Role of gonadotrophins and progesterone in the regulation of morphological remodelling and atresia in the monkey peri-ovulatory follicle

Charles L. Chaffin1 and Richard L. Stouffer1,2,3

1 Division of Reproductive Sciences, Oregon Regional Primate Research Center, 505 NW 185th Ave, Beaverton, Oregon 97006 2 Department of Physiology and Pharmacology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peri-ovulatory progesterone plays an indispensable role in ovulation and luteinization, possibly by controlling tissue remodelling of the ovulatory follicle. This study was designed to evaluate gonadotrophin- versus progestinmediated changes to the morphology of the follicle wall during luteinization. Ovaries were obtained from macaques undergoing ovarian stimulation either before (0 h) or up to 36 h following administration of an ovulatory human chorionic gonadotrophin (HCG) bolus with or without a 3ß-hydroxysteroid dehydrogenase inhibitor and a non-metabolisable progestin. Morphological changes occurred within 12 h of HCG in the theca, and around 24 h in the granulosa layer and basement membrane. Steroid depletion resulted in follicles that did not luteinize during the 36 h interval, or alternatively, those that exhibited premature luteinization by 12 h post-HCG. Progestin replacement restored normal morphology, although the presence of antral blood suggested acceleration of normal tissue remodelling. A proportion of pre-ovulatory follicles became atretic after the HCG bolus, although progestin treatment reduced the percentage of atretic follicles. Ovarian stimulation resulted in the development of multiple pre-ovulatory follicles which are heterogeneous in their response to the HCG bolus and local progestin action. Nevertheless, this model supports both anti-atretic and pro-differentiative actions of progesterone in promoting follicular health and remodelling during the development of the corpus luteum.

Key words: atresia/monkey/morphology/peri-ovulatory/progesterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The morphological characteristics of the luteinizing follicle and developing corpus luteum were described for the rhesus monkey in 1945 by Corner, Sr, (Corner, 1945Go) and for the human in 1956 by Corner, Jr (Corner, 1956Go). The findings of the Corners were later extended (Koering, 1969Go; Mori et al., 1978Go) to include pre-ovulatory and atretic follicles, and correlations with serum hormones respectively. However, difficulty in defining the onset of the ovulatory gonadotrophin surge during spontaneous menstrual cycles precludes the precise timing of events within the peri-ovulatory follicle in primates. Although studies using hormonally stimulated rats demonstrated changes in follicular wall morphology at precise points following the administration of an ovulatory stimulus (Szoltys et al., 1994Go), this approach has not been applied to primates, despite the widespread use of ovarian stimulation cycles in research and clinical settings.

In primates, follicular rupture occurs 36–40 h following the onset of the midcycle gonadotrophin surge (Fritz et al., 1992Go). During this peri-ovulatory interval, granulosa cells undergo changes in response to the ovulatory stimulus that result in terminally differentiated luteal cells. While differentiating (luteinizing) granulosa cells synthesize large amounts of progesterone (Chaffin et al., 1999aGo), the discovery that these cells express the progesterone receptor (PR; Hild-Petito et al., 1988; Iwai et al., 1990; Chaffin et al., 1999b) led to the hypothesis that progesterone acts in a local manner to mediate ovulation and luteinization. Indeed, several laboratories have demonstrated an obligate role for progesterone in the ovulatory process of rats (Snyder et al., 1984Go; Brännström and Janson, 1989Go; Espey et al., 1990Go) and primates (Hibbert et al., 1996Go). While the mechanism of progesterone action remains unclear, recent evidence points to the regulation of several genes thought to be important in tissue remodelling, such as the matrix metalloproteinases (Iwamasa et al., 1992Go; Chaffin and Stouffer, 1999Go). Progesterone was also reported to act as an anti-apoptotic/cell survival factor for human and rat luteinized granulosa cells during culture (Chaffkin et al., 1992Go, 1993Go; Peluso and Pappalardo, 1994Go), via either nuclear PR (Peluso and Pappalardo, 1994Go) or a novel progesterone-binding protein (Peluso and Pappalardo, 1998Go). However, it is not known whether progesterone acts as a survival factor in the primate follicle in vivo.

Studies were designed to test the hypothesis that progesterone regulates changes in the morphology of the peri-ovulatory follicle, and acts in vivo as an anti-atretic factor in peri-ovulatory follicles during ovarian stimulation of rhesus monkeys. Whole ovaries and granulosa cells were obtained from rhesus monkeys undergoing ovarian stimulation before (0 h) and at 12, 24 or 36 h after the administration of an ovulatory human chorionic gonadotrophin (HCG) bolus. This model provides multiple large pre-ovulatory follicles capable of ovulating following an HCG bolus (Hibbert et al., 1996Go; Chaffin et al., 1999bGo). In addition, the intrafollicular steroid milieu can be controlled using a 3ß-hydroxysteroid dehydrogenase (3ß-HSD) inhibitor and progestin replacement during the peri-ovulatory interval (Chaffin and Stouffer, 1999Go), permitting the analysis of local progestin actions on follicular morphology and atresia.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal protocols
The general care and housing of monkeys at the Oregon Regional Primate Research Center (ORPRC) was described previously (Wolf et al., 1996Go). Animal protocols and experiments were approved by the ORPRC Animal Care and Use Committee, and studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Adult female rhesus monkeys were stimulated with recombinant human gonadotrophins (r-hFSH ± r-hLH for 9 days; Laboratoires Serono SA, Aubonne, Switzerland) in order to promote the development of multiple pre-ovulatory follicles. A gonadotrophin-releasing hormone (GnRH) antagonist (Antide; Laboratoires Serono SA) was administered daily to prevent endogenous LH/FSH secretion. These protocols have been described in detail elsewhere (Chaffin et al., 1999bGo). Animals were assigned randomly to receive either no ovulatory stimulus, or 1000 IU r-HCG (Laboratoires Serono SA) by single i.m. injection, to initiate peri-ovulatory events. Ovaries were removed during laparotomy of anaesthetized animals either the morning after the last LH/FSH treatment (0 h), or at 12, 24 and 36 h following administration of 1000 IU r-HCG (n = 3–4 monkeys at each time point, except n = 2 at 24 h). An additional group (n = 3–4 monkeys per time point) of animals was stimulated in an identical fashion, but also received the 3ß-HSD inhibitor trilostane (TRL; Sanofi Research Division, Malvern, PA, USA) orally [1 g in 8 ml orange Kool-Aid (Kraft General Foods, Inc., White Plains, NY, USA) containing 1% (w/v) gum tragacanth (Sigma, St Louis, MO, USA)] beginning 4 h before HCG administration and for every 12 h thereafter until the time of ovariectomy. It has been shown previously that this treatment attenuates completely the peri-ovulatory rise in intrafollicular progesterone concentrations (Chaffin and Stouffer, 1999Go). A third group of animals (n = 3–4 per time point) received TRL plus the progestin R5020 (Promegestrone; DuPont/NEN, Boston, MA, USA) 2.5 mg in sesame oil, s.c., once daily starting at the time of HCG. R5020 binds to and activates the progesterone receptor, but is not metabolized to androgens or oestrogen.

Morphological classification of peri-ovulatory follicles
Ovaries were placed into ice-cold phosphate-buffered saline (PBS), transported to the laboratory, and subsequently one ovary per pair was bisected. Half of the ovary was placed in 10% formalin overnight before embedding in glycol methacrylate. The remaining ovarian tissue was processed for other studies. A single section (3 µm) of each ovary was taken from near the point of bisection, roughly equal to the largest cross-section and stained with haematoxylin and eosin. Follicles >=4 mm diameter were identified by direct measurement using a low-power light microscope. Identification of morphologically luteinizing follicles was based upon the presence of an expanded, hypertrophic granulosum (Corner, 1945Go; Koering, 1969Go; Mori et al., 1978Go). Follicles were classified as non-atretic or atretic according to Corner (1945), Koering (1969), Mori et al. (1978) and Chikazawa et al. (1986). Extensive debris in the antrum, consisting largely of unadhered, pyknotic granulosa cells and/or extensive pyknosis of adhered granulosa cells was used as a marker of atresia. The total number of healthy or atretic follicles was pooled among animals within a treatment and expressed as the percentage of total follicles per treatment group (generation of individual animal means was precluded as some sections contained only a single follicle >=4 mm).

Statistical analysis
The percentage of abnormal and atretic follicles over time and between treatment groups was compared by {chi}2 analysis. The TRL and TRL + R5020 data for atretic follicles were compared to time-matched HCG-treated animals at the 12 and 36 h time points by {chi}2. Differences were considered significant at P < 0.05, and data were presented as mean ± SEM.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Follicular morphology of non-atretic follicles from control monkeys treated with HCG only
The follicular wall morphology of control ovaries removed before (0 h) and at 12, 24 or 36 after an ovulatory HCG bolus is shown in Figure 1Go. Before HCG, the avascular granulosa layer was compact, and generally comprised fewer than 10 cell layers (Figure 1AGo). The number of granulosa cell layers was consistent throughout the follicle, giving a `smooth' appearance to the granulosa–antral boundary. The basement membrane was smooth, and abutted by vessels of relatively small diameter (Figure 1AGo). In healthy follicles, the theca interna layer was generally thin, with oblong cells running parallel to the basement membrane. The morphology of the granulosa cell layer did not change within 12 h of HCG administration; the only notable change was an increase in the number of small vessels in the theca layer abutting the basement membrane compared with 0 h animals (Figure 1BGo). Theca interna had begun to hypertrophy at this time point, as shown by the presence of round nuclei, although a considerable number of oblong theca cells remained. The follicle wall underwent dramatic change by 24–36 h post-HCG (Figure 1CGo, E and F), although at 24 h not all follicles appeared morphologically luteinized (Figure 1DGo). Notably, the granulosa cells took on a fusiform shape with a hypertrophic cytoplasm. In general, cells appeared to be `streaming' into the antrum, with fewer connections to other cells or the basement membrane. The basement membrane was irregular but mostly contiguous, with protuberances (`infolding'; Koering, 1969) into the antrum, often associated with large vessels. At 24 h post-HCG, the theca interna had variable characteristics that were associated with the degree of morphological changes visible in the granulosa layer. In follicles that were luteinized to a greater extent (based on granulosa cell characteristics; Figure 1CGo), extensive vasculature was present in the theca layer, and theca cells were round and hypertrophic. In contrast, in less luteinized follicles (Figure 1DGo), the vasculature was not as prevalent in the theca layer and theca cells were less hypertrophied, but round in shape. By 36 h after an ovulatory stimulus, theca cells were very large and round, and generally one to three layers deep. At several points around the follicle, it was difficult to distinguish clearly the theca cells from the granulosa cells. However, there was no evidence of vascularization of the granulosum, although the vasculature was very pronounced within the infoldings of the follicle wall (Figure 1EGo). No macroscopic differences were noted between the left and right ovaries upon ovariectomy, and no ovulatory stigmata were observed at any time point (Figure 1FGo).



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Figure 1. Morphology of the follicle wall from control (HCG only) animals before (0 h) and at 12, 24 or 36 h after administration of an ovulatory HCG bolus. (A) 0 h (no HCG; scale bar = 20 µm; also applicable to panels B–E); (B) 12 h; (C) 24 h (luteinizing follicle), infolding of the follicle wall is apparent, and associated with vascular elements, and granulosa cells have begun to hypertrophy and expand into the antrum; (D) 24 h (less luteinized follicle), this follicle does not show infolding or granulosa hypertrophy, and thus demonstrates the transitional nature of follicles at this time point; (E) 36 h; (F) ovarian section at 36 h (scale bar = 5 mm).

 
Morphology of non-atretic follicles following steroid depletion and progestin replacement
At 12 h after HCG administration, 53% of the follicles in TRL (steroid-depleted)-treated animals were indistinguishable morphologically from control (HCG-only) follicles (Table IGo). A second group of follicles (47%) had an extensively infolded granulosa layer often detached from the basement membrane (Table IGo; Figure 2AGo). The granulosa layer of these follicles was compact, but many of the theca interna cells were rounded and hypertrophic rather than oblong-shaped, as seen in control follicles. Notably, the vasculature around these follicles was extensive compared with that in controls, both in terms of vessel size and number (Figure 2AGo).


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Table I. Follicular characteristics in control (CTRL, HCG only), Trilostane (TRL)- and TRL + R5020-treated monkeys before (0 h) and at 12 or 36 h after HCG administration
 


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Figure 2. Morphology of non-atretic follicles from steroid-depleted [trilostane (TRL)-treated] ± progestin (R5020) replaced animals 12 or 36 h after administration of an ovulatory HCG bolus. (A) 12 h + TRL; (B) 36 h + TRL; insets are low-magnification images of those depicted in (A) and (B). (C) 12 h + TRL + R5020; (D) 36 h + TRL + R5020. Arrows in (C) and (D) indicate red blood cells in the antrum. Atretic follicles (E) 12 h and (F) 36 h after HCG. Scale bar = 20 µm (applies to all panels).

 
By 36 h post-HCG, 66% of non-atretic follicles from steroid-depleted monkeys had a basement membrane lacking the characteristic infolding of time-matched controls and a severely restricted vascular presence in the theca layer (Table IGo; Figure 2BGo). Both the overall follicular and cellular morphology of these follicles were highly reminiscent of 0 h (no HCG) control follicles (see above).

Co-administration of TRL and R5020 did not change follicular morphology at 12 h post-HCG compared with the TRL treatment group, although two follicles (from separate animals) had traces of red blood cells in the antrum, particularly where the granulosa layer had detached from the basement membrane (Figure 2CGo). The addition of R5020 prevented the effects of steroid depletion at 36 h post-HCG, resulting in only 23% of follicles with morphological similarities to 0 h follicles (Table IGo). However, while progestin replacement typically returned follicle morphology to that of time-matched 36 h controls, blood was present in the antrum of 54% of follicles (Table IGo; Figure 2DGo). Theca interna from follicles with antral blood were difficult to differentiate from granulosa cells, i.e. the basement membrane was no longer intact. This was especially prevalent at the antral tips of the infolded portions of the follicle wall (Figure 2DGo).

Incidence and characteristics of atresia
In control animals, the percentage of grossly atretic follicles was low (<10%) both before and soon after (12 h) HCG, but increased (P < 0.05) to over one-third of the large follicles 36 h after the administration of an ovulatory gonadotrophin bolus (Figure 3Go). Steroid depletion did not significantly change the proportion of atretic follicles 12 h post-HCG, but appeared to increase (not significant; P = 0.18) the percentage of atretic follicles at 36 h to ~70% of all follicles. Progestin replacement (R5020) significantly lowered the percentage of atretic follicles, compared with TRL treatment groups at 36 h (P < 0.05), to values comparable with those of controls.



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Figure 3. Proportion of atretic follicles before (0 h) and at 12, 24 or 36 h after administration of an ovulatory HCG bolus ± steroid depletion and progestin replacement. The total number of follicles and number of atretic follicles analysed in control (CTRL; HCG alone), Trilostane (TRL) and TRL + progestin (R5020) animals are summarized in Table IGo. Data are mean values analysed by {chi}2. Letters above bars indicate significant differences in CTRLs (HCG alone) across time; lines with asterisks indicate significant (P < 0.05) differences between groups within time points; NS = not significant.

 
At 0–12 h post-HCG, large numbers of unadhered, pyknotic granulosa cells were found in the antrum in grossly atretic follicles, and the theca interna layer was exceedingly thin. Nuclei of theca interna cells were very small and spindle-shaped, and the orientation of the nuclei was disorganized (i.e. did not necessarily run parallel to the basement membrane) (Figure 2EGo). By 24–36 h post-HCG, pyknotic granulosa cells were similar to those at 0–12 h, while the theca interna layer was visually healthy, except for a more limited vascular presence than in healthy counterparts (Figure 2FGo). No apparent differences in the characteristics of atretic follicles were noted between control, TRL and TRL + R5020 treatments.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study details for the first time the morphological changes that occur in the follicle wall during the pre-ovulatory interval in primates undergoing ovarian stimulation. During the 36 h period following the ovulatory HCG stimulus, approximately two-thirds of the large antral follicles in rhesus monkeys exhibited changes in the theca and granulosa layers that were consistent with differentiation into the luteinized tissue of the corpus luteum. In contrast, the other third become grossly atretic. We reported previously that ovarian stimulation in macaques results in approximately 15 pre-ovulatory follicles, of which an average of 10 will ovulate following an HCG bolus (Hibbert et al., 1996Go). The subgroup of atretic follicles identified in the current study may be analogous to the fraction that do not ovulate. Our study also provides novel evidence in non-human primates that locally produced progesterone has both pro-differentiative and anti-atretic actions. The data support the concept that progesterone produced in response to the midcycle gonadotrophin surge plays an important role in promoting follicular health and remodelling, leading to corpus luteum formation.

The first indices of remodelling in the macaque follicle wall occur in the theca layer; by 12 h after HCG, theca interna cells began to hypertrophy and vascular elements became more prominent. These data are consistent with the report by Corner (1956) of theca cells with large, round nuclei in peri-ovulatory follicles in women. Although others (Fujita et al., 1981Go) have suggested that theca interna cells regress during luteinization and reappear in the mature corpus luteum of women, we found no evidence of pyknotic nuclei in the theca interna of macaque follicles. The increased vascular presence was consistent with increased levels of angiogenic factors, such as vascular endothelial growth factor (VEGF; Hazzard et al., 1999), within the follicle within 12 h after an ovulatory stimulus. Although detailed molecular and cellular studies are needed, the early time-course of morphological changes supports the contention that luteinization proceeds from outside (theca) to inside (granulosa) the follicle in primates (Conley and Bird, 1997Go).

Recognized as a peri-ovulatory event for over 50 years (Corner, 1945Go), the current study demonstrates that granulosa cell hypertrophy does not occur until between 12 and 24 h after the ovulatory HCG bolus. Indeed, by 24 h post-HCG, ovaries contain follicles with either non-luteinized or luteinized (hypertrophied) granulosa cells. The data support the hypothesis that cells display a granulosa-, intermediate- and luteal-phenotype at 12, 24 and 36 h after HCG respectively. We reported that a number of genes markedly change their expression pattern in macaque granulosa cells by 24 h after HCG, including steroid and aryl hydrocarbon receptors (Chaffin et al., 1999bGo), cellular components for cholesterol uptake and steroidogenesis (Chaffin et al., 2000Go), matrix metalloproteinases and their inhibitors (Chaffin and Stouffer, 1999Go), plus angiogenic factors (Hazzard et al., 1999Go). Nevertheless, there are early (12 h) changes in mRNA levels in granulosa cells before cellular hypertrophy or breakdown of the basement membrane. It is likely that early gene expression [e.g. progesterone receptor, interstitial collagenase (Chaffin and Stouffer, 1999Go; Chaffin et al., 1999bGo)] is critical for changes in cell sensitivity or activities that are initial events in cell luteinization or follicular remodelling, or for processes that require a longer time period to complete.

Steroid depletion is associated with profound changes in follicular morphology during the peri-ovulatory interval, although the nature of these changes is dependent upon the duration of HCG exposure. Steroid depletion for 36 h following HCG results in a large proportion of follicles lacking theca and granulosa cell hypertrophy or basement membrane infolding. These follicles are morphologically similar to those observed before (0 h) exposure to an ovulatory stimulus, and thus are presumed to be non-luteinized. Steroid depletion results in decreased mRNA levels in granulosa cells, including those for various proteases [interstitial collagenase, gelatinase B, tissue inhibitor of metalloproteinase (TIMP)-2], angiogenic factors (angiopoeitin-2) and progesterone receptor, by 36 h post-HCG (Chaffin and Stouffer, 1999Go; Chaffin et al., 1999bGo; Hazzard et al., 1999Go). Thus, multiple factors associated with tissue remodelling and differentiation are suppressed in concert with the lack of morphological luteinization observed by steroid depletion. However, analyses of follicles from earlier (12 h) in the peri-ovulatory interval suggest that a cohort of follicles undergoes abnormal premature luteinization in response to the depleted steroid milieu. Although ~50% of the follicles were non-luteinized as expected by 12 h after HCG plus steroid depletion, other follicles displayed theca, but not granulosa, cell hypertrophy. The theca layer was highly vascularized and, in some cases, the intact granulosa layer was separated from the theca layer, suggesting that breakdown of the basement membrane had occurred. Since steroid-depleted ovaries were not collected at 24 h post-HCG, and these abnormal follicles were not observed by 36 h, the fate of such steroid-depleted follicles is unknown. However, it is reasonable to speculate that they form a portion of the atretic follicles observed at 36 h post-HCG. Thus, following steroid depletion, follicles appear to follow one of two fates: namely, that they may prematurely luteinize as seen at 12 h post-HCG; or that they do not luteinize, as seen by 36 h post-HCG. It is not known what dictates a follicle's response to steroid depletion, but it may be related to the maturity (or post-maturity) of the follicle at the time of HCG administration.

In contrast, progestin (R5020) replacement during steroid depletion typically restored the luteinized morphology in theca and granulosa cells by 36 h after HCG. Likewise, we reported recently that progestin replacement restored many of the changes in gene expression in macaque granulosa cells elicited by steroid depletion (Chaffin and Stouffer, 1999Go; Chaffin et al., 1999bGo; Hazzard et al., 1999Go). These findings support the concept that progesterone plays a local role in the structural remodelling and cellular differentiation occurring during formation of the corpus luteum. However, ~50% of the follicles exposed to TRL and R5020 had red blood cells in the antrum by 36 h post-HCG. Antral blood is only observed in recently ruptured follicles in natural cycles (Corner, 1956Go), and never in control follicles by 36 h exposure to HCG alone in ovarian stimulation cycles (current study). Thus, unopposed progestin (in the absence of oestrogens or androgens) may elicit premature vascularization of the follicle, or more likely, premature weakening of the basement membrane. Notably, steroid depletion and progestin replacement restored interstitial collagenase mRNA levels in granulosa cells, whereas TIMP-2 mRNA levels were further reduced compared with steroid depletion alone (Chaffin and Stouffer, 1999Go). Therefore, it is possible that unopposed progestin creates a situation in which proteolytic activity is not well balanced by endogenous inhibitors. A role for other steroids, for example in preventing premature luteinization at 12 h post-HCG or modulating progestin action at 36 h, cannot be ruled out since there is dynamic expression of oestrogen receptor ß and androgen receptor in macaque granulosa cells during the peri-ovulatory interval (Chaffin et al., 1999bGo).

Ovarian stimulation is used both clinically to produce multiple oocytes for assisted reproduction treatment protocols, and experimentally to study peri-ovulatory and luteal events. While certain end-points appear consistent between ovarian stimulation and natural cycles (Chaffin et al., 1999aGo), the present study demonstrates substantial follicular heterogeneity, notably in the high proportion of atretic follicles near the time of ovulation (36 h post-HCG). This is surprising in light of the fact that only presumptive pre-ovulatory follicles (i.e. >=4 mm diameter) were analysed, although it has been reported (Szoltys et al., 1994Go) that superovulatory protocols in rats result in follicles with a reduced, degenerated granulosa layer compared with those in natural cycles. Several possibilities could explain the incidence of atresia, including: (i) a pharmacological effect of multiple follicular development; (ii) gonadotrophin deprivation during the peri-ovulatory interval (due most likely to pre- or post-mature follicles that are unable to respond to HCG); or (iii) excessive intrafollicular levels of HCGstimulated cAMP (Aharoni et al., 1995Go). Further studies comparing the single dominant follicle in spontaneous cycles and the multiple follicles from ovarian stimulation cycles are needed to understand the events leading to atresia of large antral follicles in primates.

Notably, the proportion of atretic follicles in steroid-depleted animals 36 h following the HCG bolus was almost 70%, while progestin replacement reduced the percentage of atresia to <15%. It is interesting to note that unopposed progestin tended to reduce the proportion of atretic follicles to below the 36 h control values, underscoring the significance of this steroid in maintaining the health of follicles during luteinization. This is consistent with reports that progesterone inhibits apoptosis of rat granulosa cells in vitro after several days in culture (Peluso and Pappalardo, 1994Go), and decreases Fas mRNA and cell death in rat and bovine corpus luteum (Kuranaga et al., 2000Go; Rueda et al., 2000Go). It was also suggested (Duffy et al., 1994Go) that progesterone has an autocrine role in maintaining the structure and function of the primate corpus luteum. We postulate that follicular health is maintained by gonadotrophins in granulosa cells (0–24 h post-HCG), while progesterone acts to prevent atresia following luteinization of the follicle (12–36 h after an ovulatory stimulus), and that this role for progesterone extends into the luteal phase of the cycle.

In summary, the current study demonstrates changes in follicular morphology following an ovulatory stimulus given to rhesus monkeys undergoing ovarian stimulation. Morphological changes occur within 12 h of HCG in the theca layer (hypertrophy, vascular growth), and around 24 h in granulosa layer (hypertrophy, expansion). Infolding of the follicle wall occurs 24–36 h after the ovulatory stimulus in control animals (HCG). Whereas HCG alone elicited changes consistent with luteinization of the follicle wall, steroid depletion resulted in two clearly defined groups of follicles: (i) those which do not luteinize during the 36 h interval; and (ii) those which exhibit incomplete, premature luteinization by 12 h post-HCG and presumably undergo atresia by 36 h. Progestin replacement restored the luteinizing features in follicles, although the presence of antral blood suggests acceleration of normal tissue remodelling. Regardless of treatment group, a proportion of pre-ovulatory follicles became atretic after the HCG bolus, although progestin treatment reduced the percentage of atretic follicles. Thus, ovarian stimulation results in the development of multiple pre-ovulatory follicles which are heterogeneous in their response to the ovulatory HCG bolus and local progestin action. Nevertheless, this model supports both anti-atretic and pro-differentiative actions of progesterone in promoting follicular health and remodelling during the development of the corpus luteum.


    Acknowledgments
 
The authors appreciate the expert service provided by the Division of Animal Resources and the surgical team of Dr John Fanton, the Endocrine Services Core Laboratory, the Assisted Reproductive Technology Core, and the Molecular Biology Core Laboratory. Recombinant human LH, FSH, HCG and Antide were generously provided by Ares Advanced Technology, Inc., a member of the Ares-Serono Group. Trilostane was graciously supplied by Sanofi Pharmaceutical Inc., Great Valley, Malvern, PA, USA. These studies were supported by NIH/NICHD HD20869 (R.L.S.), U54 HD18185 (R.L.S.) as part of the Specialized Cooperative Centers Program in Reproductive Research, HD8302 (C.L.C.), and RR00163.


    Notes
 
3 To whom correspondence should be addressed at: Division of Reproductive Sciences, Oregon Regional Primate Research Center, 505 NW 185th Ave, Beaverton, Oregon 97006.E-mail: stouffri{at}ohsu.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Chaffin, C.L., Stouffer, R.L. and Duffy, D.M. (1999b) Gonadotropin and steroid regulation of steroid receptor and aromatic hydrocarbon receptor mRNA in macaque granulosa cells during the periovulatory interval. Endocrinology, 140, 4753–4760.[Abstract/Free Full Text]

Chaffin, C.L., Dissen, G.A. and Stouffer, R.L. (2000) Hormonal regulation of steroidogenic enzyme expression in granulosa cells during the peri-ovulatory interval in monkeys. Mol. Hum. Reprod., 6, 11–18.[Abstract/Free Full Text]

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Submitted on May 18, 2000; accepted on August 18, 2000.