The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system

Biochimica et Biophysica Acta 883 (1986) 567-573

567

Elsevier BBA 22370

The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system Mark S. Siegel, Dana S. Bechtold, Carrie I. Kopta and Kenneth L. Polakoski Department of Obstetrics and Gynecology, Washington University School o/Medicine, 4911 Barnes Hospital Plaza, St. Louis, MO 63110 (U.S.A.)

(Received April 15, 1986)

Key words: Proacrosin; Acrosin; Proteinase; (Human spermatozoa) A rapid and efficient procedure was developed for obtaining highly purified human proacrosin. Ejaculated spermatozoa were washed via centrifugation through 1 M sucrose containing 50 m M benzamidine and acid-extracted in the presence of benzamidine. The solubilized material was dialyzed then lyophUized. The sample was resuspended in 8 M guanidine hydrochloride in acetic acid (0.5 M) pH 2.5 and then subjected to gel permeation chromatography with an automated fast protein liquid chromatography system utilizing two Pharmacia Superose 12 columns set in tandem that were equilibrated in the same buffer. The proaerosin eluted as an individual peak that was well separated from another proteinase zymogen referred to as sperminogen. The proacrosin preparation was determined to be highly purified when observed on silverstained SDS-polyacrylamide gels as well as on gelatin-SDS-polyacrylamide gels. The proacrosin appeared as a doublet ( M r = 55000 and 53000) on both of these systems. The autoconversion of proacrosin to acrosin at pH 8 resulted in a typical sigmoidal autoactivation curve. Following protein staining of SDS-polyacrylamide gels, it was shown that upon activation of purified proacrosin preparations the 55 000 and 53 000 molecular weight proteins were initially degraded to a 49000 form and then to several lower molecular weight forms ( M r = 40000-34 000). Similar findings with regard to proteolytic digestion were observed following gelatinSDS-polyacrylamide zymography except that an increase with time in proteinase intensity between 58000 and 53000 was also observed. Cobalt and calcium were found to be potent inhibitors of the conversion of proacrosin into acrosin, while sodium resulted in much less inhibition of this process. Calcium was found to markedly enhance the proteolytic activity of human acrosin, while it had no observable influence on the acrosin hydrolysis of benzoylarginine ethyl ester. Thus, the described purification procedure resulted in a highly purified proacrosin preparation in sufficient yields to allow for its partial characterization.

Introduction Acrosin (EC 3.4.21.10) is a trypsin-like serine proteinase that has been shown to be required for Abbreviation: BzArgOEt, benzoylarginineethyl ester. Correspondence address: Dr. K.L. Polakoski, Department of Obstetrics and Gynecology, Washington University School of Medicine, 4911 Barnes Hospital Plaza, St. Louis, MO 63110, U.S.A.

several processes involved in mammalian fertilization [1]. The majority of the biochemical characterization data of acrosin and its zymogen precursor, referred to as proacrosin, has been obtained from studies which have utilized boar sperm as a model system [2]. Unfortunately, in spite of the obvious implication regarding possible contraceptive development, little detailed biochemical information is currently available concerning either proacrosin or acrosin from human spermatozoa.

0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

568

Furthermore, there is considerable variation in the reported findings for both the molecular weight and properties of the zymogen and active enzymes. For example, human proacrosin has been reported to have a relative molecular mass of 75000 and 40000 [3]. Human acrosin has been estimated to have a molecular weight of 75 000 [4]; 70 000 [5]; 68 000, 38 000, 25 000, 15 000 and 12 000 [6]; 49000 and 34000 [7]; 30000 [8]. In a recent study [9] we utilized a newly developed gelatinSDS-polyacrylamide gel electrophoresis technique to demonstrate that unpurified extracts of human sperm contain several proacrosin forms with molecular weights between 47000 and 54000. It was also shown that upon activation there is an increase in proteinase activity in this molecular weight range which is followed by digestion in the 34000-38000 range. This allowed for a more accurate comparison of the human proacrosinacrosin system to that which has been obtained for other species (reviewed in Ref. 1). We have also shown that human sperm contain an additional trypsin-like enzyme system referred to as sperminogen-spermin which has a molecular weight of 32 000-36 000 [1]. Consequently, in order to more accurately characterize the human proacrosin and acrosin, it was first necessary to purify the components. This p a p e r describes a procedure for the rapid purification of human proacrosin via Superose 12 fast protein liquid chromatography in 8 M guanidine. The eluted fractions were dialyzed and evaluated for the presence of proacrosin and acrosin by using synthetic substrates and the gelatin-SDS-polyacrylamide zymographic system. The highly purified proacrosin was shown to sequentially autodegrade into several forms of acrosin which were partially characterized. Thus, the utilization of newly developed techniques have allowed the purification and the partial characterization of the human proacrosin-acrosin system. It is anticipated that this information will be useful in determining the role that proacrosin and acrosin have in male fertility as well as their potential use in the development of a male contraceptive.

Materials and Methods

Materials Ultrapure Tris(hydroxymethyl)aminomethane (Tris), triethanolamine, gelatin and the electrophoretic supplies were purchased from Bio-Rad Laboratories (Richmond, CA). Azocoll is a product of Calbiochem-Behring Corp. (La Jolla, CA). All other chemicals were from either Sigma Chemical Co. (St. Louis, MO) or were reagent grade. Solutions were prepared with glass-distilled deionized water. Collection and extraction of sperm Individual human semen samples were collected by self-masturbation, allowed to liquefy and then pooled. Benzamidine (final concentration of 50 mM) was added to semen and the semen samples were centrifuged and overlayed on top of 10% sucrose containing 50 mM benzamidine. The samples were centrifuged at 6000 x g for 20 min at room temperature. The sperm were resuspended in 10% glycerol containing 50 mM benzamidine adjusted to pH 3 with 1 M HC1 and extracted overnight. The samples were then centrifuged and the resulting supernatant was dialyzed against a 1000-fold excess of 1 mM HC1 and lyophilized. Column chromatography An aliquot of the lyophilized sperm extract was resuspended in 8 M guanidine HC1/0.5 M acetic acid (pH 2.5) and had a protein concentration of 16 mg/ml. After a 1 h incubation at ambient temperature, the mixture was centrifuged at 12 000 x g for 30 rain. A volume of 125/~l of the clear supernatant was injected onto a Pharmacia fast protein liquid chromatography (FPLC) system. The samples were eluted through two Pharmacia Superose 12 columns (10 mm x 30 cm) which were connected in tandem and equilibrated with 8 M guanidine HC1/0.5 M acetic acid buffer. The samples were eluted at a flow rate of 0.1 ml per min and fractions of 1.0 ml were collected. Individual fractions were dialyzed for 16 h against a 500-fold excess of 1 mM HC1 at 4°C prior to further analysis.

569

Proacrosin-acrosin determination Proacrosin was assayed by determining the amount of acrosin activity which was produced from the zymogen after autoactivation at pH 8.0 in final buffer of 50 mM T r i s / 5 0 mM calcium chloride. 1 unit of proacrosin is equivalent to 1 unit of acrosin [11]. The rate of the conversion is estimated by the tl/2 value which corresponds.to the time required for one half of the zymogen to convert to active enzyme. Acrosin activity was determined spectrophotometrically in 1-cm cuvettes using the substrate benzoylarginine ethyl ester (BzArgOEt, 172/~g/ml) in 50 mM Tris or 50 mM triethanolamine and 50 mM calcium chloride [12] unless otherwise specified. 1 unit of acrosin activity is defined as the quantity of enzyme required to catalyze the hydrolysis of 1 ~ M / BzArgOEt/min. The proteolytic activity of acrosin against Azocoll was measured by the method of Parrish and Polakoski [3,14].

Polyacrylamide gel electrophoresis SDS-polyacrylamide slab gels (12.5% acrylamide, 0.75 mm thick) were prepared and the samples were electrophoresed according to the method of Laemmli [15]; the gels were stained for protein using the highly sensitive silver staining procedure of Wray and his co-workers [16]. The values used for the molecular weights of the reference proteins were as follows: bovine serum albumin, 68000; aldolase, 40000; carbonic anhydrase, 29000 and cytochrome c, 12 800. Gelatin-SDS-polyacrylamide gels were prepared and processed according to the method of Huessen and Dowdle [17] modified by Siegel and Polakoski [9]. Briefly, non-reduced samples of protein were electrophoresed at a constant current of 20 m A / g e l at 4°C in 12.5% acrylamide gels containing 0.1% SDS and 0.1% gelatin. After the tracking dye migrated to the end of the resolving gel (2.5 h) the gels were incubated for 1 h in 2.5% ( v / v ) Triton X-100 in distilled water to remove SDS. The Triton X-100 was removed via three washes of 200 ml of distilled water. The gels were incubated in 50 mM Tris (pH 8.0) for 2-3 h at 37°C to allow for the proteolytic digestion of the gelatin. The gels were fixed and stained for protein for at least 1 h in a 0.1% solution of amido black in methanol/acetic a c i d / w a t e r (30: 10: 60)

and destained in methanol/acetic a c i d / w a t e r (30:10:60). Proteolysis appeared as a clear zone in a darkly stained background. Results

Purification of human proacrosin The acid-solubilized extracts from approx. 2.8. 107 washed cells were chromatographed through a tandem of two Pharmacia Superose columns as described in the Materials and Methods section. A typical elution profile is shown in Fig. 1. Approx. 60% of the autoactivatable activity was recovered in a single protein band (fractions 21 and 22).

Molecular weight determinations of human proacrosin Aliquots of selected fractions were analyzed via SDS-polyacrylamide gel electrophoresis (Fig. 2) to obtain an estimation of their purity and the molecular weight of the protein. The major proteins in fractions 21 (lane B) and 22 (lane C) consisted of a doublet. Comparisons of their relative migration to that of standard proteins yielded corresponding relative molecular masses of approx. 53000 and 55000. Proteinase activity in these fractions was determined by a gelatin-SDSpolyacrylamide zymographic system as described in the Material and Methods section. The results

Q3

75

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t

10

15

q

20 25 30 35 FRACTION NUMBER

25I

•

I 40

~-45

--

50

Fig. 1. Gel filtration of human proacrosin. 125 ~1 of sample (see Materials and Methods) in 8 M guanidine-HCl was applied to two Pharmacia Superose 12 columns set in tandem which were pre-equilibrated in 8 M guanidine at pH 2.5. Fractions (1 ml) were eluted in 8 M guanidine and collected at a rate of 0.1 m l / m i n .

570

It

i

7-

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in Fig. 3 show that the p r o t e i n a s e activity present in these fractions is limited to relative molecular masses between 52000 a n d 56 000. The proteinase activity that h a d a relative m o l e c u l a r mass of b e t w e e n 31000 a n d 35000 in lanes D a n d E is referred to as s p e r m i n o g e n [11].

Autoactiuation of human proacrosin

CA-

CYT CA

B

C

D

E

'OF

Fig. 2. SDS-polyacrylamide gel electrophoresis of purified human proacrosin (PRO). Approx. 3/~g of protein from fractions 20-24 (lanes A-E) in Fig. 1 were individually electrophoresed (O = origin; DF = dye front) and the gels were silver stained for protein. The standard proteins were bovine serum albumin (BSA, Mr = 68000), aldolase (ALD, M r = 40000), carbonic anhydrase (CA, Mr = 29000 and cytochrome c (CYT c, Mr = 12800/.

M e a s u r a b l e a m o u n t s of e n z y m a t i c activity were not o b s e r v e d in a n y of the fractions if the purified p r e p a r a t i o n s were stored at 4 ° C , p H 3.0 for up to 2 weeks. However, a r a p i d a p p e a r a n c e of enz y m a t i c activity o c c u r r e d if the p H of fraction 21 (see Fig. 1) was a d j u s t e d to neutrality. The time course for this activity at p H 8.0 in 50 m M t r i e t h a n o l a m i n e buffer followed a sigmoidals h a p e d curve (Fig. 4), typical of p r o a c r o s i n autoconversion [10]. A l i q u o t s were r e m o v e d at various time intervals a n d subjected to S D S - p o l y a c r y l a m i d e gel electrophoresis (Fig. 5). E x a m i n a t i o n of the entire a u t o a c t i v a t i o n time course d e m o n s t r a t e s that purified h u m a n p r o a c r o s i n is sequentially aut o c o n v e r t e d into several forms of acrosin through limited p r o t e o l y t i c d e g r a d a t i o n . U n d e r the conditions utilized the a u t o a c t i v a t i o n of purified h u m a n p r o a c r o s i n resulted in a r a p i d loss of protein in the m o l e c u l a r weight range of 53 0 0 0 - 5 5 000 (gels A - E , Fig. 5). A c c o m p a n y i n g this loss of p r o t e i n staining m a t e r i a l is the c o r r e s p o n d i n g increase in p r o t e i n with a r e d u c e d m o l e c u l a r weight e s t i m a t e d to be 47000 (gels B - E ) . A f t e r approx. 15 min two a d d i t i o n a l b a n d s with m o l e c u l a r weights a b o u t 34000 and 30000 a p p e a r a n d the intensity at the 100 80

>-

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20

ib Fig. 3. Gelatin-SDS-polyacrylamide zymographs of purified human proacrosin. Approx. 3/~g of protein (lanes A-E) from fractions 20-24 in Fig. 1 were electrophoresed in SDS-polyacrylamide gels containing gelatin and were then analyzed for proteinase digestion as described in the Materials and Methods. For identification of standard proteins see Fig. 2.

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{Minutes}

Fig. 4. Time course of proacrosin activation into acrosin nalyzed by BzArgOEt hydrolysis. The purified proacrosin was ,~ctivated in triethylamine (50 mM) pH 8.0 at 22°C. At specified time intervals, aliquots were removed and assayed for BzArgOEt hydrolysis at 30°C.

571

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THE EFFECT OF CALCIUM AND IONIC STRENGTH ON THE ACTIVATION OF H U M A N PROACROSIN

q/2 value is the time required for one half of the zymogen to be converted into active proteinase.

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TABLE 1

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Fig. 5. SDS-polyacrylamide gel electrophoresis analysis of the time course of proacrosin conversion into acrosin. 6-/~1 aliquots (1 p.g) of purified proacrosin were incubated with 6 /~1 triethanolamine (0.1 M) pH 8.0 for 0 min (lane A), 1 min (lane B), 2 min (lane C), 3 min (lane D), 5 min (lane E), 15 min (lane F), 30 min (lane G) and 60 min (lane H). The reaction was stopped by the addition of 6 ~1 of 2% SDS/2% mercaptoethanol (pH 2.8) and incubated for 2 rain at 100°C. 15-p.1 aliquots of each sample were electrophoresed (O = origin; DF = dye front) on SDS-polyacrylamide gels and stained for protein as described in the Materials and Methods. For identification of standard proteins see Fig. 2.

47000 band is lost. Proteinase activity in corresponding samples were detected by the zymographic gelatin-SDS-polyacrylamide system as described in the Materials and Methods section. The results shown in Fig. 6 demonstrate that proteolytic activity was readily observed in the molecular weight ranges that accompany each of the major

Treatment

tl/2 (min)

50 50 50 50 50

0.8 0.9 3.8 2.2 6.8

mM mM mM mM mM

triethanolamine triethanolamine + 75 mM NaCI triethanolamine + 25 mM CaCI 2 triethanolamine + 150 mM NaCI triethanolamine+ 50 mM CaCI 2

protein bands seen in Fig. 5. Additionally, an increase in intensity of the digested bands was initially seen in the 52000-58 000 range of molecular weights. Furthermore, it was noted that additional bands with molecular weights between 34000 and 38000 were seen at 3 min (gel C).

Factors influencing proacrosin autoconversion and the enzymatic activity of acrosin The results in Table I show that calcium chloride retards the rate of proacrosin (Fig. 1, fraction 21) conversion into acrosin. Concentrations of 25-50 mM resulted in a 4-8-fold delay in the conversion. Sodium chloride at equivalent ionic strength resulted in noticeably less inhibition. In results not shown, the divalent cation Co 2÷ as cobalt chloride in concentrations as low as 5 mM completely prevented the autoconversion of proacrosin over a 90 min incubation. Complete in1000

BzArgOEt HYDROLYSIS

7 AZOCOLL HYDROLYSIS

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Fig. 6. Time course of proacrosin conversion into acrosin analyzed by gelatin-SDS-polyacrylamide gel electrophoresis. Highly purified proacrosin was incubated under identical conditions as described for Fig. 5. However, the reaction was stopped by the addition of 6 p.1 of 2,% SDS at pH 2.8. 15-~1 aliquots of the respective samples were electrophoresed on gelatin-SDS-polyacrylamide gels and processed for proteinase digestion as described in the Materials and Methods. For identification of standard proteins see Fig. 2.

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Fig. 7. The effect o f calcium chloride on the hydrolysis of

BzArgOEt and azocoll by human acrosin. The enzymatic activity in the presence of calcium chloride (hatched bars) and sodium chloride (open bars) is expressed relative to that obtained in the triethanolamine buffer control (solid bars).

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hibition over the same time interval was also observed with either 20 mM spermidine or 50 mM benzamidine. The results, shown in Fig. 7, demonstrate that there is very little observable effect of increasing the ionic strength or calcium ion concentrations on the hydrolysis of BzArgOEt by acrosin. However, there are significant effects of both ionic strength and calcium ions on the acrosin hydrolysis of azocoll, an insoluble protein substrate used for studying proteolytic enzymes. Discussion

The results of the present study show that human proacrosin can be obtained from sperm in a highly purified form. The storage of the unpurified extracts in a lyophilized form was advantageous, since it allowed for the maintenance of the proacrosin-acrosin system (unpublished results) and concentrated the extracts. Resuspension in 8 M guanidine allowed for the dissociation of protein-protein interactions which interferes with the gel filtration of proacrosin [10]. In the absence of the guanidine, the majority of the proacrosin adhered to the Superose 12 resin (unpublished resuits). Gel filtration through the Pharmacia automated FPLC system using the two Superose 12 columns set in tandem was a rapid and efficient procedure for purifying human proacrosin. Following SDS-polyacrylamide gel electrophoresis and silver staining (Fig. 2, lane B) the final product, while not homogeneous, was highly purified and the major protein bands had relative molecular weights of approx. 52000-55000. These major bands of protein probably represent proacrosin for the following reasons. First, there was no observable enzymatic activity (Fig. 4) in the initial sample, indicating that at zero time there was only proacrosin present. Second, the only proteolysis that was observed with the gelatin-SDS-polyacrylamide gel electrophoresis system (Fig. 3, lane B) corresponded to the major proteins in Fig. 2, lane B. It is therefore most likely that the purified human proacrosin has a molecular weight in the range 52000-55000, which is very similar to that previously found for boar proacrosin [11]. The above estimations have been obtained with SDS-

polyacrylamide gel electrophoresis and are different from those previously reported for human proacrosin in less purified extracts using estimates from gel filtration (75000 and 42000; [3]). The proacrosin preparation was determined to be highly purified when analyzed on silver-stained SDS-polyacrylamide gels (Fig. 2) as well as on gelatin-SDS-polyacrylamide zymography (Fig. 3). The proacrosin appeared as two or three closely migrating bands on both of these systems. The autoconversion of proacrosin to acrosin at pH 8.0 resulted in a typical sigmoidal autoactivation curve (Fig. 4). It should be noted that the ll/2 of proacrosin autoconversion occurred in approx. 2-5 min (see Fig. 4). This is of interest because the t~/z for sperminogen conversion was about 60 min (unpublished results). Other differences between proacrosin and sperminogen included molecular weights (Fig. 3, compare lanes B and E), immunochemica! properties and influences of several effectors on the autoconversion rates (detailed resuits to be published elsewhere). SDS-polyacrylamide gel electrophoresis analysis of the autoconversion process (Fig. 5) showed that upon activation the 52 000 and 55 000 molecular weight proteins disappeared with an initial appearance of a 49 000 form which was subsequently followed by several lower molecular weight forms (M r = 40000-34000). The molecular weight heterogeneity of the proacrosin and acrosin activities was further observed by the gelatin-SDS-polyacrylamide zymograph (Fig. 6). Upon activation to acrosin there was an initial enhancement of the proteinase intensity in the 55000-53000 range and the appearance of a 49000 form. This was followed by several beta-forms with molecular weights between 32000 and 38000. Perhaps this heterogeneity of acrosin which resulted from the limited proteolysis accounts for the numerous molecular weight species that have previously been reported. For example, Elce and McIntyre [7] also reported the predominance of a 49000 molecular weight form of acrosin as well as the presence of a 34000 form. Zaneveld and co-workers [8] found a 30000 form and Schleuning et al. [6] reported forms of acrosin with molecular weights of about 25000, 15000 and 12000. However, these results do not account for the larger forms of human

573

acrosin that have been reported ( M r = 75 000 [4] and M r -- 70000 [5]). The properties of human proacrosin are illustrated in Fig. 3 and Table I. Proacrosin was completely autoactivated in 5 min at pH 7.5 in triethylamine buffer, which agrees with results previously reported for unpurified acid extracts from human sperm [18,19]. Calcium ions (50 mM) markedly inhibited the rate of the purified human proacrosin activation, as was previously shown for purified boar proacrosin [10]. Calcium has been reported to have little, if any, influence on the esterase activity of human acrosin [5]. Since calcium inhibited the conversion of human proacrosin it was of interest to reinvestigate the possible effects that calcium might have on the activity of acrosin. Similar to the previous finding, calcium had no observable effect on acrosin hydrolysis of BzArgOEt (Fig. 7). However, calcium ions produced a pronounced (8.5-fold) enhancement of acrosin hydrolysis of the protein substrate azocoll (Table I). A somewhat similar finding had been observed with purified boar acrosin where calcium resulted in a 46-fold increase in proteolytic activity but only a 3.5-fold increase in esterase activity [14]. The results presented in this paper demonstrate that human proacrosin and acrosin can be readily purified in amounts that permit biochemical characterization. This is of particular importance since the basic analysis of the components of this rather complex system is an important step in the eventual understanding of the regulation of this key enzyme system. In addition, it is anticipated that the results from this report may be useful for clinical investigations of male infertility as well as in elucidating an anti-enzymatic approach to contraception.

Acknowledgments We thank Ms. Cynthia Bahr and Ms. Bonnie Baskett for typing this manuscript. We also thank

Mr. Daniel Kopta for his photographs used in this search was supported by Health Grants HD-0422, 00296.

help in preparing the manuscript. This reNational Institute of HD-12863 and HD-

References 1 Polakoski, K.L. and Siegel, M.S. (1986) in The ProacrosinAcrosin System in Andrology: Infertility and Sterility (J.D. Paulson et al., eds.), pp. 359-375, Academic Press, New York 2 Parrish, R.F. and Polakoski, K.L. (1979) Int. J. Biochem. 10, 391-395 3 Tobias, P.S. and Schumacher, G.F.D. (1977) Biochem. Biophys. Res. Commun. 74, 434-439 4 Gilboa, E., Elkana, Y. and Rigbi, M. (1973) Eur. J. Biochem. 39, 85-92 5 Anderson, R.A., Jr., Beyler, S.A., Mack, S.R. and Zaneveld, L.J.D. (1981) Biochem. J. 199, 307-316 6 Schleuning, W.D., Hell, R. and Fritz, H. (1976) Hoppe Seyler's Z. Physiol. Chem 357, 855-865 7 Elce, J.S. and Mclntyre, E.J. (1982) J. Biochem. 60, 8-14 8 Zaneveld, L.J.D., Dragoje, B.M. and Schumacher, G.F.B. (1972) Science 177, 702-703 9 Siegel, M.S. and Polakoski, K.L. (1985) Biol. Reprod. 32, 713-720 10 Siegel, M.S. and Polakoski, K.L. (1984) Fed. Proc. 43, 2066 11 Polakoski, K.L. and Parrish, R.F. (1977)J. Biol. Chem. 252, 1888-1894 12 Polakoski, K.L. and McRorie, R.A. (1973) J. Biol. Chem. 248, 8183-8188 13 Parrish, R.F. and Polakoski, K.L. (1977) Biol. Reprod. 17, 417-422 14 Parrish, R.F. and Polakoski, K.L. (1981) J. Reprod. Fertil. 62, 417-422 15 Laemmli, U.K. (1970) Nature 227, 680-685 16 Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Anal. Biochem. 118, 197-203 17 Heussen, C. and Dowdle, E.D. (1980) Anal. Biochem. 102, 196-202 18 Goodpasture, J.C., Polakoski, K.L. and Zaneveld, L.J.D. (1979) J. Androl. 1, 16-27 19 Siegel, M.S. and Polakoski, K.L. (1984) Biol. Reprod. 30 (Suppl. 1), 1977