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PURPOSE: The purpose of this study was to localize fibronectin on human sperm and correlate its distribution with the morphological and functional integrity of sperm. MATERIALS AND METHODS: Semen samples were collected and sperm fractionated by swim-up. Subsets of the swim-up sperm were capacitated and acrosome reacted. Damage to swim-up sperm was induced by freezing and thawing. The presence of fibronectin on the surface of sperm was determined by immunocytochemistry. RESULTS: FN immunoreactivity was variable but staining on the sperm tail was consistently highest, whereas FN immunoreactivity over the acrosome and equatorial band was consistently lowest. Capacitation and acrosome reaction did not substantially change the distribution of FN staining. However, swim-up sperm had significantly less FN immunoreactivity (4%) than sperm that were unable to swim-up (12%; p < 0.01). Sperm that were deliberately damaged by freeze/thaw showed significantly increased FN binding (p < 0.01). FN immunoreactivity was inversely correlated with sperm viability (r = -0.68), motility (r = -0.70), and morphology (r = -0.63). CONCLUSIONS: This study demonstrates that only a minority of the sperm in an ejaculate stain positive for FN and the localization of FN in positive sperm is primarily to the tail. Inferior sperm stain more frequently for FN leading to an inverse correlation between FN staining and sperm quality. Taken together, these results do not support a role for FN in sperm-egg binding. However, FN staining may provide a method for selecting the highest quality sperm for use in assisted reproduction techniques.

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It has been demonstrated that opioid peptides are involved in the regulation of mammalian reproduction. In our previous studies we demonstrated direct effects of opioids on preimplantation mouse embryos, and hypothesized the existence in preimplantation embryos of receptors similar to opioid receptors in the central neuronal system of adult animals. In the present study we addressed this issue by employing immunocytochemical staining for mu-opioid receptors using antisera raised against the C-terminal portion of the cloned mu-opioid receptors (MOR1, NHQLENLEAETAPLP, 384-398) predicted from the cloned receptor. Diffuse MOR1 immunoreactivity of moderate intensity has been revealed in one-cell embryos, while in follicular cells MOR1 staining was of high intensity and appeared to be associated with plasma membrane. No MOR1 immunoreactivity has been observed in two-cell to morula stages of development. However, blastocysts displayed intense MOR1-labeling that was particularly prominent in cells within the inner cell mass. MOR1-staining was most likely specific because preincubation of MOR1 antisera with cognate peptide completely abolished the staining. Our findings suggest the presence of opioid receptors during preimplantation development, long before the formation of the nervous system. Embryonic opioid receptors may play a role in the regulation of preimplantation development and implantation.

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OBJECTIVE: To determine if TEST-yolk buffer, consisting of TES (N-tris [hydroxymethyl]-methyl-2-aminoethanesufonic acid), Tris (Tris[hydroxymethyl]aninomethane), and chicken egg yolk, affects the presence of antisperm antibodies on the sperm surface as detected by the immunobead test. DESIGN: A prospective study of antisperm antibodies on sperm surface before and after incubation in TEST-yolk buffer. Direct immunobead test and indirect immunobead test were done the day of collection of the semen sample to detect the presence of human immunoglobulin class G (IgG) and immunoglobulin class A (IgA); immunobead tests were repeated on the same sperm samples after 24 hours of storage in TEST buffer. SETTING: Academic tertiary institution. PARTICIPANTS: Patients undergoing evaluation for infertility. RESULTS: There was no significant difference in the outcome of the direct immunobead test after extending semen samples with TEST-yolk buffer for 24 hours at 4 degrees C. Eleven samples that were initially negative for IgG and 13 samples that were negative for IgA remained negative after 24-hour storage in TEST-yolk buffer. Eleven samples that were positive for IgG and nine samples that were positive for IgA by the direct immunobead test the first day remained positive the next day. Five extended sperm samples used in the indirect immunobead test with IgG positive serum gave positive results and four of five used with IgA positive serum gave positive results. CONCLUSIONS: These findings suggest that TEST-yolk buffer can be used to extend semen without affecting the presence of antibodies on the sperm surface as indicated by the direct immunobead test. The higher variability of the indirect immunobead tests indicates there may be some alteration of sperm antigens after storing in TEST-yolk buffer. These findings indicate that TEST-yolk buffer can be used to store semen for batched processing of samples or as a transport medium for delivery to a central laboratory for antibody testing.

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Sexual dysfunction and infertility are common problems following spinal cord injury. Most men with complete spinal cord lesions do not ejaculate during coitus. Vibratory stimulation applied to the frenulum of the penis in six quadriplegic male subjects produced ejaculates for intrauterine inseminations. Pregnancies occurred in five of the six partners. Vibratory stimulation is a relatively safe and effective means to produce an ejaculation in men with quadriplegia.

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A large body of evidence suggests multiple forms of 17 beta-hydroxysteroid oxidoreductase (17-HOR) regulate estrogen and androgen levels within gonadal and peripheral tissues. Two kinetically-differing 17-HOR activities have been detected in placental homogenates. 17-HOR type 1, found mainly in the cytosol, is highly reactive with estradiol-17 beta (E2) and estrone (E1) but not testosterone (T) (high E2/T activity ratio). Microsomal 17-HOR type 2 is reactive with both E2 and T (low E2/T activity ratio). In this study, 17-HOR activity of cytosol and microsomes from term placenta, ovarian stroma and granulosa-luteal cells was assayed under conditions which specifically differentiate between the two forms of the enzyme. Placenta had the highest activity with either E2 or T in both cytosol and microsomes and stroma the lowest. The highest specific activity with E2 and E1 was cytosolic in all samples. The highest specific activity with T was microsomal in placenta and ovarian stroma. E2/E1 activity ratios were comparable for cytosol and microsomes while E2/T activity ratios were comparable for placenta and stroma, but markedly elevated in granulosa-luteal (G-L) cell cytosol and microsomes. The results indicate trophoblast and ovarian stroma have more 17-HOR type 2 relative to type 1. G-L cells, in contrast, are relatively enriched in 17-HOR type 1 and thus have a greater capacity for net conversion of E1 to E2 under physiologic conditions. These differences may contribute to increasing serum and follicular fluid E2/E1 ratios during development of the dominant follicle.

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The specific activity of 17 beta-hydroxysteroid oxidoreductase (17-HOR) with estradiol-17 beta (E2), estrone (E1) and testosterone (T), as well as that of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) were measured in homogenates of CF-1 mouse placenta during the latter half of pregnancy. 17-HOR activity with E2 and T increased over 100-fold between days 9 and 12, and 3- to 4-fold between days 15 and 19, with no further change to day 21. In contrast, activity with E1 increased 39-fold between days 9 and 12, 3.8-fold between days 15 and 19 but then decreased between days 19 and 21. The E2/T activity ratio was constant while the E2/E1 ratio increased between days 9 and 21. LDH increased 2-fold between days 9 and 12 with no further increase to day 19. MDH was constant from day 9 to 19. Activity with E2 was inhibited by T, 5 alpha-dihydrotestosterone (5 alpha-DHT) and DHA but not by E1, androstenedione (A) or 20 alpha-dihydroprogesterone. Activity with T was inhibited by E2, 5 alpha-DHT and DHA, but not by A. In contrast, activity with E1 was inhibited by A and DHA but not by E2, T or 5 alpha-DHT. The results suggest placental 17-HOR is developmentally regulated. Although the results are also suggestive of multiple forms of 17-HOR, a single enzyme with an ordered kinetic mechanism cannot be ruled out.

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OBJECTIVE: To study the effect of human follicular fluid (FF) and the specific contribution of its epidermal growth factor (EGF) component on the in vitro maturation of cumulus-enclosed mouse oocytes. DESIGN: A previously described mouse oocyte model system was used to study the effect of FF on oocyte maturation before and after extraction of EGF by immunoprecipitation. Follicular fluid specimens enclosing both mature and immature human oocytes were tested. MAIN OUTCOME MEASURES: The endpoints assessed were the percentage of oocytes undergoing germinal vesicle breakdown (GVBD) and polar body one formation at different intervals over a 24-hour period and the final degree of cumulus expansion achieved. RESULTS: A concentration-related stimulatory effect of mature FF was noted when compared with the spontaneous increase of GVBD and polar body one formation observed for the EGF-free control medium. Overall, the effect of immature FF was inhibitory. After extraction of EGF from FF by immunoprecipitation from both immature and mature FF, the rates of GVBD and polar body one formation were decreased in both groups. The addition of 5 ng/mL of EGF to the extracted groups reversed this effect on polar body one formation. Cumulus expansion was maximal for oocytes incubated with mature FF and minimal for those incubated with EGF-free media. CONCLUSIONS: The positive effect of mature human FF on mouse oocyte maturation and cumulus expansion is to a large extent because of the presence of EGF.

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OBJECTIVE: Immature mammalian oocytes cultured in vitro undergo inadequate cytoplasmic maturation and hence have a limited potential for fertilization. Our primary objective was to determine if the addition of epidermal growth factor (EGF) to the in vitro culture system would have a positive effect on oocyte cytoplasmic maturation. DESIGN: We studied the effect of different EGF concentrations on both denuded and cumulus-enclosed mouse oocytes cultured in vitro. MAIN OUTCOME MEASURES: The percentage of oocytes undergoing germinal vesicle breakdown (GVBD) and polar body one formation over time as a function of EGF concentration was determined. RESULTS: A dose-related positive effect of EGF on both GVBD and polar body one formation over time was observed for mouse oocytes. As well, a similar effect of EGF was seen on immature human oocytes that had not been stimulated with exogenous gonadotropins. CONCLUSIONS: The use of EGF may allow for the performance of successful in vitro fertilization procedures using immature human oocytes retrieved during unstimulated cycles.

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Nicotine at concentrations found in the cervical mucus of female smokers appeared to enhance in vitro human sperm penetrability through ovulatory bovine cervical mucus. Sperm motility parameters were not affected by the addition of nicotine to semen samples incubated with BWW medium. Overall, these results suggest that a direct inhibitory effect of nicotine on sperm penetrability through cervical mucus is not responsible for the apparent increase in cervical factor infertility present in smoking women.

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Cumulus enclosed primary oocytes from 2 to 4-mm bovine follicles were matured in vitro in Minimum Essential Medium containing follicle-stimulating hormone (0, .1, 1, 10, 50, or 100 micrograms/ml) or human chorionic gonadotropin (0, .1, 1, or 10 IU/ml) for 48 h at 37 degrees C under paraffin oil. Cumulus mass expansion comparable to that seen in vivo occurred in 18% of the control oocytes, 39% of those cultured in human chorionic gonadotropin, and 56% of those cultured in follicle-stimulating hormone. The optimum follicle-stimulating hormone concentration for cumulus expansion was 1 microgram/ml, and this was then used to mature oocytes individually or in groups of 5 for in vitro fertilization. Ejaculated bovine semen, extended 1:10 with yolk-TES-Tris extender and stored 24 to 48 h at 4 degrees C, was warmed, washed once with Minimum Essential Medium, and 500,000 motile sperm/ml were used to inseminate the matured oocyte-cumulus cell complexes. Criteria for fertilization was cleavage to the two-cell stage 48 h after insemination. Oocytes, inseminated individually, cleaved with a frequency of 5%, whereas 15% of those inseminated in groups of 5 cleaved, perhaps as the result of cumulus factors enhancing capacitation. The cleavage rate for the parthenogenetic control with killed spermatozoa was 0%. Therefore, primary oocytes matured in vitro to secondary oocytes were successfully fertilized in vitro and cleaved to at least the two-cell stage in the Minimum Essential Medium. Individual differences between bulls in ability to fertilize in vitro were noted.

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The relation of extra-embryonic fluid weight to litter number and foetal and placental weight was studied in mice on the 18th day of pregnancy, in litters both of experimentally reduced and of normal number. Partial regression analysis showed that litter number and foetal weight both exerted a negative effect on fluid weight; placental weight had no significant effect. Increased foetal weight reduced weight locally; on the other hand the effect of litter number was exerted systemically, throughout both horns of the uterus.

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Fetal fluid was studied in mice of the Q, C57BL/McL, C3H/Bi/McL and JU/Fa strains. The amount increases to day 16 of gestation, and then decreases. Amniotic fluid and fetal plasma contain three transferrins, five alpha-fetoproteins and albumin. Protein concentration in both increases during gestation; glucose decreases in amniotic fluid although it rises in fetal plasms; urea levels remain constant. The regulation of volume and biochemical composition of amniotic fluid, and its relation to the yolk-sac membrane, are discussed.

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