We compared fertility potential measurements by the zona-free hamster egg bioassay with the in vitro fertilization of human eggs. Sperm from 24 husbands participating in an in vitro fertilization-embryo transfer program were used in 27 inseminations of the wives' eggs and in simultaneous inseminations of hamster eggs. In seven cases, a positive fertility assessment was obtained in the absence of fertilization of the wives' eggs attributable to egg immaturity or in one case to equipment failure. In the remaining 20 cases, at least one egg was fertilized, for a level of 73%. In 90% of the husbands who fertilized their wives' eggs, a positive fertility assessment was obtained. Two husbands gave false-negative assessments. To account for these, alternative culture conditions were examined; sperm exposure to longer preincubations or at higher concentrations usually improved assessments. These results indicate that fertility assessment with this bioassay is highly correlated with the fertilization of human eggs in vitro.

In an attempt to maximize the success of in vitro fertilization (IVF) and embryo transfer (ET) as a treatment for human infertility, we have examined the relationship of follicular size and number to the rates of oocyte recovery, fertilization, cleavage, and ET in clomiphene citrate-stimulated cycles. The recovery of oocytes from follicles less than 20 mm in diameter was significantly reduced over that from larger follicles, and those oocytes that were obtained from smaller follicles showed a significantly lower rate of fertilization and cleavage. In addition, the overall chance that a patient would undergo ET was greater in a cycle in which more than one follicle 20 mm or larger was developing than in a cycle in which a single large follicle was developing. This latter observation suggests that attempts at laparoscopic oocyte retrieval should be confined to cycles in which more than one accessible large follicle is developing, thereby maximizing the success rate while minimizing the risk and expense for the patient.

The interaction of sperm with the zona-free hamster egg was studied. Hamster sperm were capacitated in Tyrodes media, containing 50% heat-inactivated serum and used to inseminate zona-free eggs in BWW containing 10% serum. Capacitated sperm began fusing with eggs within the first hour of insemination and by 3 h penetration had ceased as indicated by the absence of further changes in the mean number of sperm incorporated per egg. Penetration by capacitated hamster sperm was linearly related to the log of the motile sperm concentration at concentrations above 10(4) cells/ml. The viability of sperm and eggs in culture was limited in these studies to approximately 3-5 h. The existence of a block to unlimited sperm penetration of the zona-free egg was sought in reinsemination experiments. A relatively low sperm concentration was used to initiate egg responses, followed, at timed intervals, by reinsemination with a high challenge concentration of sperm. Subsequent polyspermy levels reflected the presence or absence of the egg's block to polyspermy response. In order to circumvent the problems arising from the rapid aging of hamster sperm in culture, mouse sperm were employed, a convenience afforded by the lack of species specificity in this egg. Reinseminated eggs incorporated additional sperm during the challenge; therefore, the hamster egg is not capable of preventing unlimited sperm penetration. The implications of these findings to the use of the zona-free hamster egg test in fertility evaluation is discussed.

Zona-free mouse eggs inseminated with capacitated epididymal sperm in a modified Krebs-Ringer bicarbonate medium exhibited unusual kinetics of sperm incorporation. At a sperm concentration of 105 cells/ml or higher, the mean number of sperm per egg reached a maximum and then decreased with time. This decrease was correlated with the abstriction of sperm in cytoplasmic blebs which formed during or slightly after second polar body abstriction, 1.5–2.5 hr postinsemination. A correlation was apparent between the degree of polyspermy and the total number of sperm lost by this mechanism. Of 82 dispermic eggs studied, 36 underwent sperm loss by blebbing, a process that restored the monospermic condition. The sequential steps in the abstriction process are depicted in micrographs of whole mounts of fixed eggs. A sperm head or male pronucleus could be seen in isolated blebs. The prevention of bleb formation by exposure of penetrated eggs to cytochalasin B largely eliminated any difference in sperm number when the mean number of sperm per egg was compared at 2, 4, and 6 hr postinsemination. Sperm abstriction may be a novel sperm exclusion mechanism employed by mammalian eggs. Evidence is also presented that an unknown mechanism of sperm exclusion is operative in mouse eggs, since sperm loss by abstriction did not account for all sperm loss.

An in vitro penetration assay utilizing human sperm and zona-free hamster eggs was employed to evaluate human sperm fertilizing capacity for 36 patients from the infertility clinic and 9 donors of proven fertility. The infertility patients were grouped according to the normality of their semen analyses. Test results were different for the three groups: a mean penetration level +/- standard deviation of 81% +/- 26% was obtained for proven fertile donors, while a value of 14% +/- 17% was observed for infertile couples with an abnormal semen analysis. A mean +/- standard deviation penetration level of 48% +/- 33% was associated with infertile couples in which the semen analysis was normal. These groups were statistically different (P less than 0.02) when inseminations were conducted at equivalent concentrations of motile sperm. No correlation was obvious between penetration test results and any of the parameters of the semen analysis; however, penetration test results did not correlate positively with the survival index (sperm survival at the end of insemination). These results are discussed in relation to further clinical application of the test.

Human sperm become capable of penetrating zona-free hamster eggs after preincubation in an appropriate culture medium. This observation has led to the development of an assay for characterizing the fertilizing capacity of human spermatozoa. In the present study, the incorporation of sperm by zona-free hamster eggs was quantitated, and several parmeters that contribute to penetration were evaluated. The importance of the motile sperm concentration was established; no penetration was seen at concentrations lower than 6 x 10(5) motile cells/ml, whereas above this level the mean number of incorporated sperm per egg was linearly related to concentration. Freeze-thawed sperm, although capable of penetrating zona-free hamster eggs, did so with lesser frequency than did fresh sperm at equal concentrations of motile sperm. Kinetic experiments indicated that eggs were maximally penetrated after 5 hours of exposure to capacitated sperm and that the cessation in sperm incorporation seen at this time resulted from egg-related changes that occurred during aging in vitro. A protocol for evaluating "fertilizing capacity" of human sperm samples was outlined incorporating the findings from the present study. Using these conditions, reproducible penetration levels were obtained when several ejaculates obtained from the same donor over a 3-month interval were tested at similar motile sperm concentrations.

ABSTRACT: A liquid‐phase radioimmunoassay was used to quantitate sperm‐associated IgG in a population of patients with unexplained infertility. Plasma IgG antisperm antibody activity was identified in a subpopulation of these patients. Using the human sperm/hamster egg assay, a unique functional test of human sperm penetrating capacity, we identified a subset of these positive patients whose plasma significantly and reproducibly inhibited penetration. This study implies that antigen‐antibody reactions may prevent sperm responses that are critical to sperm‐egg fusion.

Human cervical mucus was collected from seven donors during the follicular, ovulatory and luteal phases of the ovulatory menstrual cycle. Individual mucus samples were solubilized and fractionated on Sepharose columns into excluded mucins and low-molecular-weight proteins. Mucin fractions were highly purified, as evidenced by the presence of a single N-terminal amino acid residue, threonine, and by the absence of contaminating plasma proteins. Amino acid compositions of mucins isolated during different menstrual phases of a single donor or from different donors were similar. Mucin carbohydrate compositions were also similar, except for the sialic acid-to-fucose ratio, which varied significantly between donors but not within the menstrual cycle of a single donor. An analysis of variance was applied to evaluate the contribution of mucin composition to viscoelasticity, as quantitated by microrheometry. Viscoelasticity was dependent on the donor, on the percent nondialyzable solids and on the mucin content, but not on the phase of the menstrual cycle during which the sample was collected. These findings suggest that mucus function (viscoelasticity) is reflected in carbohydrate composition and/or structure and that this menstrual relationship is unique for each donor. Furthermore, the absence of menstrual phase-dependent differences in mucins suggests that mucin concentration and not composition changes in response to alterations in the hormonal milieu.

Three cytologically distinct cell populations were identified, in addition to ciliated cells, when a unit gravity sedimentation procedure was applied to pronase-dispersed rabbit endocervical cells. Two of these cell populations contained histochemically distinguishable (periodic acid- Schiff [PAS]) mucoproteins and were designated vacuolated and granular PAS-positive cells. The third, designated as vacuolated PAS-negative, did not contain secretory granules. Cell integrity was confirmed by trypan blue dye exclusion, [(3)H]leucine incorporation, and ultrastructural analysis. To demonstrate hormonal modulation of endocervical cell morphology, cell distribution profiles were compared from animals in different hormonal states. In the absence of estrogen dominance, PAS- positive cells from 5-d pseudopregnant rabbits were reduced 50 percent, while vacuolated PAS-negative cells increased fourfold as compared with estrous cell populations. The PAS-positive cells sedimented toward the top of the gradient where the bovine serum albumin concentrations were lower, consistent with a reduction in the number of secretory granules. In the sustained absence of ovarian steroid hormones, the number of PAS-positive mucous cells from ovariectomized rabbits was reduced to only 4 percent of the total endocervical cell population. The biosynthetic capacity of isolated endocervical cells was determined by incubating the three nonciliated cell populations from estrous and 5-d pseudopregnant rabbits for 36 h with the mucin precursor, [(14)C]N-acetyl- D-glucosamine. Only PAS-positive cells incorporated significant amounts of labeled precursor. This study indicates that steroid hormones influence cervical secretions by modulating the type of endocervical cells.

Mucus viscoelasticity on individual samples obtained from patients using combination oral contraceptives was quantitated by microrheometry. These results, in conjunction with mucus chemical characterization, indicate that combination oral contraceptive use eliminates the cyclic variations in mucus chemical, physicochemical, and rheologic properties associated with the ovulatory menstrual cycle. A correlation was demonstrated between the mucus elastic modulus and mucus nondialyzable dry weight, and the mucins produced during oral contraceptive therapy were shown to be similar to those recovered from ovulatory donors. Differences in mucus properties were noted when donors using estrogenic contraceptives were contrasted with those using androgenic contraceptives. On the basis of established relationships between sperm penetrability and mucus solids content, it was concluded that the use of contraceptives, as examined in this study, provided a secondary degree of fertility control at the cervical level.

Human sperm fertility potential, expressed as a quality index (QI), was evaluated objectively from considerations of sperm velocity, percentage of motile forms, sperm density, and ejaculate volume. Turbidimetry was applied in the quantitation of sperm velocity. High QIs (700) were characteristic of semen samples that were capable of penetrating cervical mucus in capillary tube penetration tests, while low QIs (80) were associated with specimens that did not penetrate mucus. The rate of decline in QI as a function of time postejaculation was determined for samples stored at 37 degrees C and at ambient temperature, providing correction factors for the comparative evaluation of semen samples analyzed at different times. The penetrability of cervical mucus was determined by capillary tube penetration testing. Subsequent chemical analysis of sperm-penetrable and impenetrable samples indicated that the concentrations of mucus nondialyzable solids (NDS), mucins, and soluble proteins were significantly higher in impenetrable specimens. These characteristics were also observed in impenetrable mucus obtained from patients with cervical factor infertility. These studies indicate that it is now feasible to employ mucus [NDS] in the diagnosis of cervical factor infertility and turbidimetry in the evaluation of human sperm quality.

The role of cortical granules in the mouse egg's plasmalemma block to polyspermy was investigated by examining the effect of premature granule loss on egg fertility. Granule loss, quantitated by transmission electron microscopic examination, was induced in zona-free eggs by exposure to the divalent cation ionophore, A23187, or by mechanical removal of zonae. Egg exposure to ionophore led to the loss of approximately 50% of the egg's complement of granules in the absence of nuclear activation (parthenogenesis), while complete cortical granule loss accompanied the parthenogenetic activation seen in a limited population of mechanically stimulated eggs. Aged eggs underwent nuclear activation without a dramatic reduction in granule complements. The fertility of treated zona-free eggs was identical to that of controls, as measured by the percentage of eggs penetrated and by the mean number of sperm recovered per egg. Moreover, both ionophore-treated and aged eggs subsequently underwent a normal sperm-induced block response. Exposure of zona-intact eggs to ionophore was also without effect on egg fertility. These results indicate that cortical granules are not involved in the plasmalemma block to polyspermy in the mouse.

The spontaneous maturation of intact, cumulus-free and zona-free oocytes of the mouse was studied in complex and in simple media. The rates and frequencies of maturation from the germinal vesicle to the metaphase II stage were similar for all oocytes, indicating that these investments are not critical to maturation. The penetration characteristics of zona-free oocytes were examined; before germinal vesicle breakdown (GVB), evidence for penetration was obtained only when the ionophor, A23187, was included in the medium. After GVB, oocytes readily incorporated spermatozoa and were usually polyspermic when inseminated with 10(5) capacitated epididymal spermatozoa/ml. The kinetics of sperm incorporation indicated that primary oocytes were capable of a plasmalemma block to penetration similar to that seen with tubal eggs.

The sperm binding properties of the mouse egg plasmalemma were assessed following an in vitro challenge of zona-free eggs with capacitated epididymal sperm. Unpenetrated eggs were capable of binding more sperm than were their in vivo or in vitro penetrated counterparts over a wide range in sperm concentration. Only capacitated sperm bound and binding was physiologic because it culminated in penetration of the vitellus. Kinetic analysis of these phenomena indicated that sperm detached after the egg was penetrated and it is suggested that this is a cause and effect relationship reflecting early aspects of the block to polyspermy response.

AbstractConditions were established for routine cinemicrographic examination of sperm incorporation by living zona‐free mouse eggs employing oil immersion objectives and Nomarski optics. Initial sperm attachment to the egg plasma membrane, which was reversible and appeared to require flagellar activity, involved localized areas of the head corresponding approximately to the position of the equatorial segment. Penetrating sperm lay flat on the egg and, during incorporation, appeared to sink into the egg cytoplasm, accompanied by short bursts of flagellar activity and subsequent rotation of the flagellum around its insertion point. Ensuing sperm head decondensation involved dissociation of individual particulate structures and a dramatic localized clearing in the egg cytoplasm. The normalcy of the penetration process and the potential applicability of this approach was attested to by the observations that polar body extrusion, male and female pronuclear formation, and migration through the egg cytoplasm in preparation for syngamy occurred in several sequences followed for extended time periods.

AbstractThe anionic fluorescein dye, Erythrosin B, inhibited the penetration of mouse eggs in vitro. High concentrations of the dye (0.5–1.0 mM) inhibited the penetration of cumulus‐intact, cumulus‐free, and zona‐free eggs at sperm concentrations of approximately 2 × 105 cells per milliliter. Examination of gamete viability indicated that the dye did not cause gross, irreversible changes in either amete, and we therefore concluded that dye‐dependent inhibition may have resulted from dye association with cell membranes. The dye concentration required to give 50% inhibition, apparent I50, of 500 μM obtained for zonafree eggs was artificially elevated by the presence of bovine serum albumin (BSA) (2%) in the culture medium. A corrected I50 of 11 μM was estimated in the absence of BSA.

The minimal requirements and characteristics of epididymal sperm binding to the zona pellucida of the mouse egg were investigated using a new stop-fix centrifugation technique. This assay provided a precise physical definition of the association between the spermatozoon and the zona and permitted quantitation of the binding reaction at short time intervals. The results demonstrated that Ca2+ is an essential physiological component required for binding to occur. Sperm preincubated for 60 min in a simplified medium lacking Ca2+ did not acquire the ability to bind to eggs. In contrast, if sperm preincubation occurred in this medium supplemented with 1.7 mM Ca2+, binding was identical to that observed following sperm preincubation in the complete culture medium which supports both capacitation and fertilization in vitro. The Ca2+-dependent binding reaction was rapid, reversed by EGTA, specific for Ca2+, and did not require the transport of Ca2+ into the cell. Sperm bound to the zona surface following preincubation with Ca2+ were capable of fertilization in vitro when the eggs were subsequently transferred to the culture medium. It is proposed that this binding reaction represents a part of capacitation and not the acrosome reaction.