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
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Abstract |
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Key words: atresia/monkey/morphology/peri-ovulatory/progesterone
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Introduction |
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In primates, follicular rupture occurs 3640 h following the onset of the midcycle gonadotrophin surge (Fritz et al., 1992). 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., 1999a
), 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., 1984
; Brännström and Janson, 1989
; Espey et al., 1990
) and primates (Hibbert et al., 1996
). 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., 1992
; Chaffin and Stouffer, 1999
). 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., 1992
, 1993
; Peluso and Pappalardo, 1994
), via either nuclear PR (Peluso and Pappalardo, 1994
) or a novel progesterone-binding protein (Peluso and Pappalardo, 1998
). 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., 1996; Chaffin et al., 1999b
). 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, 1999
), permitting the analysis of local progestin actions on follicular morphology and atresia.
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Materials and methods |
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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, 1945
; Koering, 1969
; Mori et al., 1978
). 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 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
2. Differences were considered significant at P < 0.05, and data were presented as mean ± SEM.
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Results |
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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 2C). 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 I
). 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 I
; Figure 2D
). 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 2D
).
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 3). 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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Discussion |
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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., 1981) 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, 1997
).
Recognized as a peri-ovulatory event for over 50 years (Corner, 1945), 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., 1999b
), cellular components for cholesterol uptake and steroidogenesis (Chaffin et al., 2000
), matrix metalloproteinases and their inhibitors (Chaffin and Stouffer, 1999
), plus angiogenic factors (Hazzard et al., 1999
). 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, 1999
; Chaffin et al., 1999b
)] 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, 1999; Chaffin et al., 1999b
; Hazzard et al., 1999
). 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, 1999; Chaffin et al., 1999b
; Hazzard et al., 1999
). 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, 1956
), 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, 1999
). 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., 1999b
).
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., 1999a), 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 (Szo
tys et al., 1994
) 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., 1995
). 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, 1994), and decreases Fas mRNA and cell death in rat and bovine corpus luteum (Kuranaga et al., 2000
; Rueda et al., 2000
). It was also suggested (Duffy et al., 1994
) 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 (024 h post-HCG), while progesterone acts to prevent atresia following luteinization of the follicle (1236 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 2436 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.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 18, 2000; accepted on August 18, 2000.