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SUMMARY Establishing reference norms for semen parameters in fertile men is important for accurate assessment, counselling and treatment of men with male factor infertility. Identifying temporal or geographic variability in semen quality also requires accurate measurement of semen parameters in well-characterized, defined populations of men. The Study for Future Families (SFF) recruited men who were partners of pregnant women attending prenatal clinics in Los Angeles CA, Minneapolis MN, Columbia MO, New York City NY and Iowa City IA. Semen samples were collected on site from 763 men (73% White, 15% Hispanic/Latino, 7% Black and 5% Asian or other ethnic group) using strict quality control and well-defined protocols. Semen volume (by weight), sperm concentration (hemacytometer) and sperm motility were measured at each centre. Sperm morphology (both WHO, 1999 strict and WHO, 1987) was determined at a central laboratory. Mean abstinence was 3.2 days. Mean (median; 5th–95th percentile) values were: semen volume, 3.9 (3.7; 1.5–6.8) mL; sperm concentration, 60 (67; 12–192) 9 10 6 /mL; total sperm count 209 (240; 32–763) 9 10 6 ; % motile, 51 (52; 28–67) %; and total motile sperm count, 104 (128; 14–395) 9 10 6 respectively. Values for sperm morphology were 11 (10; 3–20) % and 57 (59; 38–72) % normal forms for WHO (1999) (strict) and WHO (1987) criteria respectively. Black men had significantly lower semen volume, sperm concentration and total motile sperm counts than White and Hispanic/ Latino men. Semen parameters were marginally higher in men who achieved pregnancy more quickly but differences were small and not statistically significant. The SFF provides robust estimates of semen parameters in fertile men living in five different geographic locations in the US. Fertile men display wide variation in all of the semen parameters traditionally used to assess fertility potential.

Uterotubal junction tissues (n=12) recovered from young nulliparous mares 4 h after insemination were examined for spermatozoa in situ using scanning electron microscopy. Mares were inseminated before ovulation with semen containing 66-85% morphologically abnormal spermatozoa and approximately 1 x 10(9) progressively motile spermatozoa. Spermatozoa were found in all the tissues and displayed a variety of relationships to the epithelium, depending on regional location, spatial constraints and epithelial surface characteristics. Spermatozoa were most abundant in the epithelial folds on the uterine side of the uterotubal junction papilla. More than 90% of the spermatozoa had normal morphology. These observations indicate that morphologically normal equine spermatozoa develop specific associations with the luminal epithelium of the reproductive tract during transport to the oviduct. Furthermore, the uterine papilla of the uterotubal junction may be a site of preovulatory sperm storage. The scarcity of morphologically abnormal spermatozoa, despite their abundance in the inseminate, indicates that most abnormal spermatozoa either do not reach the uterotubal junction or do not develop normal associations with the epithelium of the uterotubal junction. This observation provides evidence for a mechanism of sperm selection during transport. Hence, both normal morphology and progressive motility of spermatozoa should be considered in determining insemination doses, particularly for subfertile stallions.

Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anti-calpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also localized within the same cytoplasmic region as calpains. The antibodies used for ultrastructural localization were also used to probe Western blots of sperm extracts. Antibodies to either the mu- or m-calpain recognized an 80-kd protein, which is similar to the molecular weights of other ubiquitous calpains described. The small subunit (30 kd) was also recognized with a specific monoclonal antibody. An antibody to calpastatin recognized a major band at 78 kd and a lighter band at 45 kd, while the antibody to the testis-specific isoform of calpastatin (TCAST) recognized a 110-kd protein. We hypothesize that this cysteine protease system may be functional in cynomolgus macaque sperm during capacitation, the acrosome reaction, or both.

In this study, we investigated the functions of PH-20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm-zona binding in other species. Anti-macaque PH-20 IgG, anti-pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD-46, which is also located on the inner acrosomal membrane, but has no known function in sperm-zona pellucida interaction. After labeling with anti-acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH-20 and CD-46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti-acrosin IgG nor anti-CD-46 IgG affected sperm penetration of the zona at concentrations up to 300 microg/ml, but zona penetration was blocked completely when anti-PH-20 IgG (100 microg/ml) was present during sperm-oocyte interaction. Ultrastructural observations of oocytes incubated with anti-PH-20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti-PH-20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm-zona binding, rather than primary sperm-zona binding or the zona-induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida.

Capacitated cynomolgus macaque sperm have a surface hyaluronidase (PH-20) that is evenly distributed over the entire head and can be visualized at the ultrastructural level using a secondary antibody labeled with colloidal gold . Exposure of sperm to mono-specific, bivalent polyclonal antibodies to PH-20 causes a rapid clustering of PH-20. The predominant morphological consequence of PH-20 redistribution is its aggregation along the lateral edge of the sperm head. Monovalent Fab fragments of the anti-PH-20 antibody bound to the sperm head but did not induce a change in PH-20 distribution. PH-20 aggregation was observed in almost all sperm following treatment with the polyclonal antibody, but only about 20% of the sperm had morphological acrosome reactions, regardless of the time of exposure or the concentration of antibody. There was morphological evidence of swelling of the acrosomal matrix in over 50% of the sperm following exposure to anti-PH-20 antibodies. Anti-PH-20 Fab fragments did not induce the acrosome reaction or acrosomal matrix swelling. Sperm bound to macaque zona pellucida also showed aggregation of the PH-20 protein as soon as 30 sec after sperm-zona interaction. This aggregation was not observed when macaque sperm were bound to hamster zona pellucida. When macaque sperm were surface-labeled with biotin and then incubated with anti-PH-20 antibodies or macaque zona pellucida, there was no evidence of a global surface protein rearrangement, although PH-20 protein was aggregated on the surface of the same sperm cells. An increase in levels of internal sperm Ca++ was measured in association with the antibody-induced PH-20 aggregation. Fab fragments did not increase Ca++ levels, but when they were crosslinked with anti-Fab antibody there was a significant Ca++ increase and induction of acrosome reactions. Anti-PH-20 Fab fragments did not block macaque sperm binding to macaque zona pellucida or the zona-induced acrosome reaction. We conclude that PH-20 on the sperm surface is involved in sperm-zona pellucida interaction and the zona-induced acrosome reaction.

Preparations of sperm membranes (plasma membranes and outer acrosomal membranes) and denuded sperm heads were isolated from macaque sperm, and the PH-20 proteins present were characterized by Western blotting, hyaluronic acid substrate gel analysis, and a microplate assay for hyaluronidase activity. Because we have shown previously that PH-20 is located on the plasma membrane and not on the outer acrosomal membrane, the PH-20 in the membrane preparations was presumed to be plasma membrane PH-20 (PM-PH-20). PM-PH-20 had an apparent molecular weight of 64 kDa and the optimum pH for its hyaluronidase activity was 6.5. The PH-20 associated with denuded sperm heads was localized by immunogold label to the persistent inner acrosomal membrane (IAM) and was presumed to be IAM-PH-20, which included a major 64 kDa form and a minor 53 kDa form. The 53 kDa form was not detected in extracts of denuded sperm heads from acrosome intact sperm that were boiled in nonreducing sample buffer, but was present in extracts of sperm heads from acrosome reacted sperm and in the soluble material released during the acrosome reaction, whether or not the samples were boiled. Substrate gel analysis showed that the hyaluronidase activity of the 53 kDa form of PH-20 was greatest at acid pH, and this activity was probably responsible for the broader and lower optimum pH of IAM hyaluronidase activity. When hypotonic treatment was used to disrupt the sperm acrosome and release the acrosomal contents, less than 0.05% of the total hyaluronidase activity was released. The PH-20 protein released by hypotonic treatment was the 64 kDa form and not the 53 kDa form, suggesting that its source might be the disrupted plasma membranes. Our experiments suggest that the soluble form of hyaluronidase, which is released at the time of the acrosome reaction, is derived from the IAM. This soluble hyaluronidase is composed of both the 64 kDa form and 53 kDa form of PH-20. The 53 kDa form appears to be processed from the 64 kDa form at the time of the acrosome reaction.

A microplate assay for hyaluronidase and a heterologous cumulus penetration assay were used to determine the effects of four flavonoids (tannic acid, kaempferol, quercetin, and apigenin) on the function of cynomolgus monkey sperm. All four flavonoids inhibited the activity of hyaluronidase extracted from monkey sperm in a concentration-dependent manner over the range of 50-200 microM. Tannic acid and apigenin had lower inhibitory effects than kaempferol and quercetin. Kaempferol, quercetin, and apigenin at 100 microM were shown to significantly inhibit monkey sperm penetration into hamster cumulus. There was a significant linear relationship between the capacity of the flavonoids to inhibit monkey sperm hyaluronidase activity and their inhibitory effects on hamster cumulus penetration (r = 0.97). Tannic acid was observed to reduce sperm motility, and it was not used in the cumulus penetration assay. The other three flavonoids tested in the cumulus penetration assay did not affect sperm motility, nor did they induce acrosome reactions. The results demonstrate that the flavonoids are useful tools for assessing the involvement of hyaluronidase in the functions of monkey sperm that are involved in fertilization.

In this study we investigated the ultrastructure of macaque sperm induced to acrosome-react with calcium ionophore A23187, and the interaction between these acrosome-reacted sperm and the macaque zona pellucida. Transmission electron microscopy revealed that the majority of ionophore-treated sperm retained the vesiculated acrosomal cap or "shroud." Untreated, acrosome-reacted sperm on the zona had a similar ultrastructural appearance. In sperm-zona binding experiments, a mean of 4.5 ionophore-treated sperm were bound per zona after 1 min of coincubation compared with 41 sperm per zona in the solvent control. Vigorous pipetting was used to remove the acrosomal shrouds from approximately 50% of acrosome-reacted sperm before incubation with oocytes. Significantly more of these mechanically treated sperm were bound to the zona after a 4-min coincubation compared with acrosome-reacted sperm that were not pipetted. The number of mechanically treated sperm bound to the zona was the same whether the sperm and oocytes were coincubated in calcium-free medium or in control medium. The percentage of mechanically treated sperm that were acrosome-reacted on the zona also was not different in the two media. We conclude that macaque sperm that undergo the acrosome reaction on the zona surface are bound by the acrosomal shroud before zona penetration. When sperm acrosome-react before interaction with the oocyte, their zona binding capacity is significantly reduced. Removal of the acrosomal shroud and exposure of the inner acrosomal membrane increases the affinity of sperm for the zona. This sequence occurs naturally during the transition from primary binding to secondary binding on the zona surface.

PH-20 is a sperm plasma-membrane protein that has been shown to have hyaluronidase activity in several mammalian species including nonhuman primates. In this investigation, the PH-20 protein was characterized in noncapacitated human sperm and in capacitated human sperm. Two forms of PH-20 were observed in immunoblots of sodium dodecylsulfate polyacrylamide-gel electrophoresis (SDS PAGE) using a polyclonal antibody to recombinant PH-20: a major band of 64 kDa appeared in noncapacitated and capacitated sperm extracts and a 53-kDa band that appeared only in the acrosome-reaction supernatant of acrosome-reacted sperm. Using hyaluronic acid substrate gel analysis, we demonstrated that noncapacitated sperm extracts, capacitated sperm extracts, and the acrosome-reaction supernatant had hyaluronidase activity at neutral pH (pH 7) and acid pH (pH 4). The 64-kDa form in all samples had hyaluronidase activity at both neutral and acid pH, but the 53-kDa form was only active at acid pH. Total hyaluronidase activity, as measured by a microplate assay, was higher at pH 7 than at pH 4. Very low hyaluronidase activity was detected in the acrosome-reaction supernatant. Transmission electron microscopy and immunogold labeling showed that PH-20 of acrosome-intact human sperm was located on the plasma membrane over the entire head but not on the sperm midpiece and tail. After the acrosome reaction, PH-20 was also located on the inner acrosomal membrane. The biochemical characteristics and the ultrastructural localization of PH-20 in human sperm suggest that this protein is the human sperm hyaluronidase and, therefore, has an important function during fertilization.

A model system consisting of cynomolgus macaque sperm and ovulated hamster ova-cumulus complexes (OCCs) was utilized to study the role of the sperm protein PH-20 in cumulus penetration. The hyaluronidase activity of solubilized macaque sperm PH-20 was evaluated using an ELISA-like microplate assay prior to and following the addition of the hyaluronidase inhibitors heparin (0-100 microg/ml) and apigenin (250 microM), as well as the Ig fraction of a polyclonal antibody raised against purified recombinant macaque PH-20 (R10; 10-400 microg/ml). Sperm motility following exposure to enzyme inhibitors was evaluated using computer-aided sperm motility analysis. Macaque sperm were labeled with the permeant fluorescent nuclear dye, Hoechst 33342, and were coincubated with ovulated hamster OCCs for 30 min at 37 degrees C. The addition of heparin, apigenin, or R10 antibody to solubilized sperm extracts resulted in a linear dose-dependent decrease in hyaluronidase activity (P.01). In the heterologous cumulus penetration assay, fluorescently labeled macaque sperm that were pretreated with heparin (1-100 microg/ml), apigenin (250 microM), or R10 antibody (Ig fraction, 10-400 microg/ml) demonstrated a dose-dependent decrease in the ability to penetrate hamster OCCs (P0.01), in the absence of effects on sperm motility. In the homologous assay, experiments using macaque OCCs and fluorescently labeled macaque sperm confirmed that the same concentrations of heparin and R10 antibody similarly suppressed spermatozoal cumulus penetration (P.01). These results suggest that macaque sperm PH-20-derived hyaluronidase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete interaction.

Fluorescence microscopy and transmission electron microscopy (TEM) were used to determine the location of the membrane protein PH-20 on spermatozoa of cynomolgus macaques. Rabbit antiserum raised against recombinant cynomolgus macaque sperm PH-20 was used as the primary antibody, and the second antibody was goat anti-rabbit IgG conjugated with either fluorescein isothiocyanate or 15 nm gold particles. Spermatozoa were evaluated before capacitation and after capacitation and induction of acrosome reactions with calcium ionophore A23187. In sperm suspensions with a high percentage of intact acrosomes, fluorescence labeling was observed uniformly over most of the sperm head. The sperm midpiece and tail were not labeled. In sperm suspensions with a high percentage of acrosome reactions, most spermatozoa labeled intensely over the anterior sperm head, but labeling of the posterior sperm head was greatly reduced. TEM of acrosome-intact spermatozoa revealed gold particles distributed uniformly on the plasma membrane overlying the acrosome, the equatorial segment, and most of the post-acrosomal region. After the acrosome reaction, gold label was present on the inner acrosomal membrane and on the plasma membrane overlying the equatorial segment. Very little label was present on the plasma membrane in the post-acrosomal region of acrosome-reacted spermatozoa. The location of PH-20 on the surface of macaque spermatozoa suggests a function for this protein in primary and/or secondary binding to the zona pellucida. The apparent decrease in amount of PH-20 on the posterior head of macaque spermatozoa following the acrosome reaction is consistent with the migration of this protein to the inner acrosomal membrane, as demonstrated previously for the homologous PH-20 protein of guinea pig spermatozoa.

Mammalian sperm that have completed capacitation are capable of undergoing the acrosome reaction in response to a number of biological and chemical stimuli. In the present report, we have investigated the ability of progesterone to stimulate acrosome reactions of stallion sperm capacitated in vitro. Motile sperm were selected by a two-layer Percoll gradient centrifugation and were incubated in TALP medium modified by the 1:1 (v/v) addition of TEST-yolk medium for 5 hours at 39 degrees C, under 5% CO2 in humidified air. Sperm incubated in vitro in TALP-TEST medium had a higher percentage of acrosome reactions following the addition of progesterone (3.18 mumol/L) compared to controls (P0.05). Furthermore, sperm from stallions classified as fertile on the basis of breeding history had higher percentages of progesterone-induced acrosome reactions in comparison with stallions classified as subfertile (P0.05). Acrosome reactions were assessed routinely by fluoresceinated lectin binding, but the physiological appearance of induced acrosome reactions was confirmed at the ultrastructural level by transmission electron microscopy. We conclude that 1) TALP-TEST medium supports stallion sperm capacitation in vitro, 2) progesterone-induced acrosome reactions are physiological, and 3) sperm from fertile stallions may be more responsive to progesterone-induced acrosome reactions than those of subfertile stallions. This is the first report in a nonhuman species that differences exist between the sperm of fertile and subfertile males in the ability to capacitate and acrosome react in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

This review of the currently recommended clinical and laboratory approach to the evaluation of male reproductive problems describes history-taking, physical examination, evaluation of semen, evaluation of abnormalities of the endocrine system, and specialized laboratory tests such as bioassays, biochemical tests of sperm function, and tests for antisperm antibodies.

It has previously been shown that type B spermatogonia in male mice treated with 0.05 Gy of X rays undergo an alteration expressed by progeny embryos as a cellular proliferation disadvantage in a chimera assay. We wished to obtain information on the assay's detection limit to ionizing radiation and on the radiosensitive target in male germ cells. Male mice were briefly irradiated with 137Cs gamma rays at nominal absorbed doses of 0.0, 0.0015, 0.005, 0.010, or 0.05 Gy and then mated for the next 8 weeks to untreated females. Four-cell embryos from treated males (experimental embryos) were paired with FITC-labeled embryos from untreated males (control embryos) to form aggregation chimeras. The chimeras were cultured for 30-40 h and examined under phase-contrast and UV illumination for the number of unlabeled cells (from the experimental embryo) and total chimera cell number, which were then expressed as "proliferation ratios" (No. unlabeled cells/total chimera cell No.). Significant decreases in proliferation ratios were observed at postirradiation weeks 4, 6, and 7 for the 0.01-Gy dose group and at weeks 5-6 for the 0.05-Gy dose group. In addition, significantly lower ratios were observed with early and mid four-cell embryos, but not with late four-cell embryos. These results suggest that mouse male germ cells express a radiosensitive target(s) whose detection limit by the assay lies at an absorbed dose between 0.005 and 0.010 Gy for brief gamma irradiation and whose effect on embryonic cell proliferation might decay by the second cleavage.

It is well known that cervical mucus restricts penetration of morphologically abnormal human sperm, both in vitro and in vivo. However, the mechanisms of such restriction are not well understood. Using videomicrography to simultaneously analyze the motions and morphology of individual human sperm, we analyzed differential penetration of normal and abnormal sperm into fresh human cervical mucus. Abnormal sperm swam slower in mucus than the normal sperm, but their flagellar beat parameters were not commensurately different. Multivariate statistical analysis of the relationship between individual sperm velocity and flagellar beat parameters indicated that the heads of the abnormal sperm experienced greater resistance from the mucus than did normal heads. Differential mucus resistance, more than altered motile vigor, appears to be responsible for the restriction of abnormal sperm during migration through mucus.

In the present study of 15 fertile men, a mean of 89 +/- 3% of ejaculated cells had nuclei which were stable after treatment with 1% sodium dodecyl sulphate (SDS). Examination of repeated ejaculates demonstrated the constancy of the response of ejaculated spermatozoa from different men to SDS treatment. After 5 cycles of washing, the percentages of cells with stable nuclei after SDS treatment declined from 85 +/- 3 to 46 +/- 6% and ranged from 12 to 66% for different men. The supernatant derived from the first wash inhibited the nuclear chromatin decondensation of washed spermatozoa. Treatment of washed spermatozoa with 1 mM-zinc (which binds to -SH groups) or 1 mM-copper (which promotes oxidation of -SH groups to -S-S-) resulted in a significant increase in the proportion of cells with stable nuclear chromatin. However, the stabilizing effects of zinc could be reversed with continued washing of the cells, whereas the nuclei of copper-treated cells remained stable to SDS after additional washing. We conclude that (1) the number of spermatozoa with nuclei that are insufficiently stabilized by disulphide bonds is much higher in the semen of fertile men than was previously thought, (2) significant differences exist among fertile men in the proportions of ejaculated spermatozoa extensively stabilized by disulphide bonds, and (3) removal of seminal plasma and/or cellular contaminants (zinc) is necessary to unveil the instability of the nuclear chromatin in the ejaculated spermatozoa of fertile men.

Epididymal spermatozoa were compared with ejaculated spermatozoa from the white rabbit to determine if there were differences in the extent of embryonic mortality. 2 New Zealand White female rabbits were inseminated in each experiment. Ejaculated and epididymal spermatozoa from the same male were injected respectively into the cranial third of contralateral uterine horns. 100 IU human chorionic gonadotropin (HC G) was given to induce ovulation. Cleaved embryos were placed in 10% serum-Tyrodes solution and transferred into the oviducts of recipient females injected with HCG at the same time as the embryo donors. Ova fertilized by each kind of sperm were transferred in equal numbers to contralateral oviducts of the same recipient. 1-cell ova were examined for evidence of fertilization. Naturally mated controls were maintained. Spermatozoa from the lower corpus epididymidis inseminated into the uterus fertilized 76.5% of 204 ova in comparison with 75.9% of 199 ova fertilized by ejaculated spermatozoa. 27 hours after insemination and HCG injection the 4-cell stage was reached by 11.4% of the ova fertilized by lower corpus sperm vs. 17.4% fertilized by ejaculated sperm. When transferred to contralateral oviducts of synchronized recipients 60.3% of 63 ova fertilized by epididymal spermatozoa implanted and 50.8% developed normally to term. This compares with 57.1 and 47.6% respectively for those ova feritilized by ejaculated spermatozoa. Differences between the 2 kinds of spermatozoa seem insignificant.

Equal numbers of rabbit spermatozoa from the proximal cauda epididymidis and ejaculate of the same male were mixed and inseminated intravaginally. Their competitive performance in the same female was evaluated by identification of the two populations with x-irradiation marking (fertilizing spermatozoon) and fluorescein isothiocyanate labelling (spermatozoa in the uterus and egg vestments). Following such mixed inseminations, epididymal spermatozoa from thirteen males fertilized 48% of 251 eggs recovered 50 hours after HCG injection and comprised 55%, 59% and 73% of spermatozoa in the perivitelline space, zona pellucida and mucin layer. When mixed spermatozoa from eight other males were inseminated 5 hours after HCG and the eggs were recovered 8 hours later (13 hours after HCG), epididymal spermatozoa accounted for 53% of the perivitelline and zona spermatozoa in 108 eggs and more than 50% of the uterine spermatozoa in seven of fifteen females. Cleavage of eggs fertilized by spermatozoa from the distal corpus epididymidis and ejaculate of the same male (eight males) was compared by insemination of the respective sperm populations into contralateral oviducts of the same female (sixteen females). There was no significant difference in the level of fertilization or the stage of cleavage 27 hours after insemination and HCG, whether eggs were fertilized by epididymal or ejaculated spermatozoa. These results indicate that once epididymal spermatozoa have acquired the capacity to penetrate the egg, they do not differ from ejaculated spermatozoa in their transport, capacitation, fertilizing ability or competence to sustain development of the egg to the sixteen cell stage.

We have used two approaches to test the ability of the human zona pellucida to induce acrosome reactions in human sperm. First, nonviable human oocytes were incubated for 1 min in a suspension of capacitated sperm (of which fewer than 5% were acrosome-reacted) to allow binding of about 200 sperm per oocyte. Some of the oocytes were fixed immediately, and the remainder were fixed after a further 1-h incubation without free-swimming sperm. As determined by light microscopy, sperm on the zona were only 3 +/- 2% (avg. +/- SD) acrosome-reacted at 1 min, and the incidence increased to 46 +/- 15% during the next hour. Electron microscopy confirmed that most sperm on the zona at 1 min were acrosome-intact. A few sperm were in an early stage of the acrosome reaction. Acrosome reactions occurring on the zona during the subsequent hour appeared to be morphologically normal. Second, treatment of sperm in suspension with acid-disaggregated zonae (2 to 4 zonae/microliter) increased the incidence of acrosome-reacted sperm from 3 +/- 1% to 24 +/- 4%. We conclude that the human zona pellucida, or material intimately associated with it, can induce acrosome reactions in human sperm.

The capability to analyze semen is not new but until recently was a clinical art practiced well by only a few experts. The development of objective, quantitative methods for semen evaluation has brought these diagnostic capabilities to every clinical laboratory. These methods are described, and an interpretation of semen parameters is presented for the reader.

In an in vitro assay human spermatozoa penetrated the zona pellucida of 38.8% of 773 human oocytes recovered from the ovaries of cadavers. Zona penetration was not observed until 8 hours had elapsed. Oocytes examined with the electron microscope were surrounded mainly by sperm with intact acrosomes, but contained sperm in the zona and perivitelline space which had lost the outer acrosomal membrane and acrosomal contents. Sperm entry into the ooplasm was never observed. Spermatozoa from 11 of 16 patients with suspected infertility penetrated the zona, although the penetration rate was lower than that with sperm from fertile donors (12.9% versus 46.4%). When oocytes were incubated with mixed suspensions containing equal numbers of motile sperm from donors and patients, the donor sperm (identified by a fluorescent label) penetrated 50.0% and the patient sperm only 12.7%. These data suggest that human sperm penetrate the zona pellucida of nonliving human oocytes and mature living ova in a similar manner. This method is a potential diagnostic and investigative tool which avoids the ethical and technical problems associated with human in vitro fertilization.

Motile spermatozoa with lifted or absent acrosomal caps (“reacted acrosomes”) were observed by phase contrast microscopy exclusively in the oviduct of the rabbit reproductive tract during the first 16 hours post coitum (p.c.). They were first identified at six hours p.c. in flushings from the lower isthmus of the oviduct and were present in both the lower and upper isthmus two hours later. Motile spermatozoa with reacted acrosomes appeared in the lower ampulla (the site of fertilization) at ten hours p.c. Such spermatozoa were obtained from all regions of the oviduct at 12 and 16 hours p.c. but they always constituted a minority of the freely swimming spermatozoa recovered. The percentage of spermatozoa with activated motility was higher following the acrosome reaction, and their apparent swimming speeds were lower than those of intact spermatozoa. The role of ovulation in the induction of acrosome reactions was studied in females artificially inseminated per vaginam (AI), with or without the induction of ovulation, and in females mated either two hours before or two hours after ovulation. The proportion of motile spermatozoa with reacted acrosomes in the isthmus and ampulla of non-ovulatory females 12 hours after AI was similar to their proportions in these regions 12 hours after mating or 12 hours after AI with induced ovulation. Four hours after delayed matings, i.e., two or six hours after ovulation, motile spermatozoa were confined to the tubal isthmus and none had undergone the acrosome reaction. These findings indicate that the products of ovulation and the endocrine changes associated with ovulation in the rabbit need not be directly involved in the induction of the acrosome reaction in vivo.

Cervical mucus was collected from 35 women after artificial insemination. Mucus collections were performed at 1 h, 1 day, 2 days, or 3 days following insemination. Sperm viability was greater than 80% at all recovery times as assessed by exclusion of the supravital dye Hoechst 33258. Virtually 100% of the viable sperm were acrosome-intact at all times as assessed with a fluorescein isothiocyanate-conjugated pea lectin. Sperm were recovered from the mucus after migration into the Biggers, Whittin, and Whittingham medium in vitro. Sperm did not undergo the acrosome reaction in response to human follicular fluid immediately after migration from the mucus but did respond to this agonist after 6 h of incubation in vitro. Sperm recovered at all times after insemination had the same pattern of response to follicular fluid. Sperm that penetrated a column of cervical mucus in vitro also responded to follicular fluid with an increase in acrosome reactions after migration from the mucus and incubation for 6 h in vitro. Unlike the sperm that migrated from cervical mucus, sperm that were separated from semen by Percoll density centrifugation did not undergo the acrosome reaction when challenged with follicular fluid after 6 h but did respond after 24 h incubation. Sperm that migrated from cervical mucus had a similar increase in acrosome reactions after 6 h incubation, regardless of whether the acrosome reaction agonist was follicular fluid or disaggregated human zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)

The motility of human spermatozoa and their ability to penetrate zona-free hamster eggs were examined after dilution of the semen with TES-Tris (TEST) yolk buffer and storage for 48 hours at 2 degrees C to 5 degrees C. Semen samples from 10 fertile donors and 19 infertility patients were studied. More than 65% of the spermatozoa which were initially motile in the TEST yolk buffer remained active after storage. During storage, the mean swimming speed of the sperm declined to approximately 60% of the prestorage value. The percentage of zona-free hamster eggs that were penetrated by spermatozoa from patients and donors increased significantly following 48 hours of storage at 2 degrees C to 5 degrees C. Normal semen and abnormal semen were equally preserved by this storage method. This procedure may be used to ship semen samples by commercial transportation to specialized laboratories for testing. Low temperature storage in the TEST yolk buffer appears to enhance the fertilizing capacity of human spermatozoa in vitro.

Few spermatozoa were present in the ampullae of females 12 h after intravaginal artificial insemination (AI) when there was no ovulation-inducing stimulus. When ovulation was induced, sperm distributions in the female tract 12 h after AI did not differ from those observed 12 h after natural mating. The number of spermatozoa in the oviductal isthmus was similar in all 3 groups as was the percentage of isthmic spermatozoa exhibiting 'activated' motility. When fertile mating was delayed for 8 or 12 h after coitus with a vasectomized male (i.e. 2 h before or after ovulation), spermatozoa were not present in the ampulla 4 h later. The numbers of spermatozoa recovered from the cranial isthmus after delayed matings and 12 h after natural matings did not differ, but after delayed matings the motility of isthmic spermatozoa was non-progressive or poorly progressive and none exhibited 'activated' motility. Flagellar activity of isthmic spermatozoa recovered 4 h after delayed matings and after natural matings was similarly depressed. These observations indicate that sperm ascent to the tubal ampulla in the sustained phase of transport, though enhanced by ovulation, must also depend on changes in flagellar activity and a specific pattern of motility, both of which appear only after spermatozoa have resided for more than 4 h in the female tract.

Male infertility is a common and distressing problem in which reproductive abnormalities frequently play an important role. Assessment requires an understanding of the control of spermatogenesis and factors responsible for normal sperm function. Standard tests for assessment of semen quality frequently fail to detect impaired function, but newer tests are now available to measure sperm movement and their ability to penetrate the ovum. Algorithmic approaches based on laboratory data can be used to characterize subgroups of infertile men, but many patients have subtle abnormalities. Treatment of male infertility is ideally directed to a specific pathogenic mechanism; nonspecific therapies have produced disappointing results. Surgery is indicated for certain types of ductal obstruction, but whether internal spermatic vein ligation should be used to treat varicocele remains uncertain.

Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as "mean ratio") was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group). However, cell numbers of singly cultured experimental embryos differed from those of singly cultured control embryos for just Week 7 for the 0.29 and 1.73 Gy dose groups, even though the mean ratios of heterologous chimeras had differed significantly from those of homologous chimeras for 3 weeks prior to and 1 week following Week 7. We conclude that sublethal changes sustained by sperm in vivo from only 0.05 Gy of X irradiation can be inherited by the embryo as a proliferative disadvantage that becomes expressed if challenged by direct cell contact with an unirradiated embryo in an aggregation chimera.

Human spermatozoan oxygen consumption was measured in semen serially diluted with its own plasma, and shortly after serial dilution of washed spermatozoa in an artificial medium. In both media, per-sperm oxygen uptake rose significantly when sperm concentration fell below the range of 15-25 X 10(6) cells/ml. Oxygen uptake was higher in the artificial medium, regardless of sperm concentration. Sperm oxygen consumption in the artificial medium declined over times short enough that no change in the percentage of motile spermatozoa was detected.