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BOR - Papers in Press, published online ahead of print February 25, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.023390
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BIOLOGY OF REPRODUCTION 71, 366–373 (2004)
DOI: 10.1095/biolreprod.103.023390
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Progesterone Promotes Oocyte Maturation, but Not Ovulation, in Nonhuman Primate Follicles Without a Gonadotropin Surge1

Sherri M. Borman3, Charles L. Chaffin4, Kristine M. Schwinof3, Richard L. Stouffer3,5, and Mary B. Zelinski-Wooten2,3,5

Oregon National Primate Research Center,3 Beaverton, Oregon 97006 Department of Physiology,4 Medical College of Georgia, Augusta, Georgia 30912 Department of Physiology and Pharmacology,5 Oregon Health and Science University, Portland, Oregon 97239


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the periovulatory interval, intrafollicular progesterone (P) prevents follicular atresia and promotes ovulation. Whether P influences oocyte quality or maturation and follicle rupture independent of the midcycle gonadotropin surge was examined. Rhesus monkeys underwent controlled ovarian stimulation with recombinant human gonadotropins followed by a) experiment 1: an ovulatory bolus of hCG alone or with a steroid synthesis inhibitor (trilostane, TRL), or TRL + the progestin R5020; or b) no hCG, but rather sesame oil (vehicle), R5020, or dihydrotestosterone (DHT). In experiment 1, the majority of oocytes remained immature (65% ± 20%) by 12 h post-hCG. However, the percentage of degenerating oocytes increased (P < 0.05) with TRL (42% ± 22% vs. 0% controls), but was reduced (P < 0.05) by progestin replacement (15% ± 7%). By 36 h post-hCG, the majority of oocytes in all three groups reached metaphase II (MI). In experiment 2, no evidence of follicle rupture was observed in the vehicle, R5020, or DHT groups. Despite the absence of hCG, a significant (P < 0.05) percentage of oocytes resumed meiosis to metaphase I in R5020- (41 ± 9) and DHT- (36 ± 15) but not vehicle- (4 ± 4) treated animals. Only oocytes from R5020-treated animals continued meiosis in vivo to MII. More (P < 0.05) oocytes fertilized in vitro with R5020 (40%) than with vehicle (20%) or DHT (22%). Thus, P is unable to elicit ovulation in the absence of an ovulatory gonadotropin surge; however, P and/or androgens may prevent oocyte atresia and promote oocyte nuclear maturation in primate follicles.

in vitro fertilization, meiosis, ovulation, ovum, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In primates, the midcycle gonadotropin surge initiates events leading to oocyte nuclear maturation, luteinization, and rupture of the follicle 36 h later [1, 2]. Intrafollicular progesterone (P) mediates some LH-initiated periovulatory events through autocrine/paracrine mechanisms. Increasing levels of P and nuclear P receptor (R) expression occur in the macaque follicle within 12 h of the ovulatory stimulus, supporting an early role for P in periovulatory events [3]. In many species, P is absolutely required for follicle rupture (review, [4]). Acute administration of a 3ß-hydroxysteroid dehydrogenase (3ß-HSD) inhibitor or a PR antagonist prevents ovulation in rodents [5–7] and monkeys [4]. Additionally, mice lacking nuclear PR do not ovulate [8]. Although the precise mechanism whereby P promotes follicular rupture remains unknown, there is mounting evidence for a link between P and its regulation of matrix metalloproteinases (MMPs), the tissue inhibitors of MMPs (TIMP-1; [3]), other proteases [9] and tissue remodeling.

A local role for intrafollicular P, following the midcycle gonadotropin surge, as a luteotropin for promoting the development of the primate corpus luteum from the ovulatory follicle in primates is also emerging [3, 10]. Experiments in rhesus monkeys undergoing controlled ovarian stimulation (COS) to develop multiple preovulatory follicles revealed that steroid depletion via concomitant treatment with the steroid synthesis inhibitor, trilostane (TRL) did not affect antral follicular growth [4, 11]. However, P promotes follicle luteinization and prevents atresia in the follicle 12– 36 h after the ovulatory gonadotropin stimulus, suggesting a role for P as a survival factor during the periovulatory interval [12, 13]. Furthermore, locally produced P is both prodifferentiative and antidegenerative in human [14, 15], rat [16], and primate [12] luteinizing granulosa cells in vitro.

Interestingly, a role for steroids within the follicle on its enclosed oocyte in vivo remains unclear. In macaques, suppression of steroid synthesis with TRL during COS did not affect oocyte meiosis but impaired fertilization, suggesting a role for steroids in oocyte cytoplasmic maturation [4, 11]. Earlier studies in women [17] and rabbits [18] exposed acutely to antiprogestins just before the hCG bolus during COS cycles also suggested that P was not necessary for the final stages of oocyte maturation but was required to protect oocytes from premature atresia during the periovulatory interval. Whether P can initiate ovulatory events or influence oocyte quality and maturation independent of the midcycle gonadotropin surge in primates is unknown. Therefore, the present study was designed to determine if P alone can mimic the effects of the midcycle gonadotropin surge to cause ovulation, prevent oocyte atresia, and reinitiate meiotic maturation in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

The general care and housing of rhesus monkeys (Macaca mulatta) at the Oregon National Primate Research Center (ONPRC) was described previously [19]. Adult females exhibiting regular menstrual cycles of approximately 28 days were monitored daily for menses. Daily blood samples were collected by saphenous venipuncture from anesthetized animals throughout the treatment interval until the next onset of menses. Serum was separated and stored at –20°C until assayed for hormone concentrations. All protocols were approved by the ONPRC Institutional Animal Care and Use Committee and were conducted in accordance with National Institutes of Health guidelines for the Care and Use of Laboratory Animals.

Controlled Ovarian Stimulation

To promote the development of multiple preovulatory follicles, all animals received a sequential regimen of recombinant (r) human (h) gonadotropins. Beginning within 3 days of menses, female macaques received 6 days of r-hFSH (Serono Reproductive Biology Institute, Rockland, MA), followed by 3 days of r-hFSH + r-hLH (30 IU, twice daily, i.m.; Serono Reproductive Biology Institute). During the 9 days of gonadotropin treatment, animals also received the GnRH antagonist Antide (5 mg/kg body weight, once daily s.c.; Serono Reproductive Biology Institute) daily to prevent an endogenous LH surge [10]. Transabdominal ovarian ultrasonography was performed on the eighth day of follicular stimulation to assess the number and diameter of large (3–6-mm) preovulatory follicles [11]. Animals were assigned randomly to one of the following experiments to receive either hCG or steroid replacement (no hCG) as the ovulatory stimulus.

Experiment 1: Gonadotropin (hCG) as the Ovulatory Stimulus: Oocyte Maturation

On the final day of gonadotropin treatment, oocyte maturation and periovulatory changes in the follicle were initiated by r-hCG (1000 IU, i.m.; Serono Reproductive Biology Institute). One group of monkeys received no further treatment, i.e., controls (CTR; n = 4 per time point, i.e., 12 and 36 h). A second group of animals (n = 3 per time point) received the 3ß-HSD inhibitor, trilostane (Sanofi Research Division, Malvern, PA) orally (1 g in 8 ml of orange Kool-Aid [Kraft General Foods, Inc., White Plains, NY] containing 1% [w/v] gum tragacanth [Sigma, St. Louis, MO]) beginning 4 h before hCG administration and every 12 h thereafter until follicle aspiration. A third group of animals (n = 3 per time point) received TRL plus the nonmetabolizable progestin, R5020 (2.5 mg in sesame oil, s.c.; NEN, Boston, MA) once daily starting at the time of hCG until follicle aspiration. Follicles ≥4 mm were aspirated by laparoscopy of anesthetized animals to retrieve the enclosed oocytes [11] at either 12 or 36 h post-hCG.

Experiment 2: Replacement of the Ovulatory Stimulus with Steroids: Follicle Rupture, Oocyte Maturation, and In Vitro Fertilization

The traditional ovulatory bolus of hCG (–hCG) was not given, but rather was replaced with a single daily i.m. injection of (n = 3/group) a) sesame oil (vehicle); b) R5020 (2.5 mg in sesame oil, s.c.); or c) the nonmetabolizable androgen, 5{alpha}-dihydrotestosterone propionate (DHT; 5 mg in sesame oil, s.c.; Steraloids, Whilton, NH). Each treatment began on the ninth day of COS and continued for 2 additional days. To maintain follicular integrity, low doses of gonadotropins (r-hFSH + r-hLH, 30 IU each twice daily, i.m.) were continued along with vehicle and steroid treatment until surgery. The androgen treatment was included to test the specificity of the progestin treatment. Seventy-two hours after the initial vehicle or steroid (R5020 or DHT) injection, anesthetized animals underwent paramedian pelvic laparotomy to observe the presence or absence of ovulatory stigmata and to retrieve oocytes from unruptured follicles via follicular aspiration [4].

Evaluation of Oocyte Maturation and In Vitro Fertilization

Oocytes were isolated from all animals in both experiments, counted, and evaluated for nuclear maturity following brief exposure to hyaluronidase (0.1%; Sigma Chemical Co., St. Louis, MO) to remove the cumulus cells [4]. Oocytes were classified as germinal vesicle (GV)-intact, metaphase I (MI, no GV and no polar body), metaphase II (MII, extruded polar body in perivitelline space), or degenerate (presence of fragmentation or vacuoles in ooplasm and/or aspherical shape) at the time of collection and again 6 h later [20, 21].

Semen from rhesus males was collected by penile electroejaculation and washed, sperm motility and density were determined, and sperm were activated before insemination as described previously [4]. Mature oocytes (MII at collection or MI at collection that matured to MII in vitro) from experiment 2 were inseminated with 5 x 104 activated motile sperm. Fertilization was confirmed 16 h following insemination by the appearance of two pronuclei and the extrusion of the second polar body.

Hormone Assays

Serum concentrations of estradiol and progesterone were determined by specific electrochemoluminescent assay using a Roche Elecsys 2010 analyzer by the Endocrine Services Core Laboratory, ONPRC [22]. Hormone concentrations were validated against previous RIAs in this laboratory [23, 24].

Data Analysis

Using Sigma Stat (San Rafael, CA) for statistical analysis, one-way ANOVA followed by Student-Neuman-Keuls tests were used to compare a) the peak levels of serum estradiol and progesterone among treatments, b) the total number of oocytes collected per animal and the average percentages of oocytes at each stage of nuclear maturation among treatments, and c) the percentages of oocytes collected at each stage of nuclear maturation within treatment at 12 h and 36 h post-hCG and within steroid treatment. Fishers exact test was used to compare the total proportion of oocytes that fertilized in vitro among groups not receiving an ovulatory stimulus. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Gonadotropin (hCG) as the Ovulatory Stimulus: Oocyte Maturation

The follicular response to COS in the CTR, TRL, or TRL + R5020 groups with hCG as the ovulatory stimulus was similar, as indicated by serum levels of estradiol. In all groups, serum levels of estradiol were similar during COS and increasing progressively, reaching peak levels the day before hCG administration (12-h groups: control, 2555 ± 710 pg/ml [mean ± SEM] TRL, 1051 ± 144 pg/ml; TRL + R5020, 1061 ± 272; 36-h groups: control, 2795 ± 673 pg/ml [mean ± SEM]; TRL, 1960 ± 131 pg/ml; TRL + R5020, 1051 ± 151]). The pattern and levels of circulating estradiol observed in the present study were similar to those previously reported [4, 11]. In addition, the numbers of large (≥4 mm) preovulatory follicles observed by ultrasound on the eighth day of COS were similar among groups (data not shown). Consistent with midcycle hCG administration, serum progesterone levels during the periovulatory period and subsequent luteal phase were within the normal range in both control groups [4] and exhibited similar peak levels during midluteal phase (22.6 ± 2.8 ng/ml [mean ± SEM], 24.3 ± 9.4; 12-h and 36-h groups, respectively). As previously reported [4], serum progesterone levels were suppressed during midluteal phase following TRL (6.9 ± 3.8; 1.4 ± 0.9) and TRL + R5020 treatment (1.5 ± 0.7; 2.5 ± 0.3).

The total number of oocytes collected from rhesus monkeys in the presence of hCG as the ovulatory stimulus following COS in the presence or absence of the 3ß-HSD inhibitor (TRL) and steroid replacement is shown in Table 1. Similar numbers of oocytes were retrieved within time between groups. Regardless of treatment, more (P < 0.05) oocytes were collected per animal at 36 h post-hCG relative to 12 h post-hCG.


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  		TABLE 1. Oocytes collected from rhesus monkeys following an ovulatory stimulus (+hCG) during controlled ovarian follicular stimulation alone (CTR), or in the presence of a 3ß-HSD inhibitor (TRL) and steroid replacement (TRL + R5020)

The percentages of oocytes collected at various stages of nuclear maturation 12 h after the administration of hCG alone, with steroid depletion and progestin replacement, are summarized in Figure 1. While the majority (P < 0.05) of oocytes in the control group remained immature (GV intact), more (P < 0.05) metaphase I (MI) than degenerating oocytes were retrieved. In the TRL-treated group, equal proportions of degenerating and immature (GV-intact) oocytes, relative to few (P < 0.05) metaphase I (MI) oocytes, were recovered. In contrast, a greater (P < 0.05) proportion of MI oocytes were collected in the TRL + R5020 group relative to degenerating oocytes, but this was not different from immature oocytes (GV intact). Additionally, similar percentages of GV-intact oocytes were observed in the control, TRL, and TRL + R5020 groups. Compared with controls, the percentage of degenerating oocytes increased (P < 0.05) following steroid depletion with TRL but was reduced (P < 0.05) by progestin replacement. Furthermore, the percentage of MI oocytes retrieved increased (P < 0.05) in the TRL + R5020 group relative to the control or TRL groups (Fig. 1). MII oocytes were not obtained within 12 h of hCG administration in any treatment group.



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  		FIG. 1. Role of P on oocyte maturation or degeneration 12 h post-hCG. Bars indicate the percentages of oocytes collected as degenerating (open bars), immature GV intact; (closed bars), or metaphase I (MI, hatched bars) following the administration of an ovulatory hCG bolus alone (CTR; n = 4), with steroid depletion (TRL; n = 3), or steroid depletion plus progestin replacement (TRL + R5020; n = 3). Data represent mean ± SEM. Different letters above the bars indicate significant (P < 0.05) differences between the percentages of oocytes at a given stage of nuclear maturity within a group. Different symbols above the bars indicate significant (P < 0.05) differences among the percentages of oocytes within a given stage of nuclear maturity among groups

The percentages of oocytes collected at each stage of nuclear maturity 36 h after administration of an ovulatory hCG bolus in the absence or presence of steroid depletion and progestin replacement are summarized in Figure 2. Mostly MII oocytes and relatively few degenerating and GV-intact oocytes were collected in each group, and the percentages within each group did not differ. However, the percentage of MI oocytes following steroid depletion with TRL and replacement with R5020 was greater (P < 0.05) relative to control. Similar percentages of MII oocytes were retrieved in all groups. The percentages of degenerating and GV-intact oocytes collected in the TRL and TRL + R5020 groups 36 h post-hCG (Fig. 2) were lower (P < 0.05) than those observed at 12 h post-hCG (Fig. 1). In contrast with 12 h post-hCG (Fig. 1), by 36 h post-hCG, the majority (P < 0.05) of oocytes collected were MII in each group (Fig. 2).



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  		FIG. 2. Role of P on oocyte maturation or degeneration 36 h post-hCG. Bars indicate the percentages of oocytes collected as degenerating (open bars), immature (GV intact; closed bars), metaphase I (MI, hatched bars), or metaphase II (MII, striped bars) following the administration of an ovulatory hCG bolus alone (CTR; n = 4), with steroid depletion (TRL; n = 3), or with steroid depletion plus progestin replacement (TRL + R5020; n = 3). Data represent mean ± SEM. Different letters above the bars indicate significant (P < 0.05) differences between the percentages of oocytes at a given stage of nuclear maturity within a group. Different symbols above the bars indicate significant (P < 0.05) differences among the percentages of oocytes within a given stage of nuclear maturity among groups

Experiment 2: Replacement of the Ovulatory Stimulus with Steroids: Oocyte Maturation, Follicle Rupture, and In Vitro Fertilization

The follicular response to COS in the vehicle group and the groups that received progestin (R5020) or androgen (DHT) as the ovulatory stimulus did not differ as indicated by serum levels of estradiol. In all groups, estradiol levels were similar and increased progressively, reaching peak levels before the initial administration of the steroid treatment (vehicle, 820 ± 166 pg/ml [mean ± SEM]; R5020, 910 ± 274 pg/ml; DHT, 1288 ± 680 pg/ml). In addition, similar numbers of large (≥4 mm) preovulatory follicles as observed by ultrasonography were recruited among groups (data not shown).

There was no evidence of follicular rupture 72 h after the onset of vehicle or the steroid (R5020 or DHT) treatment used as the ovulatory stimulus in the absence of hCG (data not shown). However, in macaques undergoing COS, ovulatory stigmata are typically evident 72 h post-hCG [4]. Consistent with the lack of midcycle hCG administration, serum progesterone levels in all groups remained at baseline (<1 ng/ml) during the periovulatory period and thereafter, as previously reported (data not shown) [4].

The total number of oocytes per animal collected following aspiration of unruptured follicles was similar among all treatment groups (Table 2). The percentage of oocytes collected at various stages of nuclear maturity following treatment with sesame oil, R5020, or DHT to replace the typical ovulatory bolus of hCG are shown in Figure 3. GV-intact oocytes were predominantly collected in the vehicle group with fewer (P < 0.05) degenerating and MI oocytes retrieved. Additionally, only 1/3 of the animals receiving vehicle yielded MI oocytes; no MII oocytes were collected from any vehicle-treated animal. Similar proportions of GV-intact and MI oocytes but few (P < 0.05) degenerating oocytes were collected in both the R5020 and DHT groups. However, MII oocytes were only collected in the R5020 group. No differences were observed in the numbers of degenerating oocytes between all treatment groups (Fig. 3). However, significantly fewer (P < 0.05) GV-intact oocytes were recovered following R5020 treatment relative to both the vehicle and DHT groups. Conversely, 3/3 females in both the R5020 and DHT groups yielded more (P < 0.05) MI oocytes relative to vehicle. Oocytes collected from the R5020 group were spherical in shape but had grainy ooplasm with dark centers (not shown). Likewise, oocytes collected from the DHT group were spherical in shape but had large perivitelline spaces and grainy, dark centers.


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  		TABLE 2. Nuclear maturity of oocytes collected from gonadotropin-stim ulated macaques treated with vehicle or steroids (R5020 or DHT) as the ovulatory stimulus



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  		FIG. 3. Percentages of oocytes collected as degenerating (open bars), GV intact (closed bars), metaphase I (MI, hatched bars), or metaphase II (MII, striped bars) using vehicle, progestin (R5020), or androgen (DHT) to replace the typical ovulatory bolus of hCG. Data represent mean ± SEM (n = 3/group). Different letters above the bars indicate significant (P < 0.05) differences between the percentages of oocytes at a given stage of nuclear maturity within a group. Different symbols above the bars indicate significant (P < 0.05) differences among the percentages of oocytes within a given stage of nuclear maturity among groups. Mature (metaphase I, MI) oocytes were only produced in 1 of 3 females in the vehicle group and in 3 of 3 females in the R5020 and DHT groups. NC, None collected—no MII oocytes were collected in the vehicle or DHT groups

MI oocytes from all (n = 3) R5020-treated animals continued maturation to MII in vitro within 6 h of collection (Table 2), compared with a lower proportion (P < 0.05) in the vehicle and DHT groups. Oocytes matured from MI to MII in vitro in only 1/3 and 2/3 animals in the vehicle and DHT groups, respectively (Table 2). The percentage of oocytes collected at MI that matured in vitro to MII within 6 h of retrieval was less in the vehicle (33 ± 33) compared with the DHT group (43 ± 18).

Oocytes from 1/3 females in the vehicle and DHT groups and 3/3 females in the R5020 group fertilized in vitro. In the vehicle group, 10 MI oocytes were retrieved but only 2 matured to MII in vitro, 1 within 6 h and 1 after more than 6 h from collection, and subsequently fertilized (Table 3); moreover they did not cleave (data not shown). In contrast, of the 40 maturing oocytes (MI + MII) retrieved from the R5020 group, 30% of those collected as MI that completed meiosis to MII in vitro as well as 75% of those collected at MII fertilized in vitro, yielding an overall higher (P < 0.05) fertilization rate of 40% relative to vehicle and DHT (Table 3). All resultant zygotes from the R5020 group cleaved and formed embryos, but all arrested at the morula stage (data not shown). Of the total number of oocytes collected at MI in the DHT group, a greater proportion (P < 0.05) continued meiosis to MII within 6 h of collection relative to the R5020 group. Fertilization occurred in oocytes that resumed meiosis to MII in vitro, with an overall fertilization rate of 22% (Table 3). The zygotes cleaved, but all arrested at the eight-cell stage (data not shown).


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  		TABLE 3. In-vitro fertilization rates of oocytes collected from gonadotropin-stimulated macaques treated with vehicle or steroid (R5020 or DHT) replacement of the ovulatory stimulus


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study examined for the first time whether the replacement of the ovulatory gonadotropin stimulus by P would permit periovulatory events, particularly reinitiation of oocyte meiotic maturation and follicular rupture/ovulation in primates. In the present study, the capacity of macaque follicles induced by COS to produce biologically active steroids was blocked using a 3ß-HSD inhibitor, TRL [11]. This model was successfully used in macaques to inhibit the activity of 3ß-HSD and effectively suppress P, androgen, and estradiol production, resulting in atretic periovulatory follicles [12]. The specificity of P to reinitiate oocyte meiotic maturation and cause ovulation was examined by in vivo replacement of progestin (R5020) and androgen (DHT).

Steroid depletion increased the percentage of degenerating oocytes recovered 12 h post-hCG. Progestin replacement prevented oocyte degeneration and increased the percentage of mature, MI oocytes recovered. Similar effects were not observed by 36 h post-hCG, when limited oocyte degeneration was noted. Despite progestin replacement, fewer oocytes were collected 12 h relative to 36 h post-hCG. This may be due to the increased oocyte degeneration observed after only 12 h or the lack of expansion of cumulus-oocyte complexes, which occurs later during the periovulatory interval. Twelve hours post-hCG, limited (<10%) follicular atresia was noted in macaques undergoing COS, whereas an increase in follicular atresia was observed 36 h post-hCG [12]. By 12 h post-hCG, remodeling of the follicle is evident in the theca layer, wherein cellular hypertrophy and angiogenesis appear [12, 25]. These morphological changes are not typically seen in the granulosa layer during the early periovulatory interval; rather, granulosa cells display a variety of phenotypes, i.e., nonluteinized, intermediate, or luteinizing after 12, 24, and 36 h post-hCG [12]. Additionally, patterns of gene expression change during this interval of changing phenotypes [3]. Steroid depletion significantly alters follicular morphology and gene expression during the periovulatory interval [12]. Follicles lacking theca and granulosa cell hypertrophy or basement membrane infolding, typical of follicles that are nonluteinized are observed, along with decreased expression of many genes [12]. In the present study, the oocytes collected 12 h post-hCG in the TRL group may have originated from a cohort of follicles that are undergoing abnormal premature luteinization in response to the depleted steroid milieu [12], accounting for the increased collection of degenerating oocytes. Because this observation did not translate to an increase in the number of degenerating oocytes 36 h post-hCG, the final fate of these follicles is unknown. However, it appears follicles from steroid-depleted animals may have two fates: either they prematurely luteinize, as seen 12 h post-hCG, or they do not luteinize at all, as observed 36 h post-hCG [12]. Progestin replacement during steroid depletion restored follicular luteinization by 36 h post-hCG, thereby reducing the number of atretic follicles [12]. These findings support a role for P, not only in structural remodeling and cellular differentiation of the periovulatory follicle [12], but also in promoting follicular [12] and oocyte health during the early periovulatory interval in primates.

P may have multiple roles in oocyte health and meiosis as well as cytoplasmic maturation and fertilization during the periovulatory interval in primates. In the absence of an ovulatory gonadotropin stimulus, a significant percentage of oocytes reinitiated meiosis in vivo in animals receiving R5020 and DHT but not vehicle treatment. Our data would suggest a small percentage may undergo spontaneous maturation in vivo during COS in the absence of a gonadotropin bolus. It is not known whether these oocytes are derived from atretic follicles, were exposed to a spontaneous LH surge in vivo, or responded to the pharmacologic dose of LH given during COS. Nevertheless, oocytes obtained from gonadotropin-stimulated macaques before an ovulatory hCG bolus are typically at the GV stage. Reinitiation and continuation of oocyte nuclear maturation triggered by the gonadotropin surge appears not to require increasing intrafollicular P in vivo before follicle rupture [4]. Because R5020 and DHT produced significantly more MI oocytes in the absence of hCG, resumption of meiosis (i.e., germinal vesicle breakdown) in vivo may not require a gonadotropin surge. However, despite evidence of oocyte nuclear maturity, the oocytes retrieved in the absence of hCG, but with progestin or androgen treatment, did not morphologically resemble healthy oocytes that typically fertilize following hCG administration in macaques [20, 21]. Thus, in addition to preventing oocyte degeneration during the periovulatory interval, steroids may enable reinitiation of meiosis, i.e., germinal vesicle breakdown, the transition from GV to MI.

It seems both progestin and androgen elicit events in the MI oocyte in vivo that can subsequently support oocyte maturation to MII in vitro in the absence of hCG. However, only progestin, but not androgen, permitted the continued progression of meiosis from MI to MII in vivo in the absence of hCG. In contrast with the inability of androgen to support the continuation of meiosis in vivo in the absence of an ovulatory stimulus in the present study, MII oocytes were recovered from unruptured follicles 72 h post-hCG in macaques undergoing COS with steroid depletion (TRL) and DHT replacement [4]. However, exposure of mouse [26] and porcine [27] oocytes to androgens in vitro prevented germinal vesicle breakdown. Whether suppression of meiosis by androgens in nonprimate species was due to an excess of androgen in relation to other steroids or the in vitro conditions used is unknown. While a high intrafollicular androgen/estrogen ratio during the preovulatory interval was associated with inhibition of oocyte nuclear maturation in macaques receiving an aromatase inhibitor during COS [28], the intrafollicular levels of androgen achieved with exogenous DHT replacement in the present study were presumably relatively lower. Furthermore, oocyte nuclear maturity in rhesus monkeys was correlated with the degree of follicular luteinization [29]. Mature oocytes within a follicle were associated a with high intrafollicular P:estradiol ratio, but not with DHT levels [29]. Perhaps in primates, androgen, like progestin, can support resumption of meiosis in vivo but, unlike P, is unable to sustain meiosis in the absence of the gonadotropin surge.

In the present study, replacement of the ovulatory bolus of hCG with P or androgen in vivo yielded a greater proportion of mature oocytes capable of fertilization and cleavage in vitro relative to vehicle. While embryos derived from the progestin replacement group developed in vitro to the morula stage, embryos from the androgen-treated females arrested at the eight-cell stage. Thus, oocytes retrieved in the absence of a gonadotropin surge but in the presence of P or androgen are not developmentally competent in vitro [20, 21]. Inhibition of steroid synthesis with TRL [21] or P action with a PR antagonist [30] during COS including a bolus of hCG in macaques did not affect antral follicular growth and oocyte meiosis but impaired fertilization and early embryonic development. In addition, in vitro fertilization of ovine [31] and hamster [32] oocytes was suppressed in the presence of steroid synthesis inhibition and restored with steroids. Therefore, P and other steroids may have a role in oocyte cytoplasmic maturation that impacts oocyte competence [11, 21]. Whether the resultant embryos from P or androgen replacement in the absence of hCG would develop in vivo is unknown. In contrast, our previous study demonstrated the ability of androgen, but not P, replacement to support fertilization of oocytes derived from TRL-treated macaques undergoing COS with a bolus of hCG [4]. Although the dose of R5020 used restored ovulation [4], it is possible that P requirements differ for promoting oocyte fertilization in the presence of TRL. Perhaps androgen confers some protective effects on oocytes in the presence of TRL or has undefined mechanisms for promoting fertilization in the presence of hCG in primates. In support of the latter, the antiandrogen flutamide suppressed fertilization and embryogenesis in vivo when administered to superovulated rats [33]. Thus, our data suggest there may be a gonadotropin surge-independent/steroid- (P and androgen) regulated component of the continuation of meiosis in primates, analogous to regulation of meiotic maturation by P in nonmammalian species [34]. However, our data support the concept that both the gonadotropin surge and steroids (P and androgen) production during the early periovulatory interval are required for oocyte quality and competence to undergo normal embryogenesis postfertilization.

In the present study, neither R5020 nor DHT, at least at the doses tested, could elicit follicle rupture during COS cycles in the absence of hCG. Likewise, in a previous study, hCG in a steroid-depleted milieu could not cause ovulation. However, R5020, but not DHT, replacement during steroid depletion in the presence of hCG in macaques could restore multiple ovulations, as evidenced by the presence of stigmata on the ovaries, as typically observed with hCG alone [4]. Similarly, an absolute requirement for P in follicle rupture has been documented in rodents wherein inhibition of P synthesis or action prevents ovulation that can be restored with P [5–7]. P-induced follicular rupture appears to be mediated via the classical mechanism because mice lacking nuclear PR do not ovulate [8]. Interestingly, androgen replacement following steroid depletion restored ovulation in rats [35, 36], but this was not observed in macaques [4]. Thus, ovulation requires both hCG priming and P, but not androgens, for successful rupture of the follicle in primates.

Whether P is acting directly on the oocyte or indirectly through the granulosa or theca cells to promote oocyte health and meiotic or cytoplasmic maturation during the periovulatory interval remains unclear. Specific mechanisms of P action in the oocyte may involve action though classical nuclear PR either indirectly via granulosa, cumulus, or theca cells or directly via the oocyte; alternatively, P may act nonclassically via PR localized to the plasma membrane of granulosa or theca cells or the oocyte. Classical, nuclear PR are induced by the gonadotropin surge in granulosa cells of the periovulatory follicle in several species [3]. Thus, P may indirectly activate the oocyte via nuclear PR induced by hCG in the granulosa and cumulus cells. Reinitiation of meiosis observed in porcine cumulus-oocyte complexes in response to P was speculated to occur via classical nuclear PR to disrupt gap junctions via decreased connexin 43 [37]. Evidence for direct actions of P on the oocyte via classical nuclear PR is lacking. The mRNA for nuclear PR was not detected in human [38] nor in mouse oocytes [39] after reverse transcription-PCR analysis. Detection of nuclear PR protein in mammalian oocytes and embryos awaits demonstration. Additionally, rapid, nongenomic actions of steroids on the oocyte have been identified in fish and amphibians, wherein P interaction with membrane PR regulates oocyte meiotic maturation (review, [34]) via generation of membrane-mediated second messengers. Whether nongenomic actions of P contribute to oocyte nuclear and/or cytoplasmic maturation in primate oocytes is currently unknown. However, direct nongenomic effects of other steroids, i.e., estradiol and androstenedione, on intracellular calcium release in human oocytes have been observed [40]. Alternatively, P effects on oocyte function could be manifested indirectly via nongenomic action in the somatic cells of the periovulatory follicle in primates. Putative membrane PR has been identified in rat [41] and bovine [42] granulosa cells as well as in bovine theca and luteal cells [42, 43] and associated with P regulation of intracellular calcium and mitosis in rat granulosa cells as well as luteal function in rats [44, 45]. Further studies are needed to dissect the presence and roles of nonclassical receptor-mediated pathways for P action versus the genomic PR-A/PR-B signal pathways in the oocyte and periovulatory follicle in primates (review, [46]). Last, the actions of steroids in the oocyte may involve alterations in signaling pathways necessary for germinal vesicle breakdown and resumption of meiosis (review, [47]), such as the decrease in intracellular cAMP that must occur in the oocyte before germinal vesicle breakdown and the resumption of meiosis in rodents [48, 49] or the production of meiosis-activating sterols (MAS) shown to promote oocyte maturation in rodents in the absence of gonadotropins [50]. However, exposure of mouse cumulus-oocyte complexes to P and 17-OH-P, in concentrations similar to those in follicular fluid, in the presence of hypoxanthine did not affect germinal vesicle breakdown in vitro [51].

In summary, in macaques undergoing COS with gonadotropins in the presence of an ovulatory stimulus, P prevented oocyte degeneration 12 h post-hCG. In contrast, in the absence of hCG during COS cycles, P and androgen promoted reinitiation of oocyte nuclear maturation and fertilization, but only P permitted the progression of MI oocytes to MII in vivo. However, P was unable to elicit ovulation of large preovulatory follicles in the absence of hCG. In response to the midcycle gonadotropin surge, P production plays a critical role in ovulation and luteinization of the mature follicle as well as the development and maintenance of the corpus luteum of the menstrual cycle [3]. Therefore, P is required for the success of processes initiated by the gonadotropin surge that result in ovulation; however, P cannot replace the LH surge to elicit follicle rupture in primates. Although P is not required for reinitiation of meiosis during the periovulatory interval, steroids (P or androgen) can stimulate meiotic maturation in the absence of the gonadotropin surge. P may also promote continued health/maturation of the oocyte and zygote. In addition, early actions of P in the oocyte may prevent atresia and drive meiosis. However, as yet unknown periovulatory events induced by the midcycle gonadotropin surge appear to be necessary for the mature oocyte to undergo normal preimplantation embryogenesis after fertilization. The cellular and molecular mechanisms whereby P contributes to oocyte maturation and competence, either directly or indirectly, in primates remain to be explored.


   ACKNOWLEDGMENTS
 
Serono Reproductive Biology Institute (Rockland, MA) generously provided the recombinant human gonadotropins. The authors are grateful to the dedicated and conscientious staff of the Surgical Department and the Division of Animal Resources for their enthusiastic participation in this study. We also thank Jessica Vance, Drs. Kelly Young and Ted Molskness for technical assistance; and Dr. David Hess, Mark Poroli, and Rachel Dykman of the Endocrine Services Laboratory for hormone analyses.


   FOOTNOTES
 
1 Supported by NICHD/NIH though cooperative agreement U54 HD18185 as part of the Specialized Cooperative Centers Program in Reproductive Research and National Institute of Health grants 2T32 HDO7133 (S.M.B.), RR00163 (R.L.S.), and HD20869 (R.L.S.). Back

2 Correspondence: Mary B. Zelinski-Wooten, Oregon National Primate Research Center, 505 NW 185th Ave., Beaverton, Oregon 97006. FAX: 503 690 5563; zelinski{at}ohsu.edu Back

Received: 17 September 2003.

First decision: 16 October 2003.

Accepted: 24 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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