Hovey Lambert

In vitro studies of the golden hamster sperm acrosome reaction: Completion on the zona pellucida and induction by homologous soluble zonae pellucidae☆

We have studied the occurrence of the golden hamster sperm acrosome reaction (AR) in vitro during interaction with the oocyte investments: the cumulus cell matrix and the zona pellucida. Hamster sperm were capacitated in a defined medium that does not induce the AR. These spermatozoa were allowed to interact with the ovum vestments, the events of which were recorded using high-speed videomicrography. Frame-by-frame analysis revealed that sperm did not complete the AR in the cumulus cell matrix, but did so on the zona pellucida. Furthermore, a higher percentage of sperm completed the AR on the zona pellucida of cumulus-invested than on cumulus-free eggs. We also investigated the effect of solubilized hamster and mouse zonae pellucidae on the hamster sperm AR. Addition of solubilized hamster zonae to capacitated sperm elicited the AR within 15 min. Solubilized mouse zonae were significantly less effective, indicating that the zona-induced AR in hamster sperm may be species specific. These results suggest that the hamster zona pellucida is an inducer of the AR in the intact or soluble form, and that the majority of spermatozoa traverse the cumulus cell matrix without completing the AR in our in vitro system.

Synergistic effect of intraperitoneally administered calcium channel blockade and recombinant tissue plasminogen activator to prevent adhesion formation in an animal model.

Previous reports have shown the benefits of calcium channel blockers and recombinant tissue plasminogen activator to prevent postoperative adhesion formation in animal models. To assess the potential benefit of synergistic therapy for the prevention of postoperative adhesion formation, these agents were studied in a rabbit uterine horn model. Four groups of New Zealand White rabbits (n = 8 per group) had a bilateral devascularization injury to the uterine horns. Before closure saline solution, verapamil hydrochloride (2.5 mu/kg/hour), recombinant tissue plasminogen activator (4 mg total dose), or a combination of verapamil and recombinant tissue plasminogen activator at the stated doses were instilled by means of an Alzet osmotic pump x 200 hours. Adhesion scores were evaluated after this time period by estimating the total uterine horn surface involved in adhesions at a terminal laparotomy and by clinically grading the response to determine whether minimal adhesions formed. Results of the total uterine horn surface scores were (mean score +/- SE): saline solution, 44% +/- 3.7%; verapamil, 19% +/- 4.8%; recombinant tissue plasminogen activator, 11% +/- 3.6%; combined, 3% +/- 1% (p less than 0.01 to control and p less than 0.05 to single-drug therapy). Results of the number of animals per group with minimal adhesions were as follows: saline solution, 0; verapamil, 1; recombinant tissue plasminogen activator, 3; combined, 8 (P less than 0.01). These results show a synergistic benefit of verapamil and recombinant tissue plasminogen activator to prevent postsurgical adhesion formation when delivered via the intraperitoneal route.

Expression of CD15 (Lewisx) antigen on human sperm and its role in sperm-egg interaction

PROBLEM: The carbohydrate epitope 3‐fucosyl‐N‐acetyllactosamine (CD15) is a constituent of cell surface glycoconjugates that has been implicated in cell‐cell adhesion mediated by carbohydrate‐specific ligands. The present study was designed to investigate whether CD15 is present on human sperm and whether it plays a role in human sperm‐egg interaction.

Sperm binding activity of the zona pellucida of immature mouse oocytes

Immature oocytes taken from ovarian follicles are sometimes used in studies of sperm-zona interaction in species for which it is difficult to obtain ovulated eggs. As yet, however, there has been no quantitative comparison of the sperm binding capacities of immature and ovulated oocytes. We report here that in mice there is no significant difference in the numbers of sperm which bind to the zonae pellucidae of immature and ovulated oocytes in vitro. These results support the use of immature oocytes in studies of sperm-zona interaction. We have also analyzed the sources of variability in sperm binding assays, and we make suggestions for the most efficient design of experiments.

Reduction of primary posttraumatic adhesion formation with the prostacyclin analog iloprost in a rodent model

Recent evidence suggests that inhibition of postsurgical adhesion formation may be effected by modulation of the activities of inflammatory cells contributing to mesothelial repair. Iloprost, a stable analog of prostacyclin, has been shown to exert vasodilatory, antiinflammatory, fibrinolytic, and antithrombotic influences. To determine whether these properties of iloprost might protect mesothelial surfaces from perioperative damage and hence prevent adhesion formation, we evaluated the effect of iloprost on peritoneal healing in a hamster model for primary pelvic injury. Perioperative iloprost therapy significantly reduced posttraumatic adhesion formation when compared with that in vehicle-treated controls. Dose-response studies demonstrate adhesion prevention with doses ranging from 0.04 to 4 mg/kg per 8 hours given subcutaneously over the course of 3 days. These data demonstrate that iloprost is a potent positive modulator of peritoneal healing after pelvic trauma. Further studies to characterize the potential application of iloprost as an adjuvant in reproductive surgery are indicated.

Case report: Treatment of congenital vas obstruction with sperm aspiration, nonstimulated in fitro fertilization, and nonsurgical tubal embryo transfer

Among the factors that can explain differences between spontaneous and induced pregnancies, the characteristics of the infertile population can play an important role, and recently some studies (3,4) have shown that infertile couples have a higher risk of obstetrical complications than fertile couples even without IVF. We want to point out the fact that the method of calculating the duration of IVF pregnancies can also be involved. Usually, the term of IVF pregnancies is estimated by adding 14 days to the date of oocyte retrieval to obtain a theorical number of weeks of amenorrhea. This supposes that ovulation occurs at the 14th day in natural cycles on average. However, the literature is not totally consistent on this point. For example, Spira et al. (5) found a mean hypothermic phase of 18 days for 894 patients trying to obtain a natural pregnancy. In the study of 162 IVF singleton pregnancies conceived in Clamart, France, between 1987 and 1989, the prematurity rate (<37 weeks) changed from 10.7% when we added 14 days to 7.5 and 5.7% when we added 16 and 18 days, respectively. When we did the same calculation on the 916 singleton pregnancies in the French collaborative study (2), we observed the rates of 12.2, 9.3, and 7.8% when we added 14, 16, and 18 days, respectively, to the day of oocyte retrieval. In fact, the choice of retrieval day or reimplantation day as a reference can change the ratio of prematurity. A difference of 4 days in the mode of calculation of the term can halve the global prematurity rate, which can be explained by the high number of births occurring between 36 and 37 weeks of amenorrhea. It can also decrease early prematurity. Even if prematurity cannot be explained only by differences in the date of ovulation, further studies are needed to find a dating system allowing a nonbiased comparison between birth resulting from assisted and birth from natural conception. 4. Williams M, Goldman M, Mittendorf R, Monson R. Subfertility risk of low birth weight. Fertil Steril 1991 ;56(4):668-671 5. Spira A, Spira N, Papiernik-Berkauer E, Schwartz D: Pattern of menstrual cycles and incidence of congenital malformation. Early Hum Dev 1985;1l:317-324

Detection of human leukocyte antigen class I messenger ribonucleic acid transcripts in human spermatozoa via reverse transcription-polymerase chain reaction*†*Supported by grants HD24495 and HD24180 from the National Institutes of Health, Bethesda, Maryland.†Presented at the 41st Annual Meeting of The Pacific Coast Fertility Society, Indian Wells, California, April 14 to 18, 1993.

OBJECTIVE To determine by reverse transcription-polymerase chain reaction (PCR) whether human leukocyte antigen (HLA) class I messenger RNA (mRNA) is present in mature human spermatozoa. DESIGN Mature human spermatozoa was isolated from donor semen using a swim-up technique. Total RNA was extracted via guanidinium isothiocyanate-cesium chloride ultracentrifugation. By the method previously validated in our laboratory, reverse transcription-PCR was performed using primers specific for HLA class I transcripts. Positive control cells included a choriocarcinoma cell line (JEG) and human fetal tissue. Transformed peripheral blood lymphocytes (PBL) were used as a negative control for somatic cellular contamination. RESULTS Human spermatozoa were positive for HLA class I (-G and -B) mRNA by reverse transcription-PCR, consistent with the positive controls. We did not detect any mRNA for beta-actin, retinoblastoma (RB), CD4, or kappa light chain genes in the sperm complementary DNA samples, verifying that the class I mRNA detected was not due to somatic cellular contamination of the purified sperm samples. CONCLUSION These experiments provide the first evidence that mRNA for HLA class I molecules are present in mature human spermatozoa. The physiological role of these transcripts is unknown at present. Further experiments characterizing the expression of HLA class I (-G and -B) mRNA in oocytes and preimplantation embryos are in progress.

Enhanced gamete interaction in the sperm penetration assay after coincubation with pentoxifylline and human follicular fluid**Presented at the 46th Annual Meeting of The American Fertility Society, Washington, DC, October 15 to 19, 1990.

OBJECTIVE To evaluate the effect of pentoxifylline and heat-inactivated human follicular fluid (FF) on performance in the sperm penetration assay (SPA) as a paradigm for the effect of these agents on human sperm-egg interaction in vivo and in vitro fertilization. DESIGN Semen specimens from men undergoing SPA testing for evaluation of suspected male factor infertility were coincubated with neat medium or media supplemented with pentoxifylline or human FF in a nonblinded manner. PARTICIPANTS Twenty male factor infertility patients. INTERVENTIONS Semen specimens were preincubated with: [1] pentoxifylline 0.25 mg/mL; [2] 10% human FF; [3] pentoxifylline+human FF; and [4] neat Biggers, Whitten, and Whittingham medium. MAIN OUTCOME MEASURES Differences in the rate of penetration of zona-free hamster oocytes. RESULTS Preincubation with either human FF or pentoxifylline produced a significant improvement in hamster egg penetration rates. Coincubation with a combination of human FF and pentoxifylline resulted in a significant enhancement of penetration as compared with single agent treatment. CONCLUSIONS Coincubation of sperm with human FF and pentoxifylline may provide a means of enhancing sperm activity for insemination and assisted reproduction.

Heterologous transplantation of activated murine peritoneal macrophages inhibits gamete interaction in vivo: a paradigm for endometriosis-associated subfertility**Supported by a grant from the Reproductive Biology Research Foundation, Torrance, California.

Macrophage hyperactivation has been postulated to be the pathologic aberration in patients suffering from endometriosis-associated subfertility. In this report an in vivo model for macrophage-mediated infertility is described. Populations of macrophages were obtained from an inbred strain of mice (Balb/C) as follows: (1) in vivo hyperactivated macrophages (harvested from donor mice treated with intraperitoneal thioglycolate); (2) hyperactivated macrophages deactivated ex vivo with the protein synthesis inhibitor emetine; and (3) basal state (nonactivated) macrophages obtained from untreated mice. Recipient mice underwent ovarian hyperstimulation with pregnant mare serum gonadotropins; 2 x 10(6) macrophages were transferred on the afternoon of stimulation day 3 before injection of human chorionic gonadotropin (hCG) and mating. Unfertilized oocytes and 4-cell embryos were counted on day hCG +2 as a reflection of reproductive performance. Heterologous transfer of in vivo hyperactivated macrophages, but not basal state macrophages, significantly inhibited fertilization. This effect was largely reversed by pretreatment with emetine. These experiments confirm the relevance of macrophage-mediated interference with early reproductive performance and provide a model for the development of alternative therapies (e.g., immunomodulation of the peritoneal fluid environment) for endometriosis-associated subfertility.