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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.

The role of calmodulin in fertilisation events was examined in a zona-free mouse system by using a selective calmodulin inhibitor, calmidazolium (1 μM). The effects of this antagonist were studied either on the ooplasmic calcium oscillations induced by fertilisation by using the Ca2+ indicator, fluo-3/AM, or on pronucleus formation 4 h later by using the nucleic acid stain, Syto-15. When the calmidazolium treatment was applied to one or the other gamete before insemination, the fertilisation process was affected only when spermatozoa were treated: most of the oocytes were partially fertilised as demonstrated by the profile of Ca2+ oscillations and the presence of polar bodies with no typical male and female pronuclei. When the treatment was applied during insemination, more than half the oocytes were unfertilised and only a few were partially fertilised. These results demonstrate that: (1) the calmodulin-dependent events taking place in spermatozoa before insemination appear essential at least for regular Ca2+ oscillations and for pronucleus formation; (2) the inhibition of calmodulin by calmidazolium applied to metaphase II oocytes before insemination has no major impact on their fertilising ability; and (3) at the time of gamete fusion calmodulin, either from the oocyte or from the spermatozoon, is essential for fertilisation to occur.

Summary.Analysis of ovaries from 31 women with normal ovarian function permitted study of the diameter of the largest healthy and atretic follicles during the menstrual cycle. The follicle destined to ovulate is selected during the early follicular phase (Days 1–5). Throughout the cycle the diameter of the largest healthy follicles, with the exception of the dominant follicle, did not exceed, on average, 6 mm during the follicular phase and 4 mm during the luteal phase. Consequently, excluding the dominant follicle during the second half of the follicular phase, the largest follicles present in the human ovary are atretic.From these data, it was concluded that a new ovulation could not occur very soon after a spontaneous or experimentally induced premature disappearance of the dominant follicle or the corpus luteum of the cycle.

Summary.Follicular rupture was never observed when preovulatory rabbit follicles were isolated from the ovary before the ovulatory gonadotrophin discharge and placed in hormone-free culture for 10–14 h (n= 48). However, if the follicles were taken 1 h post coitumand cultured under the same conditions, 11/24 (45·8%) ruptured spontaneously. Follicular rupture under these conditions appeared to be chronologically and histologically comparable to that of ovulation in vivo. Culture in the presence of progesterone increased the proportion of follicles ovulating in vitro(17/23 = 73·9%; P< 0·05). Inhibition in vitroof the synthesis of steroids or prostaglandins suppressed ovulation, although meiotic maturation of the oocyte did take place. These findings indicate that the follicle constitutes an independent entity within the ovary from 1 h post coitum.

Summary.Ovulation was never observed, even in the presence of gonadotrophins, when preovulatory follicles isolated from oestrous does were cultured in vitro(n= 112). If the follicles were co-cultured with pieces of ovarian tissue and gonadotrophins, then 50% ovulated (P< 0·01). Attempts to replace the ovarian tissue by various steroid hormones or prostaglandins did not result in follicular rupture except when PGE-2 was used (23·8%, P< 0·05). We suggest that gonadotrophins have an immediate (<1 h) action upon the follicle and ovarian interstitial tissue simultaneously. The response of the ovarian tissue to gonadotrophins is essential for eventual follicular rupture and involves PGE-2.

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.

Summary.Detailed histological analysis of ovaries obtained from 25 women by ovariectomy for medical reasons demonstrated that the degeneration of the cyclic corpus luteum is a long process, taking several cycles.The ages of the various regressing corpora lutea were identified to permit classification of the side of ovulation during the preceding 4 or 5 cycles. In the 25 women studied, with regular menstrual cycles of 28 ± 3 days (48 cycles), ovulation occurred most often (87·6%) in an alternating manner.