Thomas T. F. Huang

Phospholipase A of Guinea Pig Spermatozoa: Its Preliminary Characterization and Possible Involvement in the Acrosome Reaction

Phospholipase A activity was demonstrated in guinea pig spermatozoa using [U‐14C] phosphatidyl choline as a substrate. The activity had a neutral pH optimum, was stimulated by Ca2+ and low concentrations of detergent, and. was inhibited by EDTA, mepacrine and p‐bromophenacyl bromide. Appropriate concentrations of mepacrine and p‐bromophenacyl bromide inhibited the acrosome reactions of capacitated spermatozoa without interfering with their motility. These results support the notion that phospholipase A is involved in the acrosome reaction of mammalian spermatozoa.

The effect of peritoneal fluid from patients with endometriosis on human sperm function in vitro

OBJECTIVE Our purpose was to evaluate the effect of peritoneal fluid from women with endometriosis on sperm motility and function in an in vitro model. STUDY DESIGN Peritoneal fluid was collected at laparoscopy from patients with and without endometriosis. Human donor sperm was diluted with this fluid, and its effect on sperm function and motility was measured was measured with the zona-free hamster egg sperm penetration assay and computer-assisted semen analysis. RESULTS The mean number of eggs penetrated by the sperm mixed with peritoneal fluid from patients with endometriosis was significantly fewer than the number penetrated by the sperm mixed with fluid from control patients (22.9 +/- 5.31 vs 44.4 +/- 4.96, p < 0.01, Student t test, n = 20). When evaluated by computer-assisted semen analysis, sperm mixed with peritoneal fluid from patients with endometriosis showed a significant decrease in mean swimming velocity compared with sperm mixed with peritoneal fluid from control patients (54.0 +/- 1.77 vs 59.2 +/- 1.05, p = 0.02, Student t test, n = 20). A significant increase in the fraction of sperm swimming at slower velocities was also found. A trend toward a positive correlation between eggs penetrated and sperm velocity was seen, but statistical significance was not achieved (correlation coefficient 0.4392, p = 0.053, n = 20). CONCLUSION These data suggest that substances found in the peritoneal fluid of patients with endometriosis could contribute to infertility through impairment of both sperm function and motion kinematics.

Impact of Vitrification on the Meiotic Spindle and Components of the Microtubule-Organizing Center in Mouse Mature Oocytes

ABSTRACT Cryopreservation of oocytes is becoming a valuable method for fertility preservation in women. However, various unphysiological alterations occur in the oocyte during the course of cryopreservation, one of which is the disappearance of the meiotic spindle. Fortunately, the meiotic spindle does regenerate after thawing the frozen oocytes, which enables completion of meiosis and further development after fertilization. Nonetheless, the mechanistic understanding of the meiotic spindle regeneration after cryopreservation is still scarce. Here, to gain insight into the mechanisms of the spindle disappearance and regeneration, we examined the status of spindle microtubules as well as the key components of the microtubule-organizing center (MTOC), specifically gamma-Tubulin, NEDD1, and Pericentrin, in mature (metaphase II) mouse oocytes at different steps of vitrification, a major cryopreservation technique. We found that the configuration of the spindle microtubules dynamically changed during the process of vitrification and that spindle regeneration was preceded by excessive microtubule polymerization, followed by reduction into the normal size and shape. Also, all three MTOC components exhibited disappearance and reappearance during the vitrification process, although Pericentrin appeared to regenerate in earlier steps compared to the other components. Furthermore, we found that the localization of the MTOC components to the spindle poles persisted even after depolymerization of spindle microtubules, suggesting that the MTOC components are impacted by vitrification independently from the integrity of the microtubules. The present study would set the stage for future investigations on the molecular mechanisms of the meiotic spindle regeneration, which may contribute to further improving protocols for oocyte cryopreservation.

Short-Term Storage of Human Spermatozoa in Electrolyte-Free Medium Without Freezing Maintains Sperm Chromatin Integrity Better Than Cryopreservation

Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage. Short-term (1 week) storage without freezing of sperm in electrolyte-free medium maintains chromatin integrity better than conventional cryopreservation.

Cytoplasmic transfer in the mouse in conjunction with intracytoplasmic sperm injection.

Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been used clinically to facilitate human pregnancies. Data reported here describe the first characterization of CT coincident with intracytoplasmic sperm injection in the mouse system. Sibling oocytes were used to transfer 2, 4, or 6 pl of ooplasm to a recipient egg along with a sperm head using piezo-actuated injection. Survival and fertilization after CT were comparable to controls at 2 pl and 4 pl, but survival was significantly reduced with 6 pl volumes. Development to the blastocyst stage was also inversely related to CT volume, with some decline beginning with the 4 pl CT group. However, some blastocysts did develop in all of the groups. The results are in contrast with human eggs, which tolerate larger CT volumes. Results indicate that the mouse system can be used to characterize the transfer of exogenous materials concomitant with sperm injection, provided that the CT volume is not excessive.

Freezing-Free Preservation of Human Spermatozoa – A Pilot Study

This study tested a method for maintaining human spermatozoa without freezing for subsequent use in intracytoplasmic sperm injection (ICSI). We demonstrated that human sperm stored in electrolyte-free solution maintain their motility and viability for at least 4 and 6 weeks, respectively. We also have shown that preserved spermatozoa are fully functional in ICSI. Sperm chromosome analysis after injection of human sperm into mouse oocytes revealed that two weeks of storage does not negatively affect sperm DNA integrity. A mouse model was used to analyze the ability of preserved sperm to participate in normal embryogenesis. Mouse sperm preserved in electrolyte-free solution in a similar manner as human sperm maintained motility for up to 3 weeks. When mouse spermatozoa stored for 1 week were injected into the oocytes by ICSI, they yielded normal blastoctysts and normal viable fetuses. The results of the study bear significance for human assisted reproduction technologies (ART) and provide clinicians and infertile patients with a new method that can simplify sperm preparation for ICSI, assisting men who are unable to provide semen on the day of assisted fertilization.

Effects of pentoxifylline on sperm motility and hyperactivated motility in vitro: a preliminary report**Presented in part at the 47th Annual Meeting of The American Fertility Society, Orlando, Florida, October 19 to 24, 1991.

Previous reports (2, 3) have suggested that pentoxifylline increases sperm motility. In this preliminary report based on five asthenozoospermic and five normal motility semen samples, we were unable to demonstrate any statistically significant effect of pentoxifylline on percent motility of human spermatozoa. However, in vitro exposure to capacitation medium with pentoxifylline may lead to an increase in total hyperactivated motility in asthenozoospermic samples, an effect not evident in the normal motility samples in this study.