Human fertilization: epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3.

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

Androgens control androgen-binding sites in rat epididymis.

Previous results suggested that the number of androgen-binding sites in rat epididymis was controlled by androgens. Using an exchange technique for receptor determination, we now report that cytoplasmic receptor number decreased to about half the control value after 4 days of castration and reached its lowest level (30% of the control value) after 12 days of castration. The Kd for [3H]methyltrienolone for cytoplasmic receptor varied from 2.8 X 10(-9) M in controls to 0.6 X (10-9) M (P less than 0.001) after 6 or more days of castration. Nuclear binding sites were reduced to 17% of the control value from the second day of castration on, but no change in the affinity for methyltrienolone was noted. The administration of testosterone propionate (400 micrograms/day) to rats castrated for 20 days significantly increased the number of nuclear and cytoplasmic binding sites from the second and third days of treatment, respectively. Labeled thymidine incorporation into DNA rose significantly after a 4-day latency period. The proportion of total binding sites capable of translocation into the nucleus was elevated during the early phase (2-4 days) of androgen treatment. These results also suggest the heterogneity of the cytoplasmic binding site population. Control epididymides were composed of 61.5% receptor-containing epithelial cells, and this proportion was significantly reduced to 53.2% after 20 days of castration. Androgen administration elevated this percentage after an 8-day latency period. Proteolytic activity in cytosol was increased over control values after castration (for 20 days) and until the fourth day after the onset of androgen treatment. However, this activity does not seem to be an important factor in the decrease in binding sites caused by orchidectomy.

Androgen-controlled synthesis of specific proteins in the rat epididymis.

Previous investigations have established the production of specific epididymal proteins (SEP) in the rat which become attached to the sperm surface as these cells pass along the duct. The present study is concerned with the regulation of SEP synthesis by androgens. For this purpose, we determined the concentrations of SEP and protein DE, one of its main components (40%), during sexual maturation, after castration with and without androgen administration, and after ligation of the efferent ductuli in rats. SEP were first detectable at 25 days of age and attained adult values at 60-90 days of age. Protein DE behaved similarly. Castration of the adult rat led to a decrease in SEP and DE concentrations. The fall was more rapid and marked in the caput than in the caudal segments. SEP synthesis seemed to stop promptly after castration; the different rates of decrease of SEP in caput and cauda may reflect different rates of exit of spermatozoa from those segments. SEP and DE concentrations in castrated rats were increased by the administration of testosterone (100 micrograms/day). The SEP concentration was increased after 4 days and restored to control values after 11 days of treatment. Testosterone and 5 alpha-dihydrotestosterone were equipotent in inducing SEP and DE synthesis, while 5 alpha-androstandiols were less potent. The effects of androgens were significantly reduced by the simultaneous administration of cyproterone acetate. We propose that SEP is a suitable marker for following the action of androgens in the epididymis.

The effect of castration and testosterone replacement on specific proteins and androgen levels of the rat epididymis.

The normal weight increase of the epididymis during sexual maturation and its maintenance through adulthood were found to be dependent on the provision of androgens. Binding of [3H]dihydrotestosterone (DHT) to the epididymal 8S cytoplasmic receptor gradually decreased after castration to become undetectable after 25 days. Binding to the androgen binding protein (ABP) was absent 4 days after castration and was not reinduced by 3 weeks of testosterone (T) administration. Unilateral castration for periods of up to 27 days showed the disappearance of ABP with preservation of the 8S receptor on the castrated side, indicating a testicular source for ABP and the epididymal origin of the 8S receptor. The tissue concentrations of T and DHT in the epididymis became undetectable 30 days after castration and were restored to normal values by administration of testosterone in large doses (1.5 mg/100 g BW). Similar results were obtained in rats castrated at 10 days of age and injected with testosterone until 60 days old. The ratio DHT/T was depressed in the castrate and increased with testosterone treatment. The protein content of the epididymis (mg of protein/g wet weight) was also found to be influenced by androgens. Our results show evidence of some mechanisms involved in the trophic effect of androgens upon the epididymis and suggest the possible androgenic control of epididymal 5alpha-reductase activity. They also indicate that a testicular factor is required for the maintenance of the 8S cytoplasmic androgen receptor. It is not known whether this factor is testosterone or some other testicular secretion.

Androgen dependence of protein N-glycosylation in rat epididymis.

The rat epididymis is known to produce and secrete glycoproteins which interact with spermatozoa during the maturation process. The synthesis of the protein core of these compounds is dependent on androgenic stimulation. As a consequence, we studied the possible androgenic control of the N-glycosylation process dependent on the dolichol (Dol) pathway. Glucosyl and mannosyl transferase activities in rat epididymal microsomes decreased by approximately 76% after only 2 days of castration with respect to intact controls. Depleted mannosyl transferase activity could be restored to control values by administration of 100 micrograms/day testosterone propionate (TP) for 4 days. The effect of 20 micrograms/day TP was blocked by the simultaneous administration of 500 micrograms/day of the antiandrogen cyproterone acetate. The addition of excess dolichyl phosphate (12 times the Michaelis-Menten constant (Km) value) to the incubation mixture did not eliminate the difference in mannosyltransferase activity between epididymal microsomes from castrated rats and these from control or testosterone-treated animals. Moreover, the endogenous pool of dolichyl phosphate was found unchanged in the different hormonal situations. Finally, the incorporation of [14C]mannose into lipid-bound oligosaccharides and into glycoproteins was decreased by approximately 60% as a result of castration and reinduced to control values by treatment with TP (50 micrograms/day for 4 days). The results demonstrate the androgen dependence of the initial steps of N-glycosylation in the rat epididymis and suggest that the hormonal regulation is exerted at the level of Dol-nucleotide sugar transferases, rather than upon the size of the endogenous Dol phosphate pool.

Androgen-controlled specific proteins in rat epididymis.

The pattern of proteins in the soluble fraction of the cytoplasm of the rat epididymis was studied by acrylamide gel electrophoresis. The components of five distinct bands, labelled A, B, C, D and E, were found to be sensitive to changes in androgen in the blood. Castration for 14 days produced a sharp decrease in the colour intensity of bands B-E when stained with Amido black. After 21 days of castration, bands D and E were undetectable, bands B and C were severely diminished and band A was more intense. Seven days of replacement with testosterone (1 mg/day) induced a return towards a normal pattern. The degree of restoration was inversely proportional to the duration of castration. Quantitation by densitometry showed that the relative contributions of bands B-E to the region A-E were 61% in the control rat, only 27% after 21 days of castration and 35% when testosterone was given between days 14 and 21 of castration. The components of bands A-E are presumed to be proteins since the electrophoretic pattern was altered by digestion with pronase but not by ribonuclease, phospholipase C or neuraminidase. Epididymides from castrated and androgen-treated castrated rats were incubated with 14C- and 3H-labelled mixed amino acids respectively. After co-electrophoresis the ratio 3H: 14C rose from a baseline of 2-5 in band B, 32 in band C and 7 in bands D and E. Molecular weights were estimated as 27900 for B, 23100 for C and 34400 for D. Band A had the same electrophoretic mobility as serum albumin. Bands B and C were also present in testicular cytosol. Bands D and E were only found in the epididymis, localized mainly within the lumen of the tubules. Bands B-E increased with age during sexual maturation, bands D and E became detectable in the 20-day-old rats. Preliminary evidence indicates that the proteins in bands C, D and E can be removed from caput spermatozoa by washing.

Effect of androgen on androgen receptors in cultured human epididymis.

Optimal assay conditions were developed to determine androgen receptor concentrations in samples of human epididymis. Pretreatment of cytosol with mersalyl, as well as the inclusion of molybdate in the homogenization buffers, resulted in a substantial increase in the number of soluble sites detected. A high yield of nuclear and microsomal binding sites was obtained by prolonged incubation (12 h) in 0.6 mol NaCl/l. Organ culture for 6 days resulted in a major loss of androgen-binding sites. In the absence of androgen in the culture media, only 20% of the original sites were found in cultured tissue. Inclusion of dihydrotestosterone (0.1 mumol/l) in the media resulted in samples containing twice as many sites as controls. It is concluded that androgens influence the number of androgen-binding sites in cultured human epididymis in a manner analogous to that described for rat epididymis in vivo.

Hormonal regulation of 5alpha-reductase activity in rat epididymis.

The specific activity of epididymal 5alpha-reductase (pmol 5alpha-reduced products mg protein-1 h-1) decreased by 17, 44, 58 and 83% of the initial value and its total activity (nmol 5alpha-reduced products organ-1 h-1) decreased by 66, 85, 94 and 98% 2, 4, 8 and 14 days respectively, after castration. The loss of total activity always exceeded the decrease in organ weight and protein content. The decline in enzymic activity could be prevented by implantation of testosterone at the time of castration. Administration of testosterone propionate (200 microgram/day) for 12 days starting 1 month after castration was associated with the weights of the accessory organs returning to the control values and although the specific activity of 5alpha-reductase was almost completely restored by this treatment, the total activity of the nuclear fraction remained at 49% of the control value. Recombination experiments demonstrated that the effect of androgens is not mediated by a factor present in the soluble fraction and the concomitant administration of androgen and either cycloheximide or actinomycin D blocked the effect of androgen. These data suggest that androgens stimulate the synthesis of epididymal 5alpha-reductase.

The organ culture of hunan epididymal tubules and their response to androgens.

Human epididymal tubules from 9 patients undergoing orchidectomy were cultured for periods of up to 8 days with preservation of the histological structure of the tissue. Addition of androgens (testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol) significantly increased the epithelial height, the incorporation of [3H]amino acids and [3H]thymidine. The effect on protein synthesis was significant 48 h after the onset of treatment and was maximal after 3 days. The effect on DNA replication was maximal during the 3rd day of treatment. In both instances maximal activity was achieved at a concentration of 10(-7) M of testosterone. Cyproterone acetate (10(-5) M) was able to negate all effects of androgens.

Isolation and characterization of specific rat epididymal proteins.

The partial purification and characterization of specific rat epididymal proteins (SEP) is reported. Starting from the cytosol fraction obtained from epididymal homogenates, protein C was purified 15-fold and proteins D--E were purified 19-fold. The molecular weight, determined by molecular sieving, of protein C was 22,400 while that of D--E was 37,000. These proteins stained as glycoproteins with periodic acid--Schiff reagent. The isoelectric point of protein D was 5.13 while that of protein E was 4.95. Protein C separated into 3 bands during isoelectric focussing. The major component focussed at 5.56 and the two minor components at pH 5.38 And 5.79. Using a specific antiserum we could confirm the organ specificity of SEP and their androgen-dependence.

Further characterization of a model system for the study of human epididymal physiology and its relation to sperm maturation.

Some preliminary speculations about the possible participation of epididymal antigens in sperm function may be supported by the above data. On the one hand, the reduction in the amount of antigens and their abnormal localization on spermatozoa from infertile patients may be coincident with our view about participation of epididymal antigens in the development of zona pellucida binding ability and fertilizing capacity by spermatozoa during maturation. This hypothesis is derived from experiments showing that immature hamster spermatozoa gain their ability to recognize and bind to zona pellucida and to penetrate homologous oocytes when exposed to preparations enriched in androgen-dependent epididymal secretory proteins or preincubated in conditions that favor their interaction with these proteins. Supporting our viewpoint for such a role in humans is evidence showing the progressive development of the ability to interact with hamster denuded oocytes as human spermatozoa pass along the epididymis. On the other hand, the apparent correlation between the loss of epididymal antigens during capacitation and the increased fertilization of human oocytes in vitro may be reminiscent of the removal of a decapacitation or acrosome stabilizing factor known to occur in many species and that must be removed prior to fertilization. Pending further understanding of their physiological role, the androgen-dependent epididymal proteins may become a useful marker of epididymal function and/or of sperm capacitation in humans. Within this context, we wish to stress the potential value of the model system that we have developed for the study of human epididymal physiology.

Abnormal distribution of epididymal antigens on spermatozoa from infertile men.

An antiserum raised against human epididymal proteins associated with ejaculated sperm was used to test the hypothesis that the amount and/or localization of these antigens may be altered in men with infertility. With the use of immunofluorescence we found that in sperm from fertile donors 88.4% of the cells had the antigens localized over the acrosomal cap only and 1.3% had most of the antigens at extraacrosomal sites. Fifteen of the 26 infertile men (P1) studied had a similar relative distribution of antigens, but the remaining 11 patients (P2) had a 38-fold increase in cells with extraacrosomal localization of the antigens (40%, P less than 0.005). Using flow cytometry to quantitate immunofluorescence, content of antigen on sperm from patients from population P1 (680 +/- 60 V X 10(-4)) was not different from that of control (835 +/- 53 V X 10(-4], whereas it was significantly lower in sperm from patients from population P2 (554 +/- 64 V X 10(-4), P less than 0.005). Differences could not be correlated with parameters measured by routine semen analysis. Our results suggest a possible relationship between the decreased amount of epididymal antigens or their altered localization on sperm and the infertility of patients from population P2.

Transport in vitro fertilization and intracytoplasmic sperm injection: results of a collaborative trial.

OBJECTIVE: To determine whether the use of a central assisted reproduction laboratory, with gamete transport to the facility (transport assisted reproduction), would decrease oocyte quality or performance in IVF-ET and intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective clinical study. SETTING: Public and private fertility clinics. PATIENT(S): A total of 467 couples underwent transport IVF, whereas 108 underwent transport ICSI. A group of 60 couples underwent conventional IVF during the same period. All methods and protocols used were similar among centers. Oocyte pick-up was performed by ultrasound-guided vaginal puncture. INTERVENTION(S): Oocytes were transported under controlled conditions, from the site of follicular aspiration to a central laboratory. MAIN OUTCOME MEASURE(S): The fertilization and cleavage rates and clinical pregnancies were compared among the study populations. RESULT(S): The differences between the fertilization and cleavage rates of ova and the rates of clinical pregnancies produced by transport and conventional methods were not statistically significant. CONCLUSION(S): Gamete transport to a central laboratory was not harmful for oocytes or for the outcome of assisted reproduction. Transport makes the use of IVF and ICSI available to physicians who are not affiliated with an assisted reproduction program, reduces costs, and increases acceptability of the procedures to patients.

Participation of human epididymal sperm coating antigens in fertilization.

A polyclonal antiserum directed against human sperm coating proteins of epididymal origin (anti-KCl) was tested for its ability to alter sperm function. Spermatozoa from normal ejaculates were selected by swim-up separation and capacitated by overnight incubation at room temperature. Exposure of these cells to anti-KCl (0.39 mg protein/ml), prior to their use in the hamster ova penetration test, reduced the penetration of denuded oocytes by 65% (P less than 0.005). Significant inhibitions of lesser magnitude were observed at lower serum concentrations (to 0.098 mg/ml). In an effort to understand the mechanism of this inhibition, other sperm function parameters thought to be related to oocyte penetration were studied. The inhibitory effect was exerted without noticeable changes in sperm motility (determined by the percentage of motile cells and their linear velocity), and in the absence of major sperm agglutination. Anti-KCl did not inhibit the occurrence of spontaneous or induced (by human follicular fluid) acrosome reaction in capacitated spermatozoa. In contrast, exposure to anti-KCl reduced the ability of capacitated spermatozoa to bind tightly to the hamster oolemma. None of these effects were elicited by a control preparation obtained from pre-immune rabbit sera. Exposure of zona-free oocytes to the antiserum did not alter their penetrability by normal sperm. These results suggest that the antigens recognized by anti-KCl participate in some specific step of the sperm-ovum interaction.

Partial characterization of epididymal 5 alpha reductase in the rat.

Epididymal 5alpha reductase activity was found distitributed in the crude nuclear fraction (44 percent) and microsomal fraction (41 percent). Spermatozoa contaminating the nuclear preparation accounted for only 3 percent of its activity. There were no regional differences in the distribution of total 5alpha reductase activity. However, the nuclear enzyme was more active in caput than in other regions. Maximal activity was found at pH 6.2 and at 32 degrees C. Both enzymes had an absolute requirement of reduced dinucleotides. The microsomal preparation could only us NADPH while the nuclear enzyme could use NADPH and NADH. The apparent Km for the microsomal preparation was 0.62 +/- 0.05 X 10(-6)M and Vmax was 555 +/- 38 pmoles/mg protein/hour. The nuclear enzyme presented similar values. The reaction was not inhibited by accumulation of product in the medium, but other steroids such as progesterone, epitestosterone (17alpha-hydroxy-4-androsten-3-one) and 3-oxo-4-androstene-17beta-carboxylic acid were potent competitive inhibitors. The reaction was strongly inhibited by Hg, Zn and Cu. The properties of the epididymal reductase are similar to those of the prostatic enzyme.

Androgen concentration and partial characterization of 5alpha reductase in the epididymis of the rhesus monkey.

The 5alpha reductase activity ofthe monkey epididymis was studied. The enzyme was found in particulate subcellular fractions, its distribution closely resembling that of the microsomal marker enzyme NADPH: cytochrome c reductase, suggesting an association of 5alpha reductase with membranes of the endoplasmic reticulum. Maximal enzyme activity was found at pH 5.4 and at 32--37 C. The crude nuclear preparation had a Km: 0.315 x 10(-6)M and Vmax: 168 pmoles/mg protein/h. The microsomal enzyme had a Km: 0.243 x 10(-6)M and Vmax: 828 pmoles/mg protein/h. Neither enzyme preparation was affected by addition to the incubation media of dihydrotestosterone (DHT) or 5alpha-androstane-3alpha,17beta-diol. The endogenous androgen concentration in the epididymides of 2 different monkeys, in ng/g wet weight was: DHT 20.81 +/- 1.98; T: 9.0L +/- 2.83; diol: 3.03 +/- 0.41.