A. Tsafriri

Gonadotropin Action on Cultured Graafian Follicles: Induction of Maturation Division of the Mammalian Oocyte and Differentiation of the Luteal Cell

Publisher Summary This chapter discusses the nature of interactions between oocyte and granulose cells and the triggering effect of luteinizing hormone (LH). The mammalian oocyte embarks on its first reduction division in prenatal life or during the early postnatal period. This division has a protracted and complicated prophase. Just before or shortly after birth, depending on the species, the germ cell reaches the stage of diplotene. By this time, the oocyte has doubled its DNA complement, the chromosomes have condensed, and homologous chromosomes have become paired and are linked by chiasmata permitting the exchange of paternal and maternal genetic information. In murid rodents, the chromosomes decondense and resume their transcriptive activity. In the adult, during each estrous cycle, a number of oocytes characteristic of the species complete their first reduction division, resulting in the abstriction of the first polar body shortly before ovulation. This resumption of meiosis and its progress to the metaphase of the second meiotic division refer to as ovum maturation. Completion of the second meiotic division, with extrusion of a second polar body, occurs only upon penetration of the oocyte by a spermatozoon.

Resumption of oocyte meiosis in mammals: On models, meiosis activating sterols, steroids and EGF-like factors

De novo synthesis of meiosis activating sterols (MAS) was stimulated by LH- and AY-9944 in rat cultured follicles and cumulus oocyte complexes (COCs), but could not be measured in denuded oocytes. Thus, MAS synthesized by the somatic compartment of the follicle could serve as a signal for the resumption of meiosis. Nevertheless, the delay in germinal vesicle breakdown (GVB) after MAS or AY-9944 stimulation as compared with gonadotropins, obtained by several groups, remains the strongest evidence against the suggested role of MAS as an essential mediator of LH in meiosis resumption. Recently several studies using mammalian COCs in culture have implied that steroids, like in fish and amphibians, serve as signals in mediating the LH/hCG stimulation of meiosis. However, in these studies there was no clear distinction between the requirement for steroids for the acquisition of meiotic competence, oocyte and follicle wellbeing or as a signal for meiotic resumption. Further, some of the authors overlooked earlier studies showing that blocking ovarian or follicular steroidogenesis does not affect GVB, the first step of meiosis resumption. Finally, in vivo and in vitro studies in the rat confirm and extend recent studies showing that locally produced and released EGF-like factors, such as epiregulin, seem to mediate at least part of the LH/hCG actions on oocyte maturation and release of ova at ovulation.

Role of fsh and oestradiol-17β in the development of meiotic competence in rat oocytes

The ability of rat oocytes to resume meiotic maturation upon isolation (meiotic competence) was shown to be abolished by hypophysectomy on day 15 post partum (p.p.). FSH (NIAMD-rat FSH-B-1; 10-20 micrograms/day), but not LH (NIAMD-oLH-21; 2-20 micrograms/day) was able to induce meiotic competence in rats hypophysectomixed on day 15 pp. Implantation of a capsule of oestradiol-17 beta, but not of progesterone or androstenedione, partially restored meiotic competence in hypophysectomized rats within 24 h of treatment. When inhibitors of steroidogenesis (aminoglutethimide, 17 beta-formamidoandrost-4-en-3-one, 1,4,6-androstatriene-3,17-dione or 4-hydroxy-4-androstene-3,17 dione) were coadministered with FSH they inhibited its effect on the development of meiotic competence. Furthermore, when oestradiol-17 beta was administered together with these inhibitors of steroidogenesis, the effect of FSH on the development of meiotic competence was not disturbed. These results strongly suggest that FSH, but not LH, is involved in the development of meiotic competence. Further, this action of FSH appears to be mediated, at least partially, by follicular oestrogen production.

Steroids and follicular rupture at ovulation

The preovulatory surge of gonadotropins stimulates follicular steroidogenesis and changes from estrogen as the major product to progesterone. We shall overview the studies dealing with the role of ovarian steroidogenesis in follicular rupture at ovulation. Several inhibitors of steroidogenesis blocked follicular rupture in vivo. Likewise, RU 38486 partially blocked ovulation triggered by hCG. Collectively, these data support the knowledge that follicular steroidogenesis is required for ovulation. Recent studies confirmed the essential role of plasminogen activator (PA) in follicular rupture. The LH stimulation of PA activity was partially blocked by several inhibitors of steroidogenesis and it could be restored by the addition of progesterone, testosterone and estradiol-17 beta, but not the non-aromatizable 5 alpha-dihydrotestosterone. Gonadotropic stimulation enhanced only the synthesis of tissue type PA (t-PA) and not that of urokinase. Likewise, inhibition of steroidogenesis, reduced only the synthesis of t-PA and was reversed by addition of estradiol-17 beta. It seems, therefore, that follicular steroids, most probably estrogen, are involved in the preovulatory rise in follicular t-PA activity.

Human follicular fluid protease and antiprotease activities: a suggested correlation with ability of oocytes to undergo in vitro fertilization

Plasminogen activator activity was determined in human follicular fluids (FFs) obtained during in vitro fertilization procedures. The fibrinolytic activity of plasminogen activator was significantly higher in fluids from follicles that contained oocytes that were later found to fertilize in vitro (group F) as compared with fluids from follicles that contained oocytes that failed to fertilize (NF). To assess whether this difference in overt plasminogen activator activity reflects differences in conversion of an inactive, latent plasminogen activator to the active enzyme, the ability of exogenous trypsin to enhance plasminogen activation was measured. The plasminogen-dependent hydrolysis of the chromogenic substrate S-2444 in presence of trasylol (Bayer, Leverkusen, Germany) was taken as a measure of plasminogen activator activity in these experiments. No activity was found in untreated FFs, while exposure to trypsin resulted in emergence of marked plasminogen activator activity. In addition, FFs exhibited trasylol-sensitive chromogenic activity indicative of serine-protease activity. Both the plasminogen activator and serineprotease levels after tryptic activation were significantly higher in NF than in F samples. Thus, while F samples have most of their plasminogen activator in an active form, NF samples have most of their plasminogen activator in a latent, trypsin-activatable form. Follicular fluids also contain inhibitory activities toward plasmin and trypsin. The inhibition of these enzymes correlates positively with the latency of plasminogen activator. These results suggest a direct relationship between the ability of oocytes to fertilize and he overt to latent plasminogen activator activity ratios in the FFs.

Control of the Development of Meiotic Competence and of Oocyte Maturation in Mammals

Fertilizability of the mammalian oocyte is achieved only after a prolonged period of development and differentiation. In most mammals, fertilization occurs at the metaphase of the second meiotic division. In these species, oocytes that have not reached this stage usually cannot be penetrated by the spermatozoa or, if penetration does occur, the sperm nucleus fails to be transformed into a sperm pronucleus (Barros and Munoz 1973; Iwamatsu and Chang 1972; Niwa and Chang 1975; Usui and Yanagnimachi 1976).

Response of follicle cells to ovulatory stimuli within the follicle and in primary culture

Cultures of mural granulosa cells (mGCs) and cumulus oocyte complexes (COCs) were employed to investigate various aspects of follicle cell function and response to gonadotropins. Yet, such studies do not reveal the intricate cell-to-cell interactions in the whole follicle. Here we compare the ovulatory responses to LH/hCG or epiregulin (ER) of rat preovulatory follicles and of mGC and COC whether they were stimulated within the follicle or in primary cell cultures. The expression of TSG-6 and COX-2 mRNA varied according to the culture system and mode of stimulation. In primary cultures stimulated with LH or ER resulted in their lower expression as compared to stimulation of follicles. LH/hCG stimulated higher follicular and mGC AR, ER and EGFR mRNA levels than in primary mGC cultures. COCs stimulated by LH/hCG in vivo responded with AR, ER and EGFR mRNA expression, but not in culture where only EGFR mRNA was stimulated. The differences in gene expression of mGCs and COCs when stimulated within their intact follicle or in primary cultures revealed here underscore the important role of cell-cell interactions in follicle physiology. Therefore, results obtained in primary mGC cultures need careful validation in models reproducing such in situ interactions for revealing mGC activity within the intact follicle.

Follicular Rupture During Ovulation: Activation of Collagenolysis

The preovulatory surge of gonadotropins induces a series of orderly sequenced changes in the mature ovarian follicle. These include resumption of oocyte meiosis, luteinization of the follicle and finally the rupture of follicle wall and release of a fertilizable ovum. While initial studies on follicular response to gonadotropins dealt primarily with changes in steroidogenesis and resumption of meiosis, recently the biochemical changes associated with follicular rupture have been investigated

Relationship between glycolysis and steroidogenesis in cultured Graafian follicles stimulated by LH or prostaglandin E2.

Abstract Graafian follicles isolated from 3-month-old rats during early pro-oestrus (8:00–12:00 a.m.) and placed in organ culture responded to luteinizing hormone (LH)(NIH-LH-S18; 5 μg/ml) with increased release of lactate and steroids (progesterone, androstenedione and oestradiol-Pβ) into the medium during 6 h incubations; addition of prostaglandin E 2 (PGE 2 ; 10 μg/ml) to the medium likewise enhanced aerobic glyeolysis and progesterone accumulation in the medium. Cyanoketone (10 −4 M) prevented the steroidogenic effects of LH and PGE 2 , and reduced the basal rate of follicular steroidogenesis, without impeding the stimulatory action of either LH or PGE 2 on aerobic glyeolysis. Aminoglutethimide (10 −3 M) also suppressed basal as well as LH- or PGE 2 -induced steroidogenesis, but did not impair the glycolytic effect of LH; the effect of PGE 2 on glyeolysis, however, was reduced or abolished. Iodoacetate (2.5 × 10 −5 M) prevented the LH effect on glyeolysis and significantly reduced, yet did not abolish, the steroidogenic action of LH. It is concluded that the hormonal stimulation of follicular glyeolysis and steroidogenesis involves divergent pathways, rather than a single sequence of events. However, the glycolytic effect reinforces the steroidogenic action of LH, perhaps by supplying NADPH needed for steroid hydroxylation.