) adshwa*pr

temporal relationship b eten hroe b diga

response. For exampletee i ula cuua

androgen and stimulation o N oyeaeI ci












testicularan seu tr s

must play a role in the trasotoirnfo etl

to the spermatocytes and semtd.Mr eety

and Clermont (1986) haveshwtatdrn4 Sertoli cells and spermato niite alzdrnse

receptor-amed iateAtd endocytossa th bseo tesmi

ep ithe 3lum.*












obscure. Furthermore thequsinc ce igth ab

the Sertoli c ell to sytesz DOa3nt a

d ir e ctl1y.

Nyquist and Holt (198)rcnl mes edteC

and subcellular distributioofDLi ra te isb

method and f ound that el ritopu fed srm

cells had very low concnrtosoiO.P












The high content ofDO InteSrolclma

a requirement for high DOI opri ai

glycoprotein biosynthesis drn h praoeei

might be possible that Sertl eladsemtgfi

may have a coordinate pattr fd ooDLboy

which is dependent on theprsneo th ohrcl

This would require that eahcl tye avtecpa












those cells. Recent data rm u lbraoy sn

dependent mannosyl transferaeso httelvlo

increases dramatically fromdy7t ay2 npe

rats (Allen & Ward, 1987) hs tm nevl C

period when the first groupotpraoei el r

through differentiation to eoesemais ts

noted that the acrosomal enyeicuigtegyo












g ain ed f rom, the assessmenif dhdo DL P

ac t ivi* tie s tn S er t ol celnd dfern1y

spermatogen ic cells may prvdsoe iigtl

mechanism of biochemical cnrl o elfnto

d if ferent iat ion. Th is marliael eueu

development of male contracetvs

















.M
L
DEVELOP AND OPTIMIZE
DIPHOSPHATE SY',



Ir












dehydro DOL Ps ranging i ie fo 7 OC5

recently, Baba et al. (198)dsrbdteynhe
semiifeoustubules, but soe httepout


enzyme were both de'hydro DL P n-eyr Hydrolyxs.is of: both of thsIrdcs wt

phosphatase in the a'bsenceo a ilddtesm

length alcohols (C75-C90). h sltono ,-ey












nor dehydro DOL P was fore bu4rdut et

identif ied as presqualene mnpopaeacmltdi Therefore, it was necessary oetn h ale t

Baba et al. (1987) to ensrthtte sa eeo

the synthase act iv ity in cue tuua1omgnt

measuring the desired activiy

This chapter 1) showsta retetoftets












Material ad etod


MtriasMale SprgeDwe aswr i


from local suppliers. t t-Frey ihsht a

as previously described (Baa ndAln 17)(1l

Isopentenyl diphosphate an(3Porhpshri

( car r ier fr e e) in dilt H1wasprcae












Prep~aratioon of homogat.MlSp gu Dwe

were decapitated. The tested eermve n efs

enriched Krebs amRinger bicabnt eim(KB

testicular vessels. Thi prceur efetvlseo

blood cells from testes.Thtuiaabws

and seminiferous tubules weegnl xpesdunt

of the younger animals (3an 7dyso),te ets












eliminate the unwanted prdcsanolyila

quant itate dehydro DOL PP addhdoDLP pia

conditions were also establihd h eednyo

formation on Triton X-mlOO, poenioetnldpo

Larnesyl diphosphate concentainadtm eedtr

The standard assayofteezmwacrid

incubation of 100 mM TrisHl ufr H75 0m












Thin Layer Chromatogah TG fRato t

The remainder of extract (. l a ruh odye

a N2 stream. Five drops of ovn eeaddt h

residue and the tube was votxdtoouhyNh e

solution was applied to Siliagl6,npatcset werepreviously ctit

additional drops of SolventAwr sd ows h












carrier free) in dilute HC1 a re nterato

over P205 under N20 Then0. proe cysaln H30

moles triethylamine, 2pmls3 nehl3bt AO

12 moles trichioroacetonitiei]0p ctnti

added and the reaction permte top cedfr57h

room temperature. The reacto a tpe yadn

of 10 mM NH4OH. The reactinpout a eaae












hydrolysis products wereexrc dwih2mofSl

Then the lower phase was sbetdt L nlss

authentic markers, [14 C]dehdoDLPadnnrdo

DOL P were chromatographed i aall otehd

products and the developelL he a ujc

auto radio gr aphy .












fraction isolated from homoeae fsmnfru

from rat testes (Baba, et al'98)

It was necessary toopiietem hdtoac


assay the enzyme in homogenaeiftblstknfo of dif ferent ages 0 Homgnts wre peae

sonication of buffered supni s of tse ist

disruption with a glasshooei r asp vosde












32Ppisopentenyl d iph oshtan(4C iop

d iphosphatie were incubatedwtfanslipoha tubular homogenates in thestnad sayTh pruc

separated by TLC as usualadterto frd

incorporated f rom 3 an (pC-spntnldp

were determined. Since thecanlnth]ftepl

products have been establise ob tesm h







F










HOMOGENATE DOL~P A
240
LU
(I, FON
z 0
Co Lu


~I2O
U Lu
w
a w


0

SONICATE B
240 DOL- P
0, 2 0











SF





















Incorporation of 43 L'CI Isopentny ipopht ad afr2,-s
Diphosphate into Dehvr O PadDhdoDLp


Exeimn IEpeienN























2,0



cm


10
44 cm












Stimulated with increasing dtretcnetaintr the concentration range shoni h fgr hl

DOL P formation was optima t05 rtnXlO

suggests that when the Trito -O ocnrto a

tha 0.%,a previously actv

was inhi*bi*ted. The sum of dhdoDLP n eyr

production was unchangedatTioXl0cnetr i





















I st hour



00

41A cm 4 0
936 eft























v 4 LW

ion Enzyma, i c Ir( r m a t uA
Concentrate ions in












In all cases the sum o h w rdcsices

i n cre as in g t ime. Thisuprstersnc diphosphatase which is inhiie byhgrcoenat

Triton Xo=100,

The r es ult s of a smlry dsge us

experiment support the same ocuin(al -)

case none*radiol0abe led isopetnldpopaewsa












product pattern, WThe f ormato fth w rout n

linearly with time up to 60( m

Base ,,Hydrolys is ofDeyrDOP.Th

dehydro DOL PP was isolatedb L n hnsbe

saponifcto in 35M KOHi,0 ehnlfr2W

1000 CO F igure 2-5 shos tadeyrDOP

overwhelming product of bas yrlss hspo























150 moo

40


















Kinet ics of t he Enzym3h nyeasycn

were optimized. F igur e26shwteefcto v

protein concentration on thefraiofte eyr

(Panel1 A), dehydro DOL P (ae )adtesmo h

products (Panel C). Thee rsls iniaeIh

enzymati1c a ct iv ity i ncraeiery wt

concentration up to 2.4 mg poenicbtd












Peanel1 C) versus f arnesyl dpopaecnetain t ha t the apparent Km=22Mn mxO6 m

protein/mmn respectively.ThKmvlewreige those observed for dehydro DLP ytae foi
ascite s (Aair et al., 1984) hsmyrfetnns


absorption of the substratesb te rtisi h

homogenate and hydrolysis o h usrt yed





























Adwhk
tow u
"Ook M=

VOOOOO




















u
LM 00000




LM CL CY)
0
























0




10 C26 CO


























60 pow 15



























cm


- ---- .....



ANO
t=


luv












formation (Panel A) weresoehtmalrhncan dehydro DOL P formation (PanlB duigtscla

development. The sum of dhdoDLP n eyr

(Panel C) represents totalspcfcatvyof hi e

A composite of data fromthssudeuin18 shown in Fig. 2mw13, A twofodices intblra

of the synthase occurred btendy7addy2











DOL branches to chleteo athhe 1e


farnesyl diphosphate. I n vtoeprmnswtsp

diphosphate as a precursorhaeswntt23dhyr presumably one of the laein rmd tsinh

biosynthetic, pathway, couldb sytei dinp pa

from hen oviduct (Grange &Adip197,a anlvr(

& Lucas, 1979), Ehrlich tuocel(Aar &Tp












obtained from 3-m and 7-.day-l as o ntn

pooled size of ten testesfrm3dyod atis bo

size of a rice grain (notalncri!.Frh sonicat ion dntrsthe rnltaseaep L

diphosphate synthase, so tha oeo hesd rdc

the assay are eliminated (ig.21I.Terao

loss in this prenyl transferaeatvt nsncto












60 min as s-0hown in Fvig. 2,w11) eod hr seie

a de te rg e nt sensitive popaaeta cs0

diphosphate to gi ve the mophsat.Tidk experiments~ have shown a cascltm eedn

monophosphate formation ccmae odpopaefr

whereas total phosphorylatepoyenlicasdn Fourth, direct chemical exprmnswreas)efr












P and dehydro DOL PP, thrf ete dtrmn i

synthase, activity required mesr enofb hpo

not dehydro DOL P or dehydro O Paoe

DOL P is an indi~spensbecrir foioac

during glycoprotein biosynthsstefoeknw dg

availblt and the ti*ming fisboynhssdrn

stages of differentiationma beipr ntnudrs