The role of phosphocreatine kinase in the motility of human spermatozoa supported by different metabolic substrates

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1 Moleculr Humn Reproduction vol.2 no.8 pp , 1996 The role of phosphocretine kinse in the motility of humn spermtozo supported by different metbolic substrtes C.H.Yeung 1-3, G.C.Mjumder 2, C.Rolf 1, H.M.Behre 1 nd T.G.Cooper 1 institute of Reproductive Medicine of the University, Domgkstrpe 11, D Munster, Germny 2 Current ddress: Indin Institute of Chemicl Biology, Jdvpur, Clcutt 7 32, Indi 3 To whom correspondence should be ddressed In the spermtozo of some species cretine kinse (CK: E.C ) is involved in shuttling energy, in the form of cretine phosphte, between the mid-piece mitochondri nd flgellum. In this study, the effects of the CK inhibitor dinitrofluorobenzene (DNFB) on humn sperm CK ctivity, motility nd ATP concentrtions were ssessed with different energy substrtes. There ws dose-dependent inhibition of CK ctivity by DNFB but inhibition ws incomplete nd there ws no effect on the percentge of flgellting cells, irrespective of substrte. However, when lctte lone supported the cells DNFB decresed velocities nd incresed mplitude of hed displcement (fewer progressively motile forms were observed), wheres ATP concentrtions in spermtozo were unltered. Demembrnted sperm models could be rectivted by ADP plus cretine phosphte, but not to the extent cused by ATP, nd were ble to be inhibited by myokinse inhibitors. Incresed velocities, linerity (LIN) nd bet cross frequency (BCF) were demonstrted for spermtozo incubted with lctte, in contrst to glucose s sole energy source, nd higher velocities nd BCF were generted in the presence of both substrtes. This suggests tht the production of ATP by glycolysis nd respirtion re independent nd complementry. CK is not obligtory for sperm motility but supplements energy provision under certin conditions. Key words: ATP homeostsis/casa/demembrntion nd rectivtion/dnfb/enzyme ctivity Introduction The enzyme cretine phosphokinse (CK: E.C ) is involved in the shuttling of high energy phosphte from the site of ATP synthesis to the site of its utiliztion in more distl cytoplsmic comprtments in muscle nd non-muscle cell types (Wllimnn nd Hemmer, 1994). It medites the reversible genertion of cretine phosphte (CP): ATP + cretine < CK > cretine phosphte (CP) + ADP In se urchin spermtozo similr role hs been demonstrted (Tombes nd Shpiro, 198) in which two forms of the enzyme medite cretine phosphte formtion in the mid-piece (mitochondril CK-Mi) nd ATP regenertion for hydrolysis in the til (cytoplsmic CK-T). Evidence for this hs come from experiments involving dinitrofluorobenzene (DNFB) which, t low concentrtions, is specific inhibitor of cretine kinse nd inhibits coupled respirtion s well s ltering sperm motility ptterns (Tombes et l., 1987). The bility of the xoneme to generte ATP from cretine phosphte nd ADP hs lso been shown with demembrnted se urchin sperm models (Tombes nd Shpiro, 198). Less is known bout this enzyme in higher nimls, lthough both the brin type (CK-BB) nd mitochondril type (CK-Mi) isoforms hve been identified in humn nd chicken spermtozo nd in the ltter these enzymes re locted in positions (the til nd midpiece, respectively) suggestive of functionl role in energy shuttling (Wllimnn et l., 1986). Nevertheless, the existence of functionl CP shuttle system in mmmlin spermtozo hs not been exmined. There re lrge species differences in the bility of sperm motility to be supported by glycolysis or respirtion nd in the CK content of spermtozo (Kmp et l., 1996). Most literture on the isoenzymes of humn spermtozo suggest tht their presence in high ctivity is relted to retention of cytoplsmic mteril, indictive of bnormlities tht my interfere with, rther thn id, sperm function. For exmple, higher enzyme ctivity is found in spermtozo from oligozoospermic men thn from normozoospermic men nd spermtozo selected by self migrtion hve lower CK ctivities thn unselected cells (Huszr et l., 1988,b). Exmintion of the isoforms indicted the presence of CK-M (the muscle) nd CK- B (brin) isoenzymes nd demonstrted n inverse reltionship between the presence of the CK-M isoenzyme nd totl CK ctivity of the spermtozo (Huszr nd Vigue, 199). An inverse reltionship between CK ctivity nd fertility ws suggested by the higher CK ctivity of spermtozo from infertile oligozoospermic men thn tht of spermtozo from fertile oligozoospermic men nd this difference persists fter seprtion of the motile cells (Huszr et i, 199). In further study, spermtozo bering low totl CK ctivity (i.e. high proportion of CK-M) were more likely to be successful in invitro fertiliztion (IVF) thn spermtozo with higher totl ctivities (Huszr et l, 1992). Europen Society for Humn Reproduction nd Embryology 91

2 C.H.Yeung et l. Immunohistochemicl locliztion of CK on humn spermtozo reveled stining of the til, including the mid-piece, of norml spermtozo but n dditionl stining of the hed of bnorml cell types seprted by centrifugtion through Percoll (Huszr nd Vigue, 1993), lthough n erlier study found no correltion of CK ctivity with sperm morphology (Huszr et l., 1988). The reduced CK stining of spermtozo bound to the zon pellucid suggests tht spermtozo expressing high CK re less ble to bind to the zon pellucid (Huszr et l., 1994). Thus the enzyme would pper to be unrelted to functions of the sperm flgellum nd there is no reltionship between sperm CK nd sperm motility (Huszr et l., 1988) or sperm ATP content (Vigue et l., 1992). This study ws performed to exmine the role of sperm cretine phosphokinse in humn sperm flgellr motility by mesuring CK ctivity, ATP content nd sperm motion of intct cells in the presence of different energy substrtes nd the presence of the CK inhibitor DNFB s well s by using rectivted demembrnted sperm models. Mterils nd methods Ejcultes were obtined by msturbtion fter 2-7 dys of sexul bstinence from 19 ptients ttending the Miinster clinic. The ejcultes were nlysed ccording to the WHO mnul (1992) nd found to be normozoospermic. The men (± SEM) vlues for the ejculte prmeters were: volume 4.2 ±.3 ml; sperm concentrtion 77 ± 14X1 6 /mi; % motile 63 ± 2, of which 6 ± 1% were grde ; ± 1% were grde b; 8 ± 1% were grde c nd 36 ± 2% were grde d motility. There were 24 ± 2% morphologiclly norml forms. Incubtion medi nd sperm preprtion Chemicls were from Sigm Chemie, Deisenhofen, Germny, unless otherwise stted. Three different medi were prepred by modifying Biggers-Whitten-Whittinghm(BWW) medium (Biggerse/ l, 1971) contining. mm glucose, 2 mm lctte,.2 mm pyruvte nd 4 mg/ml bovine serum lbumin (BSA). Glucose-free nd lctte/ pyruvte-free medi were prepred by substituting 2.7 mm NCl for glucose nd 2 mm NCl for lctte nd pyruvte respectively. A third medium (2. mm lctte,.2 mm pyruvte nd mm glucose) mimicking the energy substrtes present in humn tubl fluid (Lippes et l., 1972; Lopt et l., 1976) ws lso prepred. Sperm motility mesurements Spermtozo were wshed by centrifugtion t g for min through 3/6% Percoll grdient. The sperm pellet ws wshed gin in 2 ml medium contining 4 mg/ml BSA nd centrifuged t 44 g for min. Spermtozo were resuspended in. ml medium for n initil ssessment of sperm concentrtion nd diluted to xl 6 /ml nd incubted t 37 C in % (v/v) CO 2. Antimycin A (2 um, Ford nd Hrrison, 1981) ws dded to the lctte-free medium to block respirtion of metbolized glucose. The cretine phosphokinse inhibitor DNFB ws dissolved in isopropnol s stock solution. Sperm suspension (in 4 ul liquots) ws trnsferred to tubes contining 2 ul DNFB yielding finl concentrtions of,, nd um nd incubted t 37 C in % (v/v) CO 2. Control incubtions were mde in the presence of.% (v/v) vehicle. After 1 h incubtion (il smple ws trnsferred to Mkler chmber (Sen" Medicl Instruments, Hif, Isrel) nd video-recorded with Sony cmer from n Olympus microscope employing X1 negtive phse objective with X3.3 photo-oculr for 3 min. The HT-Mster C 92 system (Hmilton-Thorn Softwre Version 1.6H, Beverley, MA, USA) ws used to nlyse sperm kinemtics: curviliner (VCL), stright line (VSL), nd verge pth (VAP) velocities; derived vlues (linerity LIN = 1XVSL/VCL; strightness STR = 1XVAP/ VCL), the mplitude of the lterl displcement of the hed (ALH) nd bet cross frequency (BCF). Anlysis ws mde with the following settings: mgnifiction 1.6, frme rte 2 Hz, frme cquisition 2, minimum contrst 7, minimum size 8, minimum trck points 1, motility threshold (im/s VAP. The percentge motility ws ssessed by direct microscopic observtion since previous vlidtion of this mchine indicted uncceptble errors in estimtes of percentge motility (Yeung nd Nieschlg, 1993). The definitions of kinemtic prmeters re given in WHO (1992). ATP ssy At the end of the incubtion 1- ul sperm suspension (2-4 X1 6 spermtozo) were pelleted t 3 g for min t 4 C, resuspended in (J.1 phosphte-buffered sline (PBS; Gibco, Eggenstein, Germny) nd stored t - C. ATP ws estimted by luminescence ssy bsed on McLughlin et l. (1994). After thwing nd vortexing, the spermtozo were deproteinized by ddition of 1 ul ice-cold 1 M perchloric cid for min on ice nd centrifuged t 13 g for min t 4 C. Superntnt ( ul) ws neutrlized with (il 2.3 M K 2 CO 3 in.7 M 2-(A'-morpholino)-ethnesulphonic cid (MES) on ice for 6 min nd the superntnt obtined from centrifugtion t 13 g for min t 4 C ws used in the ssy. Medium used s ssy blnk contined 4 ml PBS, 2 ml 1 M perchloric cid nd.6 ml 2.3 M K2CO3. Duplicte }il smples mixed with 1 ul blnk medium, or 1 ul medium lone were incubted with 32 ul.1 M Tris contining 1 mm EDTA, ph 7.7 nd 2 ul luciferin-luciferse (reconstituted Sigm preprtion contining Tris-sprtte buffer, Ct. No. LI761). ATP stndrds (4 nd nm in the blnk medium) were mesured in the sme ssy. An LB92 Luminometer (EG & G Berthold, Bd Wildbd, Germny) ws used to register counts integrted over 1 s. ATP stndrds (4 nm) were dded to sperm cells to check for recovery (14 ± %) in the ssy. The inter-ssy coefficient of vrition ws 9 ± 1%. Cretine phosphokinse ssy A totl of JLLI incubted suspension (~4X 1 6 spermtozo) ws centrifuged t 3 g for min t 4 C nd the pellet ws resuspended in ul CK extrction buffer (1 mm NCl, 1% (v/v) Triton X-1,.1 mm dithiothreitol, mm Tris buffer, ph 8.). The sperm concentrtion ws ssessed in Mkler chmber nd fter extrction on ice for min the smples were frozen t - C until ssy. CK ctivity ws mesured with regents from Sigm kit before nd fter removl of the CK-B isoform by ion exchnge chromtogrphy (Mercer, 1974) using the btch method of Smeitink et l. (1992). The remining ctivity ws termed mitochondril CK (CK-Mi) becuse of its seprtion from CK-MM on electrophoretic gels. Demembrntion nd rectivtion of spermtozo Ejculted spermtozo were wshed through Percoll s bove nd the pellet ws wshed with rectivtion nd demembrntion medium (Yeung et l, 1988: mm Tris-HCl, ph 8.1 contining.23 M sucrose,.1 M K succinte, 2 mm MgSO 4,. mm EDTA, 1 mg/ml polyvinylpyrrolidone-4) nd resuspended in this medium to concentrtion of ~X1 6 /ml. After min t room temperture ul rection mixture (see below) ws dded to 4 ul sperm suspension for 1 s before estimtion of the percentge of motile cells. For this (il liquot ws plced on siliconized slide nd viewed under n 18X18 mm cover slip with X positive phse contrst objective.

3 Cretine kinse nd humn sperm motility 7 6 b J, _ 1 _ V de de (d) UN(%) b cd\ -A \ de > ef V u f Concentrtion of DNFB 4b 3 1 " b 8" - b ^-x ' 1 8- be d (c) VSL(»m/s) $ b O. V cde ( BCFfHz) cd d -O be de - \ \ \ bd N dv"^ s dd Figure 1. Effects of the cretine phosphokinse inhibitor dinitrofluorobenzene (DNFB; x xis) on the motility (men ± or + SEM; y xis) of humn ejculted spermtozo incubted in medi contining. mm glucose (V), 2 mm lctte (O) nd both glucose ( mm) nd lctte (2. mm) ( ) () Percentge motility; (b) curviliner velocity; (c) stright-line velocity; (d) linerity; (e) mplitude of lterl hed displcement; nd (f) bet cross frequency. Vlues bering the sme superscripts re not significntly different. All the rection mixtures tested contined Triton X-1 (.8%, v/v), cyclic AMP ( um) nd dithiothreitol (1 mm) with either (i) ATP (1 mm), (ii) ADP (2 mm) lone or (iii) ADP (2 mm) together with cretine phosphte (1 mm), with (ii) nd (iii) being exmined with or without mixture of AMP ( mm) nd didenosine pentphosphte (.2 mm) s inhibitors of myokinse (Tombes nd Shpiro, 198). Sttisticl nlysis Vlues for CK ctivity nd ATP contents were expressed s the men ± SEM. For ech kinemtic prmeter the medin vlue of ech smple ws obtined from individul trck dt. For velocities, ALH nd BCF, the men ± SEM ws clculted for ech group. For derived prmeters, nd percentge motility, dt were first trnsformed (rcsin squre root of percentge in rtio) nd the mens + SEM were clculted from the trnsformed dt. Sttisticl tests were performed using one wy nlysis of vrince followed by lest squres differences s multiple rnge test. Differences were considered significnt when P <.. Results Effect of different substrtes on sperm velocities There were no significnt differences in the percentge of motile cells when spermtozo were incubted in medi contining different substrtes. However, ll types of velocities were highest when spermtozo were supported by both glucose nd lctte, such tht they were more thn double the vlues obtined in the presence of glucose lone nd ~% higher thn those supported by lctte lone (Figure 1 t xm DNFB). A similr trend to substrte support of BCF ws observed. For the derived prmeters STR (not shown) nd LIN, the lctte-contining medi provided eqully high vlues Tble I. Cretine kinse (CK) ctivity (miu/1 8 cells) of humn spermtozo incubted in different substrtes. Vlues re men ± SEM Substrte DNFB (UM) CK isoforms CK Totl Glucose-free with lctte (2 mm) (n = 3).3 ± ± ± ±.4 Lctte-free with glucose (. mm) (n = 4) 4.67 ± ± ± ±.64 CK-Mi < :K-B 3.14 ± ± ± ± it.3.47 it.1.43 it..32 dt ± ± ± ± ii.4.9 it.8.34 jt.3.19 ±.27 Lctte (2. mm) nd glucose ( mm) (n = 4) 6.19 ± ±.96 ; >.4 ± ± ± ± ± ± ±.8.81 ±.24.1 ±:.33.2 ±.32 tht glucose lone could not support, wheres ALH ws uninfluenced by the substrtes. Effect of DNFB on sperm CK ctivity DNFB inhibited the CK ctivity of humn spermtozo in dose-dependent mnner, irrespective of the substrte supporting motility (Tble I). In Figure 2, the dt re pooled from experiments with different substrtes nd indicte tht the totl enzyme ctivity ws inhibited between - (im DNFB with little further inhibition when the DNFB concentrtion ws incresed to (im. Inhibition ws incomplete, declining to 6% of control vlues. More of the CK-B form ws inhibited 93

4 C.H.Yeung et l. ' Concentrtion DNFB (um) Figure 2. Inhibition by dinitrofluorobenzene (DNFB; x xis) of the totl (O), mitochondril (V) nd B-isoenzyme ( ) ctivity of cretine phosphokinse of humn ejculted spermtozo (men ± SEM; y xis). For ech tretment vlues bering the sme superscripts re not significntly different. (4% t um) thn the CK-Mi form (3% t (Higher concentrtions were not tested s inhibition of myokinse occurs (Tombes nd Shpiro, 198).) Effect of DNFB on ejculted humn sperm motility When incubtion medium contined the respirtory inhibitor ntimycin A nd glucose ws sole substrte, DNFB hd no effect on percentge motility nor ny kinemtic prmeter. When both lctte nd glucose were present, DNFB ws lso ineffective in ltering the percentge motility nd inhibition of most kinemtic prmeters ws only detectble t the highest dose tested (Figure 1). In glucose-free medium contining lctte the overll motility of sperm cells (i.e. the percentge exhibiting flgelltion) ws only decresed t the highest concentrtions of DNFB t which no further decline in CK ctivity ws noted (see Figures l nd 2). However, of the motile cells, there ws dosedependent decline in velocity in the presence of DNFB in the rnge - jim, gin with fi.m cusing the lrgest drop in vlues. By contrst ALH vlues incresed with incresing DNFB concentrtion in the presence of both substrtes but declined when lctte lone ws present. Effect of DNFB on ATP levels in spermtozo The ATP content of humn spermtozo ws independent of the nture of the substrtes supporting motility, lthough there ws trend towrds higher ATP vlues when both glucose nd lctte were present. DNFB only reduced intrcellulr ATP vlues t the highest concentrtion tested when spermtozo were supported by lctte lone (Tble II). Rectivtion of demembrnted humn spermtozo As previously shown (Yeung et l., 1988) humn spermtozo becme instntneously immotile fter demembrntion unless 94 Tble II. Intrcellulr ATP concentrtions of humn ejculted spermtozo fter 1 h incubtion in medi contining different substrtes nd the cretine kinse inhibitor dinitrofluorobenzene (DNFB). Vlues re men ± SEM Substrte DNFB (um) ATP (pmol/1 6 spermtozo) Glucose-free with lctte (2 mm) (n = ) 62.3 ± ± ± ± 9.2* Lctte-free with glucose (. mm) (n = 6) 68.9 ± ± ± ± 14. Lctte (2. mm) nd glucose ( mm) (n = 6) 12.8 ± ± ± ± 18.9 *P <. compred with um DNFB f ADP+CP TREATMENT ADP c ADP+CP+MK INK Figure 3. Effects of exogenous nucleotides (ATP 1 mm, ADP 2 mm), cretine phosphte (CP, 1 mm) nd myokinse inhibitors (MK INH, AMP mm + didenosine pentphosphte.2 mm) upon the rectivtion of demembrnted humn spermtozo (men + SEM). Vlues bering the sme superscripts re not significntly different. ATP ws supplied to the cells. Motility, lbeit extremely feeble, ws lso initited when the cells were given ADP nd cretine phosphte, lthough only bout one third of the response to ATP ws observed (Figure 3). However, ADP in the bsence of cretine phosphte lso rectivted similr percentge of the xonemes. Rectivtion of sperm cells in the presence of ADP nd cretine phosphte ws prevented by ddition of the myokinse inhibitors didenosine pentphosphte nd denosine monophosphte. Discussion Cretine kinse of humn spermtozo ws inhibited by DNFB, s is the cse for tht of se urchin spermtozo (Tombes nd Shpiro, 198), but the inhibition ws less, nd totl inhibition ws never chieved over the concentrtion rnge tht is selective of CK. The CK-Mi isoenzyme ws ffected less by the inhibitor thn the brin type, the ltter presumbly being the form in the cytoplsm.

5 If CK were to be involved in the trnsport of energy in the form of cretine phosphte from the mitochondri to the flgellum, it should be demonstrble under conditions of respirtory-supported motility. Experiments were thus designed to exmine the substrte-dependence of sperm motility. It ws evident tht, with the exception of percentge motility nd ALH, ll kinemtic prmeters were of significntly higher mgnitude in the presence of lctte thn observed in glucosesupported motility. The bility of humn spermtozo to generte sufficient ATP from glycolysis to swim for some time in the presence of ntimycin A ws reported by Ford nd Hrrison (1983) but only the percentge motility ws ssessed. The present study demonstrtes tht higher velocities were chieved in the presence of lctte thn glucose nd even higher velocities nd bet cross frequencies were displyed in the presence of both substrtes t concentrtions found in humn tubl fluid (Lippes et h, 1972; Lopt et h, 1976). This shows tht energy derived seprtely from respirtion nd glycolysis is complementry in the support of mximl velocity. Despite the mrked differences in velocities with different substrtes, net intrcellulr ATP concentrtions were similr. The lck of reltionship of the sperm ATP content to motility nd velocity, suggests tht there is coupling of ATP supply nd demnd in humn spermtozo, so tht ATP homeostsis results. However, insofr s mitochondrilly-derived ATP hs to diffuse the length of the flgellum, n energy shuttle, such s tht provided by CK, my be necessry to supply ATP to the cytoplsm t rte commensurte with its utiliztion. Indeed, CK does pper to be prtilly involved in the provision of energy for motility, s judged by the reduction in kinemtic prmeters of spermtozo supported by lctte nd pyruvte nd incubted with the perment CK inhibitor DNFB. However, the most mrked inhibition of sperm kinemtic prmeters ws observed when the DNFB concentrtion ws rised from to \im, i.e. n increse in concentrtion tht did not further lower CK ctivity. This my indicte tht DNFB t the higher concentrtion is cting on other components involved in flgellr ctivity. On the other hnd, tht inhibition of motility by DNFB ws only demonstrted when motility ws supported by respirtion lone rgues tht spects of the sperm mitochondri were being interfered with by the inhibitor. The effect of DNFB on intrcellulr ATP content ws demonstrble t (im but not t lower concentrtions, nd only when spermtozo were supported by respirtion lone. In the se urchin, inhibition of CK by DNFB reduces respirtion of coupled mitochondri s consequence of limiting supply of ADP. The low level of functionl significnce of CK ctivity in sperm motility ws lso demonstrted in the demembrnted models. Here the rectivtion of permebilized humn spermtozo by extrcellulr ADP with cretine phosphte ws not due to CK ctivity, since it could be mimicked by ADP lone nd could be inhibited by myokinse inhibitor. Thus genertion of ATP in the demembrnted models ws more efficient by myokinse thn by CK nd in this regrd differs mrkedly from observtions in the se urchin where genertion of ATP from ADP ws not only sufficient for flgelltion but Cretine kinse nd humn sperm motility could be inhibited by DNFB (Tombes nd Shpiro, 198; Tombes et h, 1987). This indictes tht CK in humn spermtozo is less importnt, lthough it cnnot be ruled out tht CK my be lost more esily from humn spermtozo thn from se urchin spermtozo under the conditions of extrction. As glucose (in the presence of Antimycin A to prevent oxidtion of ny glycolysed sugr) ws fully ble to support motility in the presence of the inhibitor, sperm motility is independent of their CK ctivity in conditions where glycolysble substrtes previl, lthough the velocity chieved ws sub-mximl. This independence is to be expected if ATP were supplied by glycolysis in the cytoplsm, close to the site of energy utiliztion. Wheres the CK shuttle system is best demonstrted in se urchin spermtozo, whether these spermtozo re similr to humn sperm cells in mintining their motility by glycolysis independent of CK ctivity hs not been studied. On the other hnd the cretine kinse, s well s glycolytic enzyme ctivities, of spermtozo from different species re remrkbly different (Kmp et h, 1996). For exmple, CK hs reltively high ctivity in se urchin spermtozo but is undetectble in bovine spermtozo, with the converse being true for glycolytic enzymes, especilly the key enzyme glycerldehyde 3-phosphte dehydrogense (GAPDH: see Kmp et h, 1996). In trnsgenic mice deficient in ubiquitous CK-Mi, sperm motility nd kinemtics re not compromised, demonstrting lck of role for CK in this spect nd these nimls lso hve norml fertility (Steeghs eth, 199). The recent demonstrtion tht GAPDH is bound to structurl components of the vertebrte sperm xoneme (Westhoff et h, 1996) suggest tht glycolyticlly-derived ATP is generted loclly ner the xoneml dynein ATPse. If demonstrble in mn, this could compenste for energeticlly inefficient glycolysis nd obvite the need for n energy shuttle from the mitochondri utilizing CK. In conclusion, the humn spermtozoon cn mke use of cretine phosphokinse in the provision of energy for motility when supported by respirtory substrtes. Under conditions in which glycolysis predomintes motility cn still be supported in CK-independent mnner, lthough the swimming velocities chieved re lower. Mximl velocities were ttined with both respirtory nd glycolytic substrtes. Therefore CK ctivity is supplementry to, but not essentil for, humn sperm motility. Acknowledgements This reserch ws supported by the Deutsche Forschungsgemeinschft, Confocl Reserch Group 'The mle gmete: production, mturtion, function', Grnt Ni 13/1. DAAD supplied Trvel Grnt to GCM. We thnk Brbr Kloke for technicl ssistnce. References Biggers, J.D., Whitten, W.K. nd Whittinghm, D.G. (1971) The culture of mouse embryos in vitro. In Dniel, J.C. (ed.), Methods in Mmmlin Embryology, pp Freemn, Sn Frncisco. Ford, W.C.L. nd Hrrison, A. (1981) The role of oxidtive phosphoryltion in the genertion of ATP in humn spermtozo. /. Reprod. Fertii, 63,

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