Brigitte Lefèvre

LEAD ACCUMULATION IN THE MOUSE OVARY AFTER TREATMENT-INDUCED FOLLICULAR ATRESIA

Although the main target of lead (Pb) toxicity is the red blood cell, Pb-associated changes in the nervous system, the kidney, and the reproductive system have also been described. The few Pb studies conducted on females revealed mostly miscarriages, premature delivery, and infant mortality in humans and animals. This study was done to correlate Pb accumulation in the ovary with damage to folliculogenesis. Pb burden was assayed by atomic absorption spectrometry in bone, liver, adrenal glands, ovary, and fetuses taken from mice exposed according to 2 protocols: intraperitoneal (i.p.) injection of Pb(NO(3))(2) 10 mg/kg/day for 15 days or 10 mg/kg/week for 15 weeks. Ovaries were examined histologically. Pb accumulation in the various soft tissues of acutely exposed mice was similar, and significantly higher than in the organs of chronically exposed mice. A low Pb concentration in the ovary caused dysfunction of folliculogenesis, with fewer primordial follicles and an increase in atretic antral follicles.

Nuclear calcium release by InsP3-receptor channels plays a role in meiosis reinitiation in the mouse oocyte

Our purpose was to investigate the presence of nuclear specific elements of the phosphoinositide pathway, and the link between nuclear calcium events and the first step of meiosis resumption, i.e. germinal vesicle breakdown (GVB) in mouse immature oocytes. Using confocal laser scanning microscopy, we analyzed the effects of nuclear microinjection of inositol 1,4,5-trisphosphate (InsP3), heparin and anti-InsP3 receptor monoclonal antibodies on both spontaneous nuclear and cytoplasmic calcium oscillations, as well as the effects of these components on the GVB. First we observed that nuclear Ca2+ events were dependent upon both nucleoplasmic and cytoplasmic InsP3 levels, highlighting a cross-talk between the GV and the cytoplasm concerning the Ca2+/InsP3 pathway. We demonstrated also that: 1) type 1 InsP3 receptors were localized at the nuclear membrane level while type 3 were absent from the nucleus; 2) calcium release from nuclear stores was mediated by type 1 rather than type 3 InsP3 receptor associated channels; 3) the anti-InsP3 R-1 mAB microinjected into the nucleus inhibited the GVB. These results demonstrate that reinitiation of meiosis requires an increase in nuclear phosphoinositide dependent Ca2+. Thus, the role of nuclear Ca2+ homeostasis is discussed with particular emphasis on nuclear envelope dynamics.

The nucleolus of the maternal gamete is essential for life.

The mammalian oocyte is a round cell arrested at prophase I of meiosis. It is characterized by the presence of a large nucleus, called the germinal vesicle, in the middle of which is the nucleolus. Before it can be fertilized, the oocyte must resume meiosis, enter metaphase II and be ovulated. The nucleolus is dissolved during this process. However, the nucleoli of the male and female pronuclei in the zygote are both of maternal origin. A recent paper1 demonstrates that the maternal nucleolus, together with other nucleoplasmic elements, is essential for early embryonic development. These nucleolar and nucleoplasmic factors remain undetermined.

Factors affecting human in vitro fertilization: a multifactorial study

The influence of four factors on the cleavage rate of 705 mature oocytes submitted for in vitro fertilization (IVF) was assessed with the use of a multifactorial method, i.e., logistic regression. The studied factors were (1) the regimen for cycle stimulation, (2) cumulus cell mass appearance, (3) semen quality, and (4) the time of incubation of oocytes before insemination. The logistic function permitted testing the respective influence of each factor on the cleavage rate, with the level of the other factors taken into account. The most important factor was the stimulation treatment, the association of human menopausal gonadotropin with clomiphene citrate (CC) giving better results than CC alone. Time of incubation was demonstrated to have no influence on the cleavage rate, whereas semen quality had an influence. A problem was raised by the existence of an interaction between the stimulation treatment used and the appearance of the cumulus cell mass. It can be concluded that adequate cycle treatment and eventually couple selection with respect to the semen quality of the male partner are to be considered in view of obtaining better results in IVF attempts.

Natural uranium disturbs mouse folliculogenesis in vivo and oocyte meiosis in vitro.

We investigated whether uranium intoxication affects female fertility by assessing its effects on ovarian function and on the oocyte. We treated two groups of female mice for 15 weeks with 5, 50 or 400 mg/L of uranyl nitrate in drinking water. In the first group, mice were euthanized immediately after intoxication. Mice of the second group were paired after intoxication with untreated males. Dams and their female pups were euthanized 3 months after the end of intoxication. We assayed the kidneys, femurs and one ovary per female for U content and collected the other ovary for histology. The number and size of all the ovarian follicles were analyzed. Mice from the first group and female pups had significantly fewer large antral follicles (Ø > 200 microm) than the untreated mice. By contrast, dams in the second group had more secondary and early preantral follicles (Ø 70-110 microm) than untreated mice. However, U had no effect on follicle atresia. We then analyzed the in vitro effects of U on oocyte maturation and fragmentation. GV-oocytes were cultured in the presence of 1mM uranyl acetate and observed for 72 h. Oocyte maturation was slowed down by U during resumption of meiosis and at metaphase II. However, the rhythm and rate of oocyte fragmentation were similar to those of control mice. Our findings demonstrate that U induces changes in folliculogenesis and oocyte maturation in mice and could consequently represent a risk for women who are chronically exposed.

Review: Lamin A/C, caspase-6, and chromatin configuration during meiosis resumption in the mouse oocyte.

After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific caspase-6 inhibitor to determine the presence of lamin A/C and caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of caspase-6 in the oocyte apoptotic pathway. Lamin A/C and caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.

Effect of a gonadotropin-releasing hormone agonist and gonadotropins on ovarian follicles in cynomolgus monkey: a model for human ovarian hyperstimulation

STUDY OBJECTIVE To examine the effect on large follicles (greater than or equal to 2 mm) of human menopausal gonadotropin (hMG) and buserelin acetate, a gonadotropin-releasing hormone agonist in monkeys. DESIGN Experimental. SETTING Reproductive research laboratory. ANIMALS Fourteen cyclic cynomolgus monkeys receiving hMG alone for 8 days or buserelin acetate plus 8 (group 1), 12 (group 2), or 16 (group 3) days of hMG administration always starting from day 1 of the cycle. RESULTS The different treatments were effective in over-riding the specific ovulatory quota of 1, and more large follicles developed in treatments involving long duration and higher doses of hMG. In buserelin acetate plus hMG treatments, the frequency of dissociated follicles and follicles in late atresia were, respectively, lower and higher than in hMG alone treatment. The numbers of recoverable mature oocytes (germinal vesicle breakdown) were similar to the numbers of such oocytes recovered after hyperstimulation performed for human in vitro fertilization and embryo transfer (IVF-ET). However, the number of mature oocytes enclosed in typically preovulatory follicles was very low because there were numerous dysmature follicles. CONCLUSION These data suggest a deleterious effect of buserelin acetate plus hMG treatments on the recruitable follicles at the time when treatments start. The implications of these observations in the field of human IVF-ET are discussed.

Absence of predictive value of follicular inhibin on the results of human in vitro fertilization.

During the periovulatory period, inhibin did not have a discriminant role in oocyte fertilizability. A possible role in the gonadotropin surge was not revealed by analysis at the time of follicular rupture. Furthermore, inhibin activity appears to be very low. But inhibin could still have an effect upon growth and atresia of the follicles of the following cycles.

Whole-body or isolated ovary 60Co irradiation: Effects on in vivo and in vitro folliculogenesis and oocyte maturation

For the first time, the effects of low doses of gamma-radiation were studied on folliculogenesis and on isolated oocytes. After irradiation of adult mice, even at the lowest dose, a drastic loss of primordial follicles was observed in serial sections of ovaries, with, in opposite, no effect on the other follicle stages. Moreover, oocytes freshly recovered from the largest antral follicles of irradiated adult ovaries exhibited significantly less regular Ca(2+) oscillations than controls. Finally, in vitro folliculogenesis demonstrated a smaller diameter of preantral follicles recovered from irradiated juvenile ovaries compared to control, and an increase in follicle atresia. Further on, PLC-beta1 localization was not affected in the enclosed oocytes whereas chromatin configuration revealed that a quarter of them had prematurely resumed meiosis or was fragmented. These results raise the question of the risk of genetic and teratogenic effects on women submitted to chronic exposure even of very low radiation.

Oocyte Ca2+ spike acquisition during in vitro development of early preantral follicles: influence of age and hormonal supplementation.

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.