GnRH agonist action on granulosa cells at varying follicular stages

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Abstract

In the present review, we attempt to summarize our recent research related to comparative effects of gonadotropin-releasing hormone (GnRH) agonist on the proliferation, apoptosis and differentiated function of cultured porcine granulosa cells from varying follicular stages. The inhibitory effect of GnRH agonist on Proliferating cell nuclear antigen positive rate of cultured cells was prominent in granulosa cells from small and medium but not from large follicles. By contrast, the inhibitory effect of GnRH agonist on 17β-estradiol and progesterone secretion by cultured cells was prominent in granulosa cells from large but not from small and medium follicles. The stimulatory effect of GnRH agonist on apoptosis positive rate of cultured cells was, however, uniform regardless of the stages of follicular growth. These results demonstrate that GnRH agonist exerts diverse actions on granulosa cells over the course of follicular growth. One downregulates granulosa cell proliferation in immature follicles as well as steroidogenesis in mature follicles, and the other upregulates apoptosis of granulosa cells regardless of the stages of follicular growth.

Introduction

During ovarian follicular development, only limited numbers of follicles are selected for ovulation, whereas the remaining majority of follicles undergo atresia. It is now evident that follicular atresia is associated with internucleosomal fragmentation of DNA of granulosa cells (Tilly et al., 1991, Hsueh et al., 1994). Homeostatic control of follicle growth is thought to be the result of the dynamic balance between cell proliferation and cell death. Proliferating cell nuclear antigen (PCNA) is a cell cycle-related nonhistone nuclear protein with a molecular weight of 36 kDa. Elevated levels of PCNA appear in the late G1 phase and become maximal during the S phase of proliferating cells but are not detectable in resting cells (Kurki et al., 1986). Immunocytological PCNA labeling has proven useful in evaluating the proportions of proliferating cells. On the other hand, in situ DNA 3′-end labeling with nonradioactive digoxigenin-dideoxy-UTP (dig-ddUTP) and DNA fragmentation assay have been used to evaluate the occurrence of apoptosis (Billig et al., 1993, Hsueh et al., 1994).

In the late 1980s, gonadotropin-releasing hormone (GnRH) agonist was introduced as a means of downregulating the pituitary to prevent premature ovulation, which, in the past had necessitated canceling in vitro fertilization cycles (Droesch et al., 1989, Meldrum et al., 1989). The downregulating effects of GnRH agonist, as opposed to the stimulatory effects of GnRH, are related to the frequency of administration and the prolonged occupation of GnRH receptors by the agonist (Yen, 1983). Since this introduction of GnRH agonist, pregnancy rates have increased because of the opportunity to retrieve cycles that would have been lost to early ovulation and because of the increase in the number of oocytes obtained in GnRH agonist cycles (Hughes et al., 1992). In addition to the downregulating effects, much evidence suggests direct effects of GnRH and its agonist on the ovary. GnRH and its agonist affect steroidogenesis in granulosa cells and luteal cells (Ying and Guillemin, 1979, Hsueh and Erickson, 1979, Clayton et al., 1979, Otani et al., 1982). The direct effects of GnRH and its agonist on the ovary have been supported by the demonstration of specific, high-affinity binding sites for GnRH and its agonist (Latouche et al., 1989, Harwood et al., 1980a, Harwood et al., 1980b).

In the present review, we first describe our current understanding on the changes in proliferative activity and apoptosis of granulosa cells and theca interna cells during follicular growth and regression in the human ovary. Then, we presents some of the evidence that GnRH agonist exerts diverse actions on granulosa cell proliferation and apoptosis over the course of follicular growth in a porcine model.

Ovarian folliculogenesis is a dynamic and complex process. It is during this process when follicles undergo tremendous growth and maturation. Commencing as a single layer of pregranulosa cells surrounding the immature oocyte, granulosa cells actively proliferate and differentiate until ovulation. Granulosa cell proliferation is of ultimate importance not only for growth of the follicle but also for the creation of a unique microenvironment for oocyte maturation.

Follicles begin to grow from a pool of primordial follicles. Follicular development involves two phases. The basal follicular growth or the first phase is characterized by slow growth of the follicles and the growth rate is tightly related to the proliferation of granulosa cells. FSH may exert an indirect mitogenic effect on these granulosa cells by the enhancement of expression of growth factors or growth factor receptors. These growth factors of endocrine and paracrine origin play determinant roles in antral follicular development. They act with gonadotropins and modulate survival, proliferation and differentiation of granulosa cells. The second phase (the terminal follicular growth phase) is characterized by rapid follicular growth and antral enlargement. This phase is strictly dependent on gonadotropins.

The growth of the primordial follicle to the preantral follicle stage appears to be largely independent of gonadotropic stimulation. However, little is known about the factors responsible for the autonomous growth of the immature follicles in early follicular development. Thus, we have evaluated the proliferative potential of granulosa cells during follicular growth by immunocytochemical staining of PCNA (Maruo et al., 1999). PCNA expression was first apparent in a very small number of granulosa cells of preantral follicles and became abundant during the course of follicular growth (Fig. 1).

Determination of the mean percentage of PCNA-positive nuclei revealed that PCNA-positive rate of granulosa cells was lowest in the primary follicles and significantly increased with the advance of follicular growth (Fig. 2). In comparison with healthy antral follicles, the PCNA-positive rate of granulosa cells in the early stage of atresia of antral follicles was significantly lower but still remained half of that in healthy antral follicles and declined dramatically as atresia advanced (Fig. 3).

Since several lines of evidence suggest a role of the myc oncogene in cell proliferation, we investigated the involvement of the myc oncogene in ovarian follicular development. Immunolocalization of myc protein in the human ovaries was apparent only in primordial and preantral follicles, but not in the antral follicles (Li et al., 1994). Actually, Northern blot analysis of total RNA prepared from porcine granulosa cells revealed that c-myc mRNA was expressed abundantly in small follicle granulosa cells, but not detectable in either medium follicle or large follicle granulosa cells (Sato et al., 1994). Thus, the stage-limited expression of c-myc mRNA in the immature granulosa cells may suggest the possible participation of c-myc in the autonomous growth of immature follicles. Furthermore, we have shown that the erb A oncogene, which encodes a thyroid hormone receptor, is also expressed more abundantly in small follicle granulosa cells. Scatchard analysis of iodinated triiodothyronine binding to porcine granulosa cell nuclei revealed that the number of granulosa cell receptors was higher in small follicle granulosa cells compared to that of large follicle granulosa cells. Accordingly, the increased expression of the erb A oncogene in immature granulosa cells may be responsible for the increase in the number of triiodothyronine receptors in less-mature granulosa cells (Maruo et al., 1992). Thus, the expression of these oncogenes in granulosa cells early in follicular growth may play a physiological role in the autonomous growth of immature granulosa cells in early folliculogenesis, seemingly independent of pituitary gonadotropin stimulation.

In order to assess internucleosomal DNA fragmentation, we have used the in situ DNA 3′-end labeling method in human ovarian tissue sections (Maruo et al., 1999). Apoptosis was observed in the majority of oocyte nuclei in primordial and preantral follicles, whereas oocyte nuclei in growing antral follicles were negative for DNA fragmentation. In granulosa cells of primordial and preantral follicles, apoptosis was almost negative, but in growing antral follicles apoptosis became positive only in a very small number of granulosa cells (Fig. 4).

Determination of the mean percentage of apoptosis-positive nuclei revealed that apotosis-positive rate of granulosa cells in primary and secondary follicles was almost zero, whereas in antral follicles, apoptosis-positive rate of granulosa cells slightly increased (Fig. 5).

In the early stage of atresia of antral follicles, apoptosis was present in granulosa cells, whereas in the late stage of atresia of antral follicles, apoptosis became apparent in the theca interna cells rather than in granulosa cells (Fig. 6).

Determination of the mean percentage of apoptosis-positive nuclei revealed that apoptosis-positive rate in atretic antral follicles was remarkably higher in granulosa cells in the early stage of atresia, but was remarkably higher in the theca interna cells in the late stage of atresia in comparison with that in healthy antral follicles (Fig. 7). This indicates that apoptosis in the early stage of atretic follicles occurs in granulosa cells, whereas apoptosis in the late stage of atretic follicles occurs in theca interna cells.

Consistent with these findings, we have noted that the Fas antigen, a mediator of apoptotic DNA fragmentation, is abundantly expressed in the granulosa and theca cells of atretic antral follicles. In the early stage of atresia of primordial and primary follicles, only the degenerating oocyte showed Fas antigen expression, whereas at the early stage of atresia of antral follicles, the degenerating granulosa cell exhibited Fas antigen expression. In the late stage of antral follicle atresia, Fas antigen expression was apparent only in the hypertrophied theca cells (Kondo et al., 1996). It is therefore possible that, during the course of primordial and primary follicle atresia, apoptosis may occur initially in the oocyte, whereas during the course of antral follicle atresia apoptosis may occur initially in the granulosa cell and subsequently in the theca cell layer. Thus, during the process of atresia, two distinct patterns are discernible: one involves prominent degenerative changes in the oocyte, while alterations in granulosa cells are secondary; the other is typified by distinctive degenerative changes in granulosa cells, with an almost unchanged oocyte. The former type of atresia predominates in preantral follicles, while the latter type of atresia dose so in antral follicles.

Section snippets

Direct effects of GnRH agonist on the proliferative activity, apoptosis and steroidogenesis in cultured porcine granulosa cells at varying follicular stages

Compative analyses of porcine granulosa cells from varying follicular stages to respond to GnRH agonist were performed in terms of PCNA expression, occurrence of apoptosis, and 17β-estradiol (E2) and progesterone (P4) secretion (Takekida et al., 2000). PCNA expression was examined by the avidin/bitin immunoperoxidase method with a monoclonal antibody to PCNA, and apoptosis was assessed by in situ DNA 3′-end labeling method and DNA fragmentation analysis. E2 and P4 were measured by

Discussion

In the ovary, more than 99% of the follicles undergo a degenerative process called atresia during reproductive life. It is now evident that apoptotic cell death is the molecular mechanism underlying follicular atresia (Hsueh et al., 1994). The factors that trigger apoptosis are cell specific, and different cell types may follow diverse early steps, ultimately leading to a common activation of endonuclease and the irreversible apoptotic cell death. Gonadotropins, insulin-like growth factor-I (

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