Release of norepinephrine from human ovary: coupling to steroidogenic response.

We investigated the possibility that norepinephrine from the human ovary is released after nerve stimulation and that this neurotransmitter is coupled to a steroidogenic response. Biologically significant levels of both norepinephrine and dopamine were found in human ovarian biopsies. [3H]norepinephrine incorporated in vitro was readily released by electrical stimulation in a Ca2+-dependent process. Ovarian membrane preparations exhibited specific binding sites for the beta-adrenergic antagonist [3H]dihydroalprenolol. Displacement of [3H]dihydroalprenolol with zinterol (a specific beta2-agonist) indicated that 72% of these sites were type beta2-receptors. beta-receptors were also present on granulosa cells. Stimulation of granulosa cells with luteinizing hormone or the beta-agonist isoproterenol increased the release of progesterone after 4 d in culture. These results suggest that the sympathetic nerves present in human ovary are coupled to beta-adrenergic receptors present in endocrine cells and, as in nonprimate mammals, appear to participate in the regulation of ovarian function.

Effect of copper ion on the motility, viability, acrosome reaction and fertilizing capacity of human spermatozoa in vitro.

The objective of this study is to provide additional information on the effect of copper ion (Cu2+) in preventing pregnancy. Human spermatozoa, selected by the swim-up method, were incubated for 0, 5 or 24 h in the presence of 10 ng, 1 microgram, 10 micrograms or 100 micrograms of Cu2+ mL-1 in BWW culture medium, and then evaluated in terms of their motility, viability, acrosome reaction (AR) and the capacity to penetrate zona-free hamster eggs. AR and penetration in zona-free hamster eggs were assessed at 5 h of incubation. Motility, viability and AR in sperm incubated for 5 h were significantly affected by Cu2+ at a concentration of 100 micrograms mL-1, but not at the lower concentrations. Incubation for 24 h did not affect motility and viability of sperm incubated in the presence of concentrations of Cu2+ ranging from 10 ng mL-1 to 10 micrograms mL-1, but a concentration of 100 micrograms mL-1 caused a significant decrease in both parameters. In contrast, the penetration rate of zona-free hamster oocytes significantly decreased compared with that of controls, when only sperm were incubated in the presence of concentrations of Cu2+ ranging from 10 ng mL-1 to 10 micrograms mL-1, and no penetration was observed in the presence of 100 micrograms mL-1 of Cu2+ . When only oocytes were exposed to Cu2+, the penetration rate dropped to 50% of that of the controls. Finally, when both gametes were exposed to Cu2+ before co-incubation, the penetration rate fell to zero for every concentration tested. Results showed that copper, at concentrations similar to those released from intrauterine devices (IUD), affects the fertilizing capacity of human gametes in vitro and interferes with the sperm-oocyte interaction leading to fertilization. These effects suggest that the principle action of Cu2+ released from Cu-IUD is to act as a preconception contraceptive agent when delivered in endometrial and oviducal fluids.

First pregnancies and livebirths from transfer of sodium alginate encapsulated embryos in a rodent model.

OBJECTIVE: To determine the effect of sodium alginate encapsulation of rodent embryos on in vitro embryonic cleavage rates, implantation rates, and livebirth rates, and to find the in vivo degradation time for the capsules. DESIGN: Studies were conducted using both CB6F1 mice and Golden Syrian hamsters. RESULTS: Capsules made with 3.0% sodium alginate degraded in vivo within 24 to 48 hours after transfer. In vitro embryonic cleavage of encapsulated embryos was not impaired, nor were implantation rates in CB6F1 mice. Finally, 8.6% of transferred encapsulated embryos resulted in livebirths. CONCLUSIONS: Encapsulation of rodent embryos in 3.0% sodium alginate is not detrimental to embryonic development, implantation rates, or fetal development. Because the capsule degrades within 48 hours after transfer, encapsulating embryos may be beneficial for human in vitro fertilization and embryo transfer.

[Simplified management of an in vitro fertilization program]. / Manejo simplificado de un programa de fertilización in vitro.

The development of in vitro fertilization has accelerated in a dramatic way the understanding on gamete physiology. Results obtained with the technique are easily reproducible and consistent between different centers. It is well known that cumulative pregnancy rates with 5 or 6 cycles can reach up to 60% of couples being pregnant. However, the main limitations to successive attempts have been the cost of the procedure and the surgical transfer of the embryos. In an attempt to overcome this difficulties, efforts has been made to simplify the monitoring of induction of ovulation, use of GnRh analogs and non surgical transfers whenever it is possible. Results presented in 47 of these cycles show non significant differences, with data published elsewhere, on oocyte maturity, fertilization and cleavage rates. Moreover pregnancy rates per aspiration are 28.6% and per transfer 33.3%. We can conclude that ultrasonography alone to monitor ovulation induction as well as uterine transfers do not affect the outcome of the cycle.

Pregnancy rate in an oocyte donation program.

Oocyte donation programs offer an alternative treatment for infertile women with ovarian failure or abnormal ovarian function. Seventeen cycles of in vitro fertilization and embryo transfer with donated oocytes were performed in 13 women, with a mean age of 34.8 years. The hormonal replacement therapy consisted of a fixed dose of oral estradiol valerate, 6 mg daily, and intramuscular progesterone in oil, 100 mg daily. Estrogen and progesterone were continued for 10 more weeks after embryo transfer if pregnancy was established. After 13 embryo transfers, 8 pregnancies were obtained, for a pregnancy rate per transfer of 61.5%. Today seven pregnancies are progressing normally, including one set of twins. This results suggest that an oocyte donation program using a fixed and simple hormonal replacement therapy is an adequate treatment for these infertile couples.

Fertilization rate in couples with unexplained infertility.

A group of 24 couples with unexplained infertility was scheduled for in-vitro fertilization and tubal embryo transfer between May 1989 and September 1990. In the same period, in-vitro fertilization and intrauterine transfer of embryos was planned in a control group of 44 women with tubal infertility. The mean age and duration of infertility were similar in both groups and the same scheme of ovarian stimulation was used. No statistically significant difference was obtained comparing oestradiol levels and numbers of mature oocytes retrieved between the group of patients with unexplained infertility and those with tubal infertility. The fertilization rate of the oocytes obtained from women with unexplained infertility (60.4%) was significantly lower (P less than 0.001) than that of the oocytes obtained from patients with tubal infertility (87.3%). There was no statistically significant difference in the cleavage rates between patients with unexplained infertility and those with tubal infertility. It is concluded that lack of fertilization is an unexplored cause of infertility in couples with unexplained infertility.

Effect of RU486 on development and implantation of rat embryos.

This study evaluated the effects of postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0, 2.0, or 3.0 mg/rat of RU486 were injected subcutaneously on days 1, 1 + 2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose-related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12). Treatment on day 4 was the least effective. Blastocysts recovered from RU486-treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486-treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486-treated pseudopregnant recipients failed to implant completely when the dose was greater than or equal to 1.0 mg. The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.

High potassium concentration and the cumulus corona oocyte complex stimulate the fertilizing capacity of human spermatozoa.

Progressively motile spermatozoa were incubated for 24 hours in culture media containing 4.7 or 25 mM K, in the presence or absence of hamster cumulus oophorus. The percentage of spermatozoa with progressive motility was significantly higher at 24 hours in the presence of cumulus corona oocyte complexes, irrespective of K concentration. A significant decrease in sperm mortality was observed with the association of 25 mM K and cumulus cells. A higher percentage of acrosome reaction was observed in spermatozoa incubated in 25 mM K when compared with 4.7 mM K, irrespective of time and the presence or absence of cumulus. The percentage of penetrated oocytes at 2 and 5 hours of incubation was higher when sperm had been incubated in 25 mM K than in 4.7 mM K. The presence of cumulus in the culture medium induced an additional significant increase in the percentage of penetrated oocyte. Although at 24 hours of incubation the percentage of acrosome reaction was higher than at 2 and 5 hours, the percentage of penetrated oocytes did not increase proportionally.

Cumulus cell dispersion induced by estradiol in mouse oviduct in vitro.

Dispersion of cumulus cells in nonmated mice is completed in the oviduct 15-20 h after ovulation. Oviducts, isolated 1 h after ovulation (13 h post-human chorionic gonaditropin), were cultured in vitro for 40 h. In these oviducts, denuded oocytes were first seen at 30 h of culture, indicating that cumulus dispersion proceeded at a slower rate in vitro. Oocyte denudation was accelerated in a dose-dependent manner by the addition of estradiol to the culture medium in which oviducts were incubated. The addition of progesterone or cycloheximide to the culture medium strongly inhibited oocyte denudation even in the presence of estradiol. When isolated cumuli were incubated in the absence of oviductal tissue, the rate of cell dispersion was slower than that of cumuli incubated inside the oviduct and the addition of estradiol to the culture failed to accelerate this process. On the basis of these data, we propose that cumulus cell dispersion is accelerated by an estrogen-dependent protein produced by the oviduct and that this effect of estrogen is antagonized by progesterone.

High potassium concentration improves the rate of acrosome reaction in human spermatozoa.

Progressively motile spermatozoa recovered by swim-up method from semen of two fertile men were incubated for 24 hours in culture media containing either 4.7, 15, or 25 mM of potassium (K). Aliquots of each culture condition were obtained at 0, 1, 5, 10, and 24 hours of incubation for the assessment of progressive motility, percentage of dead spermatozoa, and percentage of acrosome reaction (AR), as measured by triple-stain technique. A total of ten experiments including each K concentration were analyzed. The results of this study showed no effect of K concentration on the percentage of progressively motile spermatozoa, irrespective of the time of incubation. The percentage of live spermatozoa was significantly greater in culture medium containing 25 mM K (P less than 0.05). There was a greater percentage of reacted spermatozoa with 25 mM K, as compared with 4.7 and 15 mM (P less than 0.05). Furthermore, the time taken to achieve 20% of AR was 2 hours at 25 mM K compared with 10.9 hours at 4.7 mM K.

The effect of RU486 on transport, development and implantation of mouse embryos.

Pregnancy was interrupted in Swiss-Rockefeller mice by a single subcutaneous injection of the antiprogesterone RU486 given postcoitally. A dose of 0.1 mg/animal injected on day 1, produced partial inhibition of pregnancy, a notable delay in embryonic development and a slight retention of embryos in the oviducts. When the same dose was injected on day 2, 3 or 4, no implantation sites were seen at autopsy performed on day 12 of pregnancy. Treatment with 0.5 or 1 mg/animal on day 1 produced the loss of a large proportion of the embryos from the genital tract by day 4 of pregnancy and suppressed implantation completely. These observations indicate that preimplantation phenomena in mice are highly dependent on progesterone action.

High potassium concentration improves preimplantation development of mouse embryos in vitro.

Embryo growth in vitro in culture media containing potassium (K) ions at the concentration found in common extracellular fluids proceeds at a slower pace than in vivo. Considering that oviductal fluid has an unusually high concentration of K, the effect of various concentrations of this ion on development of mouse embryos in vitro was investigated. Two-cell to 4-cell preimplantation mouse embryos were cultured in vitro for 47 hours in a medium in which NaCl was partially replaced by KCl at concentrations ranging from 4.7 to 60 mM. The number of cells per embryo increased in a dose-related fashion when the embryos were cultured in the presence of 4.7, 10, and 25 mM of K. Higher K concentrations were detrimental for development. Embryos developed in vitro under different concentrations of K were transferred to pseudopregnant recipient foster mothers as a test of viability. The highest rate of implantation was observed with embryos cultured in medium containing 25 mM K. The results indicate that a high concentration of K in the culture medium (25 mM), comparable to that found in the genital tract of the female mouse, is required for a rate of development in vitro similar to the one observed in vivo.

Efecto de la concentracion diaria de estradiol en las caracteristicas morfologicas del complejo cumulo-corona-ovocito obtenido en programas de fecundacion in vitro. / Effect of the daily concentration of estradiol on the morphological characteristics of the cumulus-corona-oocyte complex obtained in fertilization in vitro programs

El presente estudio analiza la relacion existente entre la concentracion diaria de estradiol en plasma y la morfologia del complejo cumulo-corona-ovocitos (CCO), obtenidos de aspiracion folicular en un programa de fecundacion in vitro (FIV). De un total de 99 ovocitos pertenecientes a 27 mujeres incorporadas en este estudio, 66% fueron morfologicamente del tipo II; 12% del tipo III, y 16% del tipo IV. El 80,6% de los ovocitos que se fertilizaron corespondian al tipo II, y 89,6% de los ovocitos que se segmentaron eran de esta misma caracterizacion morfologica. La concentracion diaria de estradiol en los dias que preceden a la aspiracion folicular supera al 100% cuando se obtienen ovocitos del tipo II. Sin embargo, en aquellos casos en que se obtienen ovocitos tipo III o IV el estradiol experimenta una estabilizacion en las 48 horas que preceden a la aspiracion folicular, y el incremento acumulativo total no sepera el 80%. Por otra parte, el aumento del volumen folicular total presenta un paralelismo con el incremento diario de estradiol. Cuando este paralelismo se quiebra, la probabilidad de encontrar ovocitos no fertilizables aumenta significativamente

Effect of exogenous sex steroids upon the number of germ cells and the growth of foetal ovaries grafted under the kidney capsule of adult ovariectomized hamsters.

The effect of estradiol E, progesterone P and testosterone propionate T upon growth and germ cell population of foetal ovaries transplanted under the kidney capsule of adult ovariectomized hamsters was examined. Foetal ovaries were obtained 15 days postcoitum and the host received daily subcutaneous injections of E (1 micrograms/day), P (5 mg/day), T (500 micrograms/day) or E + P in 0.1 ml oil for 25 days beginning 5 days before grafting. One day after the last injection, the size of the ovary, the germ cell population and plasma steroid levels were assessed. The results of hormone assays indicate that daily steroid administration was able to maintain continuously elevated plasma levels of the corresponding hormone. Interconversions or steroid secretion by the graft, if any, were not reflected in the peripheral circulation. The growth of the graft was stimulated by T and inhibited by P, in comparison with E and oil-treated controls. All germ cells were at the stage of primary oocyte forming part of a primordial or growing follicle. Their absolute number was significantly increased by T and E and significantly decreased by P and E + P. The number of oocytes per mm3 of ovary was increased 80%, 48% and 40% with E, T and E + P, respectively. It is concluded that, in the hamster, exogenous sex steroids given to the host can exert specific effects upon the growth and oocyte population of a grafted foetal ovary. Whether or not the action of steroids upon the graft is a direct one and whether they influenced oogonial mitosis, the evolution of the meiotic prophase or atresia of primary oocytes remains to be determined.