Citation
Change in dehydrodolichyl diphosphate synthase during spermatogenesis in the rat

Material Information

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

Subjects

Subjects / Keywords:
Bees ( jstor )
Biosynthesis ( jstor )
Diphosphates ( jstor )
Enzymes ( jstor )
Glycoproteins ( jstor )
Ions ( jstor )
Pachytene stage ( jstor )
Rats ( jstor )
Sertoli cells ( jstor )
Testes ( jstor )
Biochemistry and Molecular Biology thesis Ph.D ( mesh )
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF ( mesh )
Muridae ( mesh )
Sertoli Cells -- physiology ( mesh )
Spermatogenesis ( mesh )
Testis -- physiology ( mesh )
Transferases -- metabolism ( 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.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Zhong Chen.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030567875 ( ALEPH )
18721388 ( OCLC )

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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







Full Text
36
those cells. Recent data from our laboratory using DOL P
dependent mannosyl transferase show that the level of DOL P
increases dramatically from day 7 to day 20 in prepuberal
rats (Allen & Ward, 1987). This time interval covers a
period when the first group of spermatogenic cells are going
through differentiation to become spermatids. It should be
noted that the acrosomal enzymes, including the glycoprotein
acrosin, are elaborated in early stages of spermatid
formation. Several other proteins required for glycoprotein
biosynthesis are also maximally expressed during this time
interval, e.g. galactosyl transferase, N-acetylglucosaminyl
transferase, and N-acety1glueosaminide fucosyltransferase in
mice testes (Letts et al., 1974a). It is true for the
androgen binding protein in rat Sertoli cell culture as well
(Rich et al., 1983). Therefore, we have measured the
specific activity of dehydro DOL PP synthase in spermatogenic
cells and Sertoli cells during testicular development in
order to determine its potential importance in the regulation
of spermatogenesis.
A knowledge of changes in the concentration of
intermediates and activities of enzymes in DOL metabolism is
important if we are to understand the regulation of
glycoprotein biosynthesis. Spermatogenesis offers a complex
but good model system to study control mechanisms of DOL
metabolism and DOL function in the biosynthesis of specific
glycoproteins during cell differentiation. Information


Figure 2-2. Triton X-100 Dependency on the Formation of
Dehydro DOL PP and Dehydro DOL P.
Incubations containing 100 mM Tris-HCl buffer
(pH7.5), 10 mM MgCl2, the indicated percentage of Triton
X-100, 250 /zM t,t-farnesyl diphosphate, 1.6 mM ATP, 50
mM NaF, 36 /zM [ 1 ] isopentenyl diphosphate, and 1.0
mg of enzyme protein in a final volume of 0.25 ml were
carried out at 37 C for 60 minutes. The formation of
[]-dehydro DOL PP (Panel A) and [^^C]-dehydro DOL P
(Panel B) were estimated by the method described before.
Panel C represents dehydro DOL PP synthase activity
(A+B).


126
peaked.
The number of pachytene cells relative to other
spermatogenic cells is maximum, and there are few if any
spermatids present by this time.
The number of Sertoli cells
has
become
relatively constant.
Therefore,
the
total
activity of synthase is due to the sum of Sertoli cell,
pachytene spermatocytes and other spermatocytes preceding the
pachytene stage
The specific activity is optimal at this
time, because Sertoli cell specific activity is highest at
this
po int
and
the
relative
percentage
of pachytene
spermatocytes
(the
spermatogenic
cell
with
the
highest
spec
ac tivity)
i s
also highe s t
th i s
time
o f
development.
Actually, it is the only time period in the
rat's life span that the relative percentage of pachytene
spermatocytes reaches a peak value (Fig. 3-6).
At 30 days of age, the pachytene spermatocytes still
have active synthase activity but the relative number of
these cells present in the seminiferous tubules is a smaller
fraction of the total spermatogenic cell population than at
day 23. The Sertoli cell number is constant but its synthase
specific activity has decreased by day 30. The contribution
of spermatids to the total activity is relatively low at this
time, since the number of spermatids is small although
increasing rapidly. Our data also indicate that as pachytene
spermatocytes
differentiate
into
spermatids,
the
enzyme
specific activity decreases by 30 % (Fig. 3-4). There was,
however, no net change in total enzyme activity during the


44
Thin Laver Chromato
TLC
Reaction Products.
The remainder of extract (4.5 ml) was brought to dryness with
a N2 stream. Five drops of Solvent A were added to the dried
residue and the tube was vortexed thoroughly. The resulting
solution was applied to Silica gel 60 on plastic sheets which
were previously cut into 4 cm
x
20 cm sections.
Five
additional drops of Solvent A were used to wash the sample
tube and this wash was added to the sample at the origin of
the TLC sheets .
The TLC sheets were then developed in a
chamber with Solvent C.
The developed TLC sheets were subjected to either
scanning with a radiochromatogram scanner (Packard model
7201) (Fig. 2-1-1) or autoradiography for 3-5 days on X-omat
AR
Kodak film (Fig. 2-1-II). The positions of migration of
authentic DOL P standard and the radiolabeled products were
correlated.
Sections corresponding to the migration of
dehydro
DOL
PP
and
dehydro
DOL
P
were
scraped into
scintillation vials and 10 ml of scintillation fluid was
added to each vial for radiochemical analysis.
The level of
was
expressed
m
pmoles
o f
[l^C]-isopentenyl
diphosphate incorporated into dehydro DOL PP and dehydro DOL
P /mg protein.
Preparation
of Isopentenyl
LqlJL
32
PI
Diphosphate.
32
Isopentenyl [a,/3- ^ P] diphosphate was synthesized fro
3-
methy1-3-buten-1-o1 and ^Pi according to the procedure of
Cramer and Bohm ( 1959). [ ^^P ] -Orthophosphoric acid (0.5 mCj_,


117
Table 3-1
Dehydro Dolichyl Diphosphate Synthase Activity in
Enriched Spermatogenic Cellsa
Experiment Activity Ratio
pmoles/10^ Cell hr
Pachytene (P)
Spermatid (S)
P/S
1
34.9
7.9
4.4
2
33.1
7.9
4.1
3
22.9
5.9
3.9
4
25.0
4.5
5.6
Average
28.9 + 2.9
6.5 4* 0.8
4.5 + 0.4
aEnzyi
described
activity was measured in cellular sonicates as
in the Materials and Methods and expressed
isopentenyl diphosphate incorporated per 10^ cell per
cell numbers were calculated by using the conversion factors 258
protein 10^ cells and 83 /g protein = 10^ cells, for
and
pmoles of
hour. The
Mg
pachytene
spermatocytes
unpublished data).
spermatids, respectively (from L. J. Romrell's


124
at day 60 near that seen at day 7.
The increase in DOL P
levels is consist with the observations of Nyquist and Holt
(1986), who showed an increase in DOL concentrations in rat
testes during this time period and the work of Potter et al.
(1981b), who showed a high rate of DOL biosynthesis from
by
mouse pachytene spermatocytes.
Potter and
coworkers
also
showe d
that
hydroxymethyl
glutary1
CoA
reductase activity was high in pachytene spermatocytes.
Nyquist and Holt (1986)
have
also
reported
that
DOL
concentration was high in Sertoli cells and suggested that
DOL may be synthesized in the spermatogenic cell then
transported to and accumulated in the Sertoli cell.
This
conclusion was supported by the observation by James and
Kandutsch (1980c) that testes of x-irradiated mice or testes
of mutant
ice severely deficient in spermatogenic cells (but
with apparently normal Sertoli cells) incorporated acetate
into DOL at a 20 fold lower rate than normal testes
However, the results reported here show that both the
spermatogenic
cells
and
the
Sertoli
cells
contribute
substantially to the dehydro DOL PP synthase activity of rat
seminiferous
tubules during early
o f
testicular
development.
This is not surprising considering the active
glycoprotein biosynthesis occurring in both cell types, the
rapid changes occurring in the spermatogenic cell during
differentiation and the active role of the Sertoli cell
in
supporting this development.


141
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Biochem.
Keller, R, K.,
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L. W., 1985, Anal. Biochem. 147:
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Kornblatt, M. J., Knapp, A., Levine, M., Schachter, H., and
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Kornfeld, R., and Kornfeld, S. 1980, in The Biochemistry of
Glycoproteins and Proteoglycans (Lennarz, W. J., ed.).
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Commun. 58: 287-295.
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Endocrinol. 9: 227-236.
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Commun. 141: 861-869.


pinoles Isopentenyl Diphosphate Incorporated/ mg protein hr
no
10 20 30 40 50 60
DAYS AFTER BIRTH


40
nor dehydro DOL P was formed, but a product, tentatively
identified as presqualene monophosphate accumulated instead.
Therefore, it was necessary to extend the earlier studies of
Baba et al ( 1987 ) to ensure that the assay developed for
the synthase activity in crude tubular homogenates was
measuring the desired activity.
This chapter 1) shows that treatment of the testicular
homogenate by sonication yielded good dehydro DOL PP synthase
activity while greatly reducing the formation of farnesol via
another prenyl transferase activity; 2) describes the optimal
parame ters
for
the
dehydro DOL PP synthase
3)
demonstrates
unequivocally
the
precursor product
relationship between dehydro DOL PP and dehydro DOL P, and 4)
elucidates a change in the enzymatic activity for dehydro DOL
PP synthase in the seminiferous tubules during early stages
of development.
A possible role of the dehydro DOL PP
synthase in regulating the biosynthesis of DOL is discussed.
The two-fold increase in the specific activity of this
synthase between day 7 and day 23 and a similar decrease in
activity between day 23 and day 60 shown in this chapter
provided the impetus to evaluate (in Chapter III) the enzyme
specific activity in different cellular populations of rat


143
Nyquis t,
s.
E. ,
and
Holt, S.
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D .
A. ,
and
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C. F. 1984,
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Parvinen, M. 1982, Endocrine Rev. 3: 404-417.
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Nature. 186: 470-472.
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, S. R., and Rothman, J. E. 1987, Ann. Rev. Biochem.
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Chem, 256: 3762-3769.


130
Duraiswami
(1986)
have
concluded,
based
o n
a n
autoradiographic study, that the protein synthetic potential
of Sertoli cells is greater than that of spermatogenic cells
at any stage of differentiation.
The parallel results seen
between that study and results represented here may be highly
significant and suggest a key role for dehydro DOL PP
synthase in regulating glycoprotein biosynthesis in Sertoli
cells during spermatogenesis in rats.
The reason for the rise and fall of the enzyme specific
activity in Sertoli cells in the immature testes is not well
unders tood.
Enzyme activities and protein secretion in
rodent seminiferous tubules have been shown to peak at about
day 20 to day 23 in several cases (Parvinen,1982). This has
been attributed in some cases to the appearance of pachytene
spermatocytes with their constituent enzymes and in other
cases to the onset of new protein synthesizing activities in
Sertoli cells.
In the current study, the results showed that
both Sertoli cell and spermatogenic cells are contributing to
the increase of the dehydro DOL PP synthase activity.
The
possibility that the presence of the pachytene spermatocyte
may cause an increase in Sertoli cell synthase activity is an
interesting conjecture, which has been postulated for other
systems (LeMagueresse & Jegou, 1986; Ireland & Welsh, 1987)
and requires further study.


35
The high content of DOL in the Sertoli cell may reflect
a requirement for high DOL P to permit a rapid rate of
glycoprotein biosynthesis during the spermatogenesis. It
might be possible that Sertoli cell and spermatogenic cells
may have a coordinate pattern of de novo DOL biosynthesis
which is dependent on the presence of the other cell type.
This would require that each cell type have the capacity to
synthesize DOL without relying on intercellular DOL
transport. In this way the Sertoli cell can independently
regulate the level of DOL P and hence the synthesis and
secretion of glycoproteins. Consequently, Sertoli cells can
regulate spermatogenesis. Therefore, it was of interest to
determine the capacity of Sertoli cells for de novo
biosynthesis of DOL P in order to have a better understanding
of the relationship between the Sertoli cells and
spermatogenic cells during testicular development.
Significanee
Dehydro DOL PP synthase is obviously a prime candidate
as a regulated enzyme in the DOL biosynthesis and
glycoprotein synthesis. It was of interest to determine if
increased dehydro DOL PP synthase activity correlated with
the increased rate of DOL synthesis in specific types of
spermatogenic cells and the high DOL content in Sertoli
cells, particularly since glycoprotein synthesis is active in


132
Chapter II support the hypothesis that increasing DOL
synthesis observed during testicular development (Potter et
al., 1981b) is due at least in part to an increase in the
dehydro DOL PP synthase activity.
The cellular localization of this increased synthase
activity was of interest because of the multicellular nature
of the testicular tubules. The specific activity of synthase
in homogenates of protease treated seminiferous tubules, cell
fractions enriched in spermatogenic cells or Sertoli cells
peaked in rats aged 23 days, as shown with non-protease
treated cells. Homogenates of cell fractions enriched in
pachytene spermatocytes, spermatids or Sertoli cells had
higher synthase activity than a whole testicular homogenate
or a mixture of cells prepared by protease treatment of
tubules. Enzymatic activity in pachytene spermatocytes
expressed per mg protein, was about 5.3 fold higher than
spermatogonia, 1.7 fold higher than in spermatids and about
8.3 fold higher than in spermatozoa. Therefore, the increase
of the synthase activity in spermatogenic cell before day 23
can be accounted for by the appearance of the pachytene
spermatocytes. Little net increase in enzyme occurred during
or after meiotic cell division of pachytene spermatocytes
into spermatids. The enzymatic activity decreased remarkably
during the differentiation of spermatids into spermatozoa.
It is reported for the first time that Sertoli cells
have the potential to synthesize DOL P. The enzymatic


133
activity in enriched Sertoli cells was 1.5 to 2.3 fold higher
than in the enriched spermatogenic cells between day 14 and
day 30 .
The increase in synthase activity in spermatogenic
cells and Sertoli cells indicates that both are contributing
to changes in the enzymatic activity in seminiferous tubules.
In
addition, and perhaps more significantly, the work
presented here provides evidence that dehydro DOL PP synthase
may be important in regulating the availability of Dol P for
glycoprotein synthesis during early stages of spermatogenesis
in rat
Future research on the role that dehydro DOL PP
synthase plays in spermatogenesis in rat will focus on
several fundamental questions. In vitro studies on the
incorporation of radiolabeled probe, such as mevalonate,
into DOL with enriched Sertoli cells will determine whether
synthase activity function in these cells in vivo. Synthase
activity would also be measured in co-cultures of Sertoli
cells and different spermatogenic cells. This could test
the role of cell-cell interaction with respect to the
function of DOL P in regulating spermatogenesis. Activators
or inhibitors of this enzyme may also be produced as a
result of cell-cell interactions. Such questions can now be
approached with the techniques and information presented in
this dissertation.


LIST OF TABLES
Table Page
2-1 Incorporation of A^-fl-^^C] Isopentenyl Diphosphate
and [ a,^9-]-Isopentenyl Diphosphate into
Dehydro DOL PP and Dehydro DOL P 52
2-2 Formation of Enzymatic Product at Different Triton
X-100 Concentrations in Pulse-Chase Experiment 58
3-1 Dehydro Dolichyl Diphosphate Synthase Activity in
Enriched Spermatogenic Cells 117
3-2 Estimated Specific Activities of Dehydro DOL PP
Synthase in Pure Sperma togenic Cells 120
3-3 Estimated Specific Activities of Dehydro DOL PP
Synthase in "Pure" Sertoli Cells From Rats of
Different Ages 121
vi


Figure
PP .
2 5 Product of
Hydrolysis of Dehydro DOL
Dehydro DOL PP and dehydro DOL P were prepared by
biosynthesis and isolated by TLC as described in the
Methods. (A) Dehydro DOL PP was saponified. The
hydrolysis product was extracted and chromatographed on
Silica 60 F254 in Solvent C as described in the text.
(B) as a control, dehydro DOL P was extracted from
Silica 60 TLC sheets and chromatographed as described in
A. The position of migration of authentic DOL P is
shown by the arrow I. The position of dehydro DOL PP is
shown by the arrow II.


123
The timing of the secretion of glycoproteins such as
androgen binding protein and plasminogen activator by the
Sertoli cell, has been described in several laboratories
(Parvinen,
1982) .
Furthermore,
there are now numerous
reports which show that the secretion of androgen binding
protein may be regulated by the type of spermatogenic cells
associated with the Sertoli cell at different times during
the spermatogenic cycle (LeMagueresse et al 1980; Ritzen et
al., 1982; Galdier, 1984).
Recent studies indicated that the mammalian testis
exhibits unusually high rates of DOL synthesis. This could
be related to high rates of glycoprotein biosynthesis, and to
temporally regulated synthesis of acrosomal enzymes in late
pachytene spermatocytes
o r
early spermatids
(J ames
&
Kandutsch, 1980c; Wenstrom & Hamilton, 1980; Potter et al.,
1981b). Acrosomal enzymes may also represent end products of
dolichol-mediated glycosylation in the testis since many of
these constituents are glycoproteins (Flechon, 1979; Mukerji
& Meizel, 1979).
Since DOL P has such a critical role in N-
linked glycoprotein biosynthesis, its availability is also
potentially regulatory factor of glycoprotein biosynthesis
during spermatogenesis. The results of previous work (Allen
6c Ward, 1987; Chapter II) showed that the level of DOL P and
dehydro DOL PP synthase increased in parallel between day 7
and day 23 of spermatogenesis in the seminiferous tubules of
immature rats
Synthase activity then decreased to a level


94
prepuberal rats. The highest activity of this enzyme
occurred in each case with cells from rats aged 23 days.
Homogenates of cell fractions enriched in pachytene
spermatocytes, spermatids or Sertoli cells were found to have
higher synthase activity than a whole testicular homogenate
or a mixture of cells prepared by protease treatment of
tubules. The specific enzymatic activity in pachytene
spermatocytes expressed per mg protein, was about 1.7 fold
higher than in spermatids and about 8.3 fold higher than in
spermatozoa. Therefore, the increase in spermatogenic cell
synthase before day 23 can be accounted for by the appearance
of the pachytene spermatocytes. Generally speaking, little
net increase in enzyme occurred during or after meiotic cell
division of spermatocytes into spermatids. Enzymatic
activity decreased remarkably after the differentiation of
spermatids into spermatozoa. Enzymatic activity in the
enriched Sertoli cells was 1.5 to 1.7 fold higher than in the
enriched spermatogenic cells between day 15 and day 30 of
age. The increase in synthase specific activity in
spermatogenic cells and Sertoli cells indicates that both are
contributing to changes in the enzymatic activity in
seminiferous tubules. This change may be important in
regulating the availability of DOL P for glycoprotein
synthesis during early stages of differentiation.
Spermatogenesis proceeds through a precise sequence of
biochemical and morphological phases, spermatogonial, meiotic


IPP (uM)
pmoles Isopentenyl Diphosphate Incorporated/ mg protein* hr
%


137
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31
DOL P levels (day 15) it was suggested that DOL kinase may
function primarily in maintaining, adequate levels of DOL P
for glycoprotein biosynthesis after the initial burst of DOL
P biosynthesis. Therefore, DOL kinase was postulated to have
little or no effect in regulating the rise in DOL P levels
during the initial phases of differentiation during
spermatogenesis. It was suggested instead that dehydro DOL
PP synthase or DOL PP phosphatase may be the putative
regulatory enzyme which directly affects the levels of DOL P
in spermatogenic cells at different stages of development.
The studies described here will extend our understanding of
the regulation of DOL P metabolism in the testicular system.
Glycoproteins in Sertoli Cells
Sertoli cells are histologically and physiologically
fundamental for spermatogenesis, since they are the only
somatic epithelial component of the seminiferous tubules
(Fawcett, 1975).
The close physical association of Sertoli
cells with the spermatogenic cell and the organization of
this association into a cyclic pattern have been described in
detail (Clermont & Perey, 1957).
The characterization of
Sertoli cells as nursing cells of testis was based originally
on the morphological cellular relationship in the testis.
The concept of Sertoli cells functioning as a support or
regulatory factor has been confirmed by both biochemical and
endocrine
s tudie s.
It has
been postulated
that
the


I /V (hr/pmoles)
72


BIOGRAPHICAL SKETCH
Zhong Chen was born in Beijing, China, in 1944.
1962, Zhong began his premedical education at Peking
In
University. Later Zhong studied medicine at China Medical
College (the former Peking Union Medical College founded by
the Rockefeller Foundation) and he received the M.D. degree
in 1968. After graduation he practiced medicine for several
years and specialized in ophthalmology. In 1981, Zhong
visited the Royal Hospital (Rigshospitalet) and the
University of Copenhagen in Denmark as a visiting scholar in
the Departments of Ophthalmology and Virology. During that
period of time, Zhong realized that biochemistry and
molecular biology are the keys to open the mysterious kingdom
of medicine.
So
he decided to brush up on biochemistry and
use it as a tool to explore the unanswered questions in
medicine.
After finishing his Ph.D., Zhong will move to Oklahoma
t
City, Oklahoma, and the laboratory of Dr. Jordan J. N. Tang
in the Oklahoma Medical Research Foundation to continue his
training.
146


I /V (hr/pmoles)
0
10 20
30 40 50
1/ FPP
(.mM)


103
Dehydro POL PP Synthase Assay. The enzyme activity was
determined by the same assay method described in chapter II.
The enzyme protein (1 mg) from homogenats of enriched cell
populations or various cell mixtures were assayed as
indicated. The products, dehydro DOL PP and dehydro DOL P
were extracted with CHCI3/CH3OH (2:1), isolated by TLC and
quantitated as described in Chapter II. The level of
enzymatic activity was expressed as the sum of the pmoles of
isopentenyl diphosphate incorporated/mg protein. The
relationship of these two products were extensively discussed
in the previous chapter.
Results
Synthase Activity in Protease Treated Seminiferous
Tubules.
The synthesis of dehydro DOL PP and dehydro DOL P
from farnesyl diphosphate and [ ^C] isopentenyl diphosphate
was compared in sonicates of pro tease treated and untreated
tubules from rats aged 7-65 days (Fig. 3-2).
It is necessary
to determine if the enzyme in mixed tubular cell populations
has the same enzymatic properties as that in the isolated
tubules.
In another words, did proteases treatment change
the enzymatic properties of
the separated tubular cells?
The fluctuation in enzyme specific activities in the protease
washes. Therefore, the enzymatic activities measured in the
study do not need to be corrected.


Figure 2-13. Dehydro DOL PP Synthase Activity in
Sonicates of Tubules from Rats of Different Ages.
The enzymatic activity was assayed under standard
conditions with sonicates of seminiferous tubules as
described in the Methods. The data ^s presented as the
mean + standard deviation (x +
parentheses indicate the number
prepare the tubules.
) .
Numbers in
animals used to


Therefore, it is likely that the pathway of DOL synthesis may
have its own regulatory point which is independent of
cholesterol biosynthesis. This could make dehydro DOL PP
synthase a rate limiting step in DOL biosynthesis. This,
coupled with the finding that DOL synthesis in developing
systems is greatly enhanced relative to cholesterol
biosynthesis, makes it likely that large increases in dehydro
DOL PP synthase activity might accompany or precede an
increase in glycoprotein synthesis.
Studies Related to DOL P Biosynthesis and Spermatogenesis
The Role of Dehydro DOL PP in DOL Metabolism
Testicular tissues contain large quantities of DOL and
are actively engaged in glycoprotein synthesis. Early
studies on human tissues by Rupar and Carroll (1978) using
gravimetric methods for determining DOL concentrations
suggested that testis contained more DOL than any other
organ.
James and Kandutsch (1980b) suggested that one or more
of the spermatogenic cell types are responsible for the high
rate of DOL synthesis observed in normal testicular tissue.
Further studies of Potter et al. (1981b) showed that purified
mouse spermatogenic cell populations are capable of DOL
synthesis. The pachytene spermatocyte were the most active,
whereas the round spermatids are less active. These studies


140
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LIST OF FIGURES
Figur e
Page
1-1 The Cellular Composition of the 14 Stages of the
Cycle of the Seminiferous Epithelium in Rat 4
1-2 Schematic Drawing of Human Seminiferous Epithe1ium....9
1-3 DOL Cycle for Glycoprotein Formation in
Eucaryotic Cells 16
1-4 A Putative Pathway Showing the Relationship between
Dehydro DOL PP and Glycoprotein Biosynthesis 22
1-5 Pathway of Isoprenoid Biosynthesis 25
1-6 The Structure of Dehydro DOL PP and DOL PP 28
2-1
2-2
2-3
Separation of Enzymatic Products by TLC 49
Triton X-100 Dependency on the Formation of
Dehydro DOL PP and Dehydro DOL P 53
Dependence of Product Formation on Triton X-100
Concentration and Incubation Time 56
2-4 Time Course of Dehydro DOL PP and Dehydro DOL P
Formation 61
2-5 Product of Base Hydrolysis of Dehydro DOL PP 63
2-6 Effect of Protein Concentration on Dehydro DOL PP
and Dehydro DOL P Formation 67
2-7 Isopentenyl Diphosphate Concentration Dependency on
the Formation of Dehydro DOL PP and Dehydro DOL P....69
2-8 A Double Reciprocal Plot of the Sum of Dehydro
DOL PP and Dehydro DOL P Formation vs. Isopentenyl
Diphosphate Concentrations 71
2-9 Farnesyl Diphosphate Concentration Dependency on
the Formation of Dehydro DOL PP and Dehydro DOL P....73
vii


98
different purified spermatogenic cell populations and in
Sertoli cells. A model is presented which explains the time
dependent change of the dehydro DOL PP synthase specific
activity in tubules of prepuberal rats.
Materials and Methods
Materials.
Sprague-Dawley rats were obtained from
local suppliers.
t, t-Farnesyl diphosphate was prepared as
previously described (Baba & Allen, 1978). [^C]-A^-
Isopentenyl diphosphate was purchased from Amersham/Searle
C o r p .
Bovine
ser urn
albumin
(BSA),
tryp sin,
trypsin
inhibitor, deoxyribonuclease, and collagenase were purchased
from Sigma Chemical Corp.
All other chemicals were of
reagent grade.
Solutions.
Phosphate buffered saline, essential amino
acids (BME 50X) and MEM nonessential amino acids were
obtained from Gibco Labs.
Enriched Krebs-Ringer bicarbonate
medium (EKRB) contained 120.1 mM NaCl, 4.8 mM KC1, 25.2 mM
NaHC03, 1.2 mM KH2P04, 1.2 mM MgS04*7H20, 1.3 mM CaCl2, and
was
enriched by the addition of 11 mM glucose, 1 mM
glutamine, 10 ml/liter of essential amino acids, and 10
ml/liter nonessential amino acids. Streptomycin sulfate (100
/ig/ml) and penicillin G (K+ salt) (60 /ig/ml) were also added
to the
e dium.
The solution was prepared from a stock
solution
immediately prior
to use,
filtered (0.30 //


21
controlling DOL P level in the cells, since this pathway is
the only de novo biosynthesis pathway known and serve as a
"bridge connecting the small metabolites, such as acetyl
CoA, with the large DOL molecules.
A key enzyme of this
pathway is dehydro DOL PP synthase, which catalyzes the
synthesis of dehydro DOL PP from farnesyl diphosphate and
isopentenyl diphosphate. Dehydro DOL PP synthase could be an
important cellular regulator of glycoprotein biosynthesis as
consequence of its regulation in the DOL P de novo
biosynthesis.
A postulated pathway showing the important
role of dehydro DOL PP synthase in DOL P and glycoprotein
biosynthesis is demonstrated in Figure 1-4.
However, this
enzyme has not been well studied.
One approach to clarifying the fate of DOL in vivo has
been to inject this compound into an experimental animal and
thereafter monitor its appearance in various organs,
injection of ^H-DOL into the bloodstream of a rat
After
the
radioactivity rapidly appeared
i n
the
high
density
lipoprotein (HDL) fraction of blood, with subsequent uptake
into all tissues (Keenan et al., 1977 ).
Since Elmberger
suggested that rat liver might be the main or exclusive site
of DOL synthesis, the presence of DOL in the high density
lipoprotein fraction of blood may point to high density
lipoprotein as a DOL transporter (Elmberger et al., 1987).
The rate of clearance of
14
C-DOL from tissues of the
rat is very slow, since about half the radioactivity is


101
purity. The cells were examined by Nomarski differential
interference and phase microscopy. The samples of enriched
early spermatids (stages 1 through 8) and pachytene
spermatocytes were pooled separately, washed three times with
phosphate buffered saline solution and used immediately for
the measurement of the enzymatic activity. The cellular
purity of the pachytene spermatocyte (Fig. 3-1-A) was 70%;
the major contaminants being Sertoli cells and spermatids.
The purity of the spermatid (Fig. 3-1-B) fractions was 80%,
with pachytene spermatocytes, the primary contamination.
Spermatozoa were obtained from adult rats (3 months of
age). The cauda epididymis was removed and flushed with 1 ml
of phosphate buffered saline via the ductus deferens. The
collected spermatozoa were then washed three times in
phosphate buffered saline and used for the enzymatic assay.
Sertoli Cell Preparation. Sertoli cells were prepared
by modification of the procedure of Dorrington et al. (1975).
Sertoli cells were isolated from Sprague-Dawley rats of
specific ages. Tubules were treated with proteases as
described above except that phosphate buffered saline was
used in place of EKRB and more (3 /g/ml) DNAse was used. The
process was similar to that for spermatogenic cell
fractionation, only the Sertoli cells retained on the nylon
filter were collected as the fraction enriched in Sertoli
cells. These aggregates of 10-50 Sertoli cell was further
treated for 3-4 min with a hypotonic solution of two-fold


7
remainder of spermatogenesis (Florke et al 1983). Acrosin
is thought to be
an
essential protease required for the
proteolysis
o f
the
zona
pellucida o f
the
ovum during
o n
(Hartree,
19 7 7 ) .
In
purified rabbit
proacrosin, glucosamine, mannose, galactose and sialic acid
were found in the ratio of 3,3,1,1 per mole of proacrosin
which is consistent with the ratio expected of these sugars
m
N-linked glycoproteins
(Muke rj i
&
Me iz e1,
1979) .
Therefore, proacrosin would be expected to be synthesized via
p a thway which
invo1ve d
dolichyl phosphate (DOL P)
me tabolism.
A general study of the glycosylation of protein would
be of value in understanding factors which might regulate the
biosynthesis of these cell and stage specific proteins.
An
understanding of the origin, metabolic pathway and mechanism
of biosynthesis of these glycoproteins during spermatogenesis
may provide insight into the biochemical control of the cell
function and differentiation.
These findings may be of
significance in the design of male contraceptive agents and
in understanding molecular basis of male sterility.
Sertoli Cell
Histological Structures and Functions
The Sertoli cells are the nongerminal elements in the
seminiferous tubules of the testes. They were first


PMOLES ISOPENTENYL DIPHOSPHATE INCORPORATED/MG PROTEIN
112
*


Figure 3-5. Age Dependent Variation in Synthase
Activity in Sertoli Cells, Spermatogenic Cells and
Protease Treated Seminiferous Tubules.
Synthase
activity
was
measured
under
standard
conditions in
sonicates
o f
Sertoli
cells
( O ) ,
spermatogenic
cells filtrate
from
protease
treated
seminiferous
tubules (
A )
, and
protease
treated
seminiferous
tubules (
)
prepared from
rats of
different ages.


90
saccharides.
Berkowitz and Nyguist (1986) showed a sharp
rise in kinase activity at 21 days of age with a peak at 24
days .
Allen and Ward ( 1987 ) showed a similar change in
kinase activity, but the rise in activity appeared to be more
gradual with a peak in activity at about 30 days and with one
half maximal activity between day 20 and day 25.
It seems
unlikely that change in DOL kinase activity account for the
changes in DOL P levels.
It has been suggested that alterations in the levels of
the active, phosphorylated form of dolichol regulate the rate
of N-linked glycoprotein synthesis (Lucas, 1979; Carson &
Lennarz, 1979; Carson & Lennarz, 1981).
The present results,
which show that dehydro DOL PP synthase changes in specific
activity at early stage
of testicular development in rats
(Fig. 2-13) suggest that increased DOL levels must accompany
fluctuations in glycoprotein biosynthesis observed during
this time period (Letts et al., 1978).
The high rate of
testicular DOL synthesis shown by Kandutsch and co-workers,
as well as the temporal changes in DOL metabolism shown here
during sperm differentiation, strongly suggest that membrane
glycoprote ins
and
alterations
m
the

timing
o f
the ir
appearance may be significant regulators of spermatogenesis.
Potter et al (1981b) showed a high rate of acetate
incorporation into DOL in pachytene spermatocytes of adult
ouse testes
At the same time, they also found increased
HMG CoA reductase activity in these cells. Although HMG CoA


SYNTHASE ACTIVITY, pMOLES/MG PROTEIN HR
116
TUBULES TUBULES SPERMATO- PACHYTENE SPERMATID SPERM
PROTEASE GONIA
TREATED


127
division of pachytene spermatocytes into spermatids (Table 3-
1). Most of the enzymatic activity is lost during the
differentiation of spermatids to spermatozoa. Therefore, the
decrease in tubular synthase activity after 23 days can be
accounted for by the decreasing fraction and specific
activity of Sertoli cells and the fact that the pachytene
spermatocytes, which have the highest spermatogenic cell
specific activity, are becoming a smaller fraction of the
total spermatogenic cells population and are being replaced
by spermatids with lower specific activity.
Kumari and Duraiswami (1986) have estimated the
percentages of Sertoli and spermatogenic cell populations in
rat seminiferous epithelium at various days during early
stages of spermatogenesis. If these percentages are used in
conjunction with the enzyme specific activities observed here
for the Sertoli and mixed spermatogenic cell population, the
predicted enzymatic activities of the whole tubular
homogenate can be calculated for animals 15, 23, and 30 days
of age (Fig. 3-6). There was a good correlation between the
predicted specific activity and the observed activity from
this study except for the result on day 15.
In general, the specific activity of dehydro DOL PP
synthase in Sertoli cell was always higher than that in
spermatogenic cells measured between day 7 and day 65 of age.
This suggests that glycoprotein biosynthesis is more active
in Sertoli cell than in spermatogenic cells. Kumari and


37
gained from the assessment of dehydro DOL PP synthase
in
Sertoli
cells
and
different
types
o f
spermatogenic
cells
may provide
some
insight into
the
mechanism of biochemical control of cell function during
differentiation.
This may ultimately be useful in the
development of male contraceptives.
Obiec tive s
The main objectives of this dissertation are to study
dehydro DOL PP synthase and dehydro DOL PP phosphatase during
spermatogenesis in rat.
Specific objectives are the following:
A. Optimize the assay conditions for dehydro DOL PP
synthase.
B. Characterize the enzymatic products.
C. Determine the synthase specific activities in
homogenate of testicular tubules of different aged
D.Determine enzymatic activities in homogenate of
different spermatogenic cell types (pachytene
spermatocytes and spermatids), and Sertoli cells
from rats testes.
Chapter II of this dissertation describes the study in
achievement of objectives A through C. Chapter III describes
the work in fulfillment of objective D.


85
obtained fro
3- and 7-day-old rats
For instance, the
pooled size of ten testes from 3-day-old rats is about the
size of a rice grain (not a long grain!)
Furthermore ,
denatures
the
p r eny1
farne sy1
diphosphate synthase, so that some of the side products from
the assay are eliminated (Fig. 2-1-1).
The reason for the
loss in this prenyl transferase activity on sonication is not
clear.
Possibly, these enzymes are more sensitive to the
heat generated by the sonication, despite cooling during this
process.
It was necessary to optimize the conditions to
accurately assay the specific enzymatic activity in sonicated
seminiferous tissue
from rats
of
different
ages.
Several
pieces of evidence
support
the
premise
that
the slow
migrating TLC component (Rf=0.47) (Fig. 2-1), identified as
dehydro DOL PP, was the initial enzymatic product, which was
subsequently hydrolyzed in vitro to dehydro DOL P. Earlier
work showed that the chain length of both the mono- and
diphosphate derivatives of the polyprenyl product were the
same (C75-C90) (Baba et al 1987 ). The present study
established more clearly the precursor-product relationship
between the products dehydro DOL PP and dehydro DOL P.
First, the sum of the mono- and diphosphorylated polyprenols
increased linearly with a variety of increasing variables,
i. e. Triton X-100 concentration (0 to 0.5% as shown in Fig.
2-2), protein (0 to 2.4 mg as shown in Fig. 2-6), time (0 to


17
The
first
step
in
the
as s embly
o f
1ip id- 1inked
oligosaccharide involves the addition of GlcNAc-P from UDP-
GlcNAc to DOL P to generate DOL PP-GlcNAc.
Then, this
molecule reacts with an additional UDP-GlcNAc to form DOL PP-
(GlcNAc)2
Five mannose residues are added next from GDP-
annose to form DOL PP-(GlcNAc)2-Man5.
It had been shown
that
DOL
P-Man also
manno s e
donor
to
the
core
oligosaccharide chain. In the oligosaccharide lipid carrying
nine mannose residues, the last four of these are transferred
through DOL P (Rearick et al 1981).
The next steps are
transfers of three glucose units from DOL P-Glc to the core
oligosaccharide chain with the formation of the lipid-linked
oligosaccharide DOL PP-(GlcNAc)2Mang-Glc3.
In the final
step of this pathway, the core oligosaccharide is transferred
en bloc to newly synthesized polypeptide with the concomitant
release of DOL PP. This core protein linked oligosaccharide
is then processed by a now well described pathway involving
spec
0
glycosidases (Kornfeld & Kornfeld, 1980)
and
additional sugar residues added by DOL P independent glycosyl
Schachter
and his coworkers from studies of
glycoprotein and glycolipid metabolism during spermatogenesis
in rat and mouse testis indicated that spermatocytes and
early spermatids had highly active glycosylating systems
(Kornblatt et al., 1974; Letts et al 1974a; Letts et al.,
1974b).


60
product pattern. The formation of the two products increased
linearly with time up to 60 min.
Base Hydrolysis of Dehvdro POL PP. The putative
dehydro DOL PP was isolated by TLC and then subjected to
saponification in 3.5 M KOH in 70% methanol for 2 hr at
100 C. Figure 2-5 shows that dehydro DOL P is the
overwhelming product of base hydrolysis. This product is
consistent with this enzymatic product being dehydro DOL PP.
Saponification in aqueous 0.1 M KOH at 100 C for 30
min is used in the standard assay. This procedure makes
nonsaponifiable lipids more accessible for extraction and
provides a better resolution of the enzymatic products on
TLC, because of the hydrolysis and hence elimination of
saponifiable lipids. Saponification under these milder
conditions did not change the ratio between dehydro DOL PP
and dehydro DOL P (results are not shown).
These results indubitably showed the in vitro reaction
proceeds as follows: dehydro DOL PP synthase catalyzes the
condensation of isopentenyl diphosphate and farnesyl
diphosphate to generate dehydro DOL PP, which is the
precursor of dehydro DOL P. Since the proportion of the
diphosphate and monophosphate occasionally varied from one
experiment to another, an accurate measurement of synthase
activity required analysis of both products.


33
testicular and serum transferrin that testicular transferrin
must play a role in the transport of iron fro
Sertoli cells
to the spermatocytes and spermatids. More recently, Morales
and Clermont (1986) have shown that during spermatogenesis
Sertoli cells and spermatogonia internalized transferrin by
receptor-mediated endocytosis at the base of the seminiferous
epithe1ium.
Recent studies in the ram have shown that clusterin, a
glycoprotein with a molecular weight of 37,000-40,000 is also
synthesized de novo and secreted by Sertoli cells and
transported to the rete testis (Rosenior et al., 1987).
Collectively,
Sertoli
cells
actively
glycoproteins into the lumen of the seminiferous tubules and
regulate spermatogenesis.
Therefore,
it
relevant
presumption that the synthesis of glycoproteins must be very
active in these cells and that adequate DOL P must be
provided by biosynthesis or uptake to support this function.
The very high DOL content in Sertoli cell Golgi membrane
sugges ts
that
Sertoli
cells
actively involved
m
glycoprotein synthesis (Nyquist & Holt, 1986).
DOL in Sertoli Cells
Although a clear relationship between DOL P
concentrations and the rate of glycoprotein synthesis has
been established in many systems, the mechanism of regulation
of glycoprotein biosynthesis in Sertoli cells is still


113
homogenate with or without protease treatment (Fig. 3-4). On
the other hand, the spermatozoa and spermatogonial enriched
cell fractions had activities less than 20% of the
spermatocyte activity. When the enzymatic activity was
expressed as pmoles/mg protein/hr, pachytene spermatocytes
had an activity 1.6 fold higher than that seen with a mixture
of cells obtained from protease treated seminiferous tubules.
The enzymatic activity of the enriched spermatocytes was
about 1.4 fold higher than enriched spermatids, 4.8 fold
higher than spermatogonia and about 7.6 fold higher than
spermatozoa. The enzymatic activity of the spermatogenic
c
cells may also be expressed as pmoles product formed/100
cells/hr (Table 3-1). In this case the enzymatic activity of
spermatocytes (28.9 pmoles/10^ cells/hr) was 4.5 fold higher
than that of spermatids (6.5 pmoles/10^ cells/hr) and about
126 fold higher than that of spermatozoa (0.23 pmoles/10^
cells/hr).
Synthase Activity in Sertoli Cells. Synthase specific
activities were also measured in homogenates of Sertoli cells
and a mixed spermatogenic cell population from rats 7-65 days
old (Fig. 3-5). The fluctuations in the enzyme specific
activities for both of these cell populations were parallel
to that seen with the protease and non-protease treated
tubules. In each case, the activities peaked at day 23. The
enriched Sertoli cell specific activity ranged from 1.5-2.3


12
have been studied, such as androgen binding protein (ABP)
(Fritz et al., 1976), plasminogen activator (Lacroix et al.,
1977), testicular transferrin (Skinner & Griswold, 1980), and
sulfated glycoprotein 1 (SPG-1) and 2 (SPG-2) (Griswold et
al. 1986) .
The stage specific nature of the elaboration of one
glycoprotein, androgen binding protein, has been particularly
well described. Sertoli cells have maximum FSH binding
during stages XIII through I followed by maximum cAMP
production in stages II through VI which in turn is followed
by maximum production of androgen binding protein in stages
VII through VIII (Parvinen et al 1980; Ritzen et al. ,
1982). Since Sertoli cells are actively involved in
glycoprotein biosynthesis, we speculate that the regulation
of the N-linked glycoprotein biosynthesis may be significant
in the function of these cells.
Role of POL P in Glycoprotein Biosynthesis
The
S truc ture
and
Distribution
o f
Dolichol
and
Its
Derivatives
Dolichol (DOL)
general term for a group of
polyisoprenoid alcohols.
They contain the dimethylallyl
terminal unit (w-terminal), two trans-isoprene residues,
number of cis-isoprene residues and a terminal hydroxylated
a-saturated
isoprene
un i t
1inke d
i n
head-to-tail
orientation.
Pennock, Hemming and Morton first isolated and


58
Table 2-2
Formation of Enzymatic Product at Different Triton X-100
Concentrations in Pulse-Chase Exnerimenta
Isopentenyl Diphosphate Incorporated
(pmoles)
Time
Conditions
dehydro DOL PP
%
dehydro DOL P
%
1st hour
2% Triton
68
71
27
29
2nd hour
0.5% Triton
49
41
70
59
aIncubation conditions were the same as described in legend of Fig. 2-3
except that 4.81 mM unlabeled isopentenyl diphosphate was added to the
ruction mixture after the first hour, reducing the specific activity of
[ C]-isopentenyl diphosphate 134-fold.


28
Figure 1-6. The structure of dehydro DOL PP and DOL PP.
The a-unsaturated isoprene unit is present on the structure
of dehydro DOL PP.
The
condens at ion
o f
these
sub s t r a t e s
polymerization reaction which finally leads to the formation
of dehydro DOL PP with farnesyl diphosphate providing the
three (-terminal isoprene units and the last isopentenyl
diphosphate added providing the a-unsaturated unit bearing
the diphosphate.
The hen oviduct and Ehrlich tumor cell
enzymes are membrane associated.
Dehydro DOL P biosynthesis from isopentenyl diphosphate
and farnesyl diphosphate has also been described in
microsomal fractions of rat liver (Wong & Lennarz, 1982).


16
UDPGlcNAc
GlcNAc2P-P-Ool
5 GDPMan
IPP + FPP > > P-P-Dol Glc3Man9GlcNAc2P-P-Dol
HMG-CoA
Acetyl-CoA
Glc-3Man9GlcNAc2Protein Protein
Figure 1-3.
in eukaryotic cells.
Dolichol cycle for glycoprotein formation


CHAPTER III
DEHYDRO DOLICHYL DIPHOSPHATE SYNTHASE ACTIVITY IN
ENRICHED CELL POPULATIONS FROM RAT TESTIS
Introduction
Although DOL metabolism during testicular development
has been the subject of several studies in recent years, a
number of intriguing questions still remain unanswered. For
example, do all rat spermatogenic cell populations have
dehydro DOL PP synthase activity? Does each subpopulation of
spermatogenic cells have the same enzymatic activity for the
synthase? Is there a temporal relationship between synthase
activity and developmental stage? Do Sertoli cells also have
the synthase activity and synthesize its own dolichol? Is
there a temporal relationship between spermatogenic cell and
Sertoli cell synthase activity? Elucidating the answers to
these questions will be of value in understanding the
regulation in DOL metabolism as well as glycoprotein
biosynthesis during spermatogenesis in rat. In this chapter,
the specific activity of 2,3-dehydro DOL PP synthase in
homogenates of protease treated seminiferous tubules, cell
fractions enriched in spermatogenic cells or Sertoli cells
from testis were measured as a function of the age of
93


106
Figure 3-1. Panel A. Spermatids.


3
series of different cell associations would be
until the cycle was completed.
The time interval
between the appearance of the same cell association at
given point of the tubule is called the cycle of the
seminiferous epithelium.
The number of stages in the cycle
is constant for a given species; man has 6, mouse and monkey
have 12, the rat, 14 (Figure 1-1).
The cycle involves changes with time in the appearance
of one segment of
tubule, whereas the wave refers to the
distribution of different cellular associations along the
length of the tubule.
The segments of the tubules which
specific cellular associations occur in a sequential series
along the length of the tubule.
For example a segment
containing cells at stage V of the cycle is bordered on one
side by cells at stage IV and on the opposite side is at
s tage VI.
The average length of each tubular segment
roughly with
the
duration
o f
the
corresponding cellular association or stage of the cycle.
The sequence of segments or waves, representing the stages in
the cycle of the seminiferous epithelium, repeats itself
along the length of an individual tubule.
In the rat for
example, there is an average of 12 waves per tubule.


145
Skinner, M. K., and Griswold, M. D. 1982, Biol. Reprod. 27:
211-221.
Steinberger, A., and Steinberger, E. 1971, Biol. Reprod. 4:
84-87.
Struck, D. K., and Lennarz, W. J. 1980, in The Biochemistry
of Glycoproteins and Proteoglycans (Lennarz, W. J.
eds.). Plenum Press, New York, pp 35-83.
Tavares, I. A., Coolbear, T. and Hemming, F. W. 1981, Arch.
Biochem. Biophys. 207: 427-436.
Tollbom, 0., and Dallner, G. 1986, Br. J. Exp. Path. 67: 757-
764.
Valtersson, C., van Duyn, G., Verkleij, A. J., Chojnacki, T.,
de Kruijff, B., and Dallner, G. 1985, J. Biol. Chem.
260: 2742-2751.
Vernon, R. J., Kopec, B., and Fritz, I. B. 1974, Mol. Cell.
Endocrin. 1: 167-175.
Vitale, R., Fawcett, D. W., and Dym, M. 1973, Anat. Rec.
176: 333-344.
Wellner, R. B., and Lucas, J. J. 1979, FEBS Letts, 104: 379-
383 .
Wens tro
J C
and
23
1054-1059
Hamilton,
D. W. 1980,
Biol. Reprod.
Wong,
Wong ,
Wong ,
T. K., Decker, G. L., and
Chem. 257: 6614-6618.
T. K., and Lennarz, W. J.
710: 32-38.
T. K., and Lennarz, W. J.
6619-6624.
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Biophys. Acta.
Chem. 257:


pmoles Isopentenyl Diphosphate Incorporated/ mg protein-
IOO
80-
60-
40
DAYS AFTER BIRTH


122
Discussion
Glycoproteins are undoubtedly
components in
the spermatogenic process.
The timing of their elaboration
is thought to be
for spermatogenesis to proceed
normally.
Histochemical studies in spermatogenic cells show
th a t
the
Golgi
apparatus
o f
spermatocytes
and
early
spermatids are highly active in glycoprotein biosynthesis
(Letts
al ,
1974b) .
However,
the Golgi
apparatus
and
lost from spermatogenic
cells
spermatids mature into spermatozoa.
At least some of the
enzyme s
necessary
for
N-glycoprotein bio synthe sis
presumably lost as well.
Letts e_t al. (1974a) have shown
developmental dependent changes in the specific activities of
three glycosyl transferases involved in the late reactions of
oligosaccharide maturation
m
glycoprotein biosynthesis.
Galactosyl and N-acetylglucosaminy1 transferases were found
in spermatogonia, whereas the fucosyl transferase was not
highly active until the spermatocytes appeared.
All three
enzymes were low in spermatozoa.
A later study (Letts et
al., 1978) indicated that spermatocytes and early spermatids
were highly active in glycoprotein biosynthesis. Therefore,
it was of interest to
investigate if other biological
machineries necessary for the glycoprotein biosynthesis are
changing accordingly in these cell populations.


91
reductase may be one of the regulated enzymes in DOL
synthesis (Rodwell et al., 1976; James & Kandutsch, 1979),
the observed independent regulation of DOL and cholesterol
biosynthesis can not be well explained by the control of this
enzyme alone. A strong case is presented here for a
regulatory role for dehydro DOL PP synthase during de novo
DOL biosynthesis, because the specific activity of this
enzyme rises in parallel with a two to three fold increase in
DOL P concentration measured both directly and indirectly.
Therefore, it can be concluded that dehydro DOL PP synthase
is a regulatory enzyme responsible for controlling DOL
biosynthesis on a pathway that is independent of cholesterol
biosynthesis. This conclusion is consistent with the
findings of Keller and Adair ( 1980), that DOL P synthase or
long chain cis-prenyl transferase is a rate limiting factor
in the biosynthesis of DOL P in liver. Recently, when
radioactively labeled mevalonate was utilized to study in
vivo and in vitro cholesterol, DOL and ubiquinone
biosynthesis, considerable differences were observed between
the rate of cholesterol synthesis and the rate of DOL and
ubiquinone synthesis, while the rates of DOL and ubiquinone
synthesis were quite similar (Elmberger et al., 1987). This
observation suggested that the presence of important rate-
limiting steps in the biosynthesis of DOL and cholesterol
after mevalonate. This study suggests that dehydro DOL PP
synthase may be one of these rate 1imiting factors.


22
Is op enteny1 Diphosphate
+
Farnesyl Diphosphate
Dehvdro POL PP
Synthase
t
Dehydro DOL PP
Dehydro DOL P
t
Dehydro DOL
+
[ H2 ]
+ [H2]
+ [H2]
DOL PP
t
DOL P
A

DOL
GLYCOPROTEIN
ASSEMBLY CYCLE
SHOWN IN
Figure 1- 3.
DOL Kinase
t
DOL- esters
Figure 1-4. A putative pathway showing the
relationship between dehydro DOL PP and glycoprotein
bio synthe sis.


Figure 2-8. A double reciprocal plot of the sum of
dehydro DOL PP and dehydro DOL P formation (Fig. 2-7.
Panel C) vs. isopentenyl diphosphate concentrations is
presented.


92
Since the cellular composition of the seminiferous
tubules differs as a function of age during early stages of
differentiation, changing cell populations may partially
explain the difference in activity of dehydro DOL PP synthase
in testes from rats of different ages. For instance, in 7-
day-old rats the only spermatogenic cells are spermatogonia,
by day 23, there are spermatogonia and pachytene
spermatocytes as well, and after day 26, spermiogenic cells
start appearing in the seminiferous tubules. It is likely
that the different enzymatic activity observed during
development can be accounted for by different cell
populations with different enzymatic activity. The specific
activity of different cellular populations are elaborated in
the next chapter.


APPENDIX C
SUMMARY OF EXPERIMENTAL DATA PRESENTED IN FIGURE 3-5. AGE DEPENDENT VARIATION IN
SYNTHASE ACTIVITY IN SERTOLI CELLS, SPERMATOGENIC CELLS AND PROTEASE TREATED
SEMINIFEROUS TUBULES. SYNTHASE ACTIVITY IS PRESENTED IN UNITS OF PMOLES
ISOPENTENYL DIPHOSPHATE INCORPORATED/MG PROTEIN
Rats
Age
(Days)
Assays
Sertoli
Synthase
Activity
Rats
Assays
Tubules
Synthase
Activity
Rats
Assays
Spermatogeni'
Cells
Synthetase
Activity
7
24
4
27
+
6
12
4
32
+
2
24
4
19
+
2
15
12
4
109
+
4
12
6
61
+
14
12
4
47
+
7
23
7
4
119

4
7
6
93
+
4
7
4
69
+
2
30
4
4
89

8
4
6
76
+
7
4
4
61
+
2
40
6
4
63

7
6
4
52
+
6
6
4
49
+
5
60
4
4
30

3
4
4
24
+
1
4
4
25
3
136


KEY TO ABBREVIATIONS
BS A
bovine serum albumin
Ci
cur i e
cm
centimeter
cpm
counts per minute
C
degree centigrade
DIBK
diisobutyl ketone
DNA
deoxyribonucleic acid
DNase
deoxyribonuclease
DOL
do1icho1
DOL P
dolichyl phosphate
DOL PP
dolichyl diphosphate
dpm
disintegrations per minute
EKRB
enriched Krebs-Ringer bicarbonate medium
IPP
is openteny1 diphosphate
FPP
t,t-farnesyl diphosphate
G lc
glucose
GlcNAc
acetylglycosamine
GPP
gerany1 diphosphate
g
gram
HAc
acetic acid
hr
hour
M
molar
ix


107
Figure 3-1. Panel B. Pachytene Spermatocytes (continued).


4
Figure 1-1. The Cellular Composition of the 14
of the Cycle of the Seminiferous Epithelium in Rat.
shows
the
Each numbered column (roman numeral)
spermatogenic cell types present in cellular associations
found in cross sections of seminiferous tubules. The
cellular associations or
ano the r
tages of the
in
cycle
of the
succeed one
seminiferous
cellular association XIV
time in any given
epithelium in the rat. Following
cellular association I reappears, so that the sequence
over again. Steps in the development of the spermatids,
numbered 1 to 19 and defined by changes in the structure of
the nucleus and acrosome. are indicated with arabic numerals.
Letters are used to identify
A1 A 2
A 3
and
spermatogonia
a4
In.
represent
intermediate
spermatogonia
four
and spermatocytes.
of type A
spermatogonia; PI, preleptotene spermatocytes
generations
spermatogonia;
: L.
B
spermatocytes
Z
spermatocytes
Di
spermatocytes
and Diakinesis
P
type
leptotene
pachytene
II
, zygotene
Diplotene
spermatocytes. The subscript m indicates the occurrence
mitotic division of the spermatogonia (From Dym, M. &
Clermont, Y., 1970, Am. J. Anat., 128, 265-282,).
secondary
o f


51
SF
m
DOLP C
Figure 2-1
(continued)


CHANGE IN DEHYDRODOLICHYL DIPHOSPHATE SYNTHASE
DURING SPERMATOGENESIS IN THE RAT
BY
ZHONG CHEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988


108
Figure 3-1. Panel C.
Sertoli Cells (continued).


14
liver, kidney, testis, lung and heart) (Pullarkat et al.,
1984) .
DOL may have some effects on membrane structure and
fluidity (Valtersson et al 1985 ).
Otherwise, most DOL
apparently
has
no
direct relationship
to
glycoprotein
synthesis, since the bulk of the DOL is present in membranes
other than the endoplasmic reticulum (Wong et al., 1982;
Adair & Keller, 1982; Ekstrom et al., 1982). Some DOL may be
phosphorylated by a CTP-dependent kinase (Allen et al. 1978;
Burton et al 1979).
Dolichol is found in several combined
forms in the cell, free dolichol and esterified form with
fatty acids or mono or diphosphate. The phosphorylated forms
are important as we will see soon because they are also found
*
with different degrees of glycosylation which is important
for glycoprotein biosynthesis.
roles.
Dolichyl fatty acyl esters may have several metabolic
First, the fatty acyl esters may be a suitable and
stable for
for the storage of dolichol in lipid droplets.
Alternatively, the fatty acyl moiety might be necessary for
the transport of dolichol from its site of synthesis to
different subcellular locations.
The percentage of dolichol found in the esterified form
varies widely in different tissue.
For example, it is
reported to be 0% and 25% esterified in pig kidney and
spleen, respectively.
In another report, it is shown that
the ester form represents about 63% of total DOL in pig


ACKNOWLEDGEMENTS
Sincere appreciation is expressed to my friend, mentor,
Dr.
Charles M.
Allen,
Jr.,
for his enduring patience,
guidance, and support. I am deeply grateful for his help in
proofreading my English.
His belief in the fundamentals of
biochemistry and molecular biology and literacy influenced
the designing of each project undertaken, and I learned the
meaning of scientific integrity, creativity, and serendipity.
I
am
indebted
to
the members
o f my
supervisory
committee, Dr. Michael S. Kilberg, Dr. Rusty J. Mans, Dr.
Thomas W.
0'Brien,
and Dr.
Lynn J.
Romre11
for their
encouragement, assistance and productive criticism of my
research projects.
I am especially grateful to Dr. Lynn J.
Romrell who allowed me the privilege of working in his
laboratory and who provided support and advice through the
duration of the endeavor.
In addition, I am indebted to the faculty of the
Department of Biochemistry and Molecular Biology for both
educational and financial support. Finally, I would like to
thank William Blakeney, Mary Handlogton, Michael Campa and
William Wong for their friendship and assistance during the
course of my studies.


Figure 2-7. Isopentenyl Diphosphate Concentration
Dependency on the Formation of Dehydro DOL PP and
Dehydro DOL P.
Incubations containing 100 mM Tris-HCl buffer
(pH7 5) 10 mM MgCl2, 0.5% Triton X-100, 250 /M t,t-
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF, the
indicated concentration of [ 1 -] isopentenyl
diphosphate, and 1.0 mg of enzyme protein in a total
volume of 0.25 ml were carried out at 37 C for 60
minutes. The formation of [^C] -dehydro DOL PP (Panel
A) and [ ]- dehydro DOL P (Panel B) were estimated by
the method described before. Panel C represents dehydro
DOL PP synthase activity (A+B).


23
present in the whole rat and in the liver 24 hr after
inj ection.
Furthermore, it was still present 20 days later,
almost entirely as
14
C-DOL.
The half-life of DOL P in rat
liver has been estimated to be 7-12 days on the basis of the
size of the DOL P pool. The catabolic products derived from
labeled DOL have not yet been found (Rip et al., 1985).
The Importance of Dehydro DOL PP Synthase
Studies on developing brain (James & Kandutsch, 1980a),
and liver (Keller et al., 1979; Tavares et al., 1981; Keller,
1986) have clearly shown that the rates of cholesterol and
dolichol biosynthesis are regulated independently, although
they share in part a common biosynthetic pathway.
Although 3-hydroxy 3 methy1glutary1 CoA reductase (HMG
CoA reductase), the rate limiting enzyme in cholesterol
synthesis and whose product, mevalonate, is a metabolic
precursor of DOL, was shown to be high in these cells, the
fact that DOL and cholesterol biosynthesis can be
independently regulated would lead to uncertainty about the
potential of this enzyme as the regulatory enzyme in DOL
biosynthetic pathway.
Dolichol, cholesterol and ubiquinone are all formed
naturally from mevalonate. During the synthesis, mevalonate
undergoes a series of reactions and is converted to
isopentenyl diphosphate, which in turn, is isomerized to form
dimethylally1 diphosphate. Then there is a sequential


APPENDICES
A SUMMARY OF EXPERIMENTAL DATA PRESENTED IN
FIGURE 2-12. DEHYDRO DOL PP SYNTHASE ACTIVITY
IN SONICATES OF TUBULES FROM RATS OF
DIFFERENT AGES 134
B TYPICAL NUMBER OF RATS, TOTAL TUBULE WEIGHT,
AND NUMBER OF ASSAY USED FOR EACH EXPERIMENT
AS A FUNCTION OF RAT AGE 135
C SUMMARY OF EXPERIMENTAL DATA PRESENTED IN
FIGURE 3-5. AGE DEPENDENT VARIATION IN
SYNTHASE ACTIVITY IN SERTOLI CELLS,
SPERMATOGENIC CELLS AND PROTEASE TREATED
SEMINIFEROUS TUBULES 136
BIBLIOGRAPHY 137
BIOGRAPHICAL SKETCH 146
V


102
diluted phosphate buffered saline to remove spermatogenic
cells. Under these conditions the spermatogenic cells are
lysed but the Sertoli cells retain their integrity. The
cells were examined by Nomarski differential interference and
phase microscopy to check for purity and integrity. The
resulting cell suspension contained more than 80% Sertoli
cells with a contamination of pachytene spermatocytes and
spermatids (Fig. 3-1-C).
Homogenate Preparation. Homogenates were prepared from
seminiferous tubules, cell suspensions from tubules, enriched
spermatogenic cell populations and Sertoli cells. The
excised testicular tubules were weighed, suspended at a ratio
of 1:2 (w/v) in ice cold buffer (20 mM Tris-HCl pH 7.5 and 1
mM EDTA) and sonicated as described in Chapter II.
Homogenates of various mixed and enriched cell fractions,
were prepared similarly, except that in these cases the
packed cells were suspended in two volumes of buffer (v/v).
Protein quantitation was determined by the method of Lowry et
al (1951) before assay-*-.
i
The cellular dissociation procedure used a relatively
large amount of BSA (2-4%). There was a concern that binding
of BSA to the purified cell fractions might lead to some
error in protein determination of these isolated cells.
Therefore, it was useful to estimate the binding of BSA to
the enriched perm cells. Measurement of binding was carried
out with [-*-^I]-BSA as a probe in 2% BSA. Protease
dissociated germ cells were incubated with [^^^I]-BSA for 3
min at room temperature then washed. The radioactivity on
the cell in the beginning and the end of the test was
compared. The results showed negligible non-specific binding
of BSA to the cell surface (144 jug/14350 fig which is less
than 1%) after the STA-PUT fractionation and the buffer


18
DOL P plays a major role in the biosynthesis of N-
linked glycoprotein,
since
DOL
P
not
only
an
oligosaccharide unit carrier but also is an activator which
reacts with certain nucleotide sugars and facilitates the
sugar transfer to the core oligosaccharide chains.
The
involvement of DOL P in the production of the DOL PP-
oligosaccharide
and
the
sub sequent
o f
the
oligosaccharide to asparagine residues within the newly
formed peptide is thought to occur on the luminal surface of
the endoplasmic reticulum membrane (Pfeffer & Rothman, 1987,
for review).
Studies with chick embryo fibroblast (Hubbard &
Robbins, 1980) and canine kidney cells (Schmitt & Elbein,
1979 )
sugge s t
that oligosaccharide transfer is the rate
limiting step
i n
glycoprotein synthesis.
Sub s e quent
regeneration of DOL P permits the reinitiation of lipid
oligosaccharide biosynthesis.
Therefore, enzymes of the DOL
P pathway associated with formation or utilization of DOL P
could be very important in the control of glycoprotein
biosynthesis in spermatogenesis.
The availability of DOL P was found to be a rate-
limiting factor in some glycosylation processes (Potter et
al., 1981a; Eggens et al., 1984). Furthermore, the shortage
of the lipid intermediates influences some vital biological
processes such as embryonic development (Carson et al ,
1981) .
Carson and Lennarz showed that when DOL P synthesis


stimulated with increasing detergent concentration throughout
the concentration range shown in the figure, while dehydro
DOL P formation was optimal at 0.5% Triton X-100. This
suggests that when the Triton X-100 concentration was higher
than 0.5%, a previously active dehydro DOL PP diphosphatase
was inhibited. The sum of dehydro DOL PP and dehydro DOL P
production was unchanged at Triton X-100 concentrations of
0.5% and higher.
The product ratio of dehydro DOL P to dehydro DOL PP
shifted to favor the monophosphate when the detergent
concentration in the incubation mixture was decreased midway
through the incubation period. A part of the tubular
homogenate was incubated at 37 C with substrates in either
0.5% Triton X-100 or 2% Triton X-100. In each case the
products were analyzed after 1 hr and 2 hr. The incubation
with 0.5% Triton X-100 gave dehydro DOL P as the predominant
product at both 1 hr and 2 hr (Fig. 2-3, Panel A), whereas
with 2% Triton X-100, the slower migrating product, dehydro
DOL PP, was the predominant product at both time points (Fig.
2-3, Panel B). However, when a similar incubation was
carried out in 2% Triton X-100 for the first hour to favor
dehydro DOL PP formation and then the concentration of Triton
X-100 changed to 0.5% during the second hour of incubation,
the predominant product observed at the end of the second
hour was dehydro DOL P (Fig. 2-3, Panel C).


pmole Isopentenyl Diphosphate
Incorporated/mg protein hr
119
140
120 -
100 -
80 -
60 -
40 -
20
O Sertoli Cells
Tubules
0
0
10
20
30
40
50
60
70
Days After Birth


APPENDIX B
TYPICAL NUMBER OF RATS, TOTAL TUBULE WEIGHT, AND NUMBER OF ASSAY USED
FOR EACH EXPERIMENT AS A FUNCTION OF RAT AGE
Age
(Days)
# of Rats Used
Total Tissue Weight
(g)
Assays
3
10
0.09
2
7
4 5
0.12
2
15
2
0.20
2 3
23
2
0.85
3 or more
30
2
1.20
3 or more
40
2
1.60
3 or more
60
2
3.50
3 or more
135


39
dehydro DOL Ps ranging in size from C75 to C95
More
recently, Baba et al. ( 1987 ) described the synthase in rat
seminiferous tubules, but showed that the products of this
enzyme
were
both dehydro
DOL
PP
and
dehydro
DOL
P.
Hydrolysis of both of these products with a testicular
phosphatase in the absence of NaF yielded the same chain
length alcohols (C75.C90). The isolation of 2,3-dehydro DOL
P and DOL P in some cases instead of the diphosphate
derivatives undoubtedly
reflects
the
action
o f
diphosphatase.
The methods for the assay of dehydro DOL PP synthase,
which were described by Adair and collaborators (Adair &
Keller, 1982; Adair et al., 1984), and Wellner and Lucas
(1979) have been modified, as described below, to optimize
the analysis of synthase activity in whole homogenates of
small testicular samples obtained at different stages of
spermatogenesis.
I n
this
assay ,
[]-isopentenyl
diphosphate and t,t-farnesyl diphosphate were chosen as
substrates.
A s
poss ible
i nh i b i t o r s
o f
endogenous
phosphatase, NaF and ATP were used to protect the substrates.
Although Baba et al. (1987) showed that there were no major
changes in the extent of formation of radiolabeled enzymatic
products when NaF, MgCl2 and ATP were omitted in the assay of
partially purified subcellular membrane fraction, there were
major changes in the nature of the in vitro products.
For
example, when Triton X-100 was omitted neither dehydro DOL PP


100
the washing.
The suspension was filtered through a nylon
mesh
(135
m)
t o
remove
cell
The
cell
concentration was determined using a hemocytome ter.
This
enriched spermatogenic cell suspension was finally suspended
in EKRB containing 0.5% BSA and adjusted to a concentration
of 2 x 106 cells/ml.
Spermatogenic Cell Fractionation.
The entire cell
separation procedure was carried out
at 5 C.
Spermatogenic
cells were separated by a modification of the STA-PUT unit
gravity procedure (Romrell et al 1977 ). The sedimentation
chamber was initially filled with 70 ml EKRB. Then the cell
suspension, which contained 10^ cells in 50 ml of 0.5% BSA in
EKRB, was introduced into the chamber at a flow rate of 10
ml/min.
The sample was followed by a linear gradient of 2%
t o
4%
BSA
i n
EKRB
generated
from
two
interconnected
that contained 1100 ml of 4% BSA and 1100
1 of
2% BSA, respectively (total volume 2200 ml). Five min after
loading the cell suspension, the flow rate was increased to
40 ml/min. Eighty minutes after loading the cell suspension,
the chamber was drained in 10-ml fractions at a rate of 10
ml/min.
Cell collection was finished within 5 hr after
introducing
the
cell
suspension
to
the
chamber.
The
separated cell fractions were numbered and centrifuged at 200
g for 10 min; the supernatant were decanted; the resulting
pellets were resuspended in 0.5 ml of EKRB.
Aliquots were
taken from individual samples and checked for cell type and


19
was inhibited by compactin, a potent inhibitor of
hydroxymethyl glutaryl CoA reductase and consequently
po 1y isoprenoid biosynthesis, protein glycosylation was
impaired and the oligosaccharide chains synthesized were more
negatively charged (Carson & Lennarz, 1981). In another
report, inhibition of DOL P biosynthesis induced abnormal
gastrulation in sea urchin embryos (Carson & Lennarz, 1979).
All of the findings suggested that DOL P plays an important
role in the N-linked glycoprotein biosynthesis. Needless to
say, a good understanding in the regulation of DOL metabolism
is a necessary step to explore the regulation of the N-linked
glycoprotein biosynthesis.
Metabolism and Functions of DOL P
There are at least three pathways to generate DOL P.
First, the dephosphorylation of DOL PP could provide the main
supply of DOL P for the biosynthesis of intermediates in the
protein glycosylation reactions (Dallner & Hemming, 1981).
On the other hand, investigations in recent years have
established that increased protein glycosylation is often
accompanied by increased phosphorylation of DOL by a CTP-
dependent kinase; therefore, a second possibility is that DOL
P is supplied by direct phosphorylation of DOL (Burton et
al., 1981; Coolbear & Mookerjea, 1981). Third, when the
sugar residues are transferred to the growing oligosaccharide
chain, DOL P is released from DOL P-Man or DOL P-Glc.


DETECTOR RESPONSE DETECTOR RESPONSE
50
HOMOGENATE DOL-P
DISTANCE (cm)


pmolea Isopentenyi Diphosphate Incorporated/ mg protein-hr
129
DAYS AFTER BIRTH


5
Exocrine and Endocrine Functions of Testis
The testis has both exocrine and endocrine functions.
The exocrine function of the testis resides in the cells of
the seminiferous epithelium which produce testicular fluids
and spermatozoa.
The endocrine function of the testis
resides primarily
in
the
Leydig cell population which
synthesizes and secretes the principal circulating androgen,
testosterone (Nearly 95% of the testosterone is produced by
the testis; the rest is produced by the adrenal glands).
The
steroidogenic and spermatogenic activities of the testis are
regulated by hormonal interactions among the hypothalamus,
adenohypophysis, and the cells of the gonad
the Sertoli
cells, spermatogenic, and Leydig cells.
The Leydig cells, which are located in the space
surrounding
the
seminiferous
tubules,
to
testosterone during
the
fetal
period,
p romoting
the
development of the male reproductive tract.
Biosynthesis of Glycoproteins During Spermatogenesis in
Testis
Mammalian spermatogenesis involves extensive
morphological and biochemical transformations to produce
mature gametes that are structurally and functionally unique.
During this process, gene expression is temporally regulated
both at transcriptional and translational levels (Bellve &
O'Brien, 1983; Hecht, 1986). A variety of proteins, many of
which are unique to spermatogenic cells, are differentially


24
condensation
o f
isopentenyl diphosphate
to
the
dimethy 1 a 11y 1 diphosphate primer
consequential allylic diphosphate.
and
then
t o
the
Earlier biosynthetic
experiments confirmed
that
the
isoprene
residues
o f
polyisoprenoid alcohols are added in a stereochemically
specific
anner (Hemming, 1970).
The cis
addition to
trans. trans-farnesyl diphosphate gives rise to dolichols,
and the trans-addition to trans. trans-farnesyl diphosphate
generates the polyprenyl side chains of ubiquinones.
On the
other hand ,
the
synthesis
o f
squalene,
choles terol
precursor,
results from a reductive condensation of two
molecules of trans t trans-farnesyl diphosphate (Figure 1-5).
Many enzymes are common to the pathways of cholesterol
and DOL biosynthesis. In particular HMG-CoA reductase, which
is a major regulatory enzyme for cholesterol biosynthesis
(Faust et al., 1979; Rodwell et al., 1976),
could
also
control
the
bio synthe sis
of
isoprene units
utilized for dolichol synthesis. But, effectors that inhibit
cholesterol biosynthesis in a major way have minimal effect
on DOL biosynthesis (James & Kandutsch, 1980b).
A current
debatable hypothesis to explain these findings suggests that
dehydro DOL PP synthase
saturated at
much lower
concentration of the prenyl diphosphate substrates than the
enzymes of cholesterol pathway, and therefore, the synthase
is less subject to large changes in the availability of these
substrates (Keller et al., 1979; James & Kandutsch, 1979).


144
Rich, K. A., Bardin, C. W., Gunsalus, G. L., and Mather, J.
P. 1983, Endocrinology. 113: 2284-2293.
Rip, J. W., Rupar, C. A., Ravi, K., and Carroll, K. K. 1985,
Prog. Lipid Res. 24: 269-309.
Ritzen, E. M., Boitani, C., Parvinen, M., French, F. S., and
Feldman, M. 1982, Mol. Cell. Endocrin. 25: 25-33.
Ritzen, E. M., Hansson, V., and French, F. S. 1981, in The
Testis (Burger, H., and deKrester, D. J. eds.). Raven
Press, New York, pp 171-193.
Rodominska-Pyrek, A., Chojnacki, T., and Pyrek, J. S. 1979,
Biochim. Biophys. Res. Commun. 86: 395-401.
Rodwell, V. W., Norstrom, J. L., and Mitschelen, J. J., 1976,
Adv. Lipid Res, 14: 1-77.
Romrell, L. J. 1979, in The Spermatozoon (Fawcett, D. W., and
Bedford, J. M. eds.). Urban and Schwarzenberg,
Baltimore, pp 375-378.
Romrell, L. J., Bellve, A. R., and Fawcett, D. W. 1977. Dev.
Biol. 49: 119-131.
Rosenior, J., Tung, P. S., and Fritz, I. 1987, Biol Reprod.
36: 1313-1320.
Ross ,
M. H., and Reith, E. J. 1985, Histology: A Text and
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Rossignol, D.
J. Biol.
Che
Lennarz
m. 256:
W. J., and Waechter,
10538-10542.
1980 ,
Rupar, C. A., and Carroll, K. K.
1978, Lipids 13: 291-293.
Sanborn, B. M., Steinberger, A., Tcholakian, R. K., 1979,
Steroids. 34: 401-412.
Sanborn, B. M., Steinberger, A., Tcholakian, R. K., and
Steinberger, E. 1977, Steroids. 29: 493-502.
Schefler, W. C. 1984, Statistics for Health Professionals.
Addison-Wesley Publishing Company. Reading, Mass, pp
166 169 .
Schmitt, J. W., and Elbein, A. D. 1979, J. Biol. Chem. 254:
12291-12294.
Skinner, M. K., and Griswold, M. D. 1980, J. Biol. Chem.
255: 9523-9525.


8
described in 1865 by Sertoli to have a nursing function, to
provide mechanical support for the developing spermatogenic
cells as well to be phagocytic.
It is believed that
circulating hormones, which act on spermatogenesis, have
their effects mediated via the Sertoli cell (Hansson et al ,
1976 ) .
Sertoli cells are basically columnar cells,
which
surround the adjacent spermatogenic cells and fill the spaces
between them (Figure 1-2).
Sertoli cells form the major
structural component of the seminiferous tubules and serve a
number of functions, including (a) mediating movement of
spermatogenic cells from the basal lamina to the lumen and
the release of the late spermatids into the tubular lumen,
(b)
compartmentalizing
the
epithelium into
basal
and
adluminal compartments and forming part of the blood-testis
barrier, (c) phagocytizing degenerating spermatogenic cells
and residual bodies, (d) secreting androgen binding protein
and inhibin as well as other glycoproteins and (e) mediating
the movement of steroids, metabolites, and nutrients utilized
by spermatogenic cells across the seminiferous epithelium.
During development,
the
Sertoli
cell undergoes
fundamental changes.
The most remarkable feature of these
maturational changes is the cessation of cell multiplication
that occurs before puberty. It occurs in the rat at about 15
days of age (Steinberger & Steinberger, 1971).
After that
age, the Sertoli cells may change their metabolic activities


32
androgenic and tropic hormone action on spermatogenesis is
mediated by the Sertoli cells. These cells have both FSH and
androgen receptors (Sanborn et al., 1977; Sanborn et al.,
1979; Means & Vaitukaitis, 1972) and show an appropriate
temporal relationship between hormone binding and cell
response. For example, there is nuclear accumulation of
androgen and stimulation of RNA polymerase II activity in
cultured Sertoli cells when FSH or testosterone were added in
the media (Lamb et al., 1981). Therefore, it is possible to
envision a scenario where the regulation of spermatogenesis
is a result of the biochemical properties of Sertoli cells.
One of the possible mechanisms for Sertoli cells to
influence spermatogenic cell development is via the secretion
of proteins and glycoproteins which serve as signals or
transport vehicles. The first glycoprotein to be identified
as a Sertoli cell specific secretion product was androgen
binding protein (French & Ritzen, 1973; Vernon et al., 1974).
This protein is secreted by cultural Sertoli cells and its
synthesis is regulated by FSH, testosterone, and vitamin A
(Louis & Fritz, 1977; Karl & Griswold, 1980). The function
of androgen binding protein is not clear but it is probably
related to the capability of the protein to bind and
transport androgens to the epididymis.
Sertoli cells synthesize and secrete a testicular
transferrin. Skinner and Griswold (1982) speculate on the
basis of biochemical and immunological similarities between


6
expressed during meiosis and spermiogenesis
These include
basic
chromosomal proteins
that
unde r go
successive
during
spermatogenic
cell
(Meistrich et al., 1981), several spermatogenic ce11-specific
iso z yme s
(Goldb erg,
19 7 7 )
and
stage-specific
surface
glycoproteins
with biochemical
(Millette
&
Moulding,
1981a ;
Millette
&
Moulding,
1981b)
and
immunological probes (O'Rand 6c Romrell, 1980; Gaunt, 1982;
Fenderson et al., 1984; O'Brien 6c Millette, 1984; O'Brien 6c
Millette, 1986).
It has been suggested that many proteins
play
roles
in various
processes
including
sperm-egg
interaction,
Sertoli -spermatogenic
cell
association
capacitation and the acrosomal reaction. For example, rabbit
sperm autoantigen (RSA-1), a sialoglycoprotein located in the
postacrosomal region, plays a role in the spermatozoon's
binding to and penetration of the zona. This protein first
appears
on
the
surface
of pachytene spermatocytes
and
increases in amount throughout spermatogenesis (O'Rand 6c
Romrell, 1981; 0'Rand et al., 1984).
Such sperm specific
surface components are probably involved in antibody mediated
agglutination and immobilization of spermatozoa.
Early in spermatogenesis, glycoproteins are synthesized
and deposited in the acrosome.
One of the acrosomal
glycoproteins is proacrosin, which yields acrosin after being
activated. Proacrosin is first produced during the spermatid
stage of differentiation, and is retained throughout the


87
P and dehydro DOL PP, therefore, the determination of
synthase activity required the measurement of both products,
not dehydro DOL P or dehydro DOL PP alone.
DOL P is an indispensible carrier of oligosaccharides
during glycoprotein biosynthesis, therefore, knowledge of its
availability and the timing of its biosynthesis during early
stages of differentiation may be important in understanding
the regulation of spermatogenesis. This laboratory has shown
that DOL P, as measured indirectly by a DOL P dependent
mannosyl transferase assay, increased in immature rat testes
about two fold between day 7 and day 30 after birth (Allen &
Ward, 1987). Unpublished work of others in this laboratory
has demonstrated similar results by direct measurement of DOL
P with HPLC analysis of CHCI3/CH3OH extracts of seminiferous
tubules from different aged rats (Fig. 2-14).
Changes in dehydro DOL PP synthase during
spermatogenesis in the rat have been studied here. The
products of this synthase are undoubtedly intermediates in
DOL P(P) biosynthesis. Dehydro DOL PP synthase was shown to
increase two fold in specific activity between day 7 and day
23 after birth, and a similar decrease in activity between
the day 23 and day 60. These findings parallel the changes
in DOL P described above. Mechanisms which account for the
increase in DOL P may include the phosphorylation of DOL with
CTP dependent DOL kinase, de novo biosynthesis of DOL or as a
result of release of DOL P from pools of DOL P and DOL PP


83
formation (Panel A) were somewhat smaller than changes in
dehydro DOL P formation (Panel B) during testicular
development.
The sum of dehydro DOL PP and dehydro DOL P
(Panel C) represents total specific activity of this enzyme.
A composite of data from these studies using 118 rats is
shown in Fig. 2-13. A two fold increase in tubular activity
of the synthase occurred between day 7 and day 23 and
similar decrease in activity occurred between day 23 and day
60 .
A statistical treatment (Wilcoxon two-sample rank test)
(Schefler, 1984) of these data shows a significant difference
between 1) the 7-day-old and the-15-day old groups of rats
(p<0.005) and, 2) the 30-day-old and the 60-day-old group of
rats (p<0.01). Therefore, the peak of activity at 23 day old
rats must be significantly higher than activity in rats aged
both 7 days and 60 days.
Discussion
Early in vivo experiments have demonstrated that
mevalonic acid serves as a precursor of dolichol in pig,
rabbit and rat liver (Butterworth et al 1966; Martin &
Thorne, 1974). Using doubly and stereospecifica 1 ly
radiolabeled mevalonate and tissue slices, it was also shown
that dolichol was synthesized from all-trans-farnesvl
diphosphate by cis- addition of isoprene units (Gough &
Hemming, 1970), suggesting that the biosynthetic pathway to


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Figure 2-6. Effect of Protein Concentration on Dehydro
DOL PP and Dehydro DOL P formation.
Incubations
(pH7.5)
10
containing
M MgC12, 0.5%
100
mM Tris-HCl
buffer
Triton X-100
2 5 0 /xM t t -
farnesyl diphosphate,
] -isopentenyl diphosphate, and varying protein
concentration in a final volume of 0.25 ml was carried
14
1.6 mM ATP, 50 mM NaF, 36 [1-
and
out at 37
C for 60 minutes
The formation of
dehydro
DOL PP
(Panel A)
the
and [ -^C ]- dehydro DOL P
method described before.
B) were estimated by
C represents dehydro DOL PP synthase activity (A+B)
[it+c]-
(Pane 1
Panel


42
Preparation of homogenate.
Male Sprague-Dawley rats
were decapitated. The testes were removed and perfused with
enriched Krebs-Ringer bicarbonate medium (EKRB)
via the
testicular vessels
This procedure effectively removed all
blood cells from testes
The tunica albuginea was removed
and seminiferous tubules were gently expressed
In the case
of the younger animals (3 and 7 days old), the testes and the
tunica albuginea were removed under dissecting microscope
without perfusion.
The seminiferous tubules were weighed then suspended at
ratio of 1:2 (w/v) with ice cold buffer (20 mM Tris-HCl pH
7.5 and 1 mM EDTA) .
This mixture was sonicated in an ice
bath in a Sonic dismembrator (model 300, Fisher) three times
for 20 seconds with 20 seconds intervals without sonication.
The pink
ilky homogenate was used as the enzyme source.
Protein quantitation was made by a modified method of Lowry
et al (1951). Accurate protein quantitation was extremely
important
for
de te rmining
enzyme
specific
m m
Therefore, samples were saponified in order to solubilize all
proteins and each solubilized sample was assayed in duplicate
or triplicate for protein.
Assay for Dehydro POL PP Synthase.
The
e thod
was developed as adapted from the method described by Grange
and Adair ( 1977 ). The level of the enzyme was monitored by
measuring the formation of polyprenyl products, dehydro DOL
PP and dehydro DOL P.
It was particularly important to


11
Physiological and morphological studies have indicated
that the Sertoli cells undergo cyclic changes in their
metabolic activity which are related to specific stages in
the cycle of the seminiferous epithelium. Cytochemical
studies showed that several enzyme activities in Sertoli
cells vary depending on the stage of the cycle of the
seminiferous epithelium. For instance, the peak activity of
acid phosphatase appeared at stages VII and VIII, but little
activity was found in stages IX through II (Niemi & Kormano,
1965). A similar stage specific distribution has been
observed with the thiamine pyrophosphatase in Sertoli cells
(Hilscher et al., 1979).
Sertoli Cells Secrete Many Glycoproteins
One
o f
the
mechanisms
by
wh i ch
Sertoli
cells
biochemically influence the spermatogenic cells is through
the synthesis and secretion of glycoproteins.
At leas t 15%
o f
all
the
proteins synthesized by
Sertoli
cell
glycoproteins (Bridges
al ,
1986) .
Some
o f
the
glycoproteins secreted by the Sertoli cells are specific to
the testis
and others are similar, if not identical, to
serum proteins.
Sertoli cells secrete glycoproteins into the
lumen of the tubules, perhaps into the blood stream or lymph,
and
maybe into
the
space
between
Sertoli
cells
and
spermatogenic cells for subsequent uptake by spermatogenic
cells .
Several glycoproteins secreted by the Sertoli cells


CHAPTER II
DEVELOP AND OPTIMIZE AN ASSAY FOR DEHYDRO DOLICHYL
DIPHOSPHATE SYNTHASE FROM RAT TESTES
Introduction
Elucidation of the mechanisms that regulate the
synthesis of N-linked glycoproteins requires a clear
understanding of the biosynthesis and metabolism of DOL and
DOL P. For this reason we have undertaken studies on the
biosynthesis and metabolism of dehydro DOL PP in rat testes.
It seems that dehydro DOL PP is the precursor of DOL PP in
the de novo biosynthesis pathway. Dehydro DOL PP synthase is
responsible for the de novo biosynthesis of dehydro DOL PP
from isopentenyl diphosphate and t,t-farnesyl diphosphate.
This synthase has been demonstrated in several animal tissues
(Grange & Adair, 1977; Wellner & Lucas, 1979; Wong & Lennarz,
1982; Adair & Keller, 1982; Adair et al., 1984; Adair &
Cafmeyer, 1987a and 1987b; Baba et al., 1987), and is
membrane associated. The products of this synthase were
labile to acid and yielded petroleum ether soluble products
indicating that the a-isoprene unit was unsaturated. Adair
and Keller (1982) characterized the carbon number of the
enzymatic product in rat liver and showed they are a group of
38


13
characterized this compound from pig liver (Pennock et al.,
1960) .
Mammalian polyprenols contain a larger number of
isoprene residues Cg5-C]_]_5 (17-23 x C5) than those present in
plants and bacteria (10-12 x C5). Polyprenols found
in bacteria have an oc-unsaturated isoprene unit. However, it
is known that liver contains small amounts of a-saturated
shorter polyprenols (Mankowski et al., 1976), and in some
tissues, such as bovine pituitary gland (Rodominska-Pyrek et
al ,
1979) and hen oviduct (Hayes & Lucas, 1980),
a-
unsaturated compounds have been reported. DOL is present in
most eukaryotic cells and is found in particularly high
concentration in some human tissues, such as the adrenal
gland, pancreas, pituitary gland, testis and thyroid gland
(Rupar & Carroll, 1978; Tollbom & Dallner, 1986).
James and
Kandutsch showed that in mouse the synthesis of DOL is
uch
more active in testes than in liver (James & Kandutsch,
1980c). It seems that DOL content is relatively high in the
rapidly growing and differentiating tissues.
For example,
the DOL content of the hyperplastic liver nodules in liver is
four times higher in the homogenate and six times higher in
the microsomes than that found in normal rat liver.
In
developed hepatocareinoma the amount of DOL was found to be
doubled.
In contrast to free DOL, DOL P was found to be
greatly decreased in nodules (Eggens et al., 1984).
Other studies show an increase in DOL levels with age
although the increases vary widely with tissues (brain,


142
Letts, P. J., Hunt, R. C., Shirley, M. A., Pinteric, L., and
Schachter. H. 1978, Biochim. Biophys. Acta. 541: 59-75
Letts, P. J., Meistrich, M. L., Bruce, W. R., and Schachter,
H. 1974a, Biochim. Biophys. Acta. 343: 192-207.
Letts, P. J., Pinteric, L., and Schachter, H. 1974b, Biochim
Biophys. Acta. 372: 304-320.
Louis, B. G., and Fritz, I. B. 1977, Mol. Cell. Endocrin
7: 9-16.
Lowry, 0. H., Roseborough, N. J., Farr, A. L., and Randall,
R. J. 1951, J. Biol. Chem. 193: 265-273.
Lucas,
J.
J. .
1979, Biochem.
Biophys. Acta
. 572 :
153-159 .
J.
J. ,
and Levin,
E .
1977, J. Biol.
Chem .
252: 4330
4336

J .
J ,
and Navar,
C .
1978, Biochim.
Biophy
s. Acta.
528 :
475
-482 .
, T.
M. ,
Hallaban,
T .
W., Beach, D.
H and
Lucas, J
J. 1985, Arch. Biochem. Biophys. 238: 401-409.
Mankowski, T., Jankowski, W., Chojnacki, T., and Franke, P.
1976, Biochemistry. 15: 2125-2130.
Martin, H. G., and Thorne, K. J. I. 1974, Biochem. J. 118:
277-280.
Means, A. R., and Vaitukaitis, J. 1972, Endocrinology, 90:
39-46.
Meistrich, M. L., Trostle, P. K., and Brock, W. A. 1981,
Bioregulators of Reproduction. Academic Press, New
York, pp 151-166.
Millette, C. F., and Moulding, C. T., 1981a, Gamete. Res. 4:
317-331.
Millette, C. F., and Moulding, C. T., 1981b, J. Cell. Sci.
48: 367-382.
Morales, C., and Clermont, Y. 1986, Biol Reprod. 35: 393-405.
Mukerji, S. K., and Meizel, S. 1979, J. Biol. Chem. 254:
11721-11728.
Niemi, M., and Kormano, M. 1965, Anat. Rec. 151: 159-170.


25
Acetyl CoA
t
HMG-CoA
reductase
t
Mevalonate

Isopenteny1 Diphosphate
t
Farnesyl Diphosphate
trans- prenyl transferase
cis-prenyl transferase
t
Squalene
Polyp reny1 Diphosphate
Precursors of Ubiquinone
Dehydro DOL PP

Choles terol

Dolichol
Figure 1-5. Pathway of Isoprenoid Biosynthesis. c i s -
prenyl transferase is the same as dehydro DOL PP synthase.


Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHANGE IN DEHYDRODOLICHYL DIPHOSPHATE SYNTHASE
DURING SPERMATOGENESIS IN THE RAT
by
ZHONG CHEN
Ap ri1 19 8 8
Chairman: Charles M. Allen, Jr. Ph.D.
Major Department: Biochemistry and Molecular Biology
Dolichyl phosphate (DOL P), a carbohydrate carrier in
glycoprotein biosynthesis, may be formed from the metabolism
of dehydro dolichyl diphosphate (dehydro DOL PP). A method
has been developed to measure 2,3-dehydro DOL PP synthase
with [ ] isopentenyl diphosphate and t t-farnesyl
diphosphate. Enzymatic activity was measured in homogenates
prepared by sonication of seminiferous tubules or isolated
cell fractions. There was a 2 fold increase in tubular
activity between day 7 and day 23 and a similar decrease in
activity between day 23 and day 60. The increase in activity
paralleled an increase in DOL P concentration, suggesting a
regulatory role for the synthase in DOL P synthesis.
xi


139
Eggens, I., Chojnacki, T., Kenne, L., and Dallner, G. 1983,
Biochim. Biophys. Acta. 751: 355-368.
Ekblom, P., Thesleff, I., Saxen, A., Miettinen, A., and
Timp1, R. 1983, Proc Natl Acad Sci. USA. 80: 2651-2655.
Ekstrom, T., Eggens, I., and Dallner, G. 1982, FEBS Lett.
150: 133-136.
Ekstrom, T. J., Chojnacki, T., and Dallner, G. 1987, J. Biol.
Chem. 262: 4090-4097.
Elmberger, P. G., Kalen, A., Appelkvist, E. L., and Dallner,
G. 1987, Eur. J. Biochem. FEBS. 168: 1-11.
Faust, J. R., Goldstein, J. L., and Brown, M. S. 1979, Proc.
Natl. Acad. Sci. 76: 5018-5022.
Fawcett, D. W., 1975, in Handbook of Physiology (Hamilton,
D. W., and Greep, R. 0. eds.). American Physiological
Society, Bethesda, MD. Vol. 5, pp 21-56.
Fenderson, B. A., O'Brien, D. A., Millette, C. F., and Eddy,
E. M. 1984, Dev. Biol. 103: 117-128.
Flechon, J. E., 1979, Gamete Res. 2: 43-51.
Florke, S., Phi-van, L., Mulle Es ter1, W., Scheuter, H. P.,
and Engel, W. 1983. Differentiation. 24: 250-256.
French, F. S., and Ritzen, E. M. 1973, Endocrinology. 93: 88
95 .
Fritz, I. B., Rommerts, F. F. G., Louis, B. G., and
Dorrington, J. H. 1976, Exp. Cell Res. 123: 127-135.
Galdieri, M., Monaco, L., and Stefanini, M. 1984, J. Andol
5: 409-415.
Gaunt, S. J., 1982, Dev. Biol. 89: 92-100.
Goldberg, E. 1977, Isozymes Curr Top, Biol Med Res. 1: 79-
124 .
Gough, D. P., and Hemming, F. W. 1970, Biochem. J. 118: 163
16 6.
Grange, D. K., and Adair, W. J. 1977, Biochem. Biophys. Res.
Commun. 79: 734-740.
Griswold, M. D., Roberts, K., and Bishop, P. 1986,
Biochemistry. 25: 7265-7270.


34
obscure. Furthermore, the question concerning the ability of
the
Sertoli
cell
to
synthesize DOL was not
addressed
directly.
Nyquist and Holt (1986) recently measured the cellular
and subcellular distribution of DOL in rat testis by a HPLC
ethod and found that elutriation purified spermatogenic
cells
had very
low
concentrations
of DOL.
Pachytene
spermatocyte and round spermatids contained 25.8 and 36.5 ng
DOL/mg protein, respectively.
Washed epididymal sperm also
had a very low DOL content (18.8 ng DOL/mg protein).
In
contrast, the Sertoli cell enriched tubular fraction that was
recovered during the preparation of purified spermatogenic
cells
showed
the
highest DOL content (3450 ng DOL/mg
protein).
These results implied that the Sertoli cells
accumulate
major portion of the testicular DOL.
This
result at first appears inconsistent with the results of
Potter et al. (1981b), who found low DOL synthesis in the
testes from X-irradiated mice and spermatogenic deficient
mice, which, although they were depleted of spermatogenic
cells, were apparently normal with respect to the number and
function of the Sertoli cells. Nyquist and Holt suggested in
explanation that
the
bulk
o f
DOL may be
synthesized
in
the
spermatogenic
cell
and
subsequently
transported to the Sertoli cell.
A possible route, they
hypothesized, would be by Sertoli cell phagocytosis of
residual body cytoplasm during spermiation.


59
In all cases the
increasing
time.
diphosphatase which is
Triton X-100.
sum of the two products increased with
This supports the presence of a
inhibited by higher concentrations of
The results of a similarly designed pulse-chase
experiment support the same conclusion (Table 2-2). In this
case non-radiolabeled isopentenyl diphosphate was added to
the incubation mixture after one hour incubation in 2% Triton
X-100. The reaction was continued for an another hour with
addition of farnesyl diphosphate and enzyme but under lower
Triton X-100 concentration (0.5%). Any polyprenyl phosphate
made during the second hour of incubation, the chase phase,
would not have been radiochemically detectable under the
conditions used. Since there was a loss in radiolabeled
product migrating as the putative dehydro DOL PP concomitant
with an increase in radiolabeled dehydro DOL P, it can be
concluded that the slower migrating product is a precursor of
dehydro DOL P, and therefore it was dehydro DOL PP.
The precursor-product relationship between dehydro DOL
PP and dehydro DOL P was also shown in a more detailed time
dependent study illustrated in Fig. 2-4. The formation of
the mono- and diphosphate was determined under standard assay
conditions except that Triton X-100 concentration was raised
to 1% to partially inhibit the diphosphatase activity. The
distinct lag in the formation of dehydro DOL P relative to
the diphosphate illustrates clearly the classical precursor-


Figure 2-9. Farnesyl Diphosphate Concentration
Dependency on the Formation of Dehydro DOL PP and
Dehydro DOL P.
Incubations
(pH7.5)
10
mM
containing
0.5%
100
mM Tris-HCl
buffer
Triton X-100
concentrations of
1.6
50 mM NaF
3 6 /iM
1.0
mg of enzyme protein
were carried out at 37 C for 60 minutes. The formation
of []-dehydro DOL PP (Panel A) and []-dehydro DOL
P (Panel B) were estimated by the method described
before. Panel C represents dehydro DOL PP synthase
activity (A+B).
MgC12 >
t,t-farnesyl diphosphate,
[1-^-^C] isopentenyl diphosphate,
final volume of 0.25
varying
mM ATP,
and
m
for
ml


64
I

A
B


27
clearly demonstrated high DOL synthesis in specific
spermatogenic cell populations. However, the enzyme or
enzymes that might be responsible for this increased DOL
synthesis were not identified.
In order to understand how glycoprotein assembly is
coordinated with differentiation, it is necessary to
understand how the individual steps in the sequence of
glycoprotein assembly are modulated. We focus our attention
here on the biosynthesis of the carbohydrate carrier, DOL PP.
It is believed that dehydro DOL PP, a derivative of DOL
PP with an a-unsaturated isoprene unit, is an intermediate in
the de novo biosynthesis of DOL PP. Figure 1-6 compares the
structures of dehydro DOL PP and DOL PP.
The enzyme catalyzing the synthesis of this
intermediate is dehydro DOL PP synthase. Dehydro DOL PP
%
biosynthesis from isopentenyl diphosphate (IPP) and farnesyl
diphosphate (FPP) has been demonstrated in hen oviduct
(Grange & Adair, 1977), Ehrlich tumor cells (Adair &
Trepanier, 1980), chicken liver (Wellner & Lucas, 1979),
mouse L-1210 cells (Adair & Cafmeyer, 1987a) and yeast (Adair
& Cafmeyer, 1987b).
FPP + (IPP) dehydro DOL PP (17-23 x C5)


Figure 3-1. Purity of Enriched Cell Fractions.
The testicular tubules were dissociated with
proteases and the spermatogenic and Sertoli cells were
separated as described in the Materials and Methods.
The purity of each cell fraction, as estimated by
Nomarski differential interference microscopy (x 600),
was estimated to be 80% for spermatids (Panel A), 70%
for pachytene spermatocytes (Panel B) and 70% for
Sertoli cells (Panel C).


96
et al., 1977).
Coordinated secretion of androgen binding
protein by the Sertoli cell and the association of the
Sertoli cell with pachytene spermatocytes during the meiotic
phase
even
s ugge s t s
the
possibility
o f
intercellular
communication in regulating temporal expression of certain
Sertoli cell proteins (LeMagueresse et al., 1980; Ritzen et
al., 1982; Galdieri, 1984; LeMagueresse & Jegou, 1986).
The biosynthesis of these glycoproteins at specific
phases during testicular development requires a coordinate
func tioning
of a series of enzymes, so that cofactors and
associated biochemical apparatus must be present in the cell
at or before the time of glycoprotein expression or function.
However, only a few studies have described the regulation of
express ion
o f
these
components
possible controlling
factors of spermatogenesis.
Three of the glycosyl transferases, which are needed
for the terminal reactions in glycoprotein oligosaccharide
biosynthesis in rat and mouse testis
have been shown to
increase in specific activity in a sequential manner, that
parallels their use in the last steps of oligosaccharide
processing (Letts et al., 1974a).
The availability of DOL P, another
component
in the synthesis of the N-linked glycoproteins, has been
suggested as a rate- 1imiting factor in some developmental
processes (Lucas, 1979; Harford 6c Waechter, 1980; Rossignol
et al., 1980).
Therefore, the study of DOL metabolism will


15
liver, and 65-90% in mouse testes and preputial glands
(Malvar et al., 1985). The mechanism which determines these
distributions is not known.
POL P in Glycoprotein Synthesis
In the early 1970s, Behrens and Leloir demonstrated the
involvement of DOL P in the N-linked glycoprotein
biosynthesis (Behrens & Leloir, 1970; Behrens et al., 1971).
Although, only a small amount of the total DOL in cells is
phosphorylated (Eggens et al., 1983), the phosphorylated form
(DOL P) is an essential carbohydrate carrier in the
biosynthesis of asparagine -1inked glycoproteins (Struck &
Lennarz, 1980; Hubbard & Ivatt, 1981).
Most secretory proteins and membrane proteins, numerous
receptors and proteins related to cell recognition are
glycoproteins. A typical N-linked glycoprotein contains one
or a few oligosaccharide units linked to asparagine side
chains by N-glycosidic bonds.
The biosynthesis of N-linked glycoproteins proceeds
through a cyclic process, which requires the involvement of
DOL P, in the synthesis of the core oligosaccharide unit
(Parodi & Leloir, 1979; Hubbard & Ivatt, 1981).
Figure 1-3 represents a highly abbreviated
representation of this cyclic process and associated
reactions (Rip et al., 1985; Hemming, 1985, for review).


84
DOL branches from that to cholesterol at the level of
farnesyl diphosphate. In vitro experiments with isopentenyl
diphosphate as a precursor have shown that 2,3-dehydro DOL P,
presumably one of the later intermediates in the DOL
biosynthetic pathway, could be synthesized in preparations
from hen oviduct (Grange 6c Adair, 1977), avian liver (Wellner
& Lucas, 1979), Ehrlich tumor cells (Adair 6c Trepanier,
1980), mouse L-1210 cells (Adair & Cafmeyer, 1987a), yeast
(Adair & Cafmeyer, 1987b), and testes (Baba et al., 1987).
Testis has been shown to contain large quantities of dolichol
(Rupar & Carroll, 1978; Tollbom 6c Dallner, 1986; James 6c
Kandutsch, 19 80c) and therefore it seems to be an
appropriate tissue in which to investigate DOL biosynthesis.
Homogenates of rat seminiferous tubules have been
previously shown to catalyze the synthesis of acid labile
polyprenyl mono- and diphosphate (the a-unsaturated isoprene
unit is acid labile). The enzymatic activity was dependent
upon t,t-farnesyl diphosphate, isopentenyl diphosphate and
divalent cation (Baba et al., 1987).
The conditions for the assay of dehydro DOL PP synthase
in sonicates of rat seminiferous tubules have been
systematically characterized and optimized here. The
sonication of seminiferous tubules gives a few advantages for
the assay. The sonication method, in contrast to
homogenization with a glass homogenizer, can be easily
applied to a small amount of testicular tissue, such as


29
Adair and Keller (1982) have showed that the enzymatic
products of the liver enzyme were a group of dehydro DOL
monophosphates ranging in size from C75 to C95 (15-19x05).
Recent
data
fro
this
laboratory have
shown that
testicular homogenates and their membrane fractions will
catalyze the synthesis of ^75-035 dehydro DOL P and dehydro
DOL
PP
from t,t-farnesyl diphosphate
and
isopentenyl
diphosphate (Baba et al., 1987).
The isolation of dehydro
DOL P and DOL P in some cases instead of the diphosphate
analogues reflects the activity of diphosphatases.
A
mechanism
o f
reduction
o f
dehydro dolichyl
derivatives to DOL was suggested recently by Ekstrom et al.
(1987). They hypothesized that during the saturation of the
terminal isoprene unit, dehydro DOL PP (also referred to by
some as polyprenols to distinguish them from the saturated
counterparts, the dolichols) are elongated by an additional
isoprene residue and saturated at the same time.
The enzymes of DOL metabolism are membrane associated
and,
therefore,
most often assayed
preparations.
Solubilization and purification of individual
enzymes of the dolichol pathway have proven to be difficult
and in most cases not particularly successful.
DOL Metabolism in Spermatogenic Cells
James and Kandutsch (1980c*) studied DOL biosynthesis in
X-irradiated or genetically mutated mice that were deficient


Triton X-100 (%>
pmoles Isopentenyl Diphosphate Incorporated/ mg protein-hr
ui


The activity in homogenates of protease treated
seminife rous
tubu1e s,
and
cell
fractions
enriched
in
spermatogenic cells or Sertoli cells also changed as a
function of age in the rats
The highest enzymatic activity
occurred in each case at age 23 days.
Cell fractions
enriched in pachytene spermatocytes, spermatids or Sertoli
cells were shown to have higher synthase activity than a
whole testicular homogenate or a mixture of cells prepared by
collagenase-trypsin treatment of tubules. Enzymatic activity
in pachytene spermatocytes expressed as pmoles/mg protein was
about 5.3 fold higher than spermatogonia, 1.7 fold higher
than spermatids and about 8.3 fold higher than spermatozoa.
The enzymatic activity of pachytene spermatocytes expressed
as pmoles/10^ cells was 4.5 fold higher than spermatids and
about 126 fold higher than spermatozoa.
These studies are
the first to show that Sertoli cells have the potential to
synthesize DOL.
The increase in synthase activity in spermatogenic
cells
and
Sertoli
cells
during early stages
o f
spermatogenesis indicates that both cell populations are
contributing to the increase in activity in seminiferous
tubules.
Furthermore, the increase can be explained on the
basis of changes in the specific
of the Sertoli
cells and the different populations of spermatogenic cells.
This increase may be important in regulating the availability
of dolichyl phosphate for glycoprotein biosynthesis during
early stages of spermatogenesis.


104
treated tubules was parallel to that seen in the untreated
tubules. Although there was a decrease in specific activity
in the protease treated tubules at all ages
there was no
apparent selective loss of activity at any particular age
2
Synthase Activity in Enriched Cells from Testis.
The
level of synthase activity was also evaluated in isolated
spermatogenic cells and epididymal spermatozoa.
The time
course of formation of dehydro DOL PP and dehydro DOL P was
evaluated in the enriched pachytene spermatocytes, spermatids
and Sertoli cell (Figure. 3-3).
The results indicated that
the su
of product formation with 1 mg of protein from
homogenates
o f
each
cell
type
increased linearly with
increasing incubation time as shown in the chapter II for the
tubular homogenates.
Protease treatment did not have an
adverse effect on the linearity of time dependent assay
The
enzyme
specific activity in Sertoli cell was the highest.
Homogenates of cell fractions highly enriched for pachytene
spermatocytes (70% purity) and spermatids (80% purity) had
synthase activity equal to or higher than a whole testicular
2
The
cells
from
methodology for dissociating
seminiferous tubules was satisfactory, since 80-90% of the
enzymatic activity still remained after the
treatment. Disease, a commercial preparation of
has
also
success fully.
for
proteases
preparation of collagenase
reported to dissociate several tissues
Therefore.
Dispase,
been
dispase was
the cellular
a replacement
dissociation from
collagenase in
seminiferous tubules. About 40% of the enzymatic activity in
the collected dissociated cells was lost by this treatment
(results are not shown).


2-10 A Double Reciprocal Plot of the Sum of Dehydro
DOL PP and Dehydro DOL P Formation vs. Farnesyl
Diphosphate Concentration 75
2-11 Time Course of Incorporation of [^C]-Isopentenyl
Diphosphate into Dehydro DOL PP and Dehydro DOL P....77
2-12 Dehydro DOL PP Synthase in Testicular Homogenate
of Different Aged Rats 79
2-13 Dehydro DOL PP Synthase Activity in Sonicates of
Tubules from Rats of Different Ages 81
2-14 Comparison of Changes in DOL P Concentration,
and Dehydro DOL PP Synthase Activity as a
Function of Rat Age 88
3-1 Purity of Enriched Cell Fractions 105
3-2 Dehydro Dolichyl Diphosphate Synthase Activity in
Sonicates of Tubules from Rats of Different Ages....109
3-3 Time Course of Incomorat ion of [ ^C 1 Isooentenvl
Diphosphate into Dehydro DOL PP and Dehydro DOL p...111
3-4 Dehydro Dolichyl Diphosphate Synthase Activity in
Enriched Spermatogenic Cell Population 115
3-5 Age Dependent Variation in Synthase Activity in
Sertoli Cells, Spermatogenic Cells and Protease
Treated Seminiferous Tubules 118
3-6 The Relationship between the Dehydro DOL PP
Synthase Activity and Spermatogenesis during
Testicular Development 128
viii


Figure 3-3. Time Course of Incorporation of [^4C]-
Isopentenyl Diphosphate into Dehydro DOL PP and Dehydro
DOL p .
Incubations
(pH7.5)
containing
10 mM MgC 12 0.5%
100
mM Tris-HCl
buf fer
Triton X-100
2 5 0 /xM t
farnesyl diphosphate,
14C]-isopentenyl
1.6 mM ATP, 50 mM NaF, 36 /xM [
t-
1
diphosphate
and 1.0
sonicated of
and Sertoli
mg
enriched pachytene spermatocyte,
cell from seminiferous tubules
protein of
spermatid,
protein in a
37 0 C for the
DOL PP and
enzyme
were carried out at
indicated times. The sum of [ -*-4C ]- dehydro
[ ^4C ]- dehydro DOL P were estimated by the
described before. Sertoli cells are isolated
i 40
total volume of 0.25 ml
The
DOL P
me thod
from 23 day old rats, spermatogenic cells are fro
day old rats.


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Biology
This dissertation was submitted to the Graduate Faculty of
the College of Medicine and the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
April, 1988
Dean, College of Medicine
Dean, Graduate School


DAYS AFTER BIRTH
pmoles Isopentenyl Diphosphate Incorporated/ mg protein-hr
oo


CHANGE IN DEHYDRODOLICHYL DIPHOSPHATE SYNTHASE
DURING SPERMATOGENESIS IN THE RAT
BY
ZHONG CHEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988

This dissertation is dedicated to
my
parents, Quo-Wei Chen and Zhi-Huei Chang,
y loving wife, He-Ping Han, and my son,
Henry Hai Pei.

ACKNOWLEDGEMENTS
Sincere appreciation is expressed to my friend, mentor,
Dr.
Charles M.
Allen,
Jr.,
for his enduring patience,
guidance, and support. I am deeply grateful for his help in
proofreading my English.
His belief in the fundamentals of
biochemistry and molecular biology and literacy influenced
the designing of each project undertaken, and I learned the
meaning of scientific integrity, creativity, and serendipity.
I
am
indebted
to
the members
o f my
supervisory
committee, Dr. Michael S. Kilberg, Dr. Rusty J. Mans, Dr.
Thomas W.
0'Brien,
and Dr.
Lynn J.
Romre11
for their
encouragement, assistance and productive criticism of my
research projects.
I am especially grateful to Dr. Lynn J.
Romrell who allowed me the privilege of working in his
laboratory and who provided support and advice through the
duration of the endeavor.
In addition, I am indebted to the faculty of the
Department of Biochemistry and Molecular Biology for both
educational and financial support. Finally, I would like to
thank William Blakeney, Mary Handlogton, Michael Campa and
William Wong for their friendship and assistance during the
course of my studies.

TABLE OF CONTENTS
ACKNOWLEDGEMENTS
LIST OF TABLES..
LIST OF FIGURES.
ABBREVIATIONS...
ABSTRACT
Page
. iii
. vi
. vi i
. ix
. xi
CHAPTERS
IINTRODUCTION 1
The Spermatogenesis 1
The Sertoli Cells 7
The Role of DOL P in Glycoprotein Biosynthesis....12
Studies Related to DOL Biosynthesis and
Spermatogenesis 26
Significance 3 5
Obj ectives 37
IIDEVELOP AND OPTIMIZE AN ASSAY FOR DEHYDRO
DOLICHYL DIPHOSPHATE SYNTHASE FROM RATS TESTES....38
Introduction 38
Materials and Methods 41
Results 46
Discussion 83
IIIDEHYDRODOLICHYL DIPHOSPHATE SYNTHASE ACTIVITY
MEASURED IN ENRICHED SPERMATOGENIC CELL
POPULATIONS 93
Introduction 9 3
Materials and Methods 98
Results 103
Discussion 122
IVCONCLUSIONS AND DIRECTIONS 131
4
IV

APPENDICES
A SUMMARY OF EXPERIMENTAL DATA PRESENTED IN
FIGURE 2-12. DEHYDRO DOL PP SYNTHASE ACTIVITY
IN SONICATES OF TUBULES FROM RATS OF
DIFFERENT AGES 134
B TYPICAL NUMBER OF RATS, TOTAL TUBULE WEIGHT,
AND NUMBER OF ASSAY USED FOR EACH EXPERIMENT
AS A FUNCTION OF RAT AGE 135
C SUMMARY OF EXPERIMENTAL DATA PRESENTED IN
FIGURE 3-5. AGE DEPENDENT VARIATION IN
SYNTHASE ACTIVITY IN SERTOLI CELLS,
SPERMATOGENIC CELLS AND PROTEASE TREATED
SEMINIFEROUS TUBULES 136
BIBLIOGRAPHY 137
BIOGRAPHICAL SKETCH 146
V

LIST OF TABLES
Table Page
2-1 Incorporation of A^-fl-^^C] Isopentenyl Diphosphate
and [ a,^9-]-Isopentenyl Diphosphate into
Dehydro DOL PP and Dehydro DOL P 52
2-2 Formation of Enzymatic Product at Different Triton
X-100 Concentrations in Pulse-Chase Experiment 58
3-1 Dehydro Dolichyl Diphosphate Synthase Activity in
Enriched Spermatogenic Cells 117
3-2 Estimated Specific Activities of Dehydro DOL PP
Synthase in Pure Sperma togenic Cells 120
3-3 Estimated Specific Activities of Dehydro DOL PP
Synthase in "Pure" Sertoli Cells From Rats of
Different Ages 121
vi

LIST OF FIGURES
Figur e
Page
1-1 The Cellular Composition of the 14 Stages of the
Cycle of the Seminiferous Epithelium in Rat 4
1-2 Schematic Drawing of Human Seminiferous Epithe1ium....9
1-3 DOL Cycle for Glycoprotein Formation in
Eucaryotic Cells 16
1-4 A Putative Pathway Showing the Relationship between
Dehydro DOL PP and Glycoprotein Biosynthesis 22
1-5 Pathway of Isoprenoid Biosynthesis 25
1-6 The Structure of Dehydro DOL PP and DOL PP 28
2-1
2-2
2-3
Separation of Enzymatic Products by TLC 49
Triton X-100 Dependency on the Formation of
Dehydro DOL PP and Dehydro DOL P 53
Dependence of Product Formation on Triton X-100
Concentration and Incubation Time 56
2-4 Time Course of Dehydro DOL PP and Dehydro DOL P
Formation 61
2-5 Product of Base Hydrolysis of Dehydro DOL PP 63
2-6 Effect of Protein Concentration on Dehydro DOL PP
and Dehydro DOL P Formation 67
2-7 Isopentenyl Diphosphate Concentration Dependency on
the Formation of Dehydro DOL PP and Dehydro DOL P....69
2-8 A Double Reciprocal Plot of the Sum of Dehydro
DOL PP and Dehydro DOL P Formation vs. Isopentenyl
Diphosphate Concentrations 71
2-9 Farnesyl Diphosphate Concentration Dependency on
the Formation of Dehydro DOL PP and Dehydro DOL P....73
vii

2-10 A Double Reciprocal Plot of the Sum of Dehydro
DOL PP and Dehydro DOL P Formation vs. Farnesyl
Diphosphate Concentration 75
2-11 Time Course of Incorporation of [^C]-Isopentenyl
Diphosphate into Dehydro DOL PP and Dehydro DOL P....77
2-12 Dehydro DOL PP Synthase in Testicular Homogenate
of Different Aged Rats 79
2-13 Dehydro DOL PP Synthase Activity in Sonicates of
Tubules from Rats of Different Ages 81
2-14 Comparison of Changes in DOL P Concentration,
and Dehydro DOL PP Synthase Activity as a
Function of Rat Age 88
3-1 Purity of Enriched Cell Fractions 105
3-2 Dehydro Dolichyl Diphosphate Synthase Activity in
Sonicates of Tubules from Rats of Different Ages....109
3-3 Time Course of Incomorat ion of [ ^C 1 Isooentenvl
Diphosphate into Dehydro DOL PP and Dehydro DOL p...111
3-4 Dehydro Dolichyl Diphosphate Synthase Activity in
Enriched Spermatogenic Cell Population 115
3-5 Age Dependent Variation in Synthase Activity in
Sertoli Cells, Spermatogenic Cells and Protease
Treated Seminiferous Tubules 118
3-6 The Relationship between the Dehydro DOL PP
Synthase Activity and Spermatogenesis during
Testicular Development 128
viii

KEY TO ABBREVIATIONS
BS A
bovine serum albumin
Ci
cur i e
cm
centimeter
cpm
counts per minute
C
degree centigrade
DIBK
diisobutyl ketone
DNA
deoxyribonucleic acid
DNase
deoxyribonuclease
DOL
do1icho1
DOL P
dolichyl phosphate
DOL PP
dolichyl diphosphate
dpm
disintegrations per minute
EKRB
enriched Krebs-Ringer bicarbonate medium
IPP
is openteny1 diphosphate
FPP
t,t-farnesyl diphosphate
G lc
glucose
GlcNAc
acetylglycosamine
GPP
gerany1 diphosphate
g
gram
HAc
acetic acid
hr
hour
M
molar
ix

Man
MEM
M
H Ci
Mg
Ml
/iM
/imole
mM
mm
mmole
PBS
pmole
PP
RNA
rpm
TLC
Tris
v/v
v/w
w/v
w/w
manno s e
Eagle's minimal essential medium
micron
microcurie
microgram
microliter
micromolar
micromole
millimo1ar
millime ter
millimole
phosphate buffered saline
picomole
diphosphate
ribonucleic acid
revolutions per minute
thin layer chromatography
Tris-(hydroxymethyl)aminomethane
on a volume-to-volume basis
on a volume-to weight basis
on a we ight-to-volume basis
on a we ight-1o-weight basis
x

Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHANGE IN DEHYDRODOLICHYL DIPHOSPHATE SYNTHASE
DURING SPERMATOGENESIS IN THE RAT
by
ZHONG CHEN
Ap ri1 19 8 8
Chairman: Charles M. Allen, Jr. Ph.D.
Major Department: Biochemistry and Molecular Biology
Dolichyl phosphate (DOL P), a carbohydrate carrier in
glycoprotein biosynthesis, may be formed from the metabolism
of dehydro dolichyl diphosphate (dehydro DOL PP). A method
has been developed to measure 2,3-dehydro DOL PP synthase
with [ ] isopentenyl diphosphate and t t-farnesyl
diphosphate. Enzymatic activity was measured in homogenates
prepared by sonication of seminiferous tubules or isolated
cell fractions. There was a 2 fold increase in tubular
activity between day 7 and day 23 and a similar decrease in
activity between day 23 and day 60. The increase in activity
paralleled an increase in DOL P concentration, suggesting a
regulatory role for the synthase in DOL P synthesis.
xi

The activity in homogenates of protease treated
seminife rous
tubu1e s,
and
cell
fractions
enriched
in
spermatogenic cells or Sertoli cells also changed as a
function of age in the rats
The highest enzymatic activity
occurred in each case at age 23 days.
Cell fractions
enriched in pachytene spermatocytes, spermatids or Sertoli
cells were shown to have higher synthase activity than a
whole testicular homogenate or a mixture of cells prepared by
collagenase-trypsin treatment of tubules. Enzymatic activity
in pachytene spermatocytes expressed as pmoles/mg protein was
about 5.3 fold higher than spermatogonia, 1.7 fold higher
than spermatids and about 8.3 fold higher than spermatozoa.
The enzymatic activity of pachytene spermatocytes expressed
as pmoles/10^ cells was 4.5 fold higher than spermatids and
about 126 fold higher than spermatozoa.
These studies are
the first to show that Sertoli cells have the potential to
synthesize DOL.
The increase in synthase activity in spermatogenic
cells
and
Sertoli
cells
during early stages
o f
spermatogenesis indicates that both cell populations are
contributing to the increase in activity in seminiferous
tubules.
Furthermore, the increase can be explained on the
basis of changes in the specific
of the Sertoli
cells and the different populations of spermatogenic cells.
This increase may be important in regulating the availability
of dolichyl phosphate for glycoprotein biosynthesis during
early stages of spermatogenesis.

CHAPTER I
INTRODUCTION
Spermatogenesis
Spermatogenic Cycles and Waves
Spermatogenesis is a developmental process in which the
spermatogenic cell undergoes a series of biochemical and
orphological changes through three well described phases:
spermatogonial
renewal
and proliferation, meiosis,
and
spermiogenesis.
Spermatogenesis in the rat starts during
fetal development with appearance of gonocytes by postnatal
day 4 and continues throughout adult life.
The first
spermatozoa appear within the lumens of the seminiferous
tubules at about 45 days of ages
and all
o f
spermatogenic cycle are represented (Clermont & Perey, 1957;
Knorr et al., 1970).
The initial phase of spermatogenesis,
the spermatogonial phase, occurs in the basal compartment of
the
seminiferous epithelium
and
cons is ts
o f
mitotic
pro
ion
o f
spermatogonia
f rom
stem
cells.
The
spermatogonia divide and differentiate sequentially into type
A spermatogonia, intermediate spermatogonia, and type B
spermatogonia.
The type B spermatogonia divide to for
1
f

2
preleptotene primary spermatocytes, which undergo a final
replication of nuclear DNA before entering meiotic prophase.
The
preleptot ene
spermatocytes
m
from
the
basal
compartment
t o
the
adluminal
compartment
where
spermatogenesis
l s
completed.
The
seco nd phase
of
spermatogenesis, meiosis, occurs while the spermatocytes
remain on the adluminal side of the intercellular Sertoli
j unctions.
Meiotic prophase, which is subdivided into the
stages of the leptotene, zygotene, pachytene, diplotene, and
diakinesis, terminates in the first meiotic, or reductional,
division with the formation of secondary spermatocytes.
The
latter cells quickly enter the second meiotic, or equational
division to form the haploid spermatids. Spermiogenesis, the
final phase
o f
spermatogenesis,
cons is ts
o f
complex
morphological transformation of the haploid spermatogenic
cell, that culminates with the release of late spermatids
into the lumen of the seminiferous tubule.
Spermatogenesis has many unique features
The most
remarkable ones include the spermatogenic cycles and waves in
the seminiferous epithelium.
The various generations of
spermatogenic cells are not randomly distributed in the
seminiferous epithelium, but are organized into well-defined
cellular associations.
Certain cells are always found in
association with certain other cells.
Each of these cells
develops in synchrony with the others, so that if we could
watch one section of the tubule wall with a time-lapse

3
series of different cell associations would be
until the cycle was completed.
The time interval
between the appearance of the same cell association at
given point of the tubule is called the cycle of the
seminiferous epithelium.
The number of stages in the cycle
is constant for a given species; man has 6, mouse and monkey
have 12, the rat, 14 (Figure 1-1).
The cycle involves changes with time in the appearance
of one segment of
tubule, whereas the wave refers to the
distribution of different cellular associations along the
length of the tubule.
The segments of the tubules which
specific cellular associations occur in a sequential series
along the length of the tubule.
For example a segment
containing cells at stage V of the cycle is bordered on one
side by cells at stage IV and on the opposite side is at
s tage VI.
The average length of each tubular segment
roughly with
the
duration
o f
the
corresponding cellular association or stage of the cycle.
The sequence of segments or waves, representing the stages in
the cycle of the seminiferous epithelium, repeats itself
along the length of an individual tubule.
In the rat for
example, there is an average of 12 waves per tubule.

4
Figure 1-1. The Cellular Composition of the 14
of the Cycle of the Seminiferous Epithelium in Rat.
shows
the
Each numbered column (roman numeral)
spermatogenic cell types present in cellular associations
found in cross sections of seminiferous tubules. The
cellular associations or
ano the r
tages of the
in
cycle
of the
succeed one
seminiferous
cellular association XIV
time in any given
epithelium in the rat. Following
cellular association I reappears, so that the sequence
over again. Steps in the development of the spermatids,
numbered 1 to 19 and defined by changes in the structure of
the nucleus and acrosome. are indicated with arabic numerals.
Letters are used to identify
A1 A 2
A 3
and
spermatogonia
a4
In.
represent
intermediate
spermatogonia
four
and spermatocytes.
of type A
spermatogonia; PI, preleptotene spermatocytes
generations
spermatogonia;
: L.
B
spermatocytes
Z
spermatocytes
Di
spermatocytes
and Diakinesis
P
type
leptotene
pachytene
II
, zygotene
Diplotene
spermatocytes. The subscript m indicates the occurrence
mitotic division of the spermatogonia (From Dym, M. &
Clermont, Y., 1970, Am. J. Anat., 128, 265-282,).
secondary
o f

5
Exocrine and Endocrine Functions of Testis
The testis has both exocrine and endocrine functions.
The exocrine function of the testis resides in the cells of
the seminiferous epithelium which produce testicular fluids
and spermatozoa.
The endocrine function of the testis
resides primarily
in
the
Leydig cell population which
synthesizes and secretes the principal circulating androgen,
testosterone (Nearly 95% of the testosterone is produced by
the testis; the rest is produced by the adrenal glands).
The
steroidogenic and spermatogenic activities of the testis are
regulated by hormonal interactions among the hypothalamus,
adenohypophysis, and the cells of the gonad
the Sertoli
cells, spermatogenic, and Leydig cells.
The Leydig cells, which are located in the space
surrounding
the
seminiferous
tubules,
to
testosterone during
the
fetal
period,
p romoting
the
development of the male reproductive tract.
Biosynthesis of Glycoproteins During Spermatogenesis in
Testis
Mammalian spermatogenesis involves extensive
morphological and biochemical transformations to produce
mature gametes that are structurally and functionally unique.
During this process, gene expression is temporally regulated
both at transcriptional and translational levels (Bellve &
O'Brien, 1983; Hecht, 1986). A variety of proteins, many of
which are unique to spermatogenic cells, are differentially

6
expressed during meiosis and spermiogenesis
These include
basic
chromosomal proteins
that
unde r go
successive
during
spermatogenic
cell
(Meistrich et al., 1981), several spermatogenic ce11-specific
iso z yme s
(Goldb erg,
19 7 7 )
and
stage-specific
surface
glycoproteins
with biochemical
(Millette
&
Moulding,
1981a ;
Millette
&
Moulding,
1981b)
and
immunological probes (O'Rand 6c Romrell, 1980; Gaunt, 1982;
Fenderson et al., 1984; O'Brien 6c Millette, 1984; O'Brien 6c
Millette, 1986).
It has been suggested that many proteins
play
roles
in various
processes
including
sperm-egg
interaction,
Sertoli -spermatogenic
cell
association
capacitation and the acrosomal reaction. For example, rabbit
sperm autoantigen (RSA-1), a sialoglycoprotein located in the
postacrosomal region, plays a role in the spermatozoon's
binding to and penetration of the zona. This protein first
appears
on
the
surface
of pachytene spermatocytes
and
increases in amount throughout spermatogenesis (O'Rand 6c
Romrell, 1981; 0'Rand et al., 1984).
Such sperm specific
surface components are probably involved in antibody mediated
agglutination and immobilization of spermatozoa.
Early in spermatogenesis, glycoproteins are synthesized
and deposited in the acrosome.
One of the acrosomal
glycoproteins is proacrosin, which yields acrosin after being
activated. Proacrosin is first produced during the spermatid
stage of differentiation, and is retained throughout the

7
remainder of spermatogenesis (Florke et al 1983). Acrosin
is thought to be
an
essential protease required for the
proteolysis
o f
the
zona
pellucida o f
the
ovum during
o n
(Hartree,
19 7 7 ) .
In
purified rabbit
proacrosin, glucosamine, mannose, galactose and sialic acid
were found in the ratio of 3,3,1,1 per mole of proacrosin
which is consistent with the ratio expected of these sugars
m
N-linked glycoproteins
(Muke rj i
&
Me iz e1,
1979) .
Therefore, proacrosin would be expected to be synthesized via
p a thway which
invo1ve d
dolichyl phosphate (DOL P)
me tabolism.
A general study of the glycosylation of protein would
be of value in understanding factors which might regulate the
biosynthesis of these cell and stage specific proteins.
An
understanding of the origin, metabolic pathway and mechanism
of biosynthesis of these glycoproteins during spermatogenesis
may provide insight into the biochemical control of the cell
function and differentiation.
These findings may be of
significance in the design of male contraceptive agents and
in understanding molecular basis of male sterility.
Sertoli Cell
Histological Structures and Functions
The Sertoli cells are the nongerminal elements in the
seminiferous tubules of the testes. They were first

8
described in 1865 by Sertoli to have a nursing function, to
provide mechanical support for the developing spermatogenic
cells as well to be phagocytic.
It is believed that
circulating hormones, which act on spermatogenesis, have
their effects mediated via the Sertoli cell (Hansson et al ,
1976 ) .
Sertoli cells are basically columnar cells,
which
surround the adjacent spermatogenic cells and fill the spaces
between them (Figure 1-2).
Sertoli cells form the major
structural component of the seminiferous tubules and serve a
number of functions, including (a) mediating movement of
spermatogenic cells from the basal lamina to the lumen and
the release of the late spermatids into the tubular lumen,
(b)
compartmentalizing
the
epithelium into
basal
and
adluminal compartments and forming part of the blood-testis
barrier, (c) phagocytizing degenerating spermatogenic cells
and residual bodies, (d) secreting androgen binding protein
and inhibin as well as other glycoproteins and (e) mediating
the movement of steroids, metabolites, and nutrients utilized
by spermatogenic cells across the seminiferous epithelium.
During development,
the
Sertoli
cell undergoes
fundamental changes.
The most remarkable feature of these
maturational changes is the cessation of cell multiplication
that occurs before puberty. It occurs in the rat at about 15
days of age (Steinberger & Steinberger, 1971).
After that
age, the Sertoli cells may change their metabolic activities

9
Ad SC Ap B PT
Figure 1 2 .
Schematic Drawing of Human Seminiferous
Epithe1iu
The seminiferous epithelium recline
(BL) and a
iniferous
layer
tubule
of peritubular
Pale type A
cells
upon
(PT)
basal la
surrounds
type A spermatogonium (Ad)
located in the basal
type
and
spermatogonium
B
(Ap)
mina
the
dark
type
o f
spermatogonium
the seminiferous
epithelium below the
Sertoli cells
compartment
junctional complex
(SC); pachytene primary spermatocytes
early spermatids (ES), and late spermatids (LS)
the adluminal compartment above the junctional complex (Ross,
M. H. and Reith, E. J., 1985, Histology, 3rd printing, pp
608; Clermont, Y. 1963, Am. J. Anat., 112, 35).
(JC) between adjacent
(P) >
are seen in
0

10
under the influence of various factors, but they do not
divide any more.
Adjacent Sertoli cells are joined by the Sertoli cell
junctional complex, which is
unique structure not found in
other epithelium tissues.
This functional barrier develops
in the rat at about 16 to 19 days of age (Vitale et al.,
1973) .
The Sertoli-Sertoli junctional complex divides the
seminiferous epithelium into two compartments:
the basal
compartment and the adluminal compartment.
Actually, the
Sertoli-Sertoli junctional complexes are the site of the
blood-testis barrier, which serves an essential role in
isolating the spermatogenic cells from the immune system;
i.e. the production of unique molecules on spermatogenic
cells is recognized as foreign if these molecules come in
contact with the immune system.
Follicle stimulating hormone (FSH) and testosterone
r e gu1 ate
the
process
o f
sperm production within
the
seminiferous epithelium.
The Sertoli cells have been shown
to be the primary target for FSH and androgens. Therefore,
the Sertoli cells are considered to be the regulators of
spermatogenesis. The probable importance of Sertoli cells in
spermatogenesis
has
been emphasized
by
number
o f
investigators (Bridges et al., 1986; Fritz et al., 1976;
Griswold et al., 1986); however, their precise role in this
process is still not fully understood.

11
Physiological and morphological studies have indicated
that the Sertoli cells undergo cyclic changes in their
metabolic activity which are related to specific stages in
the cycle of the seminiferous epithelium. Cytochemical
studies showed that several enzyme activities in Sertoli
cells vary depending on the stage of the cycle of the
seminiferous epithelium. For instance, the peak activity of
acid phosphatase appeared at stages VII and VIII, but little
activity was found in stages IX through II (Niemi & Kormano,
1965). A similar stage specific distribution has been
observed with the thiamine pyrophosphatase in Sertoli cells
(Hilscher et al., 1979).
Sertoli Cells Secrete Many Glycoproteins
One
o f
the
mechanisms
by
wh i ch
Sertoli
cells
biochemically influence the spermatogenic cells is through
the synthesis and secretion of glycoproteins.
At leas t 15%
o f
all
the
proteins synthesized by
Sertoli
cell
glycoproteins (Bridges
al ,
1986) .
Some
o f
the
glycoproteins secreted by the Sertoli cells are specific to
the testis
and others are similar, if not identical, to
serum proteins.
Sertoli cells secrete glycoproteins into the
lumen of the tubules, perhaps into the blood stream or lymph,
and
maybe into
the
space
between
Sertoli
cells
and
spermatogenic cells for subsequent uptake by spermatogenic
cells .
Several glycoproteins secreted by the Sertoli cells

12
have been studied, such as androgen binding protein (ABP)
(Fritz et al., 1976), plasminogen activator (Lacroix et al.,
1977), testicular transferrin (Skinner & Griswold, 1980), and
sulfated glycoprotein 1 (SPG-1) and 2 (SPG-2) (Griswold et
al. 1986) .
The stage specific nature of the elaboration of one
glycoprotein, androgen binding protein, has been particularly
well described. Sertoli cells have maximum FSH binding
during stages XIII through I followed by maximum cAMP
production in stages II through VI which in turn is followed
by maximum production of androgen binding protein in stages
VII through VIII (Parvinen et al 1980; Ritzen et al. ,
1982). Since Sertoli cells are actively involved in
glycoprotein biosynthesis, we speculate that the regulation
of the N-linked glycoprotein biosynthesis may be significant
in the function of these cells.
Role of POL P in Glycoprotein Biosynthesis
The
S truc ture
and
Distribution
o f
Dolichol
and
Its
Derivatives
Dolichol (DOL)
general term for a group of
polyisoprenoid alcohols.
They contain the dimethylallyl
terminal unit (w-terminal), two trans-isoprene residues,
number of cis-isoprene residues and a terminal hydroxylated
a-saturated
isoprene
un i t
1inke d
i n
head-to-tail
orientation.
Pennock, Hemming and Morton first isolated and

13
characterized this compound from pig liver (Pennock et al.,
1960) .
Mammalian polyprenols contain a larger number of
isoprene residues Cg5-C]_]_5 (17-23 x C5) than those present in
plants and bacteria (10-12 x C5). Polyprenols found
in bacteria have an oc-unsaturated isoprene unit. However, it
is known that liver contains small amounts of a-saturated
shorter polyprenols (Mankowski et al., 1976), and in some
tissues, such as bovine pituitary gland (Rodominska-Pyrek et
al ,
1979) and hen oviduct (Hayes & Lucas, 1980),
a-
unsaturated compounds have been reported. DOL is present in
most eukaryotic cells and is found in particularly high
concentration in some human tissues, such as the adrenal
gland, pancreas, pituitary gland, testis and thyroid gland
(Rupar & Carroll, 1978; Tollbom & Dallner, 1986).
James and
Kandutsch showed that in mouse the synthesis of DOL is
uch
more active in testes than in liver (James & Kandutsch,
1980c). It seems that DOL content is relatively high in the
rapidly growing and differentiating tissues.
For example,
the DOL content of the hyperplastic liver nodules in liver is
four times higher in the homogenate and six times higher in
the microsomes than that found in normal rat liver.
In
developed hepatocareinoma the amount of DOL was found to be
doubled.
In contrast to free DOL, DOL P was found to be
greatly decreased in nodules (Eggens et al., 1984).
Other studies show an increase in DOL levels with age
although the increases vary widely with tissues (brain,

14
liver, kidney, testis, lung and heart) (Pullarkat et al.,
1984) .
DOL may have some effects on membrane structure and
fluidity (Valtersson et al 1985 ).
Otherwise, most DOL
apparently
has
no
direct relationship
to
glycoprotein
synthesis, since the bulk of the DOL is present in membranes
other than the endoplasmic reticulum (Wong et al., 1982;
Adair & Keller, 1982; Ekstrom et al., 1982). Some DOL may be
phosphorylated by a CTP-dependent kinase (Allen et al. 1978;
Burton et al 1979).
Dolichol is found in several combined
forms in the cell, free dolichol and esterified form with
fatty acids or mono or diphosphate. The phosphorylated forms
are important as we will see soon because they are also found
*
with different degrees of glycosylation which is important
for glycoprotein biosynthesis.
roles.
Dolichyl fatty acyl esters may have several metabolic
First, the fatty acyl esters may be a suitable and
stable for
for the storage of dolichol in lipid droplets.
Alternatively, the fatty acyl moiety might be necessary for
the transport of dolichol from its site of synthesis to
different subcellular locations.
The percentage of dolichol found in the esterified form
varies widely in different tissue.
For example, it is
reported to be 0% and 25% esterified in pig kidney and
spleen, respectively.
In another report, it is shown that
the ester form represents about 63% of total DOL in pig

15
liver, and 65-90% in mouse testes and preputial glands
(Malvar et al., 1985). The mechanism which determines these
distributions is not known.
POL P in Glycoprotein Synthesis
In the early 1970s, Behrens and Leloir demonstrated the
involvement of DOL P in the N-linked glycoprotein
biosynthesis (Behrens & Leloir, 1970; Behrens et al., 1971).
Although, only a small amount of the total DOL in cells is
phosphorylated (Eggens et al., 1983), the phosphorylated form
(DOL P) is an essential carbohydrate carrier in the
biosynthesis of asparagine -1inked glycoproteins (Struck &
Lennarz, 1980; Hubbard & Ivatt, 1981).
Most secretory proteins and membrane proteins, numerous
receptors and proteins related to cell recognition are
glycoproteins. A typical N-linked glycoprotein contains one
or a few oligosaccharide units linked to asparagine side
chains by N-glycosidic bonds.
The biosynthesis of N-linked glycoproteins proceeds
through a cyclic process, which requires the involvement of
DOL P, in the synthesis of the core oligosaccharide unit
(Parodi & Leloir, 1979; Hubbard & Ivatt, 1981).
Figure 1-3 represents a highly abbreviated
representation of this cyclic process and associated
reactions (Rip et al., 1985; Hemming, 1985, for review).

16
UDPGlcNAc
GlcNAc2P-P-Ool
5 GDPMan
IPP + FPP > > P-P-Dol Glc3Man9GlcNAc2P-P-Dol
HMG-CoA
Acetyl-CoA
Glc-3Man9GlcNAc2Protein Protein
Figure 1-3.
in eukaryotic cells.
Dolichol cycle for glycoprotein formation

17
The
first
step
in
the
as s embly
o f
1ip id- 1inked
oligosaccharide involves the addition of GlcNAc-P from UDP-
GlcNAc to DOL P to generate DOL PP-GlcNAc.
Then, this
molecule reacts with an additional UDP-GlcNAc to form DOL PP-
(GlcNAc)2
Five mannose residues are added next from GDP-
annose to form DOL PP-(GlcNAc)2-Man5.
It had been shown
that
DOL
P-Man also
manno s e
donor
to
the
core
oligosaccharide chain. In the oligosaccharide lipid carrying
nine mannose residues, the last four of these are transferred
through DOL P (Rearick et al 1981).
The next steps are
transfers of three glucose units from DOL P-Glc to the core
oligosaccharide chain with the formation of the lipid-linked
oligosaccharide DOL PP-(GlcNAc)2Mang-Glc3.
In the final
step of this pathway, the core oligosaccharide is transferred
en bloc to newly synthesized polypeptide with the concomitant
release of DOL PP. This core protein linked oligosaccharide
is then processed by a now well described pathway involving
spec
0
glycosidases (Kornfeld & Kornfeld, 1980)
and
additional sugar residues added by DOL P independent glycosyl
Schachter
and his coworkers from studies of
glycoprotein and glycolipid metabolism during spermatogenesis
in rat and mouse testis indicated that spermatocytes and
early spermatids had highly active glycosylating systems
(Kornblatt et al., 1974; Letts et al 1974a; Letts et al.,
1974b).

18
DOL P plays a major role in the biosynthesis of N-
linked glycoprotein,
since
DOL
P
not
only
an
oligosaccharide unit carrier but also is an activator which
reacts with certain nucleotide sugars and facilitates the
sugar transfer to the core oligosaccharide chains.
The
involvement of DOL P in the production of the DOL PP-
oligosaccharide
and
the
sub sequent
o f
the
oligosaccharide to asparagine residues within the newly
formed peptide is thought to occur on the luminal surface of
the endoplasmic reticulum membrane (Pfeffer & Rothman, 1987,
for review).
Studies with chick embryo fibroblast (Hubbard &
Robbins, 1980) and canine kidney cells (Schmitt & Elbein,
1979 )
sugge s t
that oligosaccharide transfer is the rate
limiting step
i n
glycoprotein synthesis.
Sub s e quent
regeneration of DOL P permits the reinitiation of lipid
oligosaccharide biosynthesis.
Therefore, enzymes of the DOL
P pathway associated with formation or utilization of DOL P
could be very important in the control of glycoprotein
biosynthesis in spermatogenesis.
The availability of DOL P was found to be a rate-
limiting factor in some glycosylation processes (Potter et
al., 1981a; Eggens et al., 1984). Furthermore, the shortage
of the lipid intermediates influences some vital biological
processes such as embryonic development (Carson et al ,
1981) .
Carson and Lennarz showed that when DOL P synthesis

19
was inhibited by compactin, a potent inhibitor of
hydroxymethyl glutaryl CoA reductase and consequently
po 1y isoprenoid biosynthesis, protein glycosylation was
impaired and the oligosaccharide chains synthesized were more
negatively charged (Carson & Lennarz, 1981). In another
report, inhibition of DOL P biosynthesis induced abnormal
gastrulation in sea urchin embryos (Carson & Lennarz, 1979).
All of the findings suggested that DOL P plays an important
role in the N-linked glycoprotein biosynthesis. Needless to
say, a good understanding in the regulation of DOL metabolism
is a necessary step to explore the regulation of the N-linked
glycoprotein biosynthesis.
Metabolism and Functions of DOL P
There are at least three pathways to generate DOL P.
First, the dephosphorylation of DOL PP could provide the main
supply of DOL P for the biosynthesis of intermediates in the
protein glycosylation reactions (Dallner & Hemming, 1981).
On the other hand, investigations in recent years have
established that increased protein glycosylation is often
accompanied by increased phosphorylation of DOL by a CTP-
dependent kinase; therefore, a second possibility is that DOL
P is supplied by direct phosphorylation of DOL (Burton et
al., 1981; Coolbear & Mookerjea, 1981). Third, when the
sugar residues are transferred to the growing oligosaccharide
chain, DOL P is released from DOL P-Man or DOL P-Glc.

20
Since DOL P is an important precursor of both DOL P-
mono s accharide
and DOL P-oligosaccharide,
i t
i s
understandable that a shortage of DOL P could have multiple
effects on the biosynthesis of 1ip id-o1igosaecharide and may
cause the production of defective glycoproteins. Chapman has
shown that a mouse lymphoma cell mutant, lacking DOL P, can
not synthesize DOL P-Man (Chapman et al. 1980) .
Kean
(1985), using microsomes from a variety of tissues, reported
that DOL P-Man, which requires DOL P for its biosynthesis,
exerts a positive allosteric effect on the enzymes that
catalyze the formation of DOL PP-GlcNac.
More recently,
Carson et al. (1987) found that during hormonal induction of
glycoprotein assembly in mouse uteri, the changes in the rate
of DOL P-Man synthesis may be an important factor in
regulating DOL P-linked oligosaccharide assembly, since uteri
contain very high levels of DOL P and DOL P
1inked
saccharides.
DOL PP, in turn, can arise via two metabolic pathways.
First, by a recycling mechanism, where DOL PP is released
from the lipid oligosaccharide when the oligosaccharide
portion is transferred to the newly synthesized polypeptide.
Alternatively, DOL PP may be derived from de novo
biosynthesis by a poorly defined pathway which undoubtedly
requires the condensation of low molecular weight precursors,
such as farnesyl diphosphate and isopentenyl diphosphate.
Regulation of this pathway could be very important for

21
controlling DOL P level in the cells, since this pathway is
the only de novo biosynthesis pathway known and serve as a
"bridge connecting the small metabolites, such as acetyl
CoA, with the large DOL molecules.
A key enzyme of this
pathway is dehydro DOL PP synthase, which catalyzes the
synthesis of dehydro DOL PP from farnesyl diphosphate and
isopentenyl diphosphate. Dehydro DOL PP synthase could be an
important cellular regulator of glycoprotein biosynthesis as
consequence of its regulation in the DOL P de novo
biosynthesis.
A postulated pathway showing the important
role of dehydro DOL PP synthase in DOL P and glycoprotein
biosynthesis is demonstrated in Figure 1-4.
However, this
enzyme has not been well studied.
One approach to clarifying the fate of DOL in vivo has
been to inject this compound into an experimental animal and
thereafter monitor its appearance in various organs,
injection of ^H-DOL into the bloodstream of a rat
After
the
radioactivity rapidly appeared
i n
the
high
density
lipoprotein (HDL) fraction of blood, with subsequent uptake
into all tissues (Keenan et al., 1977 ).
Since Elmberger
suggested that rat liver might be the main or exclusive site
of DOL synthesis, the presence of DOL in the high density
lipoprotein fraction of blood may point to high density
lipoprotein as a DOL transporter (Elmberger et al., 1987).
The rate of clearance of
14
C-DOL from tissues of the
rat is very slow, since about half the radioactivity is

22
Is op enteny1 Diphosphate
+
Farnesyl Diphosphate
Dehvdro POL PP
Synthase
t
Dehydro DOL PP
Dehydro DOL P
t
Dehydro DOL
+
[ H2 ]
+ [H2]
+ [H2]
DOL PP
t
DOL P
A

DOL
GLYCOPROTEIN
ASSEMBLY CYCLE
SHOWN IN
Figure 1- 3.
DOL Kinase
t
DOL- esters
Figure 1-4. A putative pathway showing the
relationship between dehydro DOL PP and glycoprotein
bio synthe sis.

23
present in the whole rat and in the liver 24 hr after
inj ection.
Furthermore, it was still present 20 days later,
almost entirely as
14
C-DOL.
The half-life of DOL P in rat
liver has been estimated to be 7-12 days on the basis of the
size of the DOL P pool. The catabolic products derived from
labeled DOL have not yet been found (Rip et al., 1985).
The Importance of Dehydro DOL PP Synthase
Studies on developing brain (James & Kandutsch, 1980a),
and liver (Keller et al., 1979; Tavares et al., 1981; Keller,
1986) have clearly shown that the rates of cholesterol and
dolichol biosynthesis are regulated independently, although
they share in part a common biosynthetic pathway.
Although 3-hydroxy 3 methy1glutary1 CoA reductase (HMG
CoA reductase), the rate limiting enzyme in cholesterol
synthesis and whose product, mevalonate, is a metabolic
precursor of DOL, was shown to be high in these cells, the
fact that DOL and cholesterol biosynthesis can be
independently regulated would lead to uncertainty about the
potential of this enzyme as the regulatory enzyme in DOL
biosynthetic pathway.
Dolichol, cholesterol and ubiquinone are all formed
naturally from mevalonate. During the synthesis, mevalonate
undergoes a series of reactions and is converted to
isopentenyl diphosphate, which in turn, is isomerized to form
dimethylally1 diphosphate. Then there is a sequential

24
condensation
o f
isopentenyl diphosphate
to
the
dimethy 1 a 11y 1 diphosphate primer
consequential allylic diphosphate.
and
then
t o
the
Earlier biosynthetic
experiments confirmed
that
the
isoprene
residues
o f
polyisoprenoid alcohols are added in a stereochemically
specific
anner (Hemming, 1970).
The cis
addition to
trans. trans-farnesyl diphosphate gives rise to dolichols,
and the trans-addition to trans. trans-farnesyl diphosphate
generates the polyprenyl side chains of ubiquinones.
On the
other hand ,
the
synthesis
o f
squalene,
choles terol
precursor,
results from a reductive condensation of two
molecules of trans t trans-farnesyl diphosphate (Figure 1-5).
Many enzymes are common to the pathways of cholesterol
and DOL biosynthesis. In particular HMG-CoA reductase, which
is a major regulatory enzyme for cholesterol biosynthesis
(Faust et al., 1979; Rodwell et al., 1976),
could
also
control
the
bio synthe sis
of
isoprene units
utilized for dolichol synthesis. But, effectors that inhibit
cholesterol biosynthesis in a major way have minimal effect
on DOL biosynthesis (James & Kandutsch, 1980b).
A current
debatable hypothesis to explain these findings suggests that
dehydro DOL PP synthase
saturated at
much lower
concentration of the prenyl diphosphate substrates than the
enzymes of cholesterol pathway, and therefore, the synthase
is less subject to large changes in the availability of these
substrates (Keller et al., 1979; James & Kandutsch, 1979).

25
Acetyl CoA
t
HMG-CoA
reductase
t
Mevalonate

Isopenteny1 Diphosphate
t
Farnesyl Diphosphate
trans- prenyl transferase
cis-prenyl transferase
t
Squalene
Polyp reny1 Diphosphate
Precursors of Ubiquinone
Dehydro DOL PP

Choles terol

Dolichol
Figure 1-5. Pathway of Isoprenoid Biosynthesis. c i s -
prenyl transferase is the same as dehydro DOL PP synthase.

Therefore, it is likely that the pathway of DOL synthesis may
have its own regulatory point which is independent of
cholesterol biosynthesis. This could make dehydro DOL PP
synthase a rate limiting step in DOL biosynthesis. This,
coupled with the finding that DOL synthesis in developing
systems is greatly enhanced relative to cholesterol
biosynthesis, makes it likely that large increases in dehydro
DOL PP synthase activity might accompany or precede an
increase in glycoprotein synthesis.
Studies Related to DOL P Biosynthesis and Spermatogenesis
The Role of Dehydro DOL PP in DOL Metabolism
Testicular tissues contain large quantities of DOL and
are actively engaged in glycoprotein synthesis. Early
studies on human tissues by Rupar and Carroll (1978) using
gravimetric methods for determining DOL concentrations
suggested that testis contained more DOL than any other
organ.
James and Kandutsch (1980b) suggested that one or more
of the spermatogenic cell types are responsible for the high
rate of DOL synthesis observed in normal testicular tissue.
Further studies of Potter et al. (1981b) showed that purified
mouse spermatogenic cell populations are capable of DOL
synthesis. The pachytene spermatocyte were the most active,
whereas the round spermatids are less active. These studies

27
clearly demonstrated high DOL synthesis in specific
spermatogenic cell populations. However, the enzyme or
enzymes that might be responsible for this increased DOL
synthesis were not identified.
In order to understand how glycoprotein assembly is
coordinated with differentiation, it is necessary to
understand how the individual steps in the sequence of
glycoprotein assembly are modulated. We focus our attention
here on the biosynthesis of the carbohydrate carrier, DOL PP.
It is believed that dehydro DOL PP, a derivative of DOL
PP with an a-unsaturated isoprene unit, is an intermediate in
the de novo biosynthesis of DOL PP. Figure 1-6 compares the
structures of dehydro DOL PP and DOL PP.
The enzyme catalyzing the synthesis of this
intermediate is dehydro DOL PP synthase. Dehydro DOL PP
%
biosynthesis from isopentenyl diphosphate (IPP) and farnesyl
diphosphate (FPP) has been demonstrated in hen oviduct
(Grange & Adair, 1977), Ehrlich tumor cells (Adair &
Trepanier, 1980), chicken liver (Wellner & Lucas, 1979),
mouse L-1210 cells (Adair & Cafmeyer, 1987a) and yeast (Adair
& Cafmeyer, 1987b).
FPP + (IPP) dehydro DOL PP (17-23 x C5)

28
Figure 1-6. The structure of dehydro DOL PP and DOL PP.
The a-unsaturated isoprene unit is present on the structure
of dehydro DOL PP.
The
condens at ion
o f
these
sub s t r a t e s
polymerization reaction which finally leads to the formation
of dehydro DOL PP with farnesyl diphosphate providing the
three (-terminal isoprene units and the last isopentenyl
diphosphate added providing the a-unsaturated unit bearing
the diphosphate.
The hen oviduct and Ehrlich tumor cell
enzymes are membrane associated.
Dehydro DOL P biosynthesis from isopentenyl diphosphate
and farnesyl diphosphate has also been described in
microsomal fractions of rat liver (Wong & Lennarz, 1982).

29
Adair and Keller (1982) have showed that the enzymatic
products of the liver enzyme were a group of dehydro DOL
monophosphates ranging in size from C75 to C95 (15-19x05).
Recent
data
fro
this
laboratory have
shown that
testicular homogenates and their membrane fractions will
catalyze the synthesis of ^75-035 dehydro DOL P and dehydro
DOL
PP
from t,t-farnesyl diphosphate
and
isopentenyl
diphosphate (Baba et al., 1987).
The isolation of dehydro
DOL P and DOL P in some cases instead of the diphosphate
analogues reflects the activity of diphosphatases.
A
mechanism
o f
reduction
o f
dehydro dolichyl
derivatives to DOL was suggested recently by Ekstrom et al.
(1987). They hypothesized that during the saturation of the
terminal isoprene unit, dehydro DOL PP (also referred to by
some as polyprenols to distinguish them from the saturated
counterparts, the dolichols) are elongated by an additional
isoprene residue and saturated at the same time.
The enzymes of DOL metabolism are membrane associated
and,
therefore,
most often assayed
preparations.
Solubilization and purification of individual
enzymes of the dolichol pathway have proven to be difficult
and in most cases not particularly successful.
DOL Metabolism in Spermatogenic Cells
James and Kandutsch (1980c*) studied DOL biosynthesis in
X-irradiated or genetically mutated mice that were deficient

30
in spermatogenic cells.
from these mice,
when
compared to normal controls, demonstrated markedly reduced
ratios of
cholesterol.
acetate incorporation into DOL as compared to
These results suggested that the high rate of
DOL synthesis in mouse testes may be attributed to one or
more types of spermatogenic cells, although Sertoli cells may
not be excluded. It was subsequently shown that prepuberal
mouse pachytene spermatocytes incorporate acetate into DOL at
rate which is 5 times higher than that of leptotene and
zygotene spermatocytes, and that
high rate of acetate
incorporation into DOL is maintained in adult pachytene
spermatocytes and round spermatids (Potter et al., 1981b).
A developmental study
o f
DOL kinase activity
m
sexually maturing rats
15-60 days
of age
showed the
appearance of detectable levels of activity at 21 days,
peak at 24 days and
subsequent decline to adult levels.
The
developmental
pattern of
this
enzyme
its
association with the later stages of spermatocyte development
(Berkowitz & Nyquist, 1986).
Allen and Ward (1987) also reported changes in specific
activity of DOL kinase during testicular development in the
They showed that the specific activity of kinase peaked
at day 30, whereas the level of endogenous DOL P, as measured
indirectly by the DOL P dependent mannosyl
activity, rose to a peak around day 15.
Since the optimal
activity of DOL kinase peaked later (day 30) than the peak in

31
DOL P levels (day 15) it was suggested that DOL kinase may
function primarily in maintaining, adequate levels of DOL P
for glycoprotein biosynthesis after the initial burst of DOL
P biosynthesis. Therefore, DOL kinase was postulated to have
little or no effect in regulating the rise in DOL P levels
during the initial phases of differentiation during
spermatogenesis. It was suggested instead that dehydro DOL
PP synthase or DOL PP phosphatase may be the putative
regulatory enzyme which directly affects the levels of DOL P
in spermatogenic cells at different stages of development.
The studies described here will extend our understanding of
the regulation of DOL P metabolism in the testicular system.
Glycoproteins in Sertoli Cells
Sertoli cells are histologically and physiologically
fundamental for spermatogenesis, since they are the only
somatic epithelial component of the seminiferous tubules
(Fawcett, 1975).
The close physical association of Sertoli
cells with the spermatogenic cell and the organization of
this association into a cyclic pattern have been described in
detail (Clermont & Perey, 1957).
The characterization of
Sertoli cells as nursing cells of testis was based originally
on the morphological cellular relationship in the testis.
The concept of Sertoli cells functioning as a support or
regulatory factor has been confirmed by both biochemical and
endocrine
s tudie s.
It has
been postulated
that
the

32
androgenic and tropic hormone action on spermatogenesis is
mediated by the Sertoli cells. These cells have both FSH and
androgen receptors (Sanborn et al., 1977; Sanborn et al.,
1979; Means & Vaitukaitis, 1972) and show an appropriate
temporal relationship between hormone binding and cell
response. For example, there is nuclear accumulation of
androgen and stimulation of RNA polymerase II activity in
cultured Sertoli cells when FSH or testosterone were added in
the media (Lamb et al., 1981). Therefore, it is possible to
envision a scenario where the regulation of spermatogenesis
is a result of the biochemical properties of Sertoli cells.
One of the possible mechanisms for Sertoli cells to
influence spermatogenic cell development is via the secretion
of proteins and glycoproteins which serve as signals or
transport vehicles. The first glycoprotein to be identified
as a Sertoli cell specific secretion product was androgen
binding protein (French & Ritzen, 1973; Vernon et al., 1974).
This protein is secreted by cultural Sertoli cells and its
synthesis is regulated by FSH, testosterone, and vitamin A
(Louis & Fritz, 1977; Karl & Griswold, 1980). The function
of androgen binding protein is not clear but it is probably
related to the capability of the protein to bind and
transport androgens to the epididymis.
Sertoli cells synthesize and secrete a testicular
transferrin. Skinner and Griswold (1982) speculate on the
basis of biochemical and immunological similarities between

33
testicular and serum transferrin that testicular transferrin
must play a role in the transport of iron fro
Sertoli cells
to the spermatocytes and spermatids. More recently, Morales
and Clermont (1986) have shown that during spermatogenesis
Sertoli cells and spermatogonia internalized transferrin by
receptor-mediated endocytosis at the base of the seminiferous
epithe1ium.
Recent studies in the ram have shown that clusterin, a
glycoprotein with a molecular weight of 37,000-40,000 is also
synthesized de novo and secreted by Sertoli cells and
transported to the rete testis (Rosenior et al., 1987).
Collectively,
Sertoli
cells
actively
glycoproteins into the lumen of the seminiferous tubules and
regulate spermatogenesis.
Therefore,
it
relevant
presumption that the synthesis of glycoproteins must be very
active in these cells and that adequate DOL P must be
provided by biosynthesis or uptake to support this function.
The very high DOL content in Sertoli cell Golgi membrane
sugges ts
that
Sertoli
cells
actively involved
m
glycoprotein synthesis (Nyquist & Holt, 1986).
DOL in Sertoli Cells
Although a clear relationship between DOL P
concentrations and the rate of glycoprotein synthesis has
been established in many systems, the mechanism of regulation
of glycoprotein biosynthesis in Sertoli cells is still

34
obscure. Furthermore, the question concerning the ability of
the
Sertoli
cell
to
synthesize DOL was not
addressed
directly.
Nyquist and Holt (1986) recently measured the cellular
and subcellular distribution of DOL in rat testis by a HPLC
ethod and found that elutriation purified spermatogenic
cells
had very
low
concentrations
of DOL.
Pachytene
spermatocyte and round spermatids contained 25.8 and 36.5 ng
DOL/mg protein, respectively.
Washed epididymal sperm also
had a very low DOL content (18.8 ng DOL/mg protein).
In
contrast, the Sertoli cell enriched tubular fraction that was
recovered during the preparation of purified spermatogenic
cells
showed
the
highest DOL content (3450 ng DOL/mg
protein).
These results implied that the Sertoli cells
accumulate
major portion of the testicular DOL.
This
result at first appears inconsistent with the results of
Potter et al. (1981b), who found low DOL synthesis in the
testes from X-irradiated mice and spermatogenic deficient
mice, which, although they were depleted of spermatogenic
cells, were apparently normal with respect to the number and
function of the Sertoli cells. Nyquist and Holt suggested in
explanation that
the
bulk
o f
DOL may be
synthesized
in
the
spermatogenic
cell
and
subsequently
transported to the Sertoli cell.
A possible route, they
hypothesized, would be by Sertoli cell phagocytosis of
residual body cytoplasm during spermiation.

35
The high content of DOL in the Sertoli cell may reflect
a requirement for high DOL P to permit a rapid rate of
glycoprotein biosynthesis during the spermatogenesis. It
might be possible that Sertoli cell and spermatogenic cells
may have a coordinate pattern of de novo DOL biosynthesis
which is dependent on the presence of the other cell type.
This would require that each cell type have the capacity to
synthesize DOL without relying on intercellular DOL
transport. In this way the Sertoli cell can independently
regulate the level of DOL P and hence the synthesis and
secretion of glycoproteins. Consequently, Sertoli cells can
regulate spermatogenesis. Therefore, it was of interest to
determine the capacity of Sertoli cells for de novo
biosynthesis of DOL P in order to have a better understanding
of the relationship between the Sertoli cells and
spermatogenic cells during testicular development.
Significanee
Dehydro DOL PP synthase is obviously a prime candidate
as a regulated enzyme in the DOL biosynthesis and
glycoprotein synthesis. It was of interest to determine if
increased dehydro DOL PP synthase activity correlated with
the increased rate of DOL synthesis in specific types of
spermatogenic cells and the high DOL content in Sertoli
cells, particularly since glycoprotein synthesis is active in

36
those cells. Recent data from our laboratory using DOL P
dependent mannosyl transferase show that the level of DOL P
increases dramatically from day 7 to day 20 in prepuberal
rats (Allen & Ward, 1987). This time interval covers a
period when the first group of spermatogenic cells are going
through differentiation to become spermatids. It should be
noted that the acrosomal enzymes, including the glycoprotein
acrosin, are elaborated in early stages of spermatid
formation. Several other proteins required for glycoprotein
biosynthesis are also maximally expressed during this time
interval, e.g. galactosyl transferase, N-acetylglucosaminyl
transferase, and N-acety1glueosaminide fucosyltransferase in
mice testes (Letts et al., 1974a). It is true for the
androgen binding protein in rat Sertoli cell culture as well
(Rich et al., 1983). Therefore, we have measured the
specific activity of dehydro DOL PP synthase in spermatogenic
cells and Sertoli cells during testicular development in
order to determine its potential importance in the regulation
of spermatogenesis.
A knowledge of changes in the concentration of
intermediates and activities of enzymes in DOL metabolism is
important if we are to understand the regulation of
glycoprotein biosynthesis. Spermatogenesis offers a complex
but good model system to study control mechanisms of DOL
metabolism and DOL function in the biosynthesis of specific
glycoproteins during cell differentiation. Information

37
gained from the assessment of dehydro DOL PP synthase
in
Sertoli
cells
and
different
types
o f
spermatogenic
cells
may provide
some
insight into
the
mechanism of biochemical control of cell function during
differentiation.
This may ultimately be useful in the
development of male contraceptives.
Obiec tive s
The main objectives of this dissertation are to study
dehydro DOL PP synthase and dehydro DOL PP phosphatase during
spermatogenesis in rat.
Specific objectives are the following:
A. Optimize the assay conditions for dehydro DOL PP
synthase.
B. Characterize the enzymatic products.
C. Determine the synthase specific activities in
homogenate of testicular tubules of different aged
D.Determine enzymatic activities in homogenate of
different spermatogenic cell types (pachytene
spermatocytes and spermatids), and Sertoli cells
from rats testes.
Chapter II of this dissertation describes the study in
achievement of objectives A through C. Chapter III describes
the work in fulfillment of objective D.

CHAPTER II
DEVELOP AND OPTIMIZE AN ASSAY FOR DEHYDRO DOLICHYL
DIPHOSPHATE SYNTHASE FROM RAT TESTES
Introduction
Elucidation of the mechanisms that regulate the
synthesis of N-linked glycoproteins requires a clear
understanding of the biosynthesis and metabolism of DOL and
DOL P. For this reason we have undertaken studies on the
biosynthesis and metabolism of dehydro DOL PP in rat testes.
It seems that dehydro DOL PP is the precursor of DOL PP in
the de novo biosynthesis pathway. Dehydro DOL PP synthase is
responsible for the de novo biosynthesis of dehydro DOL PP
from isopentenyl diphosphate and t,t-farnesyl diphosphate.
This synthase has been demonstrated in several animal tissues
(Grange & Adair, 1977; Wellner & Lucas, 1979; Wong & Lennarz,
1982; Adair & Keller, 1982; Adair et al., 1984; Adair &
Cafmeyer, 1987a and 1987b; Baba et al., 1987), and is
membrane associated. The products of this synthase were
labile to acid and yielded petroleum ether soluble products
indicating that the a-isoprene unit was unsaturated. Adair
and Keller (1982) characterized the carbon number of the
enzymatic product in rat liver and showed they are a group of
38

39
dehydro DOL Ps ranging in size from C75 to C95
More
recently, Baba et al. ( 1987 ) described the synthase in rat
seminiferous tubules, but showed that the products of this
enzyme
were
both dehydro
DOL
PP
and
dehydro
DOL
P.
Hydrolysis of both of these products with a testicular
phosphatase in the absence of NaF yielded the same chain
length alcohols (C75.C90). The isolation of 2,3-dehydro DOL
P and DOL P in some cases instead of the diphosphate
derivatives undoubtedly
reflects
the
action
o f
diphosphatase.
The methods for the assay of dehydro DOL PP synthase,
which were described by Adair and collaborators (Adair &
Keller, 1982; Adair et al., 1984), and Wellner and Lucas
(1979) have been modified, as described below, to optimize
the analysis of synthase activity in whole homogenates of
small testicular samples obtained at different stages of
spermatogenesis.
I n
this
assay ,
[]-isopentenyl
diphosphate and t,t-farnesyl diphosphate were chosen as
substrates.
A s
poss ible
i nh i b i t o r s
o f
endogenous
phosphatase, NaF and ATP were used to protect the substrates.
Although Baba et al. (1987) showed that there were no major
changes in the extent of formation of radiolabeled enzymatic
products when NaF, MgCl2 and ATP were omitted in the assay of
partially purified subcellular membrane fraction, there were
major changes in the nature of the in vitro products.
For
example, when Triton X-100 was omitted neither dehydro DOL PP

40
nor dehydro DOL P was formed, but a product, tentatively
identified as presqualene monophosphate accumulated instead.
Therefore, it was necessary to extend the earlier studies of
Baba et al ( 1987 ) to ensure that the assay developed for
the synthase activity in crude tubular homogenates was
measuring the desired activity.
This chapter 1) shows that treatment of the testicular
homogenate by sonication yielded good dehydro DOL PP synthase
activity while greatly reducing the formation of farnesol via
another prenyl transferase activity; 2) describes the optimal
parame ters
for
the
dehydro DOL PP synthase
3)
demonstrates
unequivocally
the
precursor product
relationship between dehydro DOL PP and dehydro DOL P, and 4)
elucidates a change in the enzymatic activity for dehydro DOL
PP synthase in the seminiferous tubules during early stages
of development.
A possible role of the dehydro DOL PP
synthase in regulating the biosynthesis of DOL is discussed.
The two-fold increase in the specific activity of this
synthase between day 7 and day 23 and a similar decrease in
activity between day 23 and day 60 shown in this chapter
provided the impetus to evaluate (in Chapter III) the enzyme
specific activity in different cellular populations of rat

41
Materials and Methods
Materials.
Male Sprague-Dawley rats were purchased
from local suppliers.
t,t-Farnesyl diphosphate was prepared
as previously described (Baba and Allen, 1978).
[1-
14C1-A3.
Isopentenyl diphosphate
and
[ ^P] orthophosphoric acid
(carrier
free)
i n
dilute
HC 1
was
purchased
from
Amersham/Searle Corp. Silica gel 60 F254 and Cellulose F254
on
plastic sheets were products of E. Merck.
All other
reagents were obtained from standard commercial sources.
Solvent Systems. The following solvents were used for
extraction or chromatography: Solvent A, CHCI3-CH3OH (2:1);
Solvent
B ,
CHCI3-CH3OH-H2O (3:48:47);
Solvent
C ,
diisobutylketone glacial acetic acid-H20 (8:5:1); Solvent D,
2-propanol-acetonitrile-0. 1
M
ammonium
bicarbonate
(45:25:30); Solvent E, 1-propanol-concentrated ammonia-H20
(6:3:1).
Animal grouping number required for analysis. In order
to have sufficient data for
analysis in each
experiment, at least two rats, but most often three or more
rats were used for each age group tested (See Appendix A).
The excised testes (six or more) from each age group were
pooled together, then two or three aliquots of the mixed
samples were taken for assay
The data presented in each
table or figure were usually means determined by two or three
similar experiments (e.g. Appendices B and C).

42
Preparation of homogenate.
Male Sprague-Dawley rats
were decapitated. The testes were removed and perfused with
enriched Krebs-Ringer bicarbonate medium (EKRB)
via the
testicular vessels
This procedure effectively removed all
blood cells from testes
The tunica albuginea was removed
and seminiferous tubules were gently expressed
In the case
of the younger animals (3 and 7 days old), the testes and the
tunica albuginea were removed under dissecting microscope
without perfusion.
The seminiferous tubules were weighed then suspended at
ratio of 1:2 (w/v) with ice cold buffer (20 mM Tris-HCl pH
7.5 and 1 mM EDTA) .
This mixture was sonicated in an ice
bath in a Sonic dismembrator (model 300, Fisher) three times
for 20 seconds with 20 seconds intervals without sonication.
The pink
ilky homogenate was used as the enzyme source.
Protein quantitation was made by a modified method of Lowry
et al (1951). Accurate protein quantitation was extremely
important
for
de te rmining
enzyme
specific
m m
Therefore, samples were saponified in order to solubilize all
proteins and each solubilized sample was assayed in duplicate
or triplicate for protein.
Assay for Dehydro POL PP Synthase.
The
e thod
was developed as adapted from the method described by Grange
and Adair ( 1977 ). The level of the enzyme was monitored by
measuring the formation of polyprenyl products, dehydro DOL
PP and dehydro DOL P.
It was particularly important to

43
eliminate the unwanted products, and only isolate and
quantitate dehydro DOL PP and dehydro DOL P. Optimal assay
conditions were also established. The dependency of product
formation on Triton X-100, protein, isopentenyl diphosphate,
farnesyl diphosphate concentration and time were determined.
The standard assay of the enzyme was carried out by
incubation of 100 mM Tris-HCl buffer pH 7.5, 10 mM MgCl2>
0.5% Triton X-100, 250 /M t, t farnesyl diphosphate, 1.6 mM
ATP, 50 mM NaF, 36 \x M [ 1 ]- isopentenyl diphosphate
(1.1x10^ dpm, 53 /Ci//i mole), and 1.0 mg of enzyme protein in
a final incubation volume of 0.25 ml for 1 hr at 37C. The
reaction was stopped by the addition of 0.25 ml of 1 M KOH.
Then the mixture was heated at 100 C for 30 min to saponify
the membrane bound lipids. Afterwards, the mixture was
cooled in an ice bath, 0.25 ml of 1 M HC1 and 1.25 ml of 2 M
KC1 were added. This mixture was extracted twice with 1 ml
aliquots of Solvent A.
The combined solvent A extract was washed first with 2
ml of deionized water, then with 2 ml of Solvent B. When the
extent of product formation was to be quantitated, a 1 ml
aliquot was taken from the organic extract, dried in a
scintillation vial and 10 ml of toluene based scintillation
fluid (Scinti Verse II, Fisher) were added for analysis of
the radioactivity. Radioactivity was then determined in a
scintillation counter.

44
Thin Laver Chromato
TLC
Reaction Products.
The remainder of extract (4.5 ml) was brought to dryness with
a N2 stream. Five drops of Solvent A were added to the dried
residue and the tube was vortexed thoroughly. The resulting
solution was applied to Silica gel 60 on plastic sheets which
were previously cut into 4 cm
x
20 cm sections.
Five
additional drops of Solvent A were used to wash the sample
tube and this wash was added to the sample at the origin of
the TLC sheets .
The TLC sheets were then developed in a
chamber with Solvent C.
The developed TLC sheets were subjected to either
scanning with a radiochromatogram scanner (Packard model
7201) (Fig. 2-1-1) or autoradiography for 3-5 days on X-omat
AR
Kodak film (Fig. 2-1-II). The positions of migration of
authentic DOL P standard and the radiolabeled products were
correlated.
Sections corresponding to the migration of
dehydro
DOL
PP
and
dehydro
DOL
P
were
scraped into
scintillation vials and 10 ml of scintillation fluid was
added to each vial for radiochemical analysis.
The level of
was
expressed
m
pmoles
o f
[l^C]-isopentenyl
diphosphate incorporated into dehydro DOL PP and dehydro DOL
P /mg protein.
Preparation
of Isopentenyl
LqlJL
32
PI
Diphosphate.
32
Isopentenyl [a,/3- ^ P] diphosphate was synthesized fro
3-
methy1-3-buten-1-o1 and ^Pi according to the procedure of
Cramer and Bohm ( 1959). [ ^^P ] -Orthophosphoric acid (0.5 mCj_,

45
carrier free) in dilute HC1 was dried in the reaction vessel
over P2O5 under N2. Then 0.5 /moles crystalline H3PO4, 6.25
/moles t r ie thy 1 amine 2 /moles 3 me thy 1 3 but en 1 o 1 and
12 /moles
in 60 /I acetonitrile were
added and the reaction permitted to proceed for 5-7 hours at
room temperature.
The reaction was stopped by adding 200 /I
of 10 mM NH4OH. The reaction products was separated by TLC
(Cellulose F254) in Solvent D.
The radioactive
32
P band
migrating beside
authentic [^C] -isopentenyl diphosphate
(Rf=0.35) was scraped from the plate, packed in a glass
column and [^^P]-isopentenyl diphosphate was eluted with 2* ml
of methanol at room temperature.
The nature of the
32
P-
labeled product was verified by TLC along with authentic
isopentenyl diphosphate by TLC on Silica 60 F254 in Solvent
E.
Base Hydrolysis of Dehvdro Pol PP. The putative
dehydro DOL PP was biosynthesized from [ ^C ]-isopentenyl
diphosphate and farnesyl diphosphate with a testicular
homogenate according to the standard assay method. Enzymatic
products were separated by TLC on Silica 60 F254 as already
described. The dehydro Dol PP region on TLC (Rf=*0.40) was
localized by autoradiography and scraped into a conical test
tube. The product bound to Silica gel was suspended in 1 ml
of 3.5 M KOH in 70% methanol and the hydrolysis was carried
out at 100 C for 2 hr. At the end of the hydrolysis, 2 ml
of water was added to the mixture and the polyprenol

46
hydrolysis products were extracted with 2 ml of Solvent A.
Then the lower phase was subjected to TLC analysis. The
authentic markers, [ ]-dehydro DOL P and non-radiolabeled
DOL P were chromatographed in parallel to the hydrolysis
products and the developed TLC sheet was subjected to
autoradiography.
Results
Optimization of Synthase Reaction and Characterization
of the Enzymatic Products.
The typical prenyl transferase
assay for the long chain polyprenyl diphosphate synthase in
bacteria
easures the amount of acid labile and organic
solvent extractable product (Keenan & Allen, 1974). However,
this method could not be satisfactorily
applied
for
the
because
of
the
quantitation of dehydro DOL PP synthase,
synthesis of other isoprenoid products with acid lability and
extractab1ity similar to the dehydro DOL phosphates.
A more
accurate method was developed which involved 1) CHCI3-CH3OH
extraction of the reaction products after saponification of
the reaction components, 2) application of TLC to
the dehydro DOL PP and dehydro DOL P from shorter chain
polyprenyl phosphates
and
free polyprenols,
and
3)
determination of the sum of the dehydro DOL PP and dehydro
DOL P formed.
This method was
applied to the
identification of dehydro DOL PP synthase in the microsomal

47
fraction isolated from homogenates of seminiferous tubules
from rat testes (Baba et al., 1987).
It was necessary to optimize the method to accurately
assay the enzyme in homogenates of tubules taken from animals
o f
different
Homogenates were prepared by
the
sonication of buffered suspensions of tissue instead of
disruption with a glass homogenizer as previous described
(Baba et al., 1987). In this study, sonication was found to
be the only satisfactory procedure for disruption of the
small amounts of tissue available from 3- and 7-day-old rats.
Sonication also had the added advantage of denaturing the
prenyl
transferase,
farnesyl diphosphate synthase,
exhibite d
by
an
e1imination
o f
radioactive
product
chromatographing with an Rf similar to exogenously added
farnesol (Fig. 2-1-1, Panel B).
*^P and Ratios in
the
Mono and Diphosphate
Products.
The carbon chain lengths of the long chain
polyprenyl products obtained by in vitro biosynthesis were
previously shown to be the same (Baba et al., 1987 ).
Since
these two compounds had the same chain length, but they
chromatographed on TLC and anionic exchange columns in
manner consistent with mono- and diphosphorylated products,
they were assumed to be the dehydro Dol P and dehydro Dol PP.
The ratios of phosphate to polyprenol chain were determined
here for the putative dehydro Dol P and dehydro Dol PP in
order to clarify this earlier assumption.
Mixtures of [a, ¡3-

48
3 2
P]-isopentenyl
diphosphate and [^ ^ C ]-isopentenyl
diphosphate were incubated with farnesyl diphosphate and
tubular homogenates in the standard assay. The products were
separated by TLC as usual and the ratios of radiolabel
incorporated
from [32
P]- and [ ]-isopentenyl diphosphate
were determined.
Since the chain lengths of the polyprenol
products have been established to be the same, the relative
o f
3 2
P
incorporated/^C incorporated into
the
polyprenyl diphosphate is expected to be twice that ratio for
the monophosphate.
The results of such a test under two
experimental incubation conditions are illustrated in Table
2-1 .
They support the hypothesis that the two products are
the dehydro DOL P and dehydro DOL PP.
Several experiments were carried out to demonstrate the
dependency
o f
the
enzyme
#
activity
on
Triton
X- 100
concentrations and incubation times as well the extent and
type of product formed. These experiments also serve to test
the precursor-product relationship between the diphosphate
and monophosphate .
Triton X-100 stimulated dehydro
DOL
PP
synthase
activity (Fig. 2-2, Panel C) with formation of dehydro DOL PP
(Panel A) and dehydro DOL P (Panel B).
The dependencies of
the extent of mono- and diphosphate product formation on the
concentration
o f
detergent
were
quite different
incubation for 1 hr. Dehydro DOL PP formation was generally

Figure 2-1. Separation of Enzymatic Products by TLC.
(I) The products from the reaction of t,t-farnesyl
diphosphate and [ ^C] isopentenyl diphosphate with
homogenates of seminiferous tubules prepared with a
glass-teflon homogenizer (Panel A) or by sonication
(Panel B) were extracted with CHCI3/CH3OH and subjected
to TLC on silica gel sheets as described in the text.
Arrows represent the position of migration of exogenous
DOL P and farnesol.
(II) A example of autoradiography from enzyme assay

DETECTOR RESPONSE DETECTOR RESPONSE
50
HOMOGENATE DOL-P
DISTANCE (cm)

51
SF
m
DOLP C
Figure 2-1
(continued)

52
Table 2-1
Incorporation of A -fl- Cl Isopentenvl Diphosphate and f a. 8-PI-Isooentenvl
Diphosphate into Dehvdro POL PP and Dehvdro POL P
Experiment 1
Experiment 2
Radiolabeled
Substrate
[ 32pi -ipp
5 nmol
(1.36 nCl)
[14C]-IPP
4 nmol
(0.21 iiCL)
RATIO
^Pcpm
^Ccpm
[3 2 p]-ipp
10 nmol
(2.72 mCI)
[14C]-IPP
4 nmol
(0.21 nCi)
RATIO
^Pcpm
^Ccpm
Radiolabeled
Product
(cpm incorporated)
(cpm incorporated)
Dehydro DOL PP
(A)
424
1489
0.28
1127
883
1.28
Dehydro DOL P
(B)
217
1526
0.14
533
936
0.57
(A)/(B)
2.0
2.2
a The enzyme assay was carried out as described in the text. The reported values of
cpm have been corrected for overlap of into the channel. (A)/(B) represents
the relative ratio of radiolabel from ^2p incorporated into the diphosphate compared
to the monophosphate.

Figure 2-2. Triton X-100 Dependency on the Formation of
Dehydro DOL PP and Dehydro DOL P.
Incubations containing 100 mM Tris-HCl buffer
(pH7.5), 10 mM MgCl2, the indicated percentage of Triton
X-100, 250 /zM t,t-farnesyl diphosphate, 1.6 mM ATP, 50
mM NaF, 36 /zM [ 1 ] isopentenyl diphosphate, and 1.0
mg of enzyme protein in a final volume of 0.25 ml were
carried out at 37 C for 60 minutes. The formation of
[]-dehydro DOL PP (Panel A) and [^^C]-dehydro DOL P
(Panel B) were estimated by the method described before.
Panel C represents dehydro DOL PP synthase activity
(A+B).

Triton X-100 (%>
pmoles Isopentenyl Diphosphate Incorporated/ mg protein-hr
ui

stimulated with increasing detergent concentration throughout
the concentration range shown in the figure, while dehydro
DOL P formation was optimal at 0.5% Triton X-100. This
suggests that when the Triton X-100 concentration was higher
than 0.5%, a previously active dehydro DOL PP diphosphatase
was inhibited. The sum of dehydro DOL PP and dehydro DOL P
production was unchanged at Triton X-100 concentrations of
0.5% and higher.
The product ratio of dehydro DOL P to dehydro DOL PP
shifted to favor the monophosphate when the detergent
concentration in the incubation mixture was decreased midway
through the incubation period. A part of the tubular
homogenate was incubated at 37 C with substrates in either
0.5% Triton X-100 or 2% Triton X-100. In each case the
products were analyzed after 1 hr and 2 hr. The incubation
with 0.5% Triton X-100 gave dehydro DOL P as the predominant
product at both 1 hr and 2 hr (Fig. 2-3, Panel A), whereas
with 2% Triton X-100, the slower migrating product, dehydro
DOL PP, was the predominant product at both time points (Fig.
2-3, Panel B). However, when a similar incubation was
carried out in 2% Triton X-100 for the first hour to favor
dehydro DOL PP formation and then the concentration of Triton
X-100 changed to 0.5% during the second hour of incubation,
the predominant product observed at the end of the second
hour was dehydro DOL P (Fig. 2-3, Panel C).

Figure 2-3. Dependence of Product Formation on Triton
X-100 Concentration and Incubation Time.
Sonicates of seminiferous tubules were assayed
under standard conditions except that Triton X-100 and
time of incubation were varied as shown. Dehydro DOL PP
and dehydro DOL P were analyzed separately in reaction
mixtures incubated for 1 hour and 2 hours in 0.5% Triton
X-100 (Panel A), 2% Triton X-100 (Panel B) and from a
reaction mixture which was incubated for 1 hour in 2%
Triton X-100 and then diluted four fold with all
reaction constituents except enzyme and Triton X-100 and
incubated for an addition 1 hour (Panel C).

Dehydro Dehydro Dehydro Dehydro
Dol PP Dol P Dol PP Dol P
i
1st hour 2nd hour

58
Table 2-2
Formation of Enzymatic Product at Different Triton X-100
Concentrations in Pulse-Chase Exnerimenta
Isopentenyl Diphosphate Incorporated
(pmoles)
Time
Conditions
dehydro DOL PP
%
dehydro DOL P
%
1st hour
2% Triton
68
71
27
29
2nd hour
0.5% Triton
49
41
70
59
aIncubation conditions were the same as described in legend of Fig. 2-3
except that 4.81 mM unlabeled isopentenyl diphosphate was added to the
ruction mixture after the first hour, reducing the specific activity of
[ C]-isopentenyl diphosphate 134-fold.

59
In all cases the
increasing
time.
diphosphatase which is
Triton X-100.
sum of the two products increased with
This supports the presence of a
inhibited by higher concentrations of
The results of a similarly designed pulse-chase
experiment support the same conclusion (Table 2-2). In this
case non-radiolabeled isopentenyl diphosphate was added to
the incubation mixture after one hour incubation in 2% Triton
X-100. The reaction was continued for an another hour with
addition of farnesyl diphosphate and enzyme but under lower
Triton X-100 concentration (0.5%). Any polyprenyl phosphate
made during the second hour of incubation, the chase phase,
would not have been radiochemically detectable under the
conditions used. Since there was a loss in radiolabeled
product migrating as the putative dehydro DOL PP concomitant
with an increase in radiolabeled dehydro DOL P, it can be
concluded that the slower migrating product is a precursor of
dehydro DOL P, and therefore it was dehydro DOL PP.
The precursor-product relationship between dehydro DOL
PP and dehydro DOL P was also shown in a more detailed time
dependent study illustrated in Fig. 2-4. The formation of
the mono- and diphosphate was determined under standard assay
conditions except that Triton X-100 concentration was raised
to 1% to partially inhibit the diphosphatase activity. The
distinct lag in the formation of dehydro DOL P relative to
the diphosphate illustrates clearly the classical precursor-

60
product pattern. The formation of the two products increased
linearly with time up to 60 min.
Base Hydrolysis of Dehvdro POL PP. The putative
dehydro DOL PP was isolated by TLC and then subjected to
saponification in 3.5 M KOH in 70% methanol for 2 hr at
100 C. Figure 2-5 shows that dehydro DOL P is the
overwhelming product of base hydrolysis. This product is
consistent with this enzymatic product being dehydro DOL PP.
Saponification in aqueous 0.1 M KOH at 100 C for 30
min is used in the standard assay. This procedure makes
nonsaponifiable lipids more accessible for extraction and
provides a better resolution of the enzymatic products on
TLC, because of the hydrolysis and hence elimination of
saponifiable lipids. Saponification under these milder
conditions did not change the ratio between dehydro DOL PP
and dehydro DOL P (results are not shown).
These results indubitably showed the in vitro reaction
proceeds as follows: dehydro DOL PP synthase catalyzes the
condensation of isopentenyl diphosphate and farnesyl
diphosphate to generate dehydro DOL PP, which is the
precursor of dehydro DOL P. Since the proportion of the
diphosphate and monophosphate occasionally varied from one
experiment to another, an accurate measurement of synthase
activity required analysis of both products.

Figure 2-4. Time Course of Dehydro DOL PP and Dehydro
DOL P Formation.
Sonicated seminiferous tubules were assayed under
standard conditions except that 1.0% Triton X-100 was
used and the time of incubation was varied as shown.
The formation of dehydro DOL PP (solid triangles),
dehydro DOL P (solid squares) and the sum of the both
(solid circles), respectively.

TIME (min)
pmoles Isopentenyl Diphosphate Incorporated/ mg protein
ON
ro
150

Figure
PP .
2 5 Product of
Hydrolysis of Dehydro DOL
Dehydro DOL PP and dehydro DOL P were prepared by
biosynthesis and isolated by TLC as described in the
Methods. (A) Dehydro DOL PP was saponified. The
hydrolysis product was extracted and chromatographed on
Silica 60 F254 in Solvent C as described in the text.
(B) as a control, dehydro DOL P was extracted from
Silica 60 TLC sheets and chromatographed as described in
A. The position of migration of authentic DOL P is
shown by the arrow I. The position of dehydro DOL PP is
shown by the arrow II.

64
I

A
B

65
Kinetics of the Enzyme. The enzyme assay conditions
were optimized. Figure 2-6 shows the effect of varying
protein concentration on the formation of the dehydro DOL PP
(Panel A) dehydro DOL P (Panel B) and the sum of these two
products (Panel C). These results indicated that the
enzymatic activity increased linearly with protein
concentration up to 2.4 mg protein incubated.
The dependency of dehydro DOL PP and dehydro DOL P
formation on substrate concentration was studied. Figure 2-7
shows that the formation of dehydro DOL PP (Panel A), dehydro
DOL P (Panel B), and the sum of these two products (Panel C)
increased with increasing isopentenyl diphosphate
concentration. A double-reciprocal plot of the sum of
dehydro DOL PP and dehydro DOL P formation (Panel C) versus
isopentenyl diphosphate concentrations showed (Figure 2-8)
that the apparent Km=32/M and Vmax=1.23 pmoles/mg protein/min
respectively.
Similarly, the dependency of the enzymatic products
formation on the substrate farnesyl diphosphate was also
studied. The experiment described (Figure 2-9) shows that
farnesyl diphosphate was incorporated into dehydro DOL PP and
dehydro DOL P. The formation of dehydro DOL PP (Panel A),
dehydro DOL P (Panel B), and the sum of these two products
(Panel C) increased with increasing concentration of farnesyl
diphosphate. In Figure 2-10, a double reciprocal plot of the
sum of dehydro DOL PP and dehydro DOL P formation (Fig. 2-9,

66
Panel C) versus farnesyl diphosphate concentrations showed
that the apparent Km=2 2 2/M and Vmax = 0.6 7 pmoles/mg
protein/min respectively. The Km values were higher than
those observed for dehydro DOL PP synthase from Ehrlich
ascites (Adair et al., 1984). This may reflect non-specific
absorption of the substrates by other proteins in the crude
homogenate and hydrolysis of the substrate by endogenous
phosphatases, although ATP and NaF were included in the assay
to minimize the action of phosphatases.
The time course of formation of dehydro DOL PP and
dehydro DOL P in the standard assay is shown in Fig. 2-11.
The formations of both dehydro DOL PP (Panel A) and dehydro
DOL P (Panel B) are increased with increasing incubation
time. Since the sum of these two products was linearly
increased up to 60 minutes (Panel C), we chose one hour as
the standard incubation time.
Changes of Dehvdro DOL PP Synthase with age. The
specific activity of dehydro DOL PP synthase in testicular
homogenates of different aged rats was studied. The
synthesis of dehydro DOL PP and dehydro DOL P from farnesyl
diphosphate and [^C]-isopentenyl diphosphate was determined
in sonicates of tubules from rats aged 3-65 days. An example
from several of these experiments is shown in Fig. 2-12.
Under standard assay condition, the changes in the specific
activity of the enzyme as measured by dehydro DOL PP

Figure 2-6. Effect of Protein Concentration on Dehydro
DOL PP and Dehydro DOL P formation.
Incubations
(pH7.5)
10
containing
M MgC12, 0.5%
100
mM Tris-HCl
buffer
Triton X-100
2 5 0 /xM t t -
farnesyl diphosphate,
] -isopentenyl diphosphate, and varying protein
concentration in a final volume of 0.25 ml was carried
14
1.6 mM ATP, 50 mM NaF, 36 [1-
and
out at 37
C for 60 minutes
The formation of
dehydro
DOL PP
(Panel A)
the
and [ -^C ]- dehydro DOL P
method described before.
B) were estimated by
C represents dehydro DOL PP synthase activity (A+B)
[it+c]-
(Pane 1
Panel

pmoles Isopentenyl Diphosphate incorporated /hr
68
Protein Cmg)

Figure 2-7. Isopentenyl Diphosphate Concentration
Dependency on the Formation of Dehydro DOL PP and
Dehydro DOL P.
Incubations containing 100 mM Tris-HCl buffer
(pH7 5) 10 mM MgCl2, 0.5% Triton X-100, 250 /M t,t-
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF, the
indicated concentration of [ 1 -] isopentenyl
diphosphate, and 1.0 mg of enzyme protein in a total
volume of 0.25 ml were carried out at 37 C for 60
minutes. The formation of [^C] -dehydro DOL PP (Panel
A) and [ ]- dehydro DOL P (Panel B) were estimated by
the method described before. Panel C represents dehydro
DOL PP synthase activity (A+B).

IPP (uM)
pmoles Isopentenyl Diphosphate Incorporated/ mg protein* hr
%

Figure 2-8. A double reciprocal plot of the sum of
dehydro DOL PP and dehydro DOL P formation (Fig. 2-7.
Panel C) vs. isopentenyl diphosphate concentrations is
presented.

I /V (hr/pmoles)
72

Figure 2-9. Farnesyl Diphosphate Concentration
Dependency on the Formation of Dehydro DOL PP and
Dehydro DOL P.
Incubations
(pH7.5)
10
mM
containing
0.5%
100
mM Tris-HCl
buffer
Triton X-100
concentrations of
1.6
50 mM NaF
3 6 /iM
1.0
mg of enzyme protein
were carried out at 37 C for 60 minutes. The formation
of []-dehydro DOL PP (Panel A) and []-dehydro DOL
P (Panel B) were estimated by the method described
before. Panel C represents dehydro DOL PP synthase
activity (A+B).
MgC12 >
t,t-farnesyl diphosphate,
[1-^-^C] isopentenyl diphosphate,
final volume of 0.25
varying
mM ATP,
and
m
for
ml

FPP (uM)

Figure 2-10. A double reciprocal plot of the sum
of dehydro DOL PP and dehydro DOL P formation (Fig. 2-9.
Panel C) vs. farnesyl diphosphate concentration is
presented.

I /V (hr/pmoles)
0
10 20
30 40 50
1/ FPP
(.mM)

Figure 2-11. Time Course of Incorporation of [^C]-
Isopentenyl Diphosphate into Dehydro DOL PP and Dehydro
DOL P.
Incubations containing 100 mM Tris-HCl buffer
(pH7 5) 10 mM MgCl2, 0.5% Triton X-100, 250 /xM t,t-
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF, 36 /zM [ 1 -
^C]-isopentenyl diphosphate, and 1.0 mg of sonicated of
seminiferous tubules as enzyme protein in a total volume
of 0.25 ml were carried out at 37 C for the indicated
times. The formation of [ ]-dehydro DOL PP (Panel A)
and [ ]-dehydro DOL P (Panel B) were estimated by the
method described before. Panel C represents dehydro DOL
PP synthase activity (A+B).

pmoles Isopentenyl Diphosphate Incorporated/ mg protein

Figure 2-12. Dehydro DOL PP Synthase in Testicular
Homogenate of Different Aged Rats.
Incubations containing 100 mM Tris-HCl buffer
(pH7.5) 10 mM MgCl2, 0.5% Triton X-100, 250 /M t,t-
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF, 36 ¡jl M [1-
^C]-isopentenyl diphosphate, and 1.0 mg of enzyme
protein frcTm indicated aged rats in a final volume of
0.25 ml were carried out at 37 C for 60 minutes. The
formation of [ ]-dehydro DOL PP (Panel A) and [^C]-
dehydro DOL P (Panel B) were estimated by the method
described before. Panel C represents dehydro DOL PP
synthase activity (A+B).

DAYS AFTER BIRTH
pmoles Isopentenyl Diphosphate Incorporated/ mg protein-hr
oo

Figure 2-13. Dehydro DOL PP Synthase Activity in
Sonicates of Tubules from Rats of Different Ages.
The enzymatic activity was assayed under standard
conditions with sonicates of seminiferous tubules as
described in the Methods. The data ^s presented as the
mean + standard deviation (x +
parentheses indicate the number
prepare the tubules.
) .
Numbers in
animals used to

pmoles Isopentenyl Diphosphate Incorporated/ mg protein-
IOO
80-
60-
40
DAYS AFTER BIRTH

83
formation (Panel A) were somewhat smaller than changes in
dehydro DOL P formation (Panel B) during testicular
development.
The sum of dehydro DOL PP and dehydro DOL P
(Panel C) represents total specific activity of this enzyme.
A composite of data from these studies using 118 rats is
shown in Fig. 2-13. A two fold increase in tubular activity
of the synthase occurred between day 7 and day 23 and
similar decrease in activity occurred between day 23 and day
60 .
A statistical treatment (Wilcoxon two-sample rank test)
(Schefler, 1984) of these data shows a significant difference
between 1) the 7-day-old and the-15-day old groups of rats
(p<0.005) and, 2) the 30-day-old and the 60-day-old group of
rats (p<0.01). Therefore, the peak of activity at 23 day old
rats must be significantly higher than activity in rats aged
both 7 days and 60 days.
Discussion
Early in vivo experiments have demonstrated that
mevalonic acid serves as a precursor of dolichol in pig,
rabbit and rat liver (Butterworth et al 1966; Martin &
Thorne, 1974). Using doubly and stereospecifica 1 ly
radiolabeled mevalonate and tissue slices, it was also shown
that dolichol was synthesized from all-trans-farnesvl
diphosphate by cis- addition of isoprene units (Gough &
Hemming, 1970), suggesting that the biosynthetic pathway to

84
DOL branches from that to cholesterol at the level of
farnesyl diphosphate. In vitro experiments with isopentenyl
diphosphate as a precursor have shown that 2,3-dehydro DOL P,
presumably one of the later intermediates in the DOL
biosynthetic pathway, could be synthesized in preparations
from hen oviduct (Grange 6c Adair, 1977), avian liver (Wellner
& Lucas, 1979), Ehrlich tumor cells (Adair 6c Trepanier,
1980), mouse L-1210 cells (Adair & Cafmeyer, 1987a), yeast
(Adair & Cafmeyer, 1987b), and testes (Baba et al., 1987).
Testis has been shown to contain large quantities of dolichol
(Rupar & Carroll, 1978; Tollbom 6c Dallner, 1986; James 6c
Kandutsch, 19 80c) and therefore it seems to be an
appropriate tissue in which to investigate DOL biosynthesis.
Homogenates of rat seminiferous tubules have been
previously shown to catalyze the synthesis of acid labile
polyprenyl mono- and diphosphate (the a-unsaturated isoprene
unit is acid labile). The enzymatic activity was dependent
upon t,t-farnesyl diphosphate, isopentenyl diphosphate and
divalent cation (Baba et al., 1987).
The conditions for the assay of dehydro DOL PP synthase
in sonicates of rat seminiferous tubules have been
systematically characterized and optimized here. The
sonication of seminiferous tubules gives a few advantages for
the assay. The sonication method, in contrast to
homogenization with a glass homogenizer, can be easily
applied to a small amount of testicular tissue, such as

85
obtained fro
3- and 7-day-old rats
For instance, the
pooled size of ten testes from 3-day-old rats is about the
size of a rice grain (not a long grain!)
Furthermore ,
denatures
the
p r eny1
farne sy1
diphosphate synthase, so that some of the side products from
the assay are eliminated (Fig. 2-1-1).
The reason for the
loss in this prenyl transferase activity on sonication is not
clear.
Possibly, these enzymes are more sensitive to the
heat generated by the sonication, despite cooling during this
process.
It was necessary to optimize the conditions to
accurately assay the specific enzymatic activity in sonicated
seminiferous tissue
from rats
of
different
ages.
Several
pieces of evidence
support
the
premise
that
the slow
migrating TLC component (Rf=0.47) (Fig. 2-1), identified as
dehydro DOL PP, was the initial enzymatic product, which was
subsequently hydrolyzed in vitro to dehydro DOL P. Earlier
work showed that the chain length of both the mono- and
diphosphate derivatives of the polyprenyl product were the
same (C75-C90) (Baba et al 1987 ). The present study
established more clearly the precursor-product relationship
between the products dehydro DOL PP and dehydro DOL P.
First, the sum of the mono- and diphosphorylated polyprenols
increased linearly with a variety of increasing variables,
i. e. Triton X-100 concentration (0 to 0.5% as shown in Fig.
2-2), protein (0 to 2.4 mg as shown in Fig. 2-6), time (0 to

60 min as shown in Fig. 2-11). Second, there is evidence for
a detergent sensitive phosphatase that acts on the
diphosphate to give the monophosphate. Third, kinetic
experiments have shown a classical time dependent lag in
monophosphate formation compared to diphosphate formation,
whereas total phosphorylated polyprenol increased linearly.
Fourth, direct chemical experiments were also performed to
establish the precursor-product relationship between dehydro
DOL PP and dehydro DOL P, such as double labeling experiment
using [cr,/?- ^P ] isopentenyl diphosphate and [ ] isopenteny 1
diphosphate (Table 2-1), and base hydrolysis (Fig. 2-5).
The mild saponification used in each assay to release
the membrane bound lipids did not change the ratio between
dehydro DOL PP and dehydro DOL P. However, during a more
vigorous saponification, dehydro DOL PP was hydrolyzed to
dehydro DOL P as previous described (Adair and Cafmeyer,
(1987). These experiments clearly showed that we can simply
sum the radioactivity in the mono and diphosphate components
as an accurate measurement of the total synthase activity.
Grange and Adair (1977) observed and therefore measured
only dehydro DOL P formation in hen oviduct, although they
commented that dehydro DOL P may be derived from dehydro DOL
PP by the action of a phosphatase. In contrast, Adair and
Cafmeyer (1987b) observed and measured only dehydro DOL PP
formation in their studies in yeast. The current study
showed that the testicular enzyme produces both dehydro DOL

87
P and dehydro DOL PP, therefore, the determination of
synthase activity required the measurement of both products,
not dehydro DOL P or dehydro DOL PP alone.
DOL P is an indispensible carrier of oligosaccharides
during glycoprotein biosynthesis, therefore, knowledge of its
availability and the timing of its biosynthesis during early
stages of differentiation may be important in understanding
the regulation of spermatogenesis. This laboratory has shown
that DOL P, as measured indirectly by a DOL P dependent
mannosyl transferase assay, increased in immature rat testes
about two fold between day 7 and day 30 after birth (Allen &
Ward, 1987). Unpublished work of others in this laboratory
has demonstrated similar results by direct measurement of DOL
P with HPLC analysis of CHCI3/CH3OH extracts of seminiferous
tubules from different aged rats (Fig. 2-14).
Changes in dehydro DOL PP synthase during
spermatogenesis in the rat have been studied here. The
products of this synthase are undoubtedly intermediates in
DOL P(P) biosynthesis. Dehydro DOL PP synthase was shown to
increase two fold in specific activity between day 7 and day
23 after birth, and a similar decrease in activity between
the day 23 and day 60. These findings parallel the changes
in DOL P described above. Mechanisms which account for the
increase in DOL P may include the phosphorylation of DOL with
CTP dependent DOL kinase, de novo biosynthesis of DOL or as a
result of release of DOL P from pools of DOL P and DOL PP

Figure 2-14. Comparison of Changes in DOL P Concentration,
and Dehydro DOL PP Synthase Activity as a Function of Rat
Age .
The specific activity of dehydro DOL PP synthase ( )
are compared with the concentration of DOL P measured
directly by HPLC (A) (unpublished observation, Allen, 1987)
or by an indirect method (), which was described in an
earlier study (Allen & Ward, 1987).

PRODUCT/mg protein
,pmol), (661(19)1 (o.pmol X10)
89
DAYS, AGE

90
saccharides.
Berkowitz and Nyguist (1986) showed a sharp
rise in kinase activity at 21 days of age with a peak at 24
days .
Allen and Ward ( 1987 ) showed a similar change in
kinase activity, but the rise in activity appeared to be more
gradual with a peak in activity at about 30 days and with one
half maximal activity between day 20 and day 25.
It seems
unlikely that change in DOL kinase activity account for the
changes in DOL P levels.
It has been suggested that alterations in the levels of
the active, phosphorylated form of dolichol regulate the rate
of N-linked glycoprotein synthesis (Lucas, 1979; Carson &
Lennarz, 1979; Carson & Lennarz, 1981).
The present results,
which show that dehydro DOL PP synthase changes in specific
activity at early stage
of testicular development in rats
(Fig. 2-13) suggest that increased DOL levels must accompany
fluctuations in glycoprotein biosynthesis observed during
this time period (Letts et al., 1978).
The high rate of
testicular DOL synthesis shown by Kandutsch and co-workers,
as well as the temporal changes in DOL metabolism shown here
during sperm differentiation, strongly suggest that membrane
glycoprote ins
and
alterations
m
the

timing
o f
the ir
appearance may be significant regulators of spermatogenesis.
Potter et al (1981b) showed a high rate of acetate
incorporation into DOL in pachytene spermatocytes of adult
ouse testes
At the same time, they also found increased
HMG CoA reductase activity in these cells. Although HMG CoA

91
reductase may be one of the regulated enzymes in DOL
synthesis (Rodwell et al., 1976; James & Kandutsch, 1979),
the observed independent regulation of DOL and cholesterol
biosynthesis can not be well explained by the control of this
enzyme alone. A strong case is presented here for a
regulatory role for dehydro DOL PP synthase during de novo
DOL biosynthesis, because the specific activity of this
enzyme rises in parallel with a two to three fold increase in
DOL P concentration measured both directly and indirectly.
Therefore, it can be concluded that dehydro DOL PP synthase
is a regulatory enzyme responsible for controlling DOL
biosynthesis on a pathway that is independent of cholesterol
biosynthesis. This conclusion is consistent with the
findings of Keller and Adair ( 1980), that DOL P synthase or
long chain cis-prenyl transferase is a rate limiting factor
in the biosynthesis of DOL P in liver. Recently, when
radioactively labeled mevalonate was utilized to study in
vivo and in vitro cholesterol, DOL and ubiquinone
biosynthesis, considerable differences were observed between
the rate of cholesterol synthesis and the rate of DOL and
ubiquinone synthesis, while the rates of DOL and ubiquinone
synthesis were quite similar (Elmberger et al., 1987). This
observation suggested that the presence of important rate-
limiting steps in the biosynthesis of DOL and cholesterol
after mevalonate. This study suggests that dehydro DOL PP
synthase may be one of these rate 1imiting factors.

92
Since the cellular composition of the seminiferous
tubules differs as a function of age during early stages of
differentiation, changing cell populations may partially
explain the difference in activity of dehydro DOL PP synthase
in testes from rats of different ages. For instance, in 7-
day-old rats the only spermatogenic cells are spermatogonia,
by day 23, there are spermatogonia and pachytene
spermatocytes as well, and after day 26, spermiogenic cells
start appearing in the seminiferous tubules. It is likely
that the different enzymatic activity observed during
development can be accounted for by different cell
populations with different enzymatic activity. The specific
activity of different cellular populations are elaborated in
the next chapter.

CHAPTER III
DEHYDRO DOLICHYL DIPHOSPHATE SYNTHASE ACTIVITY IN
ENRICHED CELL POPULATIONS FROM RAT TESTIS
Introduction
Although DOL metabolism during testicular development
has been the subject of several studies in recent years, a
number of intriguing questions still remain unanswered. For
example, do all rat spermatogenic cell populations have
dehydro DOL PP synthase activity? Does each subpopulation of
spermatogenic cells have the same enzymatic activity for the
synthase? Is there a temporal relationship between synthase
activity and developmental stage? Do Sertoli cells also have
the synthase activity and synthesize its own dolichol? Is
there a temporal relationship between spermatogenic cell and
Sertoli cell synthase activity? Elucidating the answers to
these questions will be of value in understanding the
regulation in DOL metabolism as well as glycoprotein
biosynthesis during spermatogenesis in rat. In this chapter,
the specific activity of 2,3-dehydro DOL PP synthase in
homogenates of protease treated seminiferous tubules, cell
fractions enriched in spermatogenic cells or Sertoli cells
from testis were measured as a function of the age of
93

94
prepuberal rats. The highest activity of this enzyme
occurred in each case with cells from rats aged 23 days.
Homogenates of cell fractions enriched in pachytene
spermatocytes, spermatids or Sertoli cells were found to have
higher synthase activity than a whole testicular homogenate
or a mixture of cells prepared by protease treatment of
tubules. The specific enzymatic activity in pachytene
spermatocytes expressed per mg protein, was about 1.7 fold
higher than in spermatids and about 8.3 fold higher than in
spermatozoa. Therefore, the increase in spermatogenic cell
synthase before day 23 can be accounted for by the appearance
of the pachytene spermatocytes. Generally speaking, little
net increase in enzyme occurred during or after meiotic cell
division of spermatocytes into spermatids. Enzymatic
activity decreased remarkably after the differentiation of
spermatids into spermatozoa. Enzymatic activity in the
enriched Sertoli cells was 1.5 to 1.7 fold higher than in the
enriched spermatogenic cells between day 15 and day 30 of
age. The increase in synthase specific activity in
spermatogenic cells and Sertoli cells indicates that both are
contributing to changes in the enzymatic activity in
seminiferous tubules. This change may be important in
regulating the availability of DOL P for glycoprotein
synthesis during early stages of differentiation.
Spermatogenesis proceeds through a precise sequence of
biochemical and morphological phases, spermatogonial, meiotic

95
and spermatid (Leblond & Clermont, 1952). During the second
and third phases, many immunological and biochemical changes
occur which are unique to the spermatogenic process
Among
these changes are the elaboration of spermatogenic cell or
Sertoli cell specific glycoproteins (Fenderson et al 1984;
Parvinen, 1982).
For example, the spermatogenic cells
produce an acrosomal protease precursor, proacrosin, and
unique cell surface glycoproteins, which are required for
Proacrosin is first produced during the
spermatid stage of differentiation but is retained throughout
the remainder of spermatogenesis (Florke et al. 1983 ).
Other proteins are only expressed in specific stages, so that
they may be absent or low in the spermatogonial and spermatid
phases but are abundant during the meiotic phase
(e g.
fucosyl transferase in prepuberal mouse testes) (Letts et
al. 1974a)
The Sertoli cell, a non-germinal support cell of the
seminiferous
tubule,
plays
c r i
role
in
the
spermatogenic
process
by
providing structural support,
regulating spermatogenic cell movement, and compartmentation
of spermatogenic cells from non-germinal cells, as well as
mediating the movement of hormones, metabolites and nutrients
to and from the developing spermatogenic cells (Ritzen et
al 1981) .
The Sertoli cell produces many glycoproteins
that support these functions including androgen binding
protein (Parvinen, 1982) and plasminogen activator (Lacroix

96
et al., 1977).
Coordinated secretion of androgen binding
protein by the Sertoli cell and the association of the
Sertoli cell with pachytene spermatocytes during the meiotic
phase
even
s ugge s t s
the
possibility
o f
intercellular
communication in regulating temporal expression of certain
Sertoli cell proteins (LeMagueresse et al., 1980; Ritzen et
al., 1982; Galdieri, 1984; LeMagueresse & Jegou, 1986).
The biosynthesis of these glycoproteins at specific
phases during testicular development requires a coordinate
func tioning
of a series of enzymes, so that cofactors and
associated biochemical apparatus must be present in the cell
at or before the time of glycoprotein expression or function.
However, only a few studies have described the regulation of
express ion
o f
these
components
possible controlling
factors of spermatogenesis.
Three of the glycosyl transferases, which are needed
for the terminal reactions in glycoprotein oligosaccharide
biosynthesis in rat and mouse testis
have been shown to
increase in specific activity in a sequential manner, that
parallels their use in the last steps of oligosaccharide
processing (Letts et al., 1974a).
The availability of DOL P, another
component
in the synthesis of the N-linked glycoproteins, has been
suggested as a rate- 1imiting factor in some developmental
processes (Lucas, 1979; Harford 6c Waechter, 1980; Rossignol
et al., 1980).
Therefore, the study of DOL metabolism will

97
be undoubtedly useful for the research of glycoprotein
regulation. James and Kandutsch (1980c) reported that mouse
spermatogenic cells were more active than liver cells in DOL
biosynthesis. Furthermore, Potter et al. (1981b) identified
the pachytene spermatocytes as one of the most active
spermatogenic cells in DOL synthesis. This prompted our
study of dehydro DOL PP synthase, an enzyme that could
contribute to increased DOL levels during spermatogenesis.
Chapter II described Jjl vitro assays for the synthase,
which were developed to measure changes in the potential of
seminiferous tubules to biosynthesize dehydro DOL PP, a
probable precursor in the biosynthesis of DOL P and DOL. The
temporal expression of synthase correlated well with the
increase of DOL P measured by HPLC methods in seminiferous
t
tubules during early stages of differentiation in prepuberal
rats. It was proposed, therefore, that the level of DOL P in
rat seminiferous tubules might be controlled by the
regulation of d_e_ novo dehydro DOL PP biosynthesis.
In the previous chapter, the specific activity of the
dehydro DOL PP synthase was shown to fluctuate during
testicular development; it was hypothesized that a difference
in enzymatic activities during development might due to the
presence of different cell populations with different
enzymatic activities in rat testes. In this chapter, the
methods of cell fractionation are described, the specific
activities of the dehydro DOL PP synthase are measured in

98
different purified spermatogenic cell populations and in
Sertoli cells. A model is presented which explains the time
dependent change of the dehydro DOL PP synthase specific
activity in tubules of prepuberal rats.
Materials and Methods
Materials.
Sprague-Dawley rats were obtained from
local suppliers.
t, t-Farnesyl diphosphate was prepared as
previously described (Baba & Allen, 1978). [^C]-A^-
Isopentenyl diphosphate was purchased from Amersham/Searle
C o r p .
Bovine
ser urn
albumin
(BSA),
tryp sin,
trypsin
inhibitor, deoxyribonuclease, and collagenase were purchased
from Sigma Chemical Corp.
All other chemicals were of
reagent grade.
Solutions.
Phosphate buffered saline, essential amino
acids (BME 50X) and MEM nonessential amino acids were
obtained from Gibco Labs.
Enriched Krebs-Ringer bicarbonate
medium (EKRB) contained 120.1 mM NaCl, 4.8 mM KC1, 25.2 mM
NaHC03, 1.2 mM KH2P04, 1.2 mM MgS04*7H20, 1.3 mM CaCl2, and
was
enriched by the addition of 11 mM glucose, 1 mM
glutamine, 10 ml/liter of essential amino acids, and 10
ml/liter nonessential amino acids. Streptomycin sulfate (100
/ig/ml) and penicillin G (K+ salt) (60 /ig/ml) were also added
to the
e dium.
The solution was prepared from a stock
solution
immediately prior
to use,
filtered (0.30 //

99
Millipore), and the pH adjusted to 7.3 by a 15-20 min
aeration with 5% CO2 in air. Glassware and other equipment
was siliconized before use in order to reduce damage and
adhesion of cells.
Preparation of cell suspensions. Rats aged 7-65 days
were sacrificed and testes were removed as described in
Chapter II. Testicular cell suspensions were prepared by a
modification of the two-step enzymatic method described for
mouse (Romrell, 1979). The decapsulated testes were placed
in a 50-ml Erlenmeyer flask containing 20 ml of collagenase
(1 mg/ml) and DNAse (1 /xg/ml) in EKRB. The testes were
incubated at 33C in a shaking water bath operated at 120
cycles/min, until the seminiferous tubules were freely
dispersed in the incubation medium (10-15 min). The
dispersed seminiferous tubules were allowed to sediment and
the supernatant was decanted. The isolated tubules were
washed twice with EKRB. Then fresh EKRB (20 ml) containing
trypsin (2.5 mg/ml) and DNAse (1 g/ml) was added to the
tubules and this suspension was incubated for 15 min in a
shaking water bath as just described. The resulting cell
suspension was gently pipetted approximately 50 times with a
Pasteur pipet. Trypsin inhibitor (2.5 mg/ml) was added, and
then the cell suspension was mixed with 10 ml of 0.5% BSA in
EKRB and centrifuged at 200 x g for 10 min. The resulting
pellet was washed three times with EKRB containing 0.5% BSA
and 1 g/ml DNAse and resuspended in the same solution after

100
the washing.
The suspension was filtered through a nylon
mesh
(135
m)
t o
remove
cell
The
cell
concentration was determined using a hemocytome ter.
This
enriched spermatogenic cell suspension was finally suspended
in EKRB containing 0.5% BSA and adjusted to a concentration
of 2 x 106 cells/ml.
Spermatogenic Cell Fractionation.
The entire cell
separation procedure was carried out
at 5 C.
Spermatogenic
cells were separated by a modification of the STA-PUT unit
gravity procedure (Romrell et al 1977 ). The sedimentation
chamber was initially filled with 70 ml EKRB. Then the cell
suspension, which contained 10^ cells in 50 ml of 0.5% BSA in
EKRB, was introduced into the chamber at a flow rate of 10
ml/min.
The sample was followed by a linear gradient of 2%
t o
4%
BSA
i n
EKRB
generated
from
two
interconnected
that contained 1100 ml of 4% BSA and 1100
1 of
2% BSA, respectively (total volume 2200 ml). Five min after
loading the cell suspension, the flow rate was increased to
40 ml/min. Eighty minutes after loading the cell suspension,
the chamber was drained in 10-ml fractions at a rate of 10
ml/min.
Cell collection was finished within 5 hr after
introducing
the
cell
suspension
to
the
chamber.
The
separated cell fractions were numbered and centrifuged at 200
g for 10 min; the supernatant were decanted; the resulting
pellets were resuspended in 0.5 ml of EKRB.
Aliquots were
taken from individual samples and checked for cell type and

101
purity. The cells were examined by Nomarski differential
interference and phase microscopy. The samples of enriched
early spermatids (stages 1 through 8) and pachytene
spermatocytes were pooled separately, washed three times with
phosphate buffered saline solution and used immediately for
the measurement of the enzymatic activity. The cellular
purity of the pachytene spermatocyte (Fig. 3-1-A) was 70%;
the major contaminants being Sertoli cells and spermatids.
The purity of the spermatid (Fig. 3-1-B) fractions was 80%,
with pachytene spermatocytes, the primary contamination.
Spermatozoa were obtained from adult rats (3 months of
age). The cauda epididymis was removed and flushed with 1 ml
of phosphate buffered saline via the ductus deferens. The
collected spermatozoa were then washed three times in
phosphate buffered saline and used for the enzymatic assay.
Sertoli Cell Preparation. Sertoli cells were prepared
by modification of the procedure of Dorrington et al. (1975).
Sertoli cells were isolated from Sprague-Dawley rats of
specific ages. Tubules were treated with proteases as
described above except that phosphate buffered saline was
used in place of EKRB and more (3 /g/ml) DNAse was used. The
process was similar to that for spermatogenic cell
fractionation, only the Sertoli cells retained on the nylon
filter were collected as the fraction enriched in Sertoli
cells. These aggregates of 10-50 Sertoli cell was further
treated for 3-4 min with a hypotonic solution of two-fold

102
diluted phosphate buffered saline to remove spermatogenic
cells. Under these conditions the spermatogenic cells are
lysed but the Sertoli cells retain their integrity. The
cells were examined by Nomarski differential interference and
phase microscopy to check for purity and integrity. The
resulting cell suspension contained more than 80% Sertoli
cells with a contamination of pachytene spermatocytes and
spermatids (Fig. 3-1-C).
Homogenate Preparation. Homogenates were prepared from
seminiferous tubules, cell suspensions from tubules, enriched
spermatogenic cell populations and Sertoli cells. The
excised testicular tubules were weighed, suspended at a ratio
of 1:2 (w/v) in ice cold buffer (20 mM Tris-HCl pH 7.5 and 1
mM EDTA) and sonicated as described in Chapter II.
Homogenates of various mixed and enriched cell fractions,
were prepared similarly, except that in these cases the
packed cells were suspended in two volumes of buffer (v/v).
Protein quantitation was determined by the method of Lowry et
al (1951) before assay-*-.
i
The cellular dissociation procedure used a relatively
large amount of BSA (2-4%). There was a concern that binding
of BSA to the purified cell fractions might lead to some
error in protein determination of these isolated cells.
Therefore, it was useful to estimate the binding of BSA to
the enriched perm cells. Measurement of binding was carried
out with [-*-^I]-BSA as a probe in 2% BSA. Protease
dissociated germ cells were incubated with [^^^I]-BSA for 3
min at room temperature then washed. The radioactivity on
the cell in the beginning and the end of the test was
compared. The results showed negligible non-specific binding
of BSA to the cell surface (144 jug/14350 fig which is less
than 1%) after the STA-PUT fractionation and the buffer

103
Dehydro POL PP Synthase Assay. The enzyme activity was
determined by the same assay method described in chapter II.
The enzyme protein (1 mg) from homogenats of enriched cell
populations or various cell mixtures were assayed as
indicated. The products, dehydro DOL PP and dehydro DOL P
were extracted with CHCI3/CH3OH (2:1), isolated by TLC and
quantitated as described in Chapter II. The level of
enzymatic activity was expressed as the sum of the pmoles of
isopentenyl diphosphate incorporated/mg protein. The
relationship of these two products were extensively discussed
in the previous chapter.
Results
Synthase Activity in Protease Treated Seminiferous
Tubules.
The synthesis of dehydro DOL PP and dehydro DOL P
from farnesyl diphosphate and [ ^C] isopentenyl diphosphate
was compared in sonicates of pro tease treated and untreated
tubules from rats aged 7-65 days (Fig. 3-2).
It is necessary
to determine if the enzyme in mixed tubular cell populations
has the same enzymatic properties as that in the isolated
tubules.
In another words, did proteases treatment change
the enzymatic properties of
the separated tubular cells?
The fluctuation in enzyme specific activities in the protease
washes. Therefore, the enzymatic activities measured in the
study do not need to be corrected.

104
treated tubules was parallel to that seen in the untreated
tubules. Although there was a decrease in specific activity
in the protease treated tubules at all ages
there was no
apparent selective loss of activity at any particular age
2
Synthase Activity in Enriched Cells from Testis.
The
level of synthase activity was also evaluated in isolated
spermatogenic cells and epididymal spermatozoa.
The time
course of formation of dehydro DOL PP and dehydro DOL P was
evaluated in the enriched pachytene spermatocytes, spermatids
and Sertoli cell (Figure. 3-3).
The results indicated that
the su
of product formation with 1 mg of protein from
homogenates
o f
each
cell
type
increased linearly with
increasing incubation time as shown in the chapter II for the
tubular homogenates.
Protease treatment did not have an
adverse effect on the linearity of time dependent assay
The
enzyme
specific activity in Sertoli cell was the highest.
Homogenates of cell fractions highly enriched for pachytene
spermatocytes (70% purity) and spermatids (80% purity) had
synthase activity equal to or higher than a whole testicular
2
The
cells
from
methodology for dissociating
seminiferous tubules was satisfactory, since 80-90% of the
enzymatic activity still remained after the
treatment. Disease, a commercial preparation of
has
also
success fully.
for
proteases
preparation of collagenase
reported to dissociate several tissues
Therefore.
Dispase,
been
dispase was
the cellular
a replacement
dissociation from
collagenase in
seminiferous tubules. About 40% of the enzymatic activity in
the collected dissociated cells was lost by this treatment
(results are not shown).

Figure 3-1. Purity of Enriched Cell Fractions.
The testicular tubules were dissociated with
proteases and the spermatogenic and Sertoli cells were
separated as described in the Materials and Methods.
The purity of each cell fraction, as estimated by
Nomarski differential interference microscopy (x 600),
was estimated to be 80% for spermatids (Panel A), 70%
for pachytene spermatocytes (Panel B) and 70% for
Sertoli cells (Panel C).

106
Figure 3-1. Panel A. Spermatids.

107
Figure 3-1. Panel B. Pachytene Spermatocytes (continued).

108
Figure 3-1. Panel C.
Sertoli Cells (continued).

Figure 3-2. Dehydro Dolichyl Diphosphate Synthase
Activity in Sonicates of Tubules from Rats of Different
Ages .
The enzymatic activity was
conditions with sonicates of
assayed under standard
seminiferous tubules
treated (
) and untreated (
) with
described in the Materials and Methods
presented
Numbers
the mean + standard
in parentheses indicate
used to prepare the tubules.
protease
The dat^a
deviation (x + ).
the number of animals

pinoles Isopentenyl Diphosphate Incorporated/ mg protein hr
no
10 20 30 40 50 60
DAYS AFTER BIRTH

Figure 3-3. Time Course of Incorporation of [^4C]-
Isopentenyl Diphosphate into Dehydro DOL PP and Dehydro
DOL p .
Incubations
(pH7.5)
containing
10 mM MgC 12 0.5%
100
mM Tris-HCl
buf fer
Triton X-100
2 5 0 /xM t
farnesyl diphosphate,
14C]-isopentenyl
1.6 mM ATP, 50 mM NaF, 36 /xM [
t-
1
diphosphate
and 1.0
sonicated of
and Sertoli
mg
enriched pachytene spermatocyte,
cell from seminiferous tubules
protein of
spermatid,
protein in a
37 0 C for the
DOL PP and
enzyme
were carried out at
indicated times. The sum of [ -*-4C ]- dehydro
[ ^4C ]- dehydro DOL P were estimated by the
described before. Sertoli cells are isolated
i 40
total volume of 0.25 ml
The
DOL P
me thod
from 23 day old rats, spermatogenic cells are fro
day old rats.

PMOLES ISOPENTENYL DIPHOSPHATE INCORPORATED/MG PROTEIN
112
*

113
homogenate with or without protease treatment (Fig. 3-4). On
the other hand, the spermatozoa and spermatogonial enriched
cell fractions had activities less than 20% of the
spermatocyte activity. When the enzymatic activity was
expressed as pmoles/mg protein/hr, pachytene spermatocytes
had an activity 1.6 fold higher than that seen with a mixture
of cells obtained from protease treated seminiferous tubules.
The enzymatic activity of the enriched spermatocytes was
about 1.4 fold higher than enriched spermatids, 4.8 fold
higher than spermatogonia and about 7.6 fold higher than
spermatozoa. The enzymatic activity of the spermatogenic
c
cells may also be expressed as pmoles product formed/100
cells/hr (Table 3-1). In this case the enzymatic activity of
spermatocytes (28.9 pmoles/10^ cells/hr) was 4.5 fold higher
than that of spermatids (6.5 pmoles/10^ cells/hr) and about
126 fold higher than that of spermatozoa (0.23 pmoles/10^
cells/hr).
Synthase Activity in Sertoli Cells. Synthase specific
activities were also measured in homogenates of Sertoli cells
and a mixed spermatogenic cell population from rats 7-65 days
old (Fig. 3-5). The fluctuations in the enzyme specific
activities for both of these cell populations were parallel
to that seen with the protease and non-protease treated
tubules. In each case, the activities peaked at day 23. The
enriched Sertoli cell specific activity ranged from 1.5-2.3

114
fold higher than that of the mixed spermatogenic cell
population between day 14 and day 30.
Estimated Activities in Pure Cell Populations.
Synthase specific activities in "pure" populations of the
different spermatogenic cells and the Sertoli cells were
estimated from the known purity of the enriched cell
fractions and their specific activities. Table 3-2 shows
that, after a correction was made for the contamination of
each cell fraction for other spermatogenic cells, the enzyme
specific activity in pachytene spermatocytes was 1.7, 5.3 and
8.3 fold higher than in spermatids, spermatogonia and
spermatozoa, respectively. Estimates of the specific
activities of "pure" Sertoli cells isolated from rats of
different ages is shown in Table 3-3. There was a 4.5 fold
increase in the enzyme activity of the Sertoli cell between
day 7 and day 23 and 4.3 fold decrease in activity from day
2 3 to day 6 5 .
In summary: 1) dehydro DOL PP synthase activity in
spermatogenic cells and Sertoli cells peaks in rats at age 23
days, 2) the specific activity of the enriched and pure
spermatogenic cell populations tested decreased in the
following order: pachytene spermatocyte > spermatids >
spermatogonia > sperm and 3) synthase specific activity for
Sertoli cells was 1.5-1.7 fold higher than that of
spermatogenic cells in rats between day 15 and day 30 of age.

Figure 3-4. Dehydro Dolichyl Diphosphate Synthase
Activity in Enriched Spermatogenic Cell Population.
Synthase activity was measured in sonicates of
seminiferous tubules treated and untreated with
cells
from
, speri
tubules of
atogonia
7 dav old
enriched
spermatids
spermatozoa
activity of
o f
hr .
(from
(from
the
dehydro DOL
spermatogenic
rat, pachytene spermatocytes,
day old rats) and epididymal
months old rats). The specific
enzyme is presented as the sum of pmoles
PP and dehydro DOL P formed/mg protein
day
40
3

SYNTHASE ACTIVITY, pMOLES/MG PROTEIN HR
116
TUBULES TUBULES SPERMATO- PACHYTENE SPERMATID SPERM
PROTEASE GONIA
TREATED

117
Table 3-1
Dehydro Dolichyl Diphosphate Synthase Activity in
Enriched Spermatogenic Cellsa
Experiment Activity Ratio
pmoles/10^ Cell hr
Pachytene (P)
Spermatid (S)
P/S
1
34.9
7.9
4.4
2
33.1
7.9
4.1
3
22.9
5.9
3.9
4
25.0
4.5
5.6
Average
28.9 + 2.9
6.5 4* 0.8
4.5 + 0.4
aEnzyi
described
activity was measured in cellular sonicates as
in the Materials and Methods and expressed
isopentenyl diphosphate incorporated per 10^ cell per
cell numbers were calculated by using the conversion factors 258
protein 10^ cells and 83 /g protein = 10^ cells, for
and
pmoles of
hour. The
Mg
pachytene
spermatocytes
unpublished data).
spermatids, respectively (from L. J. Romrell's

Figure 3-5. Age Dependent Variation in Synthase
Activity in Sertoli Cells, Spermatogenic Cells and
Protease Treated Seminiferous Tubules.
Synthase
activity
was
measured
under
standard
conditions in
sonicates
o f
Sertoli
cells
( O ) ,
spermatogenic
cells filtrate
from
protease
treated
seminiferous
tubules (
A )
, and
protease
treated
seminiferous
tubules (
)
prepared from
rats of
different ages.

pmole Isopentenyl Diphosphate
Incorporated/mg protein hr
119
140
120 -
100 -
80 -
60 -
40 -
20
O Sertoli Cells
Tubules
0
0
10
20
30
40
50
60
70
Days After Birth

120
Table 3-2
Estimated Specific Activities of Dehydro POL PP Synthase in
Pure Spermatogenic Cellsa
Specific Activity (pmoles/mg protein)
Spermatogenic Cell % Cell Purity Enriched
"Pure"
Spermatogonia
100
19
19
Pachytene
80
91
100
Spermatocytes
Spermatid
85
64
58
Spermatozoa
100
12
12
aSpecific activities for pachytene spermatocytes and spermatids were
estimated by assuming that each of these populations was contaminated
by the other cell population. The two simultaneous equations
on Pachytene (SA ) + Fraction Spermatid(SA )
populations of Fpermatocytes and spermatids S$lre
solved to obtain the specific activities of both pure cell
populations.

121
Table 3-3
Sertoli
Cells From Rats of Different Ases
a
Specific
Activity (pmoles/mg
Protein)
Day of
Age
%Purity of
Sertoli
Cells
Enriched
Sertoli
Cells
Enriched
Spermatogenic
Cells
"Pure"
Sertoli
Cells
7
87
27
19
28
14
88
109
47
118
23
88
119
69
126
30
86
89
61
94
40
85
63
49
66
65
81
30
25
29
aSpecific activities of "pure" Sertoli cells were estimated by
assuming that each of the enriched Sertoli cell populations was
as that
isolated
contaminated by the same mixture of spermatogenic cells
from rats of the same age. The following equation
SAt?t.*--= Fraction Sertoli cell (SA_ n ) + Fraction
Ger5rlce¥f* ) was solved to Pure
activity for eac1^Dure"^^erroll cell DODulatio
)Wtainerthe specific

122
Discussion
Glycoproteins are undoubtedly
components in
the spermatogenic process.
The timing of their elaboration
is thought to be
for spermatogenesis to proceed
normally.
Histochemical studies in spermatogenic cells show
th a t
the
Golgi
apparatus
o f
spermatocytes
and
early
spermatids are highly active in glycoprotein biosynthesis
(Letts
al ,
1974b) .
However,
the Golgi
apparatus
and
lost from spermatogenic
cells
spermatids mature into spermatozoa.
At least some of the
enzyme s
necessary
for
N-glycoprotein bio synthe sis
presumably lost as well.
Letts e_t al. (1974a) have shown
developmental dependent changes in the specific activities of
three glycosyl transferases involved in the late reactions of
oligosaccharide maturation
m
glycoprotein biosynthesis.
Galactosyl and N-acetylglucosaminy1 transferases were found
in spermatogonia, whereas the fucosyl transferase was not
highly active until the spermatocytes appeared.
All three
enzymes were low in spermatozoa.
A later study (Letts et
al., 1978) indicated that spermatocytes and early spermatids
were highly active in glycoprotein biosynthesis. Therefore,
it was of interest to
investigate if other biological
machineries necessary for the glycoprotein biosynthesis are
changing accordingly in these cell populations.

123
The timing of the secretion of glycoproteins such as
androgen binding protein and plasminogen activator by the
Sertoli cell, has been described in several laboratories
(Parvinen,
1982) .
Furthermore,
there are now numerous
reports which show that the secretion of androgen binding
protein may be regulated by the type of spermatogenic cells
associated with the Sertoli cell at different times during
the spermatogenic cycle (LeMagueresse et al 1980; Ritzen et
al., 1982; Galdier, 1984).
Recent studies indicated that the mammalian testis
exhibits unusually high rates of DOL synthesis. This could
be related to high rates of glycoprotein biosynthesis, and to
temporally regulated synthesis of acrosomal enzymes in late
pachytene spermatocytes
o r
early spermatids
(J ames
&
Kandutsch, 1980c; Wenstrom & Hamilton, 1980; Potter et al.,
1981b). Acrosomal enzymes may also represent end products of
dolichol-mediated glycosylation in the testis since many of
these constituents are glycoproteins (Flechon, 1979; Mukerji
& Meizel, 1979).
Since DOL P has such a critical role in N-
linked glycoprotein biosynthesis, its availability is also
potentially regulatory factor of glycoprotein biosynthesis
during spermatogenesis. The results of previous work (Allen
6c Ward, 1987; Chapter II) showed that the level of DOL P and
dehydro DOL PP synthase increased in parallel between day 7
and day 23 of spermatogenesis in the seminiferous tubules of
immature rats
Synthase activity then decreased to a level

124
at day 60 near that seen at day 7.
The increase in DOL P
levels is consist with the observations of Nyquist and Holt
(1986), who showed an increase in DOL concentrations in rat
testes during this time period and the work of Potter et al.
(1981b), who showed a high rate of DOL biosynthesis from
by
mouse pachytene spermatocytes.
Potter and
coworkers
also
showe d
that
hydroxymethyl
glutary1
CoA
reductase activity was high in pachytene spermatocytes.
Nyquist and Holt (1986)
have
also
reported
that
DOL
concentration was high in Sertoli cells and suggested that
DOL may be synthesized in the spermatogenic cell then
transported to and accumulated in the Sertoli cell.
This
conclusion was supported by the observation by James and
Kandutsch (1980c) that testes of x-irradiated mice or testes
of mutant
ice severely deficient in spermatogenic cells (but
with apparently normal Sertoli cells) incorporated acetate
into DOL at a 20 fold lower rate than normal testes
However, the results reported here show that both the
spermatogenic
cells
and
the
Sertoli
cells
contribute
substantially to the dehydro DOL PP synthase activity of rat
seminiferous
tubules during early
o f
testicular
development.
This is not surprising considering the active
glycoprotein biosynthesis occurring in both cell types, the
rapid changes occurring in the spermatogenic cell during
differentiation and the active role of the Sertoli cell
in
supporting this development.

125
As signment
o f
the
relative
c ontribution
o f
the
spermatogenic cells and the Sertoli cells to the changing
synthase specific activities at different times during early
spermatogenesis requires an assessment of the fraction of
each of these cells present at different ages and knowledge
of the specific
of the "pure cell types.
An
interpretation of these changes is made here on the basis of
the reported results (Fig. 3-6).
At 7 days of age the synthase activities of both the
Sertoli and spermatogenic cells are low.
At this stage of
differentiation the Sertoli cells are still dividing and the
spermatogenic cells have not yet reached the meiotic phase.
It is reasonable that the observed synthase activity from
testes at this age was low.
At 15 days of
the Sertoli cells have stopped
dividing (Steinberger and Steinberger, 1971) and at least
some
phase.
of the spermatogenic cells have entered the meiotic
Dehydro DOL PP synthase in spermatogenic cells has
increased but
the
cell
numb e r
small.
Therefore,
spermatogenic cells do not contribute in
major way to the
total specific activity of the synthase in the whole tubules.
In contrast the Sertoli cells are relatively high in both
number and in enzyme specific activity. Therefore, the total
tubular synthase activity is mainly due to Sertoli cells.
At 23 days of
the enzyme specific activities of
both early pachytene spermatocytes and Sertoli cells have

126
peaked.
The number of pachytene cells relative to other
spermatogenic cells is maximum, and there are few if any
spermatids present by this time.
The number of Sertoli cells
has
become
relatively constant.
Therefore,
the
total
activity of synthase is due to the sum of Sertoli cell,
pachytene spermatocytes and other spermatocytes preceding the
pachytene stage
The specific activity is optimal at this
time, because Sertoli cell specific activity is highest at
this
po int
and
the
relative
percentage
of pachytene
spermatocytes
(the
spermatogenic
cell
with
the
highest
spec
ac tivity)
i s
also highe s t
th i s
time
o f
development.
Actually, it is the only time period in the
rat's life span that the relative percentage of pachytene
spermatocytes reaches a peak value (Fig. 3-6).
At 30 days of age, the pachytene spermatocytes still
have active synthase activity but the relative number of
these cells present in the seminiferous tubules is a smaller
fraction of the total spermatogenic cell population than at
day 23. The Sertoli cell number is constant but its synthase
specific activity has decreased by day 30. The contribution
of spermatids to the total activity is relatively low at this
time, since the number of spermatids is small although
increasing rapidly. Our data also indicate that as pachytene
spermatocytes
differentiate
into
spermatids,
the
enzyme
specific activity decreases by 30 % (Fig. 3-4). There was,
however, no net change in total enzyme activity during the

127
division of pachytene spermatocytes into spermatids (Table 3-
1). Most of the enzymatic activity is lost during the
differentiation of spermatids to spermatozoa. Therefore, the
decrease in tubular synthase activity after 23 days can be
accounted for by the decreasing fraction and specific
activity of Sertoli cells and the fact that the pachytene
spermatocytes, which have the highest spermatogenic cell
specific activity, are becoming a smaller fraction of the
total spermatogenic cells population and are being replaced
by spermatids with lower specific activity.
Kumari and Duraiswami (1986) have estimated the
percentages of Sertoli and spermatogenic cell populations in
rat seminiferous epithelium at various days during early
stages of spermatogenesis. If these percentages are used in
conjunction with the enzyme specific activities observed here
for the Sertoli and mixed spermatogenic cell population, the
predicted enzymatic activities of the whole tubular
homogenate can be calculated for animals 15, 23, and 30 days
of age (Fig. 3-6). There was a good correlation between the
predicted specific activity and the observed activity from
this study except for the result on day 15.
In general, the specific activity of dehydro DOL PP
synthase in Sertoli cell was always higher than that in
spermatogenic cells measured between day 7 and day 65 of age.
This suggests that glycoprotein biosynthesis is more active
in Sertoli cell than in spermatogenic cells. Kumari and

Figure 3-6. The Relationship between the Dehydro DOL PP
Synthase Activity and Spermatogenesis during Testicular
Development.
The curve of the enzymatic activity of rat
testicular development is from the results in the
previous chapter. The scheme is modified from B. P.
Setchell., 1982, in Germ Cell and Fertilization, Eds. C.
R. Austin and R. V. Short, New York: Cambridge
University Press, PP. 63-101. The calculated results
are shown as ( A ).

pmolea Isopentenyi Diphosphate Incorporated/ mg protein-hr
129
DAYS AFTER BIRTH

130
Duraiswami
(1986)
have
concluded,
based
o n
a n
autoradiographic study, that the protein synthetic potential
of Sertoli cells is greater than that of spermatogenic cells
at any stage of differentiation.
The parallel results seen
between that study and results represented here may be highly
significant and suggest a key role for dehydro DOL PP
synthase in regulating glycoprotein biosynthesis in Sertoli
cells during spermatogenesis in rats.
The reason for the rise and fall of the enzyme specific
activity in Sertoli cells in the immature testes is not well
unders tood.
Enzyme activities and protein secretion in
rodent seminiferous tubules have been shown to peak at about
day 20 to day 23 in several cases (Parvinen,1982). This has
been attributed in some cases to the appearance of pachytene
spermatocytes with their constituent enzymes and in other
cases to the onset of new protein synthesizing activities in
Sertoli cells.
In the current study, the results showed that
both Sertoli cell and spermatogenic cells are contributing to
the increase of the dehydro DOL PP synthase activity.
The
possibility that the presence of the pachytene spermatocyte
may cause an increase in Sertoli cell synthase activity is an
interesting conjecture, which has been postulated for other
systems (LeMagueresse & Jegou, 1986; Ireland & Welsh, 1987)
and requires further study.

CHAPTER IV
CONCLUSIONS AND DIRECTIONS
Dehydro DOL PP synthase, which catalyzes the synthesis
of dehydro DOL PP from farnesyl diphosphate and isopentenyl
diphosphate could be very important for controlling DOL P
level in eukaryotic cells, since this is the only de novo
biosynthesis pathway known and serve as a "bridge" connecting
the small metabolites, such as acetyl CoA, with the large DOL
molecules. Therefore, dehydro DOL PP synthase could be an
important cellular regulator of glycoprotein biosynthesis as
a consequence of its regulation in the DOL P de novo
biosynthesis.
An attempt has been made to further characterize the
enzymatic products, namely, dehydro DOL PP and dehydro DOL P.
The precursor-product relationship between dehydro DOL PP and
dehydro DOL P seems clearly established.
It has been reported that the level of DOL P, a
carbohydrate carrier in glycoprotein biosynthesis, is
regulated during spermatogenesis (James 6c Kandutsch, 1980b;
Potter et al., 1981b). Temporal expression of seminiferous
tubular dehydro DOL PP synthase has been shown to correlate
well with the increase in Dol P during early stages of
differentiation in prepuberal rats. The results presented in
131

132
Chapter II support the hypothesis that increasing DOL
synthesis observed during testicular development (Potter et
al., 1981b) is due at least in part to an increase in the
dehydro DOL PP synthase activity.
The cellular localization of this increased synthase
activity was of interest because of the multicellular nature
of the testicular tubules. The specific activity of synthase
in homogenates of protease treated seminiferous tubules, cell
fractions enriched in spermatogenic cells or Sertoli cells
peaked in rats aged 23 days, as shown with non-protease
treated cells. Homogenates of cell fractions enriched in
pachytene spermatocytes, spermatids or Sertoli cells had
higher synthase activity than a whole testicular homogenate
or a mixture of cells prepared by protease treatment of
tubules. Enzymatic activity in pachytene spermatocytes
expressed per mg protein, was about 5.3 fold higher than
spermatogonia, 1.7 fold higher than in spermatids and about
8.3 fold higher than in spermatozoa. Therefore, the increase
of the synthase activity in spermatogenic cell before day 23
can be accounted for by the appearance of the pachytene
spermatocytes. Little net increase in enzyme occurred during
or after meiotic cell division of pachytene spermatocytes
into spermatids. The enzymatic activity decreased remarkably
during the differentiation of spermatids into spermatozoa.
It is reported for the first time that Sertoli cells
have the potential to synthesize DOL P. The enzymatic

133
activity in enriched Sertoli cells was 1.5 to 2.3 fold higher
than in the enriched spermatogenic cells between day 14 and
day 30 .
The increase in synthase activity in spermatogenic
cells and Sertoli cells indicates that both are contributing
to changes in the enzymatic activity in seminiferous tubules.
In
addition, and perhaps more significantly, the work
presented here provides evidence that dehydro DOL PP synthase
may be important in regulating the availability of Dol P for
glycoprotein synthesis during early stages of spermatogenesis
in rat
Future research on the role that dehydro DOL PP
synthase plays in spermatogenesis in rat will focus on
several fundamental questions. In vitro studies on the
incorporation of radiolabeled probe, such as mevalonate,
into DOL with enriched Sertoli cells will determine whether
synthase activity function in these cells in vivo. Synthase
activity would also be measured in co-cultures of Sertoli
cells and different spermatogenic cells. This could test
the role of cell-cell interaction with respect to the
function of DOL P in regulating spermatogenesis. Activators
or inhibitors of this enzyme may also be produced as a
result of cell-cell interactions. Such questions can now be
approached with the techniques and information presented in
this dissertation.

APPENDIX A
SUMMARY OF EXPERIMENTAL DATA PRESENTED IN FIGURE 2-13 DEHYDRO DOL PP
SYNTHASE ACTIVITY IN SONICATES OF TUBULES FROM RATS OF DIFFERENT AGES.
DATE
AGE
85/1/16
2/5
4/8
4/22
4/30
5/16
5/24
6/10
6/22
64.5
82.5
72.9
77.3
70.7
97.2
51.3
39.0
66.3
62.9
59.5
107.4
90.2
74.9
58.7
71.5
60.7
53.2
106.0
92.7
92.7
71.0
73.2
63.5
71.7
91.1
85.2
50.9 51.2
50.4 44.4
94.7 71.0
34.3
56.3
67.0
100.7 73.3
119.9 98.1
110.0
94.1
59.9
48.8
74.8
X
70
54
61
69

89
106
87
87
69
63
59
39
^x
23
14
8
6
14
11
13
14
5
3
12
2
N
8
12
8
4
8
8
6
14
6
6
8
n
4
6
4
2
4
4
3
7
3
3
4
1
S xApr
12
6
4
4
7
6
7
5
3
2
6
Animals
40
18
8
3
10
4
5
13
3
3
10
1
Mean x pmoles/mg protein
Standard Deviation
pmoles/mg protein
Number of Experiments
Number of Assays
134

APPENDIX B
TYPICAL NUMBER OF RATS, TOTAL TUBULE WEIGHT, AND NUMBER OF ASSAY USED
FOR EACH EXPERIMENT AS A FUNCTION OF RAT AGE
Age
(Days)
# of Rats Used
Total Tissue Weight
(g)
Assays
3
10
0.09
2
7
4 5
0.12
2
15
2
0.20
2 3
23
2
0.85
3 or more
30
2
1.20
3 or more
40
2
1.60
3 or more
60
2
3.50
3 or more
135

APPENDIX C
SUMMARY OF EXPERIMENTAL DATA PRESENTED IN FIGURE 3-5. AGE DEPENDENT VARIATION IN
SYNTHASE ACTIVITY IN SERTOLI CELLS, SPERMATOGENIC CELLS AND PROTEASE TREATED
SEMINIFEROUS TUBULES. SYNTHASE ACTIVITY IS PRESENTED IN UNITS OF PMOLES
ISOPENTENYL DIPHOSPHATE INCORPORATED/MG PROTEIN
Rats
Age
(Days)
Assays
Sertoli
Synthase
Activity
Rats
Assays
Tubules
Synthase
Activity
Rats
Assays
Spermatogeni'
Cells
Synthetase
Activity
7
24
4
27
+
6
12
4
32
+
2
24
4
19
+
2
15
12
4
109
+
4
12
6
61
+
14
12
4
47
+
7
23
7
4
119

4
7
6
93
+
4
7
4
69
+
2
30
4
4
89

8
4
6
76
+
7
4
4
61
+
2
40
6
4
63

7
6
4
52
+
6
6
4
49
+
5
60
4
4
30

3
4
4
24
+
1
4
4
25
3
136

137
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Chem. 257:

BIOGRAPHICAL SKETCH
Zhong Chen was born in Beijing, China, in 1944.
1962, Zhong began his premedical education at Peking
In
University. Later Zhong studied medicine at China Medical
College (the former Peking Union Medical College founded by
the Rockefeller Foundation) and he received the M.D. degree
in 1968. After graduation he practiced medicine for several
years and specialized in ophthalmology. In 1981, Zhong
visited the Royal Hospital (Rigshospitalet) and the
University of Copenhagen in Denmark as a visiting scholar in
the Departments of Ophthalmology and Virology. During that
period of time, Zhong realized that biochemistry and
molecular biology are the keys to open the mysterious kingdom
of medicine.
So
he decided to brush up on biochemistry and
use it as a tool to explore the unanswered questions in
medicine.
After finishing his Ph.D., Zhong will move to Oklahoma
t
City, Oklahoma, and the laboratory of Dr. Jordan J. N. Tang
in the Oklahoma Medical Research Foundation to continue his
training.
146

I
opinion it
certify
confor
that I have read this study
s tandards
and that in
s to acceptable
o f
my
scholarly
presentation
dissertation
and i s
for
fully
the degree
adequate, in
of Doctor
scope and quality, as
of Philosophy.
Charles M. Allen, Chairman
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Michael S. Kilberg
Associate Pro sor of
Biochemistry and Molecular
Biology
I certify that 1 have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Thomas W. O'Brien
Professor of Biochemistry and
Molecular Biology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Biology
This dissertation was submitted to the Graduate Faculty of
the College of Medicine and the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
April, 1988
Dean, College of Medicine
Dean, Graduate School



TIME (min)
pmoles Isopentenyl Diphosphate Incorporated/ mg protein
ON
ro
150


Figure 2-11. Time Course of Incorporation of [^C]-
Isopentenyl Diphosphate into Dehydro DOL PP and Dehydro
DOL P.
Incubations containing 100 mM Tris-HCl buffer
(pH7 5) 10 mM MgCl2, 0.5% Triton X-100, 250 /xM t,t-
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF, 36 /zM [ 1 -
^C]-isopentenyl diphosphate, and 1.0 mg of sonicated of
seminiferous tubules as enzyme protein in a total volume
of 0.25 ml were carried out at 37 C for the indicated
times. The formation of [ ]-dehydro DOL PP (Panel A)
and [ ]-dehydro DOL P (Panel B) were estimated by the
method described before. Panel C represents dehydro DOL
PP synthase activity (A+B).


60 min as shown in Fig. 2-11). Second, there is evidence for
a detergent sensitive phosphatase that acts on the
diphosphate to give the monophosphate. Third, kinetic
experiments have shown a classical time dependent lag in
monophosphate formation compared to diphosphate formation,
whereas total phosphorylated polyprenol increased linearly.
Fourth, direct chemical experiments were also performed to
establish the precursor-product relationship between dehydro
DOL PP and dehydro DOL P, such as double labeling experiment
using [cr,/?- ^P ] isopentenyl diphosphate and [ ] isopenteny 1
diphosphate (Table 2-1), and base hydrolysis (Fig. 2-5).
The mild saponification used in each assay to release
the membrane bound lipids did not change the ratio between
dehydro DOL PP and dehydro DOL P. However, during a more
vigorous saponification, dehydro DOL PP was hydrolyzed to
dehydro DOL P as previous described (Adair and Cafmeyer,
(1987). These experiments clearly showed that we can simply
sum the radioactivity in the mono and diphosphate components
as an accurate measurement of the total synthase activity.
Grange and Adair (1977) observed and therefore measured
only dehydro DOL P formation in hen oviduct, although they
commented that dehydro DOL P may be derived from dehydro DOL
PP by the action of a phosphatase. In contrast, Adair and
Cafmeyer (1987b) observed and measured only dehydro DOL PP
formation in their studies in yeast. The current study
showed that the testicular enzyme produces both dehydro DOL


APPENDIX A
SUMMARY OF EXPERIMENTAL DATA PRESENTED IN FIGURE 2-13 DEHYDRO DOL PP
SYNTHASE ACTIVITY IN SONICATES OF TUBULES FROM RATS OF DIFFERENT AGES.
DATE
AGE
85/1/16
2/5
4/8
4/22
4/30
5/16
5/24
6/10
6/22
64.5
82.5
72.9
77.3
70.7
97.2
51.3
39.0
66.3
62.9
59.5
107.4
90.2
74.9
58.7
71.5
60.7
53.2
106.0
92.7
92.7
71.0
73.2
63.5
71.7
91.1
85.2
50.9 51.2
50.4 44.4
94.7 71.0
34.3
56.3
67.0
100.7 73.3
119.9 98.1
110.0
94.1
59.9
48.8
74.8
X
70
54
61
69

89
106
87
87
69
63
59
39
^x
23
14
8
6
14
11
13
14
5
3
12
2
N
8
12
8
4
8
8
6
14
6
6
8
n
4
6
4
2
4
4
3
7
3
3
4
1
S xApr
12
6
4
4
7
6
7
5
3
2
6
Animals
40
18
8
3
10
4
5
13
3
3
10
1
Mean x pmoles/mg protein
Standard Deviation
pmoles/mg protein
Number of Experiments
Number of Assays
134


41
Materials and Methods
Materials.
Male Sprague-Dawley rats were purchased
from local suppliers.
t,t-Farnesyl diphosphate was prepared
as previously described (Baba and Allen, 1978).
[1-
14C1-A3.
Isopentenyl diphosphate
and
[ ^P] orthophosphoric acid
(carrier
free)
i n
dilute
HC 1
was
purchased
from
Amersham/Searle Corp. Silica gel 60 F254 and Cellulose F254
on
plastic sheets were products of E. Merck.
All other
reagents were obtained from standard commercial sources.
Solvent Systems. The following solvents were used for
extraction or chromatography: Solvent A, CHCI3-CH3OH (2:1);
Solvent
B ,
CHCI3-CH3OH-H2O (3:48:47);
Solvent
C ,
diisobutylketone glacial acetic acid-H20 (8:5:1); Solvent D,
2-propanol-acetonitrile-0. 1
M
ammonium
bicarbonate
(45:25:30); Solvent E, 1-propanol-concentrated ammonia-H20
(6:3:1).
Animal grouping number required for analysis. In order
to have sufficient data for
analysis in each
experiment, at least two rats, but most often three or more
rats were used for each age group tested (See Appendix A).
The excised testes (six or more) from each age group were
pooled together, then two or three aliquots of the mixed
samples were taken for assay
The data presented in each
table or figure were usually means determined by two or three
similar experiments (e.g. Appendices B and C).


Dehydro Dehydro Dehydro Dehydro
Dol PP Dol P Dol PP Dol P
i
1st hour 2nd hour


This dissertation is dedicated to
my
parents, Quo-Wei Chen and Zhi-Huei Chang,
y loving wife, He-Ping Han, and my son,
Henry Hai Pei.


CHAPTER IV
CONCLUSIONS AND DIRECTIONS
Dehydro DOL PP synthase, which catalyzes the synthesis
of dehydro DOL PP from farnesyl diphosphate and isopentenyl
diphosphate could be very important for controlling DOL P
level in eukaryotic cells, since this is the only de novo
biosynthesis pathway known and serve as a "bridge" connecting
the small metabolites, such as acetyl CoA, with the large DOL
molecules. Therefore, dehydro DOL PP synthase could be an
important cellular regulator of glycoprotein biosynthesis as
a consequence of its regulation in the DOL P de novo
biosynthesis.
An attempt has been made to further characterize the
enzymatic products, namely, dehydro DOL PP and dehydro DOL P.
The precursor-product relationship between dehydro DOL PP and
dehydro DOL P seems clearly established.
It has been reported that the level of DOL P, a
carbohydrate carrier in glycoprotein biosynthesis, is
regulated during spermatogenesis (James 6c Kandutsch, 1980b;
Potter et al., 1981b). Temporal expression of seminiferous
tubular dehydro DOL PP synthase has been shown to correlate
well with the increase in Dol P during early stages of
differentiation in prepuberal rats. The results presented in
131


52
Table 2-1
Incorporation of A -fl- Cl Isopentenvl Diphosphate and f a. 8-PI-Isooentenvl
Diphosphate into Dehvdro POL PP and Dehvdro POL P
Experiment 1
Experiment 2
Radiolabeled
Substrate
[ 32pi -ipp
5 nmol
(1.36 nCl)
[14C]-IPP
4 nmol
(0.21 iiCL)
RATIO
^Pcpm
^Ccpm
[3 2 p]-ipp
10 nmol
(2.72 mCI)
[14C]-IPP
4 nmol
(0.21 nCi)
RATIO
^Pcpm
^Ccpm
Radiolabeled
Product
(cpm incorporated)
(cpm incorporated)
Dehydro DOL PP
(A)
424
1489
0.28
1127
883
1.28
Dehydro DOL P
(B)
217
1526
0.14
533
936
0.57
(A)/(B)
2.0
2.2
a The enzyme assay was carried out as described in the text. The reported values of
cpm have been corrected for overlap of into the channel. (A)/(B) represents
the relative ratio of radiolabel from ^2p incorporated into the diphosphate compared
to the monophosphate.


pmoles Isopentenyl Diphosphate incorporated /hr
68
Protein Cmg)


TABLE OF CONTENTS
ACKNOWLEDGEMENTS
LIST OF TABLES..
LIST OF FIGURES.
ABBREVIATIONS...
ABSTRACT
Page
. iii
. vi
. vi i
. ix
. xi
CHAPTERS
IINTRODUCTION 1
The Spermatogenesis 1
The Sertoli Cells 7
The Role of DOL P in Glycoprotein Biosynthesis....12
Studies Related to DOL Biosynthesis and
Spermatogenesis 26
Significance 3 5
Obj ectives 37
IIDEVELOP AND OPTIMIZE AN ASSAY FOR DEHYDRO
DOLICHYL DIPHOSPHATE SYNTHASE FROM RATS TESTES....38
Introduction 38
Materials and Methods 41
Results 46
Discussion 83
IIIDEHYDRODOLICHYL DIPHOSPHATE SYNTHASE ACTIVITY
MEASURED IN ENRICHED SPERMATOGENIC CELL
POPULATIONS 93
Introduction 9 3
Materials and Methods 98
Results 103
Discussion 122
IVCONCLUSIONS AND DIRECTIONS 131
4
IV


CHAPTER I
INTRODUCTION
Spermatogenesis
Spermatogenic Cycles and Waves
Spermatogenesis is a developmental process in which the
spermatogenic cell undergoes a series of biochemical and
orphological changes through three well described phases:
spermatogonial
renewal
and proliferation, meiosis,
and
spermiogenesis.
Spermatogenesis in the rat starts during
fetal development with appearance of gonocytes by postnatal
day 4 and continues throughout adult life.
The first
spermatozoa appear within the lumens of the seminiferous
tubules at about 45 days of ages
and all
o f
spermatogenic cycle are represented (Clermont & Perey, 1957;
Knorr et al., 1970).
The initial phase of spermatogenesis,
the spermatogonial phase, occurs in the basal compartment of
the
seminiferous epithelium
and
cons is ts
o f
mitotic
pro
ion
o f
spermatogonia
f rom
stem
cells.
The
spermatogonia divide and differentiate sequentially into type
A spermatogonia, intermediate spermatogonia, and type B
spermatogonia.
The type B spermatogonia divide to for
1
f


pmoles Isopentenyl Diphosphate Incorporated/ mg protein


Bridges, C. D. B., Peters, T., Smith, J. E., Goodman, D. S
Fong, S. L., Griswold, M. D., and Musto, N. A. 1986,
Federation Proc. 45: 2291-2303.
Burton, W, A., Lucas. J. J., and Waechter, C. J. 1981, J.
Biol. Chem. 256: 632-635.
Burton, W. A., Scher, M. G., and Waechter, C. J. 1979. J.
Biol. Chem. 254: 7129-7136.
Butterworth, P. H. W., Draper, H. H., Hemming, F. W., and
Morton, R. A. 1966, Arch. Biochem. Biophys. 113: 646
653 .
Carson, D. D., Earles, B. J., and Lennarz, W. J. 1981, J.
Biol. Chem. 256: 11552-11557.
Carson, D. D., and Lennarz, W. J. 1979, Proc. Natl. Acad.
Sci. U.S.A. 76: 5709-5713.
Carson, D. D., and Lennarz, W. J. 1981. J. Biol. Chem. 256
4679 -4686.
Carson, D. D., Tang, J. P., and Hu, G. 1987, Biochemistry.
26: 1598-1606.
Chapman, A., Fujimoto, K., and Kornfeld, S. 1980. J. Biol.
Chem. 255: 4441-4446.
Clermont, Y. 1963, Am. J. Anat. 112: 35-51.
Clermont, Y., and Perey, B. 1957, Amer. J. Anat. 100: 241-
268 .
Coolbear, T., and Mookerjea, S. 1981, J. Biol. Chem. 256:
4529-4535.
Cramer, F., and Bohm, W. 1959, Angew. Chem. 71: 775.
Dallner, G., and Hemming, F. W. 1981, in Mitochondria and
Microsomes (Lee, C. P., Schatz, G. and Dallner, G.
eds.). Addison-Wes1ey, Reading, Mass, pp 655 -681.
Dorrington, J. H., Roller, N. F., and Fritz, I. B., 1975,
Mol. Cell. Endocrinol. 3: 59-70.
Dym, M., and Clermont, Y. 1970, Am. J. Anat. 128: 265-282.
Eggens, I., Eriksson, L. C., Chojnacki, T., and Dallner, G
1984, Cancer Res. 44: 799-805.


Figure 2-10. A double reciprocal plot of the sum
of dehydro DOL PP and dehydro DOL P formation (Fig. 2-9.
Panel C) vs. farnesyl diphosphate concentration is
presented.


I
opinion it
certify
confor
that I have read this study
s tandards
and that in
s to acceptable
o f
my
scholarly
presentation
dissertation
and i s
for
fully
the degree
adequate, in
of Doctor
scope and quality, as
of Philosophy.
Charles M. Allen, Chairman
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Michael S. Kilberg
Associate Pro sor of
Biochemistry and Molecular
Biology
I certify that 1 have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Thomas W. O'Brien
Professor of Biochemistry and
Molecular Biology


Figure 2-3. Dependence of Product Formation on Triton
X-100 Concentration and Incubation Time.
Sonicates of seminiferous tubules were assayed
under standard conditions except that Triton X-100 and
time of incubation were varied as shown. Dehydro DOL PP
and dehydro DOL P were analyzed separately in reaction
mixtures incubated for 1 hour and 2 hours in 0.5% Triton
X-100 (Panel A), 2% Triton X-100 (Panel B) and from a
reaction mixture which was incubated for 1 hour in 2%
Triton X-100 and then diluted four fold with all
reaction constituents except enzyme and Triton X-100 and
incubated for an addition 1 hour (Panel C).


114
fold higher than that of the mixed spermatogenic cell
population between day 14 and day 30.
Estimated Activities in Pure Cell Populations.
Synthase specific activities in "pure" populations of the
different spermatogenic cells and the Sertoli cells were
estimated from the known purity of the enriched cell
fractions and their specific activities. Table 3-2 shows
that, after a correction was made for the contamination of
each cell fraction for other spermatogenic cells, the enzyme
specific activity in pachytene spermatocytes was 1.7, 5.3 and
8.3 fold higher than in spermatids, spermatogonia and
spermatozoa, respectively. Estimates of the specific
activities of "pure" Sertoli cells isolated from rats of
different ages is shown in Table 3-3. There was a 4.5 fold
increase in the enzyme activity of the Sertoli cell between
day 7 and day 23 and 4.3 fold decrease in activity from day
2 3 to day 6 5 .
In summary: 1) dehydro DOL PP synthase activity in
spermatogenic cells and Sertoli cells peaks in rats at age 23
days, 2) the specific activity of the enriched and pure
spermatogenic cell populations tested decreased in the
following order: pachytene spermatocyte > spermatids >
spermatogonia > sperm and 3) synthase specific activity for
Sertoli cells was 1.5-1.7 fold higher than that of
spermatogenic cells in rats between day 15 and day 30 of age.


Man
MEM
M
H Ci
Mg
Ml
/iM
/imole
mM
mm
mmole
PBS
pmole
PP
RNA
rpm
TLC
Tris
v/v
v/w
w/v
w/w
manno s e
Eagle's minimal essential medium
micron
microcurie
microgram
microliter
micromolar
micromole
millimo1ar
millime ter
millimole
phosphate buffered saline
picomole
diphosphate
ribonucleic acid
revolutions per minute
thin layer chromatography
Tris-(hydroxymethyl)aminomethane
on a volume-to-volume basis
on a volume-to weight basis
on a we ight-to-volume basis
on a we ight-1o-weight basis
x


120
Table 3-2
Estimated Specific Activities of Dehydro POL PP Synthase in
Pure Spermatogenic Cellsa
Specific Activity (pmoles/mg protein)
Spermatogenic Cell % Cell Purity Enriched
"Pure"
Spermatogonia
100
19
19
Pachytene
80
91
100
Spermatocytes
Spermatid
85
64
58
Spermatozoa
100
12
12
aSpecific activities for pachytene spermatocytes and spermatids were
estimated by assuming that each of these populations was contaminated
by the other cell population. The two simultaneous equations
on Pachytene (SA ) + Fraction Spermatid(SA )
populations of Fpermatocytes and spermatids S$lre
solved to obtain the specific activities of both pure cell
populations.


121
Table 3-3
Sertoli
Cells From Rats of Different Ases
a
Specific
Activity (pmoles/mg
Protein)
Day of
Age
%Purity of
Sertoli
Cells
Enriched
Sertoli
Cells
Enriched
Spermatogenic
Cells
"Pure"
Sertoli
Cells
7
87
27
19
28
14
88
109
47
118
23
88
119
69
126
30
86
89
61
94
40
85
63
49
66
65
81
30
25
29
aSpecific activities of "pure" Sertoli cells were estimated by
assuming that each of the enriched Sertoli cell populations was
as that
isolated
contaminated by the same mixture of spermatogenic cells
from rats of the same age. The following equation
SAt?t.*--= Fraction Sertoli cell (SA_ n ) + Fraction
Ger5rlce¥f* ) was solved to Pure
activity for eac1^Dure"^^erroll cell DODulatio
)Wtainerthe specific


20
Since DOL P is an important precursor of both DOL P-
mono s accharide
and DOL P-oligosaccharide,
i t
i s
understandable that a shortage of DOL P could have multiple
effects on the biosynthesis of 1ip id-o1igosaecharide and may
cause the production of defective glycoproteins. Chapman has
shown that a mouse lymphoma cell mutant, lacking DOL P, can
not synthesize DOL P-Man (Chapman et al. 1980) .
Kean
(1985), using microsomes from a variety of tissues, reported
that DOL P-Man, which requires DOL P for its biosynthesis,
exerts a positive allosteric effect on the enzymes that
catalyze the formation of DOL PP-GlcNac.
More recently,
Carson et al. (1987) found that during hormonal induction of
glycoprotein assembly in mouse uteri, the changes in the rate
of DOL P-Man synthesis may be an important factor in
regulating DOL P-linked oligosaccharide assembly, since uteri
contain very high levels of DOL P and DOL P
1inked
saccharides.
DOL PP, in turn, can arise via two metabolic pathways.
First, by a recycling mechanism, where DOL PP is released
from the lipid oligosaccharide when the oligosaccharide
portion is transferred to the newly synthesized polypeptide.
Alternatively, DOL PP may be derived from de novo
biosynthesis by a poorly defined pathway which undoubtedly
requires the condensation of low molecular weight precursors,
such as farnesyl diphosphate and isopentenyl diphosphate.
Regulation of this pathway could be very important for


47
fraction isolated from homogenates of seminiferous tubules
from rat testes (Baba et al., 1987).
It was necessary to optimize the method to accurately
assay the enzyme in homogenates of tubules taken from animals
o f
different
Homogenates were prepared by
the
sonication of buffered suspensions of tissue instead of
disruption with a glass homogenizer as previous described
(Baba et al., 1987). In this study, sonication was found to
be the only satisfactory procedure for disruption of the
small amounts of tissue available from 3- and 7-day-old rats.
Sonication also had the added advantage of denaturing the
prenyl
transferase,
farnesyl diphosphate synthase,
exhibite d
by
an
e1imination
o f
radioactive
product
chromatographing with an Rf similar to exogenously added
farnesol (Fig. 2-1-1, Panel B).
*^P and Ratios in
the
Mono and Diphosphate
Products.
The carbon chain lengths of the long chain
polyprenyl products obtained by in vitro biosynthesis were
previously shown to be the same (Baba et al., 1987 ).
Since
these two compounds had the same chain length, but they
chromatographed on TLC and anionic exchange columns in
manner consistent with mono- and diphosphorylated products,
they were assumed to be the dehydro Dol P and dehydro Dol PP.
The ratios of phosphate to polyprenol chain were determined
here for the putative dehydro Dol P and dehydro Dol PP in
order to clarify this earlier assumption.
Mixtures of [a, ¡3-


46
hydrolysis products were extracted with 2 ml of Solvent A.
Then the lower phase was subjected to TLC analysis. The
authentic markers, [ ]-dehydro DOL P and non-radiolabeled
DOL P were chromatographed in parallel to the hydrolysis
products and the developed TLC sheet was subjected to
autoradiography.
Results
Optimization of Synthase Reaction and Characterization
of the Enzymatic Products.
The typical prenyl transferase
assay for the long chain polyprenyl diphosphate synthase in
bacteria
easures the amount of acid labile and organic
solvent extractable product (Keenan & Allen, 1974). However,
this method could not be satisfactorily
applied
for
the
because
of
the
quantitation of dehydro DOL PP synthase,
synthesis of other isoprenoid products with acid lability and
extractab1ity similar to the dehydro DOL phosphates.
A more
accurate method was developed which involved 1) CHCI3-CH3OH
extraction of the reaction products after saponification of
the reaction components, 2) application of TLC to
the dehydro DOL PP and dehydro DOL P from shorter chain
polyprenyl phosphates
and
free polyprenols,
and
3)
determination of the sum of the dehydro DOL PP and dehydro
DOL P formed.
This method was
applied to the
identification of dehydro DOL PP synthase in the microsomal


Figure 3-4. Dehydro Dolichyl Diphosphate Synthase
Activity in Enriched Spermatogenic Cell Population.
Synthase activity was measured in sonicates of
seminiferous tubules treated and untreated with
cells
from
, speri
tubules of
atogonia
7 dav old
enriched
spermatids
spermatozoa
activity of
o f
hr .
(from
(from
the
dehydro DOL
spermatogenic
rat, pachytene spermatocytes,
day old rats) and epididymal
months old rats). The specific
enzyme is presented as the sum of pmoles
PP and dehydro DOL P formed/mg protein
day
40
3


43
eliminate the unwanted products, and only isolate and
quantitate dehydro DOL PP and dehydro DOL P. Optimal assay
conditions were also established. The dependency of product
formation on Triton X-100, protein, isopentenyl diphosphate,
farnesyl diphosphate concentration and time were determined.
The standard assay of the enzyme was carried out by
incubation of 100 mM Tris-HCl buffer pH 7.5, 10 mM MgCl2>
0.5% Triton X-100, 250 /M t, t farnesyl diphosphate, 1.6 mM
ATP, 50 mM NaF, 36 \x M [ 1 ]- isopentenyl diphosphate
(1.1x10^ dpm, 53 /Ci//i mole), and 1.0 mg of enzyme protein in
a final incubation volume of 0.25 ml for 1 hr at 37C. The
reaction was stopped by the addition of 0.25 ml of 1 M KOH.
Then the mixture was heated at 100 C for 30 min to saponify
the membrane bound lipids. Afterwards, the mixture was
cooled in an ice bath, 0.25 ml of 1 M HC1 and 1.25 ml of 2 M
KC1 were added. This mixture was extracted twice with 1 ml
aliquots of Solvent A.
The combined solvent A extract was washed first with 2
ml of deionized water, then with 2 ml of Solvent B. When the
extent of product formation was to be quantitated, a 1 ml
aliquot was taken from the organic extract, dried in a
scintillation vial and 10 ml of toluene based scintillation
fluid (Scinti Verse II, Fisher) were added for analysis of
the radioactivity. Radioactivity was then determined in a
scintillation counter.


10
under the influence of various factors, but they do not
divide any more.
Adjacent Sertoli cells are joined by the Sertoli cell
junctional complex, which is
unique structure not found in
other epithelium tissues.
This functional barrier develops
in the rat at about 16 to 19 days of age (Vitale et al.,
1973) .
The Sertoli-Sertoli junctional complex divides the
seminiferous epithelium into two compartments:
the basal
compartment and the adluminal compartment.
Actually, the
Sertoli-Sertoli junctional complexes are the site of the
blood-testis barrier, which serves an essential role in
isolating the spermatogenic cells from the immune system;
i.e. the production of unique molecules on spermatogenic
cells is recognized as foreign if these molecules come in
contact with the immune system.
Follicle stimulating hormone (FSH) and testosterone
r e gu1 ate
the
process
o f
sperm production within
the
seminiferous epithelium.
The Sertoli cells have been shown
to be the primary target for FSH and androgens. Therefore,
the Sertoli cells are considered to be the regulators of
spermatogenesis. The probable importance of Sertoli cells in
spermatogenesis
has
been emphasized
by
number
o f
investigators (Bridges et al., 1986; Fritz et al., 1976;
Griswold et al., 1986); however, their precise role in this
process is still not fully understood.


PRODUCT/mg protein
,pmol), (661(19)1 (o.pmol X10)
89
DAYS, AGE


45
carrier free) in dilute HC1 was dried in the reaction vessel
over P2O5 under N2. Then 0.5 /moles crystalline H3PO4, 6.25
/moles t r ie thy 1 amine 2 /moles 3 me thy 1 3 but en 1 o 1 and
12 /moles
in 60 /I acetonitrile were
added and the reaction permitted to proceed for 5-7 hours at
room temperature.
The reaction was stopped by adding 200 /I
of 10 mM NH4OH. The reaction products was separated by TLC
(Cellulose F254) in Solvent D.
The radioactive
32
P band
migrating beside
authentic [^C] -isopentenyl diphosphate
(Rf=0.35) was scraped from the plate, packed in a glass
column and [^^P]-isopentenyl diphosphate was eluted with 2* ml
of methanol at room temperature.
The nature of the
32
P-
labeled product was verified by TLC along with authentic
isopentenyl diphosphate by TLC on Silica 60 F254 in Solvent
E.
Base Hydrolysis of Dehvdro Pol PP. The putative
dehydro DOL PP was biosynthesized from [ ^C ]-isopentenyl
diphosphate and farnesyl diphosphate with a testicular
homogenate according to the standard assay method. Enzymatic
products were separated by TLC on Silica 60 F254 as already
described. The dehydro Dol PP region on TLC (Rf=*0.40) was
localized by autoradiography and scraped into a conical test
tube. The product bound to Silica gel was suspended in 1 ml
of 3.5 M KOH in 70% methanol and the hydrolysis was carried
out at 100 C for 2 hr. At the end of the hydrolysis, 2 ml
of water was added to the mixture and the polyprenol


30
in spermatogenic cells.
from these mice,
when
compared to normal controls, demonstrated markedly reduced
ratios of
cholesterol.
acetate incorporation into DOL as compared to
These results suggested that the high rate of
DOL synthesis in mouse testes may be attributed to one or
more types of spermatogenic cells, although Sertoli cells may
not be excluded. It was subsequently shown that prepuberal
mouse pachytene spermatocytes incorporate acetate into DOL at
rate which is 5 times higher than that of leptotene and
zygotene spermatocytes, and that
high rate of acetate
incorporation into DOL is maintained in adult pachytene
spermatocytes and round spermatids (Potter et al., 1981b).
A developmental study
o f
DOL kinase activity
m
sexually maturing rats
15-60 days
of age
showed the
appearance of detectable levels of activity at 21 days,
peak at 24 days and
subsequent decline to adult levels.
The
developmental
pattern of
this
enzyme
its
association with the later stages of spermatocyte development
(Berkowitz & Nyquist, 1986).
Allen and Ward (1987) also reported changes in specific
activity of DOL kinase during testicular development in the
They showed that the specific activity of kinase peaked
at day 30, whereas the level of endogenous DOL P, as measured
indirectly by the DOL P dependent mannosyl
activity, rose to a peak around day 15.
Since the optimal
activity of DOL kinase peaked later (day 30) than the peak in


2
preleptotene primary spermatocytes, which undergo a final
replication of nuclear DNA before entering meiotic prophase.
The
preleptot ene
spermatocytes
m
from
the
basal
compartment
t o
the
adluminal
compartment
where
spermatogenesis
l s
completed.
The
seco nd phase
of
spermatogenesis, meiosis, occurs while the spermatocytes
remain on the adluminal side of the intercellular Sertoli
j unctions.
Meiotic prophase, which is subdivided into the
stages of the leptotene, zygotene, pachytene, diplotene, and
diakinesis, terminates in the first meiotic, or reductional,
division with the formation of secondary spermatocytes.
The
latter cells quickly enter the second meiotic, or equational
division to form the haploid spermatids. Spermiogenesis, the
final phase
o f
spermatogenesis,
cons is ts
o f
complex
morphological transformation of the haploid spermatogenic
cell, that culminates with the release of late spermatids
into the lumen of the seminiferous tubule.
Spermatogenesis has many unique features
The most
remarkable ones include the spermatogenic cycles and waves in
the seminiferous epithelium.
The various generations of
spermatogenic cells are not randomly distributed in the
seminiferous epithelium, but are organized into well-defined
cellular associations.
Certain cells are always found in
association with certain other cells.
Each of these cells
develops in synchrony with the others, so that if we could
watch one section of the tubule wall with a time-lapse


FPP (uM)


Figure 2-12. Dehydro DOL PP Synthase in Testicular
Homogenate of Different Aged Rats.
Incubations containing 100 mM Tris-HCl buffer
(pH7.5) 10 mM MgCl2, 0.5% Triton X-100, 250 /M t,t-
farnesyl diphosphate, 1.6 mM ATP, 50 mM NaF, 36 ¡jl M [1-
^C]-isopentenyl diphosphate, and 1.0 mg of enzyme
protein frcTm indicated aged rats in a final volume of
0.25 ml were carried out at 37 C for 60 minutes. The
formation of [ ]-dehydro DOL PP (Panel A) and [^C]-
dehydro DOL P (Panel B) were estimated by the method
described before. Panel C represents dehydro DOL PP
synthase activity (A+B).


Figure 2-14. Comparison of Changes in DOL P Concentration,
and Dehydro DOL PP Synthase Activity as a Function of Rat
Age .
The specific activity of dehydro DOL PP synthase ( )
are compared with the concentration of DOL P measured
directly by HPLC (A) (unpublished observation, Allen, 1987)
or by an indirect method (), which was described in an
earlier study (Allen & Ward, 1987).


Figure 3-2. Dehydro Dolichyl Diphosphate Synthase
Activity in Sonicates of Tubules from Rats of Different
Ages .
The enzymatic activity was
conditions with sonicates of
assayed under standard
seminiferous tubules
treated (
) and untreated (
) with
described in the Materials and Methods
presented
Numbers
the mean + standard
in parentheses indicate
used to prepare the tubules.
protease
The dat^a
deviation (x + ).
the number of animals


9
Ad SC Ap B PT
Figure 1 2 .
Schematic Drawing of Human Seminiferous
Epithe1iu
The seminiferous epithelium recline
(BL) and a
iniferous
layer
tubule
of peritubular
Pale type A
cells
upon
(PT)
basal la
surrounds
type A spermatogonium (Ad)
located in the basal
type
and
spermatogonium
B
(Ap)
mina
the
dark
type
o f
spermatogonium
the seminiferous
epithelium below the
Sertoli cells
compartment
junctional complex
(SC); pachytene primary spermatocytes
early spermatids (ES), and late spermatids (LS)
the adluminal compartment above the junctional complex (Ross,
M. H. and Reith, E. J., 1985, Histology, 3rd printing, pp
608; Clermont, Y. 1963, Am. J. Anat., 112, 35).
(JC) between adjacent
(P) >
are seen in
0


125
As signment
o f
the
relative
c ontribution
o f
the
spermatogenic cells and the Sertoli cells to the changing
synthase specific activities at different times during early
spermatogenesis requires an assessment of the fraction of
each of these cells present at different ages and knowledge
of the specific
of the "pure cell types.
An
interpretation of these changes is made here on the basis of
the reported results (Fig. 3-6).
At 7 days of age the synthase activities of both the
Sertoli and spermatogenic cells are low.
At this stage of
differentiation the Sertoli cells are still dividing and the
spermatogenic cells have not yet reached the meiotic phase.
It is reasonable that the observed synthase activity from
testes at this age was low.
At 15 days of
the Sertoli cells have stopped
dividing (Steinberger and Steinberger, 1971) and at least
some
phase.
of the spermatogenic cells have entered the meiotic
Dehydro DOL PP synthase in spermatogenic cells has
increased but
the
cell
numb e r
small.
Therefore,
spermatogenic cells do not contribute in
major way to the
total specific activity of the synthase in the whole tubules.
In contrast the Sertoli cells are relatively high in both
number and in enzyme specific activity. Therefore, the total
tubular synthase activity is mainly due to Sertoli cells.
At 23 days of
the enzyme specific activities of
both early pachytene spermatocytes and Sertoli cells have


97
be undoubtedly useful for the research of glycoprotein
regulation. James and Kandutsch (1980c) reported that mouse
spermatogenic cells were more active than liver cells in DOL
biosynthesis. Furthermore, Potter et al. (1981b) identified
the pachytene spermatocytes as one of the most active
spermatogenic cells in DOL synthesis. This prompted our
study of dehydro DOL PP synthase, an enzyme that could
contribute to increased DOL levels during spermatogenesis.
Chapter II described Jjl vitro assays for the synthase,
which were developed to measure changes in the potential of
seminiferous tubules to biosynthesize dehydro DOL PP, a
probable precursor in the biosynthesis of DOL P and DOL. The
temporal expression of synthase correlated well with the
increase of DOL P measured by HPLC methods in seminiferous
t
tubules during early stages of differentiation in prepuberal
rats. It was proposed, therefore, that the level of DOL P in
rat seminiferous tubules might be controlled by the
regulation of d_e_ novo dehydro DOL PP biosynthesis.
In the previous chapter, the specific activity of the
dehydro DOL PP synthase was shown to fluctuate during
testicular development; it was hypothesized that a difference
in enzymatic activities during development might due to the
presence of different cell populations with different
enzymatic activities in rat testes. In this chapter, the
methods of cell fractionation are described, the specific
activities of the dehydro DOL PP synthase are measured in


65
Kinetics of the Enzyme. The enzyme assay conditions
were optimized. Figure 2-6 shows the effect of varying
protein concentration on the formation of the dehydro DOL PP
(Panel A) dehydro DOL P (Panel B) and the sum of these two
products (Panel C). These results indicated that the
enzymatic activity increased linearly with protein
concentration up to 2.4 mg protein incubated.
The dependency of dehydro DOL PP and dehydro DOL P
formation on substrate concentration was studied. Figure 2-7
shows that the formation of dehydro DOL PP (Panel A), dehydro
DOL P (Panel B), and the sum of these two products (Panel C)
increased with increasing isopentenyl diphosphate
concentration. A double-reciprocal plot of the sum of
dehydro DOL PP and dehydro DOL P formation (Panel C) versus
isopentenyl diphosphate concentrations showed (Figure 2-8)
that the apparent Km=32/M and Vmax=1.23 pmoles/mg protein/min
respectively.
Similarly, the dependency of the enzymatic products
formation on the substrate farnesyl diphosphate was also
studied. The experiment described (Figure 2-9) shows that
farnesyl diphosphate was incorporated into dehydro DOL PP and
dehydro DOL P. The formation of dehydro DOL PP (Panel A),
dehydro DOL P (Panel B), and the sum of these two products
(Panel C) increased with increasing concentration of farnesyl
diphosphate. In Figure 2-10, a double reciprocal plot of the
sum of dehydro DOL PP and dehydro DOL P formation (Fig. 2-9,


Figure 2-4. Time Course of Dehydro DOL PP and Dehydro
DOL P Formation.
Sonicated seminiferous tubules were assayed under
standard conditions except that 1.0% Triton X-100 was
used and the time of incubation was varied as shown.
The formation of dehydro DOL PP (solid triangles),
dehydro DOL P (solid squares) and the sum of the both
(solid circles), respectively.


99
Millipore), and the pH adjusted to 7.3 by a 15-20 min
aeration with 5% CO2 in air. Glassware and other equipment
was siliconized before use in order to reduce damage and
adhesion of cells.
Preparation of cell suspensions. Rats aged 7-65 days
were sacrificed and testes were removed as described in
Chapter II. Testicular cell suspensions were prepared by a
modification of the two-step enzymatic method described for
mouse (Romrell, 1979). The decapsulated testes were placed
in a 50-ml Erlenmeyer flask containing 20 ml of collagenase
(1 mg/ml) and DNAse (1 /xg/ml) in EKRB. The testes were
incubated at 33C in a shaking water bath operated at 120
cycles/min, until the seminiferous tubules were freely
dispersed in the incubation medium (10-15 min). The
dispersed seminiferous tubules were allowed to sediment and
the supernatant was decanted. The isolated tubules were
washed twice with EKRB. Then fresh EKRB (20 ml) containing
trypsin (2.5 mg/ml) and DNAse (1 g/ml) was added to the
tubules and this suspension was incubated for 15 min in a
shaking water bath as just described. The resulting cell
suspension was gently pipetted approximately 50 times with a
Pasteur pipet. Trypsin inhibitor (2.5 mg/ml) was added, and
then the cell suspension was mixed with 10 ml of 0.5% BSA in
EKRB and centrifuged at 200 x g for 10 min. The resulting
pellet was washed three times with EKRB containing 0.5% BSA
and 1 g/ml DNAse and resuspended in the same solution after


95
and spermatid (Leblond & Clermont, 1952). During the second
and third phases, many immunological and biochemical changes
occur which are unique to the spermatogenic process
Among
these changes are the elaboration of spermatogenic cell or
Sertoli cell specific glycoproteins (Fenderson et al 1984;
Parvinen, 1982).
For example, the spermatogenic cells
produce an acrosomal protease precursor, proacrosin, and
unique cell surface glycoproteins, which are required for
Proacrosin is first produced during the
spermatid stage of differentiation but is retained throughout
the remainder of spermatogenesis (Florke et al. 1983 ).
Other proteins are only expressed in specific stages, so that
they may be absent or low in the spermatogonial and spermatid
phases but are abundant during the meiotic phase
(e g.
fucosyl transferase in prepuberal mouse testes) (Letts et
al. 1974a)
The Sertoli cell, a non-germinal support cell of the
seminiferous
tubule,
plays
c r i
role
in
the
spermatogenic
process
by
providing structural support,
regulating spermatogenic cell movement, and compartmentation
of spermatogenic cells from non-germinal cells, as well as
mediating the movement of hormones, metabolites and nutrients
to and from the developing spermatogenic cells (Ritzen et
al 1981) .
The Sertoli cell produces many glycoproteins
that support these functions including androgen binding
protein (Parvinen, 1982) and plasminogen activator (Lacroix


Figure 3-6. The Relationship between the Dehydro DOL PP
Synthase Activity and Spermatogenesis during Testicular
Development.
The curve of the enzymatic activity of rat
testicular development is from the results in the
previous chapter. The scheme is modified from B. P.
Setchell., 1982, in Germ Cell and Fertilization, Eds. C.
R. Austin and R. V. Short, New York: Cambridge
University Press, PP. 63-101. The calculated results
are shown as ( A ).


48
3 2
P]-isopentenyl
diphosphate and [^ ^ C ]-isopentenyl
diphosphate were incubated with farnesyl diphosphate and
tubular homogenates in the standard assay. The products were
separated by TLC as usual and the ratios of radiolabel
incorporated
from [32
P]- and [ ]-isopentenyl diphosphate
were determined.
Since the chain lengths of the polyprenol
products have been established to be the same, the relative
o f
3 2
P
incorporated/^C incorporated into
the
polyprenyl diphosphate is expected to be twice that ratio for
the monophosphate.
The results of such a test under two
experimental incubation conditions are illustrated in Table
2-1 .
They support the hypothesis that the two products are
the dehydro DOL P and dehydro DOL PP.
Several experiments were carried out to demonstrate the
dependency
o f
the
enzyme
#
activity
on
Triton
X- 100
concentrations and incubation times as well the extent and
type of product formed. These experiments also serve to test
the precursor-product relationship between the diphosphate
and monophosphate .
Triton X-100 stimulated dehydro
DOL
PP
synthase
activity (Fig. 2-2, Panel C) with formation of dehydro DOL PP
(Panel A) and dehydro DOL P (Panel B).
The dependencies of
the extent of mono- and diphosphate product formation on the
concentration
o f
detergent
were
quite different
incubation for 1 hr. Dehydro DOL PP formation was generally


Figure 2-1. Separation of Enzymatic Products by TLC.
(I) The products from the reaction of t,t-farnesyl
diphosphate and [ ^C] isopentenyl diphosphate with
homogenates of seminiferous tubules prepared with a
glass-teflon homogenizer (Panel A) or by sonication
(Panel B) were extracted with CHCI3/CH3OH and subjected
to TLC on silica gel sheets as described in the text.
Arrows represent the position of migration of exogenous
DOL P and farnesol.
(II) A example of autoradiography from enzyme assay


66
Panel C) versus farnesyl diphosphate concentrations showed
that the apparent Km=2 2 2/M and Vmax = 0.6 7 pmoles/mg
protein/min respectively. The Km values were higher than
those observed for dehydro DOL PP synthase from Ehrlich
ascites (Adair et al., 1984). This may reflect non-specific
absorption of the substrates by other proteins in the crude
homogenate and hydrolysis of the substrate by endogenous
phosphatases, although ATP and NaF were included in the assay
to minimize the action of phosphatases.
The time course of formation of dehydro DOL PP and
dehydro DOL P in the standard assay is shown in Fig. 2-11.
The formations of both dehydro DOL PP (Panel A) and dehydro
DOL P (Panel B) are increased with increasing incubation
time. Since the sum of these two products was linearly
increased up to 60 minutes (Panel C), we chose one hour as
the standard incubation time.
Changes of Dehvdro DOL PP Synthase with age. The
specific activity of dehydro DOL PP synthase in testicular
homogenates of different aged rats was studied. The
synthesis of dehydro DOL PP and dehydro DOL P from farnesyl
diphosphate and [^C]-isopentenyl diphosphate was determined
in sonicates of tubules from rats aged 3-65 days. An example
from several of these experiments is shown in Fig. 2-12.
Under standard assay condition, the changes in the specific
activity of the enzyme as measured by dehydro DOL PP