Dynamics of periovulatory steroidogenesis in the rhesus monkey follicle after ovarian stimulation

Charles L. Chaffin1, David L. Hess1,2 and Richard L. Stouffer1,2,3

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
The temporal relationships and regulation of events in the primate follicle during the periovulatory interval are poorly understood. This study was designed to elucidate the dynamics of steroid synthesis in the macaque follicle during ovarian stimulation cycles in which serum/follicular fluid aspirates were collected at precise intervals before (0 h) and after (up to 36 h) administration of the ovulatory human chorionic gonadotrophin (HCG) bolus. Serum concentrations of progesterone increased (P < 0.05) within 30 min, and follicular fluid progesterone concentrations were elevated 180-fold within 12 h, of HCG injection, and remained elevated until the time of ovulation. In contrast, 17ß-oestradiol concentrations increased initially, but then declined (P < 0.05) by 36 h post-HCG. Acute incubation of granulosa cells with and without steroidogenic substrates demonstrated that: (i) 3ß-hydroxysteroid dehydrogenase and aromatase activities were present in equivalent amounts before and after HCG; whereas (ii) P450 side-chain cleavage activity increased (P < 0.05) within 12 h of HCG; and (iii) exogenous low-density lipoprotein and cholesterol were not utilized for steroidogenesis. This model should be useful for further studies on ovulation and luteinization in primates, and enable elucidation of the local actions of progesterone and other steroids at specific time points during the periovulatory interval.

Key words: granulosa cell/macaque/periovulatory interval/steroid synthesis/steroidogenic enzymes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
During the ovarian cycle in mammalian species, the mid-cycle surge of luteinizing hormone (LH) signals the initiation of the periovulatory interval, during which time the follicle initiates progesterone production and begins to luteinize, the oocyte resumes meiosis, and the follicle wall ultimately ruptures to extrude a fertilizable oocyte. While the mechanisms underlying these events are complex and poorly understood, the local action of progesterone is reportedly essential in both rodents and primates (e.g. Lipner and Wendelken, 1971Go; Brännström and Janson, 1989Go; Loutradis et al., 1991Go; Lydon et al., 1995Go; Hibbert et al., 1996Go). Thus, a paracrine/autocrine feedback mechanism may exist within the mature follicle whereby locally produced progesterone stimulates the cascade of events leading to luteinization and ovulation (Fanjul et al., 1983Go; Iwamasa et al., 1992Go; Morgan et al., 1994Go; Donath et al., 1997Go; Lioutas et al., 1997Go).

In humans and Old World monkeys, ovulation generally occurs 36–38 h following the onset of the LH surge (Weick et al., 1973Go; Hoff et al., 1983Go). Unlike in many non-primate species, circulating oestrogen concentrations do not drop immediately following the onset of the LH surge in primates, but rather remain elevated for approximately 24 h. However, progesterone concentrations begin to rise after the surge in both non-primate and primate species, supporting a common function for this hormone during the periovulatory interval (Weick et al., 1973Go; Smith et al., 1975Go; Hoff et al., 1983Go; Park and Mayo, 1991Go; Sirois, 1994Go). The onset of progesterone synthesis and the increase in progesterone receptor expression following the ovulatory stimulus (e.g. Iwai et al., 1990Go; Park and Mayo, 1991Go) argues for a direct, possibly early, role for progesterone in ovulation and luteinization, although the time-frame and mechanism by which progesterone exerts this influence is not known.

While limited studies in primates have examined the production of steroids following an endogenous surge of LH (e.g. Weick et al., 1973Go; Mori et al., 1978Go; Hoff et al., 1983Go), none has yet controlled the timing of the ovulatory stimulus. The use of ovarian stimulation cycles (Wolf et al., 1989Go) provides multiple large antral follicles for study and permits analysis at precise time points after administering the ovulatory gonadotrophin bolus. In addition, the data are relevant to clinical in-vitro fertilization (IVF) settings. The present study was designed to elucidate the dynamics of steroid synthesis in the primate follicle during the periovulatory interval both in vivo and in vitro using granulosa cells aspirated before, and 12, 24, or 36 h following an ovulatory stimulus of human chorionic gonadotrophin (HCG).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
Animals
The general care and housing of rhesus monkeys at the Oregon Regional Primate Research Center was described previously (Wolf et al., 1990Go). Adult female rhesus monkeys exhibiting normal menstrual cycles of approximately 28 days were stimulated with recombinant human gonadotrophins [r-hFSH (follicle stimulating hormone), 30 IU i.m. twice daily for 8 days; r-hLH, 30 IU twice daily on days 7 and 8; Laboratoires Serono SA, Aubonne, Switzerland] beginning 1–3 days after the onset of menses in order to promote the development of multiple preovulatory follicles. Monkeys also received a daily subcutaneous injection of the gonadotrophin releasing hormone antagonist Antide [0800 h, 0.5 mg/kg body weight in propylene glycol:water (1:1); Laboratoires Serono SA] throughout the stimulation protocol to prevent an endogenous LH surge. This or comparable protocols using urinary gonadotrophin preparations were utilized by this research group to study folliculogenesis and periovulatory events in the primate, including the effects of steroid deprivation. These protocols result in an average of 18 large follicles that will ovulate (VandeVoort et al., 1989Go; Zelinski-Wooten et al., 1994Go; Hibbert et al., 1996Go). Animals were assigned randomly to receive no ovulatory stimulus, or 1000 IU r-HCG (single injection i.m.; Laboratoires Serono SA) to initiate periovulatory events. Preovulatory follicles (>=4 mm) from both ovaries of a single animal were aspirated and pooled using a 22-gauge needle during laparotomy from anaesthetized animals (Hibbert et al., 1996Go) either the morning after the last LH/FSH treatment (0 HCG) or 12, 24 or 36 h following administration of 1000 IU r-HCG (n = 3–7 monkeys/time point). No animal was duplicated within a time point, although several, but not all, animals were represented twice. Some of the granulosa cell aspirates were utilized for other experiments (e.g. mRNA), while all serum and follicular fluid samples were assayed; therefore the number of replicates was not equal between cell and fluid end-points.

Daily blood samples were obtained from unanaesthetized animals by saphenous venipuncture from the beginning of gonadotrophin treatment. The blood sample from the day of follicle aspiration was collected just prior to surgery. Serum oestradiol and progesterone concentrations were determined using specific radioimmunoassays (Wolf et al., 1990Go), and follicular growth was monitored using serum steroid concentrations and ultrasonography performed on days 6–7 of stimulation (Zelinski-Wooten et al., 1997Go). Blood samples were collected until 5 days after follicle aspiration to verify the presence of typical concentrations of serum progesterone (i.e. functional corpora lutea) during the luteal phase. Serum concentrations of bioactive LH were determined for the 3 days prior to and including the day of follicle aspiration using an in-vitro mouse Leydig cell bioassay (Ellinwood and Resko, 1980Go) to confirm the absence of an endogenous LH surge.

In order to better evaluate the changes in circulating steroids during the early periovulatory interval, serum samples from rhesus monkeys undergoing ovarian stimulation (Chandrasekher et al., 1994Go) were assayed for progesterone and 17ß-oestradiol concentrations by radioimmunoassay following a 1000 IU bolus of urinary HCG. Serum samples were collected before, or 0.5, 1, 2, 4, 6, 8, 12, or 24 h following administration of HCG.


    Granulosa cell preparation
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
Cells aspirated during laparotomy were removed from the follicular fluid by centrifugation at 277 g for 15 min (4°C), and the resulting cell-free follicular fluid was divided into volumes of 25–100 µl and stored at -80°C. Oocytes were removed from the pelleted aspirate, and the remainder of the pellet was enriched for granulosa cells as described by Christenson and Stouffer (1997). In brief, cells were pelleted at 190 g (10 min, 4°C) and resuspended in Ham's F-10 medium (Life Technologies, Grand Island, NY, USA). The resuspension was layered on to a gradient of 40% Percoll (Sigma Chemicals, St. Louis, MO, USA) and 60% Hanks' Balanced Salt Solution with 0.1% bovine serum albumin (BSA) and centrifuged at 470 g for 30 min at 4°C. The resulting layer of granulosa cells was resuspended in Ham's F-10, cell numbers were determined using a haemacytometer, and cell viability was assessed by Trypan blue exclusion.


    Granulosa cell incubation
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
Granulosa cells (4x105) were incubated in 0.4 ml Ham's F-10 medium/0.1% BSA in a gyratory waterbath at 37°C for 2 h under 95% O2/5% CO2 (Sanders and Stouffer, 1995Go). A period of 2 h was chosen in order to minimize the amount of in-vitro luteinization while allowing sufficient time for steroid production to exceed assay sensitivity concentrations. Incubations were performed in duplicate with the addition of either human low-density lipoprotein (LDL, 25 µg/ml; Sigma Chemicals), cholesterol (1 µM), 25-OH-cholesterol (a soluble cholesterol analogue, 1 µM), pregnenolone (1 µM), androstenedione (1 µM) or HCG (100 ng/ml, CR127). Granulosa cells incubated without substrate served as controls for determination of basal steroidogenesis. Following incubations, media were assayed for progesterone and 17ß-oestradiol by radioimmunoassay.


    Follicular fluid radioimmunoassay
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
Follicular fluids were assayed for progesterone, androstenedione, oestrone, and oestradiol by radioimmunoassay in the ORPRC Endocrine services Core Laboratory following chromatographic separation (Zelinski-Wooten et al., 1994Go).


    Statistical analysis
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
All steroid concentrations were subjected to a Bartlett's test, and subsequently transformed (to logarithm +10) prior to one-way analysis of variance, followed by Newman–Keuls test for comparison between means. Serum steroids obtained from Chandrasekher et al. (1994) were analysed by one-way analysis of variance (ANOVA) with repeated measures followed by Newman–Keuls test for means comparison. Granulosa cell incubation media were analysed by two-way ANOVA with one repeated measure. Differences were considered significant at P < 0.05. Values are presented as mean ± SEM.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
The mean value of serum LH bioactivity during the 3 days prior to and including aspiration was 5.1 ± 0.2 ng/ml. The presence of functional corpora lutea was verified by increasing serum progesterone concentrations during the 5 days following follicular aspiration (data not presented).


    Serum steroids
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
Circulating steroid concentrations in animals at the time of follicular aspiration, i.e. before (0 h) or 12, 24, and 36 h after the ovulatory stimulus, are depicted in Figure 1Go. Serum progesterone concentrations increased significantly within 12 h post-HCG (P < 0.05) and remained at this value for the duration of the periovulatory interval. Concentrations of serum 17ß-oestradiol did not change during the initial 12 h after HCG, but by 24 and 36 h they were significantly lower (P < 0.05) than those at 12 h post-HCG.



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Figure 1. Serum progesterone and 17ß-oestradiol concentrations at the time of follicle aspiration following an ovulatory bolus of recombinant human chorionic gonadotrophin (HCG) in rhesus monkeys. Ovarian stimulation protocols were undertaken as described in text and serum steroid concentrations were determined before (time 0), or 12, 24, or 36 h following an ovulatory bolus of recombinant HCG (1000 IU). Different superscript letters denote significant differences in progesterone between time points, while numerical superscripts indicate significant differences in 17ß-oestradiol levels. Data are means ± SEM (n = 6, 6, 6, 5; P < 0.05).

 
When more frequent sampling was performed following the administration of an ovulatory stimulus (Chandrasekher et al., 1994Go), serum progesterone concentrations increased significantly within 30 min of the administration of HCG and continued to increase for at least 18 h following the ovulatory stimulus (Figure 2Go). On the contrary, oestradiol displayed a tendency to decrease during the initial 2 h following the ovulatory stimulus, and thereafter increased until 12 h. Serum oestradiol concentrations dropped rapidly between 12 and 18–24 h following u-HCG administration (Figure 2Go).



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Figure 2. Longitudinal measurements of serum steroid concentrations following an ovulatory bolus of urinary human chorionic gonadotrophin (HCG). Serum samples were obtained before, and 0.5, 1, 2, 4, 6, 8, 12 or 24 h after the initiation of periovulatory events with 1000 IU urinary HCG. Top panel depicts serum progesterone, and the bottom panel 17ß-oestradiol. Different superscripts denote significance between (P < 0.05) time points (one-way analysis of variance, repeated measures). All data are mean ± SEM (n = 3–6/time point).

 

    Follicular fluid steroids
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
Figure 3Go summarizes steroid concentrations within follicular fluids obtained during the periovulatory interval. The mean concentration of progesterone in follicular fluid prior to the ovulatory stimulus was 31.9 ± 9.5 ng/ml and increased approximately 180-fold within 12 h following HCG (P < 0.05), remaining significantly elevated for the duration of the periovulatory interval. Androstenedione concentrations were also elevated 12 h after HCG, but returned to control values 24 h post-HCG. Likewise, 17ß-oestradiol concentrations in follicular fluid increased 12 h post-HCG (P < 0.05) and returned to baseline 24 h following HCG. However, 17ß-oestradiol continued to decline and values were significantly lower than time 0 values by 36 h after the ovulatory stimulus (P < 0.05). The pattern of oestrone concentrations was similar to 17ß-oestradiol except values were approximately one-tenth of those for oestradiol (data not presented).



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Figure 3. Follicular fluid steroid concentrations following the ovulatory human chorionic gonadotrophin (HCG) stimulus. Follicular fluid was aspirated before (time 0), or 12, 24, or 36 h following administration of 1000 IU r-HCG. Concentrations of progesterone (top), androstenedione (middle), and 17ß-oestradiol (bottom) were determined by specific radioimmunoassay following chromatography of follicular fluids. Different superscripts indicate significant differences between time points (one-way analysis of variance; P < 0.05). Data are mean ± SEM (n = 7, 5, 4, 3).

 

    In-vitro steroidogenesis
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
Progesterone
Similar to serum and follicular fluid concentrations, basal progesterone production by granulosa cells in vitro increased markedly 12 h after the ovulatory stimulus (P < 0.05; Figure 4Go). However, basal production declined by 24 h post-HCG compared with the 12 h time point, but was still elevated relative to cells collected prior to an ovulatory stimulus (0 HCG; P < 0.05). By 36 h post-HCG, basal progesterone production was intermediate to those at the 12 h and 24 h time points. Addition of LDL or cholesterol to the incubation media did not increase progesterone production above basal values at any time point. However, addition of 25-hydroxycholesterol elicited a significant increase in progesterone synthesis above basal values within each time point following, but not prior to, HCG administration (P < 0.05). The pattern of 25-hydroxycholesterol-stimulated progesterone production across time followed the basal profile: highest at 12 h post-HCG, and intermediate by 24–36 h. In-vitro progesterone production by granulosa cells incubated with pregnenolone was consistently higher (P < 0.05) than basal values at each time point, and was comparable at 0, 12 and 36 h, although a reduction was observed between 12 h and 24 h post-HCG. Interestingly, androstenedione also stimulated a significant increase in progesterone production above basal values before, as well as following (24 and 36 h), the ovulatory stimulus (data not presented). The addition of HCG to the incubation media elicited a significant increase in progesterone production above basal concentrations by granulosa cells only prior to (0 h) the ovulatory HCG stimulus (Table IGo).



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Figure 4. In-vitro progesterone production by granulosa cells collected before (time 0), or 12, 24, or 36 h after administration of 1000 IU r-HCG as an ovulatory stimulus. Cells were incubated for 2 h in the absence (basal) or presence of low-density lipoprotein (LDL), cholesterol (CHOL), 25-hydroxycholesterol (OHC), or pregnenolone (PREG) as detailed in text. Different letters above bars indicate significant differences (P < 0.05) between time points, numbers denote significant differences within time points (two-way analysis of variance). Values are mean ± SEM (n = 3, 3, 4, 4).

 

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Table I. In-vitro production of progesterone or 17ß-oestradiol before (0 HCG), and 12, 24, or 36 h following initiation of periovulatory events with 1000 IU r-HCG
 

    17ß-oestradiol
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
Basal production of 17ß-oestradiol by granulosa cells aspirated 12 h after the ovulatory stimulus was significantly higher than that by cells at 0 h (P < 0.05), while concentrations 24 and 36 h following HCG were intermediate (Figure 5Go). The addition of LDL or cholesterol to the incubation media did not increase 17ß-oestradiol production above basal values at any time point. In contrast, 25-hydroxycholesterol significantly increased 17ß-oestradiol production above basal values 12 and 24 h, but not 36 h, following the ovulatory stimulus (P < 0.05). Granulosa cell production of 17ß-oestradiol during incubation with androstenedione was 200- to 800-fold (P < 0.05) higher than basal values at each time point, but did not change across the periovulatory interval. The addition of HCG to incubation media did not alter 17ß-oestradiol production from granulosa cells at any point during the periovulatory interval (Table IGo).



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Figure 5. In-vitro production of 17ß-oestradiol by granulosa cells collected before (time 0), or 12, 24, or 36 h post-HCG. Different letters above bars indicate significant differences (P < 0.05) between time points, numbers denote significant differences within time points (two-way analysis of variance). Values are mean ± SEM (n = 3, 3, 4, 4). Cells were incubated for 2 h in the absence (basal) or presence of low-density lipoprotein (LDL), cholesterol (CHOL), 25-hydroxycholesterol (OHC) or androstenedione (A4) as detailed in text.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
This study is the first to define the time course for the shift from 17ß-oestradiol production to progesterone production in the follicle during the periovulatory interval in primates undergoing ovarian stimulation. This model, routinely used for IVF-related protocols, provides multiple preovulatory follicles, with large volumes of follicular fluid and numerous granulosa cells available for analyses. In addition, the administration of exogenous gonadotrophin (i.e. LH/HCG) as the ovulatory stimulus allows the exact timing of periovulatory follicular aspirations to be determined. Comparisons of the current data with those from natural menstrual cycles indicate that this model will be useful in further studies of the periovulatory interval of primates. The data are consistent with the hypothesis that the large increase in progesterone within 12 h of the ovulatory stimulus plays an important early role in processes leading to luteinization and ovulation of the follicle.

Administration of an ovulatory stimulus resulted in a rapid and sustained rise in progesterone concentrations in serum and follicular fluid. In-vitro studies on granulosa cells isolated from follicular aspirates suggest that these cells are a likely source of the increased progesterone production following HCG. While the mechanism for the enhanced steroidogenesis remains unknown, recent reports in rodent and primates species suggest that several key components in the pathway leading to progesterone synthesis increase upon exposure to a gonadotrophin bolus, notably P450 side-chain cleavage (P450scc) (Doody et al., 1990Go; Ronen-Furhmann et al., 1998), steroidogenic acute regulatory protein or StAR (Pollack et al., 1997Go), and LDL receptor (Golos and Strauss, 1987Go). In order to better understand the changes in enzyme activity and substrate utilization that occur within the primate follicle throughout the periovulatory interval, macaque granulosa cells aspirated at specific times following the administration of HCG were incubated for 2 h with several steroidogenic precursors. The addition of pregnenolone as a substrate for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) increased progesterone production above basal values at all time points examined, indicating that macaque granulosa cells possess significant amounts of 3ß-HSD activity before, as well as following, the ovulatory stimulus (Sasano et al., 1990Go). This strongly suggests that the rise in progesterone following the ovulatory stimulus is not due to an increase in 3ß-HSD activity. Interestingly, mRNA encoding 3ß-HSD decreased in bovine granulosa cells following the ovulatory stimulus (Voss and Fortune, 1993Go), supporting a minor role for this enzyme in the periovulatory rise of progesterone. Species differences exist with regards to the expression of 3ß-HSD in preovulatory granulosa cells: in pig and sheep, 3ß-HSD is localized only in the theca, while rat, cow and human express 3ß-HSD in both cell layers (Sasano et al., 1990Go; Voss and Fortune 1993Go; Conley et al., 1994Go, 1995Go; Donath et al., 1997Go; Garrett and Guthrie, 1997Go). The localization of 3ß-HSD in granulosa cells in the primate preovulatory follicle raises the possibility for coupling androstenedione and oestrogen synthesis together, for example, to increase the efficiency of preovulatory oestrogen synthesis (Conley and Bird, 1997Go).

The ability of isolated granulosa cells to utilize the soluble analogue 25-hydroxycholesterol was used as a marker of P450scc activity (i.e. cholesterol to pregnenolone). This compound readily enters the mitochondria without facilitation (Toaff et al., 1982Go), and hence the amount of progesterone produced should not be limited by cholesterol transport. Granulosa cells obtained prior to HCG had little P450scc activity, which is consistent with the idea that the theca cell is the primary site of cholesterol conversion to pregnenolone in the preovulatory follicle (Sasano et al., 1989Go). Within 12 h of HCG, granulosa cells acquire P450scc activity and this, along with existing 3ß-HSD activity, allows these cells to produce progesterone (Conley and Bird, 1997Go). Therefore, it does not appear that the mid-cycle gonadotrophin surge induces granulosa cell 3ß-HSD activity; rather the early periovulatory acquisition of P450scc activity by granulosa cells is an essential feature of the rise in progesterone associated with luteinization in the primate follicle.

Granulosa cells were unable to metabolize either LDL or cholesterol in vitro to produce progesterone above basal values at any time following HCG administration. There are reports that LDL-receptor mRNA in human granulosa cells is increased after 1 h of HCG exposure in vitro (Soto et al., 1984Go; Golos and Strauss, 1987Go), while macaque granulosa cells collected 27 h after the ovulatory HCG bolus increased progesterone synthesis in response to LDL within 24 h of culture (Brannian et al., 1992Go; Brannian and Stouffer, 1993Go). Thus, granulosa cell LDL usage in vitro increases following the gonadotrophin surge in primates via a LDL receptor-mediated mechanism. However, follicular fluid from both natural and stimulated menstrual cycles contains very low concentrations of LDL as compared with those of high-density lipoprotein, even 36 h after an ovulatory stimulus (Simpson et al., 1980Go; Enk et al., 1986Go), while preovulatory granulosa cells are laden with cholesterol esters (Endersen et al., 1990Go). The lack of usage of LDL and cholesterol by periovulatory granulosa cells in the current study supports the notion that esterified cholesterol is the primary steroid precursor at least until very late in the periovulatory interval, at which time LDL may become an important steroidogenic substrate.

In contrast to progesterone, both androstenedione and oestrogen concentrations in serum and follicular fluid increased in a transient fashion, with the greatest concentration 12 h following HCG. Despite the fact that oestrogen concentrations declined following the administration of an ovulatory stimulus, metabolism of androstenedione to 17ß-oestradiol by granulosa cells in vitro remained very high throughout the periovulatory interval. Consistent with previous data, the decrease in oestrogens 24 h following HCG administration was not due to a decrease in aromatase activity, but rather a reduction in aromatizable androgens (Hillier et al., 1984Go; Tamura et al., 1992Go; Foldesi et al., 1998Go). This pattern may reflect an initial increase in steroidogenesis, perhaps through increased P450scc, with a subsequent decrease in the ratio of P450c17:3ß-HSD (Conley and Bird, 1997Go), although a general decline in total steroids was observed 12–36 h post-HCG.

In the current study, granulosa cells aspirated prior to the ovulatory stimulus were able to respond to HCG exposure in vitro with a 14-fold increase in progesterone synthesis, while cells obtained at any time point following the ovulatory stimulus were not gonadotrophin responsive. The ability of gonadotrophins to support granulosa cell steroidogenesis in vitro has been established, although the responses are generally blunted in cells obtained following the ovulatory stimulus (e.g. Zelinski-Wooten et al., 1997Go). Zelinski-Wooten et al. (1997) have shown that the half-life for 1000 IU recombinant HCG is 16 h; therefore appreciable concentrations of bioactive HCG remain 36 h following injection. By 24 h post-HCG, the existing LH receptor pathways may have been occupied by HCG administered in vivo, possibly accounting for the high basal progesterone production in vitro. Alternatively, the down-regulation of LH receptor or desensitization of the LH receptor–cyclase system may account for the decrease in steroid production 24 h post-HCG via a transient reduction in several key steroidogenic enzymes (e.g. Richards et al., 1976Go; Jaaskelainen et al., 1980Go; LaPolt et al., 1990Go; Segaloff et al., 1990Go; Voss and Fortune, 1993Go). Thus, late periovulatory/luteal steroidogenesis may be linked to the reacquisition or resensitization of LH receptors.

Although it is evident that hormonally stimulated monkeys have higher concentrations of serum progesterone and oestrogen than naturally cycling monkeys or women (Weick et al., 1973Go; Hoff et al., 1983Go), the profile of serum steroids during the periovulatory interval is similar to natural cycles. In all cases, progesterone increases rapidly following the ovulatory stimulus, either plateaus or declines between 12–24 h, and begins to rise near the predicted time of follicle rupture. Oestrogen remains constant for approximately 12 h after the ovulatory stimulus before beginning to decline (Hoff et al., 1983Go), while serum androstenedione concentrations increase within 12 h of the onset of the LH surge (Hoff et al., 1983Go). These data, plus evidence that follicles will ovulate in a timely manner (e.g. Hibbert et al., 1996Go), support the idea that ovarian stimulation of monkeys results in follicles that are steroidogenically similar to follicles of the natural cycle (Weick et al., 1973Go; Hoff et al., 1983Go; Zelinski-Wooten et al., 1997Go). Thus, monkeys undergoing ovarian stimulation should prove useful models for further studies on the dynamics and regulation of periovulatory events in primates.

For example, a local role for progesterone in ovulation and luteinization has been postulated in both primate and rodent species (e.g. Lydon et al., 1995Go; Hibbert et al., 1996Go). Data from the current study demonstrate that progesterone increases within 30 min of the ovulatory stimulus, and remains at very high concentrations in the follicular fluid until the time of ovulation. In addition, progesterone receptor expression increases early in the periovulatory interval (Iwai et al., 1990Go; Park and Mayo, 1991Go; Natraj and Richards, 1993Go), for example, within 12 h of the HCG bolus during ovarian stimulation cycles in monkeys (Chaffin et al., 1998Go). Further studies are warranted to examine progesterone action, beginning early in the periovulatory interval in monkeys.

In summary, we have utilized rhesus monkeys undergoing hormonally controlled ovarian stimulation to examine changes in steroidogenesis that occur during the 36–38 h periovulatory interval. Progesterone increased rapidly following the administration of an ovulatory bolus of HCG, and remained elevated throughout the periovulatory interval. Androstenedione and oestrogen concentrations peaked 12 h following HCG administration, and thereafter declined. In-vitro studies on isolated granulosa cells collected before or after HCG demonstrate that a rise in P450scc activity drives the increase in progesterone from intracellular stores of esterified cholesterol. Collectively, these data argue for a locally mediated, early periovulatory role for the progesterone signal, perhaps to initiate or maintain the cascade of events resulting in ovulation and luteinization.


    Acknowledgments
 
The authors are grateful for the technical expertise provided by the Division of Animal Resources, the Endocrine Services Laboratory, the Assisted Reproductive Technology Core, and the skilled surgical team of Dr. John Fanton. Special thanks to Dr. Mary Zelinski-Wooten for technical and statistical assistance. Recombinant human LH, FSH, CG, and Antide were generously provided by Ares Advanced Technology, Inc., a member of the Ares-Serono Group of companies. Human CG (CR127) was provided by the National Hormone and Pituitory Program, NIDDK, Bethesda, MD. This work was supported by NIH HD-20896 (to R.L.S.), RR-00163, HD-18185, HD-08302 (to C.L.C.).


    Notes
 
3 To whom correspondence should be addressed at: Oregon Regional Primate Research Center, 505 NN 185th Ave., Beaverton, OR 97006, USA Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Granulosa cell preparation
 Granulosa cell incubation
 Follicular fluid...
 Statistical analysis
 Results
 Serum steroids
 Follicular fluid steroids
 In-vitro steroidogenesis
 17ss-oestradiol
 Discussion
 References
 
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Submitted on August 21, 1998; accepted on November 30, 1998.