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BOR - Papers in Press, published online ahead of print December 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.011494
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BIOLOGY OF REPRODUCTION 68, 1911–1917 (2003)
DOI: 10.1095/biolreprod.102.011494
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Developmental Regulation of Follicle-Stimulating Hormone Receptor Messenger RNA Expression in the Baboon Fetal Ovary1

Nicholas C. Zachos3, Reinhart B. Billiar3, Eugene D. Albrecht4, and Gerald J. Pepe2,3

Department of Physiological Sciences,3 Eastern Virginia Medical School, Norfolk, Virginia 23501 Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology,4 Center for Studies in Reproduction, University of Maryland School of Medicine, Baltimore, Maryland 21201


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the adult ovary, pituitary FSH via interaction with its receptor (FSHR) is required for follicular maturation and granulosa cell development. In humans and nonhuman primates, the pool of follicles available for adult ovarian function is established in utero. However, our understanding of the ontogeny and developmental regulation of FSHR in the ovary of the primate fetus is incomplete. Our goal was to determine whether the baboon fetal ovary expresses the full-length FSHR mRNA transcript and whether levels are developmentally regulated. Fetal ovaries were obtained at mid (Day 100) and late (Day 165) gestation (term = Day 184) from untreated baboons and on Day 165 from baboons in which fetal estrogen levels were either decreased by >95% by treatment with the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 plus estradiol benzoate administered s.c. to the mother on Days 100–164. The full-length 2088-base pair FSHR mRNA transcript was expressed in ovaries of adult and fetal baboons untreated or treated with CGS 20267 or CGS 20267 and estrogen. Mean (±SEM) FSHR mRNA levels (ratio of FSHR mRNA:18S rRNA), quantified by reverse transcription polymerase chain reaction, were increased (P < 0.05) 2-fold between mid (0.34 ± 0.06) and late gestation (0.76 ± 0.07), an increase prevented (P < 0.05) in estrogen-depleted baboons (0.44 ± 0.10) and partially restored by treatment with CGS 20267 and estrogen (0.58 ± 0.16). We previously showed that the number of follicles/0.33 mm2 in fetal ovaries of untreated baboons in late gestation was reduced 50% by treatment with CGS 20267 and restored to normal in baboons treated with CGS 20267 and estrogen. Thus, when corrected for the number of follicles/0.33 mm2, FSHR mRNA levels were similar in baboon fetal ovaries untreated (0.010 ± 0.001) or treated with CGS 20267 (0.009 ± 0.002) or CGS 20267 and estrogen (0.007 ± 0.003). We conclude that estrogen plays a major role in regulating ovarian FSHR mRNA expression in the primate fetus, and that the developmental increase in FSHR mRNA levels reflects the estrogen-dependent increase in folliculogenesis (i.e., increased number of granulosa cells and oocytes).

estradiol, follicle-stimulating hormone, follicle-stimulating hormone receptor, ovary, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary follicle-stimulating hormone (FSH) plays a central role in governing ovarian follicle growth and maturation [1, 2] through interaction with a specific FSH receptor (FSHR) [3, 4]. Female mice with targeted disruption of the gene for FSHß [5] or FSHR [6] and women homozygous for a point mutation in FSHR [7, 8] do not exhibit normal reproductive cyclicity and are infertile. In the adult ovary, FSHR is expressed in granulosa cells [9–11] and oocytes [12] of immature [13] and more advanced [14] follicles. The gene for FSHR apparently is not highly regulated [14] but once expressed is maintained by factors active in enhancing follicle maturation, e.g., FSH and estrogen. For example, estrogen increases FSHR levels in the rat ovary indirectly by promoting granulosa cell mitosis and thus the number of granulosa cells [15].

In humans and nonhuman primates [16, 17], including the baboon (Papio anubis) [18], the pool of primordial follicles available for adult ovarian function is established in utero. However, although FSHR has been detected in the fetal ovary of the pig [19] and cow [20], our understanding of the ontogeny and developmental expression of the FSHR in primates is incomplete [21, 22]. The current study was designed in part to determine whether fetal ovarian folliculogenesis is associated with expression of FSHR. Recently, our laboratories demonstrated that the baboon fetal ovary expresses estrogen receptors {alpha} and ß [23], and the number of primordial follicles in ovaries of near-term baboon fetuses deprived of estrogen in utero was decreased by approximately 50% and restored to normal in animals administered estradiol [18]. Because estrogen indirectly increases FSHR expression in adult ovary by controlling granulosa cell numbers, the current study also was designed to determine whether fetal ovarian FSHR mRNA expression was altered in baboons in which the number of primordial follicles was decreased by suppressing estrogen formation during the second half of gestation. Because fetal serum FSH serum levels in humans [24, 25] and nonhuman primates [26, 27] peak at midgestation and decline with advancing gestation while estrogen levels increase [28], we also examined correlations between fetal ovarian FSHR mRNA expression with serum levels of FSH.


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

Fetal ovaries utilized in this study were obtained from baboons previously studied [18, 23] and from two additional untreated animals in late gestation. Female baboons weighing 12–18 kg were housed individually in stainless steel cages in air-conditioned quarters and fed Purina monkey chow (Ralston Purina, St. Louis, MO) and fresh fruit and/or vegetables daily and water ad libitum. Females were paired with males for five days at the anticipated time of ovulation, and pregnancy was subsequently confirmed as described previously [29]. Fetal ovaries and umbilical venous and arterial blood samples were obtained on Day 165 of gestation from baboons untreated or treated with a highly specific aromatase inhibitor, CGS 20267 (4,4-[1,2,3-triazol-1yl-methylene] bis-benzonitrite = Letrozole; Norvartis Pharm AG, Basel, Switzerland) administered s.c. (115 µg kg body weight-1 day-1) to the mother on Days 100–165 of gestation (term = Day 184). Additional animals were injected with CGS 20267 (115 µg/kg) plus estradiol benzoate (50 µg/kg increasing the dose by 25 µg/kg at 7-day intervals to a maximum of 175 µg/kg) administered to the mother on Days 100–165 of gestation. Blood samples (3–5 ml) were obtained at 1- to 4-day intervals between Days 85 and 164 of gestation via a maternal saphenous vein after sedation with an i.m. injection of ketamine-HCl (10 mg/kg; Parke-Davis, Detroit, MI).

On Day 165 of gestation, baboons were sedated with ketamine and anesthetized with isoflurane. After maternal and umbilical venous and arterial blood samples were obtained, the placenta and the fetus were delivered by cesarean section and the fetus was killed with an overdose of sodium pentobarbital. Fetal ovaries were excised, trimmed of fat, and weighed. One ovary was stored in liquid nitrogen for mRNA studies, and the other ovary was fixed in 10% buffered formalin, embedded in paraffin, and utilized in other experiments [18, 23]. Ovaries and umbilical arterial blood samples were also obtained from baboon fetuses delivered by cesarean section on Day 100 of gestation. Ovary, kidney, adrenal gland, and endometrium samples were also collected from three adult baboons killed with sodium pentobarbital 2 or 8 days prior to or 6 days after anticipated ovulation, as judged by menstrual cycle history and daily examination of perineal turgescence [29]. Baboons were cared for and used strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals (Publication 85-23, 1985). The Institutional Animal Care and Use Committee of the Eastern Virginia Medical School approved the experimental protocol employed in this study.

RIA of Estradiol and FSH

Maternal and umbilical venous serum estradiol levels were reported previously [18] and are presented here for comparative purposes. FSH levels were determined in umbilical arterial blood samples obtained from baboons of the present study and from a previous group of animals similarly treated with CGS 20267 and estradiol [30, 31]. Serum FSH was determined essentially as described by Ramaswamy et al. [32] using homologous RIA reagents supplied by the National Institutes of Health Hormone and Pituitary Program. Recombinant baboon FSH (rec-BbFSH-RP-1; AFP 6944A) was used as the reference standard and radioiodinated tracer, and a rabbit anti-recombinant FSH antiserum (BIOQUAL 67190) was employed as the primary antibody. Gonadotropin-free ovine serum (100 µl) was added to the standard curve and unknowns, which were assayed in duplicate (10 µl and 40 µl) in a single assay. The ED50 of the assay was 0.15 ng/tube, and the minimal detectable dose was 0.009 ng/tube.

Reverse Transcription Polymerase Chain Reaction

The expression of the mRNAs for FSHR in fetal and adult ovaries was determined by reverse transcription polymerase chain reaction (RT-PCR) essentially as described previously [33, 34]. Oligonucleotide primers were synthesized by Life Technologies (Rockville, MD) and selected from cDNA sequences within exons 1 and 5 of the human FSHR [11] to yield a 321-base pair (bp) product: primer 1 (upstream), 5'-GAGAGCAAGGTGACAGAGATTCC-3'; primer 2 (downstream), 5'-GAACACTTGTAGATTCCACG-3'. The primers for 18S rRNA were selected from the human 18S rRNA gene sequence [35] to yield a 489-bp product: primer 1 (upstream), 5'-TCAAGACAAGTCGGAGG-3'; primer 2 (downstream), 5'-GGACATCTAAGGGCATCACA-3'. Total RNA (1–3 µg) was isolated from fetal and adult ovaries and nonovarian tissues and reverse transcribed, and DNA amplifications were carried out in a 50-µl reaction volume containing 5 µl (FSHR) or 1 µl (18S) of the RT, 0.2 mM each of dATP, dCTP, dGTP, dTTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1.25 U cloned Thermus aquaticus DNA polymerase (Amplitaq; Perkin-Elmer/Cetus, Norwalk, CT), and 20 pmoles each of primers 1 and 2 for FSHR or 18S. PCR was performed in a programmable thermal cycler (MJ Research, Cambridge, MA), and samples were amplified for 30 (FSHR) or 16 (18S) sequential cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min. Two negative controls, in which either RNA or RT was omitted from the reaction, were also performed. Aliquots of the PCR products were fractionated by electrophoresis in a 2% agarose gel and stained with ethidium bromide, and the expected 321-bp FSHR and 489-bp 18S PCR target products were visualized with an ultraviolet transilluminator and photographed. Quantitative analysis of the PCR products was determined using SigmaGEL Gel Analysis (Sigma, St. Louis, MO) of the agarose gel images captured on Eagle Eye II Still Video System (Stratagene, Los Angeles, CA). A preliminary study was performed in which a portion of the PCR products for FSHR were digested with restriction enzymes and subcloned, and the cDNA was sequenced by the University of Maryland Immunocytochemistry Cell Core.

To determine whether the baboon fetal ovary expressed a full-length FSHR mRNA transcript, RT-PCR was performed using the SuperScript One Step RT-PCR for Long Templates (Invitrogen Life Technologies, Grand Island, NY) and oligonucleotides that encompassed the entire open reading frame (Life Technologies): primer 1 (upstream), 5'-ATGGCCCTGCTCCTGGTCTCTTTGCTGGCATTCCTGAGCTTGGGCT-3'; primer 2 (downstream), 5'-AGGTGAATGTATGAACAGGGAGATTCAGTAAATCGGGTTTTGATT-3' (initiation and stop codons are underlined). Approximately 1–3 µg total RNA from fetal and adult baboon ovaries was added (1 µl) to 50-µl reaction mixtures containing 25 µl reaction mix (Invitrogen), 1 µl forward and 1 µl reverse primer, 2 µl RT/Plat Taq Hi Fi Mix, and 20 µl water. Reactions were incubated at 50°C for 30 min and then 94°C for 2 min and then for 35 sequential cycles of 94°C for 15 sec, 50°C for 30 sec, and 68°C for 150 sec, and a final step at 72°C for 10 min. The expected 2088-bp full-length FSHR mRNA transcript was visualized and photographed. A portion of the PCR products for the full-length FSHR was digested with restriction enzymes and subcloned, and the cDNA was sequenced by the San Diego State University Microchemical Core Facility (San Diego, CA).

Statistics

Data are expressed as an overall mean (±SEM) and were analyzed by ANOVA and the Bartlett statistic to confirm homogeneity of variance, with post hoc comparisons of the means by the Newman-Keuls multiple comparison tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Serum Estradiol and FSH

Our previously published results of maternal and umbilical serum estradiol levels in baboons treated with the aromatase inhibitor CGS 20267 or with CGS 20267 plus estradiol benzoate [18] are included here to facilitate correlation of changes in estrogen production with fetal ovarian FSHR expression and umbilical serum FSH levels. Maternal serum estradiol levels increased from approximately 1 ng/ml on Days 85–120 of gestation to 2.5–3.5 ng/ml by Day 165. Within 48–72 h of the onset of CGS 20267 treatment on Day 100, maternal serum estradiol levels decreased to and remained at approximately 0.1 ng/ml. The pattern of serum estradiol in baboons treated with CGS 20267 plus estradiol benzoate was similar to that in the control, although absolute levels were slightly greater than normal. Mean estradiol levels in umbilical venous serum on the day of delivery in untreated baboons (0.59 ± 0.13 ng/ml) were also significantly (P < 0.01) reduced by administration of CGS 20267 (0.04 ± 0.01 ng/ml) and restored (0.19 ± 0.08 ng/ml) to 30% of normal in baboons treated with CGS 20267 and estradiol.

Mean FSH in umbilical arterial serum of untreated baboons was approximately 3-fold greater (P < 0.05) on Day 100 (5.5 ± 1.2 ng/ml, n = 8) than on Day 165 (2.0 ± 0.1 ng/ml, n = 21) of gestation (Fig. 1). In contrast, serum FSH levels were not decreased on Day 165 in baboons treated with CGS 20267 (6.9 ± 1.7 ng/ml, n = 12) and thus were similar to mean values in untreated baboons on Day 100. However, in baboons treated with CGS 20267 and estrogen, umbilical arterial serum FSH levels (2.6 ± 0.2 ng/ml, n = 11) were not different from those in untreated baboons on Day 165. Similar changes in FSH levels were noted in the subpopulation of umbilical arterial samples from baboons in which fetal ovarian FSHR mRNA was also determined. Thus, mean FSH in untreated baboons was also 3-fold greater (P < 0.05) on Day 100 (7.0 ± 1.5 ng/ml, n = 5) than on Day 165 (2.1 ± 0.1 ng/ml, n = 8), increased (P < 0.05) on Day 165 in baboons treated with CGS 20267 (11.5 ± 2.1 ng/ml, n = 6), and restored to normal (P < 0.05) by treatment with CGS 20267 and estrogen (3.0 ± 0.7 ng/ml, n = 3).



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  		FIG. 1. Mean (±SEM) umbilical arterial serum FSH levels in untreated baboons on Days 100 (mid; n = 8) and 165 (late; n = 21) of gestation and on Day 165 of gestation (term = Day 184) in baboons treated on Days 100–165 with CGS 20267 (115 µg/kg; n = 12) or with CGS 20267 (115 µg) plus estradiol benzoate (50–175 µg/kg; n = 11). Values with different letter superscripts differ from each other at P < 0.05 (ANOVA; Student-Newman-Keuls statistic)

FSH Receptor mRNA

The 2088-bp full-length FSHR mRNA transcript was detected in the fetal ovary on Days 100 and 165 of gestation in untreated baboons, on Day 165 in animals treated with CGS 20267 or CGS 20267 plus estradiol benzoate, and in the adult baboon ovary but not in baboon term placenta (Fig. 2A). The full-length baboon FSHR mRNA transcript exhibited >97% homology with the published sequence of the human FSHR [11] (Fig. 3).



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  		FIG. 2. A) Representative RT-PCR of FSHR mRNA using oligonucleotide primers encoding the entire open reading frame of the full-length 2088-bp transcript in baboon adult ovary and fetal ovaries obtained on Days 100–165 of gestation from untreated baboons and on Day 165 from animals treated with CGS 20267 or with CGS 20267 plus estrogen (Fig. 1). No product was detected in RNA from baboon placenta (Plac). MWM = molecular weight marker. B) Representative RT-PCR of FSHR in baboon fetal ovary (Day 165), adult ovary collected 48 h prior to anticipated ovulation, and adrenal gland (ADR), endometrium (END), and kidney (KID) obtained from an adult baboon. One milligram of total RNA or 1 ng cDNA to human FSHR was reverse transcribed, and 5 µl of these samples was amplified for 30 PCR cycles using FSHR primers designed to detect a 321-bp product. No product was detected in samples from which RNA was omitted (No RNA) or RT was omitted (not shown)



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  		FIG. 3. Nucleotide sequence of the full-length 2088-bp FSHR product isolated from fetal baboon ovaries (Fig. 2) compared with the published sequence for human FSHR [11].

RT-PCR was employed to quantify FSHR mRNA expression. Using oligonucleotide primers specific to human FSHR, the expected 321-bp PCR product was detected in the baboon fetal (Day 165) and adult ovary and the cDNA to the human FSHR (generously provided by Dr. Aaron Hsueh) but was not detected in adult baboon adrenal gland, endometrium, or kidney (Fig. 2B). Sequence analysis confirmed that the baboon FSHR PCR product expressed in the baboon fetal ovary also was 98% homologous to the same region of the published sequence of human FSHR (data not shown).

The developmental pattern of and the effects of estrogen in utero on expression of the 321-bp FSHR mRNA and the 489-bp 18S rRNA are shown in Fig 4A. Mean FSHR mRNA levels expressed as a ratio of FSHR mRNA:18S rRNA (Fig. 4B), which did not appear to be altered by development or estrogen deprivation, were 2-fold greater (P < 0.05) in late gestation (0.76 ± 0.07) than at midgestation (0.34 ± 0.06). Moreover, FSHR mRNA levels on Day 165 of gestation in fetal ovaries of estrogen-depleted baboons (0.44 ± 0.10) were approximately 50% lower (P < 0.05) than those in ovaries of untreated fetuses and partially but not significantly restored to normal by treatment with CGS 20267 and estrogen (0.58 ± 0.16).



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  		FIG. 4. A) Representative RT-PCR of FSHR and 18S rRNA in baboon fetal ovaries obtained from untreated baboons at mid (Day 100) and late (Day 165) gestation (term = Day 184) and on Day 165 in baboons treated with CGS 20267 or CGS 20267 plus estradiol benzoate (Fig. 1). B) Mean ± SEM of the ratio of the expression of FSHR mRNA:18S rRNA determined in fetal ovaries from untreated baboons on Day 100 (n = 5) and Day 165 (n = 8) and on Day 165 following treatment with CGS 20267 (n = 6) or CGS 20267 plus estrogen (n = 3). Values with different letter superscripts differ from each other at P < 0.05 (ANOVA; Student-Newman-Keuls statistic)

We previously showed that the overall mean number of follicles/0.33 mm2 in near-term fetal ovaries of untreated baboons (77 ± 10) was reduced by 50% in animals treated with CGS 20267 (35 ± 2) and restored to normal (70 ± 4) in fetal ovaries of baboons treated with CGS 20267 and estradiol [18]. Because FSHR is expressed only in oocytes and granulosa cells of follicles in the adult ovary, we also determined the levels of FSHR mRNA relative to the number of follicles (virtually all of which were at the primordial stage [18]) that were developed in the fetal ovary by late gestation. Thus, FSHR mRNA levels per number of follicles (FSHR mRNA:18S rRNA:number of follicles/0.33 mm2; Fig. 5) were similar in fetal ovaries of animals untreated (0.010 ± 0.001), treated with CGS 20267 (0.009 ± 0.002), and treated with CGS 20267 and estrogen (0.007 ± 0.003).



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  		FIG. 5. FSHR mRNA levels (Fig. 5B) relative to the number of primordial and primarylike follicles in the baboon fetal ovary on Day 165 of gestation in animals untreated or treated with CGS 20267 or CGS 20267 plus estrogen. Follicle numbers per 0.33 mm2 of fetal ovary determined previously [18]


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the current study are the first to show that the baboon fetal ovary expresses the full-length mRNA for FSHR as early as Day 100 or at approximately the halfway point in gestation and that expression increases approximately 2-fold late in gestation. This developmental increase in FSHR mRNA levels was prevented in fetal ovaries of baboons in which the number of primordial follicles was reduced by treatment with CGS 20267, which essentially depleted the fetus of estradiol [18]. Moreover, fetal ovarian FSHR mRNA levels (this study) and the number of primordial follicles [18] were both restored to normal in baboons treated with CGS 20267 and estrogen. Therefore, estrogen appears to play a major role in regulating, directly and/or indirectly, ovarian FSHR mRNA expression in the primate fetus. Estrogen acts in synergy with FSH to increase the number of FSHRs in the adult rat ovary [15]. However, although the promoter of the human FSHR gene exhibits an imperfect 5' half-consensus estrogen-response element [14], estrogen has no effect on the number of FSHRs per cell or on the binding of FSH to granulosa cells [36, 37]. Estrogen-induced increases in FSHR levels seem to reflect a proliferative action of estrogen on granulosa cells [15] rather than a direct effect on gene transcription [14]. Based on these and previous observations [18, 23], we hypothesize that the developmental increase in fetal ovarian FSHR mRNA levels in baboons reflects the estrogen-dependent increase in folliculogenesis and presumably an increased number of granulosa cells and oocytes, whereas the decline in FSHR mRNA in estrogen-suppressed animals reflects the decrease in the number of primordial follicles. Consistent with this hypothesis is the finding that FSHR mRNA levels (per number of follicles/0.33 mm2) were similar in near-term fetal ovaries of baboons untreated or treated with CGS 20267 or with CGS 20267 plus estrogen.

FSH has both dose- and time-dependent effects on FSHR expression/binding in the rat ovary [15]. For example, although FSH increased FSHR levels in granulosa cells [36, 38], FSHR mRNA and/or binding were reduced following prolonged exposure to or treatment with elevated concentrations of FSH [15, 39]. In humans [24, 25] and nonhuman primates [26, 27], including the baboon (this study), the fetus is exposed to FSH throughout the second half of gestation. However, FSH alone or acting in concert with estrogen may not be required for fetal ovarian FSHR mRNA expression. Of two women with hypogonadism that had a mutation in the FSHß gene and thus no detectable FSH protein [40], one woman conceived after induction of ovulation with exogenous FSH [41], and the other exhibited an increase in follicle growth following treatment with exogenous FSH [42]. Thus, although FSH is critical to and plays a major role in the maturation of preantral and antral follicles and is thus essential for adult ovarian function [14], onset and development of FSHR mRNA expression in the primate fetal ovary may be gonadotropin independent. Additional studies are needed to specifically examine this possibility in the primate fetus.

Factors produced within the ovary itself (e.g., activin) and/or arriving at the gland via extrinsic innervation (e.g., nerve growth factor) can enhance formation of FSHR [43, 44]. For example, activin increased the steady-state levels of FSHR mRNA by enhancing the transcription rate of the FSHR gene and decreasing the half-life of FSHR mRNA [14, 45–47]. However, our current understanding of the developmental expression, regulation, and role(s) of activin and/or other potential intraovarian factors in the primate fetus is incomplete [22].

The baboon fetal ovary in late gestation is composed primarily of primordial follicles, i.e., oocytes surrounded by a single layer of flattened and/or cuboidal granulosa. Although adult ovarian granulosa cells exhibit FSHR mRNA/FSH binding [1], adult human and porcine oocytes also express FSHR mRNA and protein [12]. Therefore, full-length mRNA for FSHR may be expressed in the granulosa cells and/or oocytes of the primordial follicles in the baboon fetal ovary, although specific cellular localization of FSHR and whether the site(s) of expression is altered in fetal ovaries of baboons depleted of estrogen remain to be determined.

The detection of the full-length FSHR mRNA in the baboon fetal ovary at midgestation was unanticipated and indicates that expression of this gonadotropin receptor occurred in fetal ovaries composed primarily of germ cell cords, which contain oocytes and mesenchymal-epithelial cells but very few primordial follicles [18]. The mesenchymal-epithelial cells are considered pregranulosa cells [48, 49], which will surround the oocyte to form primordial follicles. Although site(s) of expression of FSHR mRNA at this point in gestation also is unknown, the results of the current study are the first to establish that pregranulosa cells and/or oocytes express the full-length FSHR mRNA transcript prior to their interaction to form a follicle. In the mouse [50] and rat [51, 52], multiple shorter FSHR mRNA transcripts can be detected very early in follicle development, and the full-length transcripts for FSHR are not detectable until Postnatal Day 5. Therefore, expression of functional FSH receptors in the rodent ovary may depend on a change in alternative splicing rather than a change in FSHR gene expression [14]. Although we have not examined fetal ovaries obtained prior to midgestation, the results of the current study demonstrate that in the primate fetal ovary, formation of primordial follicles is not immediately preceded by formation of shorter FSHR mRNA transcripts and thus may be independent of alternative splicing. Although we have detected a full-length FSHR mRNA transcript, it remains to be determined whether the mRNA is translated into a functional protein. FSH binding was not detected in human or rhesus monkey fetal ovaries obtained at early to midgestation [21] and was only detected in rhesus monkey fetal ovary in late gestation [21, 22].

The results of the current study also indicate that the levels of FSH in umbilical artery and presumably of fetal pituitary origin were modulated by the level of estrogen secreted into the fetus. Thus, FSH levels declined between mid and late gestation in association with the rise in estrogen, but FSH levels at term remained elevated in fetuses in which estrogen production was markedly suppressed and were restored in large part to normal in baboons treated with aromatase inhibitor and estrogen. Therefore, the well-established negative feedback action of estrogen on hypothalamic-pituitary function in the adult may also occur in utero in the fetus, as originally suggested by Kaplan and Grumbach [24]. A GnRH pulse-generating network appears to be organized and functional by midgestation in higher primates [53], as reflected by pulsatile secretion of pituitary gonadotropin [54, 55] and an operational pituitary-gonadal feedback loop [56].

In summary, the results of the present study are the first to show that expression of the full-length FSHR mRNA in the baboon fetal ovary increases between mid and late gestation. This increase was prevented in fetal ovaries of baboons in which the number of primordial follicles was reduced by suppressing estrogen. Moreover, fetal ovarian FSHR mRNA levels (this study) and the number of primordial follicles [18] were both restored to normal in baboons treated with CGS 20267 and estrogen. Therefore, estrogen must play a major role in regulating, directly and/or indirectly, ovarian FSHR mRNA expression in the primate fetus. Consequently, we hypothesize that the developmental increase in fetal ovarian FSHR mRNA levels in baboons of the current study reflects the estrogen-dependent increase in folliculogenesis and presumably an increased number of granulosa cells and oocytes. Collectively, these results further support the hypothesis that estrogen programs development of the primate fetal ovary and potentially impacts ovarian function and fertility in adulthood.


   ACKNOWLEDGMENTS
 
The authors greatly appreciate the supply of CGS 20267 generously provided by Norvartis Pharma AG (Basel, Switzerland). The authors thank Ms. Sandra Huband for secretarial assistance with the manuscript and preparation of the figures. The contribution of Drs. Clifford Pohl and Tony Plant (University of Pittsburgh, Pittsburgh, PA) in establishing and performing the FSH assays is sincerely appreciated.


   FOOTNOTES
 
1 This work was supported by NIH grant U54 HD 36207 as part of the NICHD Specialized Cooperative Centers Program in Reproduction Research. Back

2 Correspondence: Gerald J. Pepe, Department of Physiological Sciences, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501-1980. FAX: 757 624 2269; pepegj{at}evms.edu Back

Received: 19 September 2002.

First decision: 16 October 2002.

Accepted: 17 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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