Reproductive health

FOLLICULOGENESIS AND OVULATION

J.F. Guerin
Laboratory of Histology, Embryology and Reproductive Biology,
Faculty of Medicine Lyon Nord
8, Avenue Rockefeller - Lyon 8e, 69373 Lyon CEDEX 08

The ovaries, like the testicles, exert a double function, exocrine and endocrine, consisting of the production of gametes, the oocytes, as well as sex hormones, estrogens and progesterone. Whereas in the testicles the two functions are assured permanently from puberty onwards by two different structures, however, in the ovary they are exerted cyclically, between puberty and the menopause, and result from the evolution of a same morphological unit, the ovarian follicle, situated within the cortical stroma.

Histology of ovarian organelles

The gametogenic follicles

These follicles correspond to different stages of the evolution of primordial follicles up to the rupture of the mature follicle (ovulation). Each one contains an oocyte and is the site of oogenesis and of the production of steroid hormones.

The primordial follicle.

Around the 7^th month of embryonic development, the ovarian cortex contains a definitive stock of several million primordial follicles which progressively diminishes up to the menopause. Each follicle, within the cortical stroma, is made up of a 1^st order oocyte (oocyte 1) surrounded by a layer of flattened follicular cells, these cells being covered by a basal membrane (membrane of Slavjanski). Oocyte 1 measures about 30 µm in diameter.

The primary follicle.

It is characterized by the transformation of the flattened follicular cells into cubic cells.

The secondary follicle.

This follicle is called secondary once the multiplication of the follicular cells constitutes a second layer around the oocyte. The diameter of the follicle progressively increases up to about 180 µm. The follicular cells reach about 5000 in number and together constitute the granulosa. Oocyte 1 begins its growth and its diameter increases from 40 up to 60 µm. At the last stage of its development, the secondary follicle appears surrounded by irregularly spaced islets of differentiated epithelioid cells from stromal fibroblasts and in relation with the capillaries. Together the epithelioid cells constitute the internal theca (theca interna) of the follicle. The secondary follicle, provided with its theca interna is called a preantral follicle.

The tertiary follicle.

Also called cavitary follicle or antral follicle, it is characterized by the presence of a cavity (antrum) in the granulosa and a theca externa, a fibrous layer around the theca interna. It increases considerably in volume because of the rapid multiplication of the follicular cells which will reach about 50 million in number. At the end of its development, the follicle (roughly 2 cm in diameter) will become a preovulatory or mature follicle.

In the clumps of the granulosa there appear small drops of liquid whose confluence forms the antrum which contains the follicular fluid produced by the follicular cells. Around the oocyte, the granulosa projects into the follicular cavity—the cumulus oophorus. The theca interna, separated from the granulosa by the membrane of Slavjanski, is made up of numerous clusters of epithelioid cells. Electron microscopy reveals that these cells have the characteristics of steroidogenic cells, identical to those observed in Leydig cells. The theca externa is made up of a thick layer of collagen fibres, traversed by numerous blood capillaries; it contains myofibroblasts differentiated from fibroblasts of the stroma.

Up to the preovulatory stage of follicular evolution, the oocyte harboured in the cumulus is an oocyte 1 blocked at the end of the prophase (diakinesis stage). Cytoplasmic growth continues and the oocyte attains around 120 µm in diameter.

The preovulatory period and ovulation.

At the end of its growth, the mature follicle reacts to a discharge of gonadotropic hormones by great transformations which end in follicular rupture (ovulation). The cumulus cells secrete large quantities of hyaluronic acid which accumulates in the intercellular space and provokes the dissociation of the cumulus followed by its rupture: the oocyte surrounded by certain quantity of follicular cells is released into the follicular fluid. The apical region, the ovarian stroma, is the site of a vasoconstriction which results in an ischemia followed by necrosis, within a few hours, of the stroma and the follicular wall. The gonadotropic discharge will give rise to a release of histamine and bradykinin, leading to an edema of the theca. At the same time, the secretion of a plasminogen activator will also activate collagenases which will dissociate the theca externa, this action being reinforced by the release of prostaglandins. Lastly, the cells of the ovarian epithelium in the apical region, would appear to be subject to autolysis, leading to the release of lysosomal hydrolases and thus to the dissociation of the apex (a mechanism which could be deficient in the luteinized unruptured follicle [LUF] syndrome).

The oocyte completes its cytoplasmic and nuclear maturation in the cytoplasm, the cortical granules migrate to the periphery and attach to the plasma membrane. Meiosis resumes but is again blocked in 2^nd division metaphase (metaphase II). Ovulation commences with the rupture of the necrosed tissues of the apex (stigma). The viscous follicular fluid begins to flow. The decrease in pressure of the follicular liquid induces a series of rhythmic contractions of the myofibroblasts of the theca externa and of all the cortical stroma which lead to the expulsion of the follicular fluid and oocyte II surrounded by cumulus cells.

The corpus luteum

After expulsion of the oocyte, the follicle presents a pleated aspect. It’s then called a dehiscent follicle. The membrane of Slavjanski disappears completely and the blood capillaries of the theca rapidly invade the granulosa thereby provoking the transformation of these cells (luteinization) by the constitution of the corpus luteum.

The blood vessels completely traverse the granulosa and open up in the follicular cavity, thereby causing a circumscribed and rapidly coagulated hemorrhage (central coagulum). The granulosa cells are transformed into large luteal cells, approximately 40 µm in diameter, whose ultrastructure is the same as that of steroidogenic cells. The cells of the theca interna (hardly modified) constitute the small luteal or paraluteinic cells, situated at the periphery of the corpus luteum and forming strings that penetrate more or less deeply into the layer of the large cells.

Follicular atresia and luteolysis

Between the 7^th month of fetal life and the menopause, most of the gametogenic follicles undergo an involution (involutive or atretic follicles). Only 300-400 follicles will reach the preovulatory stage. All the involutive follicles which preserve for a certain time their theca interna are called thecogenic follicles. The theca cells of these follicles as a whole constitute the interstitial gland of the ovary.

Involution of the corpus luteum, or luteolysis, occurs most often in the form of a fibrous or fibro-hyalin degeneration with cell lysis and marked collagen fibre synthesis, which ends in the formation of a voluminous organelle called " corpus albicans ". The process is relatively slow and spread over several weeks.

Dynamics of follicular growth

In the human being, the stock of primordial follicles, called " reserve follicles ", is about 1 million at birth, and at the beginning of puberty a few hundred thousands. As already emphasized, practically all the follicles (over 99%) will be affected by the phenomenon of atresia, but at variable stages in the course of development. The interregulation of these two physiological phenomena—growth and atresia—is governed by complex mechanisms, which are now beginning to be elucidated in the human female, through the works of Gougeon in particular.

It has been established that an average of 85 days—i.e. corresponding to 3 ovarian cycles—separate the moment when a follicle becomes preovulatory (stage 8 of Gougeon’s classification) and the moment when it has differentiated its theca interna (i.e. is at stage 1 or " preantral "). This means that a preovulatory follicle enters the preantral stage 85 days earlier, in mid-cycle, at the time of the preovulatory discharge of the gonadotropic hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH).

As it is also recognised that entry into the preantral stage occurs randomly at any moment during the cycle, it may be deduced that all the follicles that differentiate their theca at a time that does not correspond to the preovulatory period will evolve more or less rapidly to atresia. A hypothesis that has been advanced is that the concentration of plasma FSH at the time of theca differentiation conditions the future quality of the theca, and more generally of the follicle to which it belongs.

It is nevertheless recognized that, up to a diameter of 2-4 mm (stage 4-5), follicular growth requires only a minimal concentration (basal) of FSH. Follicles up to 4 mm diameter may be found in impuberal girls or in women using hormonal contraception. Further follicular growth requires stimulation by gonadotropic hormones, and more especially by FSH. We can thus distinguish three steps:

1. Follicular recruitment, corresponding to entry into terminal growth of a group of follicles (stages 5 to 8).
2. Follicular selection, which will result in the emergence of the future ovulatory follicle.
3. Follicular dominance, exerted by the selected follicle and which will lead to the atretic evolution of the other follicles.

In the human female, recruitment occurs during the first days of the cycle and affects a maximum of 5 follicles per ovary, 3-5 mm in diameter (stage 5). It corresponds to an elevation in the plasma FSH level observed at the beginning of the cycle. Selection becomes more obvious shortly after: it concerns the follicle with the highest mitotic index and, generally, with the largest diameter. This follicle will continue its growth (stages 6-7) whereas the FSH level decreases (under the action of a negative feedback due to the increase in estradiol), and signs of atresia appear in the other follicles. It is of interest to note that if exogenous FSH is supplied, pure or associated with LH (human menopausal gonadotropin [hMG]) these follicles can be " recuperated " and thereby avoid atresia. It is the principle of stimulatory treatments of ovarian functions (hMG or pure FSH) which lead to multiple ovulations.

The dominance of the selected follicle is clearly evident in the second part of the follicular phase: growth continues (stages 7-8) while the level of FSH continues to decrease: such a phenomenon may account for a better uptake of FSH, but also for an amplified response to FSH, bringing into play an autocrine mechanism, corresponding to the production of growth factors, as IGF-I, by the granulosa cells. In fact, for these large follicles, evolution to continued growth or atresia is directly linked to the aromatization potentialities of the granulosa cell which will terminate in the transformation into estrogens of androgens originating from the theca interna. The dominant follicle possesses, up to the preovulatory gonadotropic discharge, a high aromatic activity. It might secrete a protein, called " regulatory ", which could perhaps inhibit the aromatase activity of the other follicles through a paracrine mechanism.

Regulation of ovarian functions

Ovarian functions are under the control of cyclic pituitary gonadotropic hormones, which in turn are subjected to stimulation by the hypothalamic peptide gonadotropin-releasing hormone (GnRH). Plasma FSH increases at the beginning of a cycle, then decreases before a peak which reaches its summit about 24 hours before ovulation (i.e. D 13) and is thus synchronous with that of LH, constituting the preovulatory discharge of gonadotrophins.

Estradiol levels rise progressively during the follicular phase: estradiol is secreted by all the follicles recruited at the beginning of the cycle, then, as atresia gradually affects the majority of these follicles, it is secreted by the dominant follicle. It is accepted that estradiol exerts first a classical negative feedback on the pituitary gland which then becomes positive as from a certain level, and then triggers the gonadotropic discharge in the 24 hours following the estradiol peak. Progesterone then begins to be secreted by the mature preovulatory follicle, and can be detected in the follicular fluid, but it is only once the corpus luteum is formed that it appears in large concentrations in the blood to reach a maximum at the 21^st day.

The important features may be summarized as follows: when the follicle reaches a diameter of approximately 5 mm (stages 5-6), the mitotic indices of the theca and granulosa cells decrease, whereas their respective secretory functions occur in a coordinated manner: stimulated by LH (only small quantities are needed), the theca cells produce increasing quantities of androgens, which are transformed into estrogens by the granulosa cells exhibiting increased aromatization capacities through FSH stimulation. FSH induces two important syntheses in these cells: the enzymatic complex responsible for aromatization on the one hand, LH receptors on the other hand.

There occurs a reciprocal slowing-down in the synthesis of progesterone and aromatization, and therefore of estradiol synthesis. Up to the gonadotropic surge, this balance is in favour of aromatization (inhibited progesterone synthesis). In contrast, in the 24-48 hours before ovulation, the LH level rises whereas the number of its receptors increases, and luteinization of the follicle begins, with slowing down of aromatization. In clinical practice it is known that luteinization of a follicle that is still immature will perturb the ovarian functions and ovulation in particular.

After constitution of the corpus luteum, the granulosa luteal cells are mainly responsible for progesterone secretion, whereas the theca luteal cells acquire the possibility to aromatize the androgens, and thus directly secrete estradiol. The granulosa cell is subjected to a complex paracrine and autocrine regulation, whose general purpose is to control aromatase activity. Among the positive effectors known, IGF-I is essentially important. Negative effectors are more numerous: progesterone, inhibin (autocrine control), epidermal growth factor and 5a-dihydrotestosterone (paracrine control).

Bibliography

1. Chappel, S.C., and Howles, C. (1991): Hum. Reprod., 6:1206-1212.
2. Driancourt, M.A., Gougeon, A., Royère, D., and Thibault, C. (1991): In: La reproduction chez les mammifères et l’homme,edited by C.Thibault, and M.C. Levasseur, pp 273-298. Ellypses INRA, Edition Marketing, Paris.
3. Gougeon,A. (1990): In: Establishing a successful human pregnancy, edited by R.G. Edwards, pp 49-62. Raven Press, New York.
4. Thibault, C., and Levasseur, M.C. (1988): Hum. Reprod., 3:513-523.

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