Pilar Vigil

Gonadotropin-Releasing Hormone-Stimulated Sperm Binding to the Human Zona Is Mediated by a Calcium Influx

Abstract The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca2+-free medium and in the presence of Ca2+ channel antagonists. We also examined the GnRH effect on the intracellular free Ca2+ concentration ([Ca2+]i). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca2+ was present in the medium. In Ca2+-free medium or in the presence of 400 nM nifedipine, 80 μM diltiazem, or 50 μM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca2+]i in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca2+]i was fast and transient, from a basal [Ca2+]i of 413 ± 22 nM to a peak value of 797 ± 24 nM. The GnRH-induced increase in [Ca2+]i was entirely due to a Ca2+ influx from the extracellular medium because the increase in [Ca2+]i was blocked by the Ca2+ chelator EGTA and by the Ca2+ channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca2+ influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca2+ influx but inhibited the progesterone-activated Ca2+ influx. Finally, the GnRH-induced Ca2+ influx was blocked by two specific GnRH antagonists, Ac-D-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3-Arg5-D-Glu(AA)6-GnRH and Ac-3,4-dehydro-Pro1,-p-fluoro-D-Phe2, D-Trp3,6-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca2+]i through T-type, voltage-operated calcium channels.

Scanning electron and light microscopy study of the cervical mucus in women with polycystic ovary syndrome

Two types of cervical mucus are recognized, oestrogenic and gestagenic. These are constituted by different subtypes, and their characteristics change depending on variations in the hormonal levels and on the existence of several pathologies. Our aim was to identify the ultrastructure and crystallization characteristics of the cervical mucus in women suffering from polycystic ovary syndrome, and to compare these characteristics with those of normal control women. Cervical mucus samples were taken from 10 women, 4 control group women (with normal ovulatory menstrual cycles) and 6 suffering from polycystic ovary syndrome (2 with ovulatory and 4 with anovulatory cycles). This mucus was characterized according to its ultrastructure and crystallization. The type of mucus obtained was related to the levels of oestradiol and progesterone present when the samples were taken. As regards mucus ultrastructure, differences were found between the control women and those with polycystic ovary syndrome and anovulatory menstrual cycles. Such variations were evident in the type of mesh and the average diameter of the mucus pores. Mucus crystallization in control women showed the usual oestrogenic disposition: fern-like (L, P2), rectilinear (S) or a hexagonal structure (P6). On the other hand, in women with polycystic ovary syndrome, indefinite mucus crystallizations were found, as well as crystallization patches resembling oestrogenic and gestagenic-like mucus. This study shows that the ultrastructure and crystallization characteristics of the cervical mucus in polycystic ovary syndrome women are different from those of control women. The latter would be dependent on their levels of oestradiol and progesterone.

Alkylating Agents and Mouse Spermatogenesis: Effects of a Single Dose of Cyclophosphamide

Summary:  Cyclophosphamide (CFA) is one of the alkylating agents which has now been used with some success in the treatment of human neoplasias and renal disease. To evaluate how this drug could affect the seminiferous epithelium, a single dose of CFA (200 mg/kg/weight) was given to twenty adult mice (strain A/Sw). The effect of the drug was compared with a control group to which physiological saline solution was injected. Mice were sacrified by cervical dislocation at four different time intervals after the drug administration in order to evaluate the action of the drug in different stages of spermatogenesis. The effect of the drug was appreciated as soon as four days after its administration. The initial damage to the epithelium was characterized by vacuolization of the Sertoli cells. Of all germ cells, primary spermatocytes showed the highest sensitivity to the drug. A high percent of teratozoospermia in the experimental group, when compared to the control group, was observed at all time intervals. The way CFA affects the morphology of mammalian spermatozoa and the usefulness of the animal model presented are discussed.

Pentoxifylline increases sperm penetration into zona-free hamster oocytes without increasing the acrosome reaction.

Summary. Several drugs have been used to stimulate human sperm motility, including 3‐deoxy‐adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona‐free hamster oocytes. Twenty‐seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim‐up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml−1 of pentoxifylline at 37 °C, 5% CO2 for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline‐free medium, at a sperm concentration of 10 × 106 cells ml−1. One hundred microlitres of each sperm suspension was then deposited under oil and 30–40 zona‐free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome‐reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona‐free hamster oocytes penetrated was increased after pentoxifylline‐treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline‐treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.