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Regular Article |
a Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7
b Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada H3A 1A3
c Department of Medicine, Université de Montréal, Montreal, Quebec, Canada H3T 1J4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Western blot) appeared to be lower in +/- ovaries at all ages. FSH-R (FSH* binding) declined steadily from 3 mo and reaching the lowest point at 1 yr. LH receptor (LH* binding) was high in the 1-yr-old ovary, and expression was localized in the stroma and interstitial cells. Our findings demonstrate that haploinsufficiency of the FSH-R gene could cause premature exhaustion of the gonadal reserves previously noted in these mice. This is accompanied by age-related changes in the hypothalamic-pituitary axis. As these features in our FSH-R +/- mice resemble reproductive failure occurring in middle-age women, further studies in this model might provide useful insights into the mechanisms underlying ovarian aging.
aging, follicle-stimulating hormone, follicular development, inhibin, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The well-known and regulated process of normal reproductive fading in women and experimental animals is influenced by pituitary hormones and, in particular, the gradual elevating levels of FSH [11]. It is generally believed that the propensity of the ovary to begin its journey toward aging becomes higher with rising basal levels of FSH. However, studies to verify this in models such as female mice that ectopically overexpress human FSH are precluded because they develop hemorrhagic and cystic ovaries and die prematurely as a result of complications such as urinary bladder obstruction [14]. Interestingly, hypogonadal (hpg) mice that lack both circulating gonadotropins have less reduction in the numbers of oocytes at 1 yr, compared with normal mice, because of a decrease in the rate of loss of follicles from the nongrowing pool [15]. In addition, it is known that hypophysectomy attenuates ovarian aging in mice [16] and reduces the rate of oocyte loss in monkeys [17].
Normal ovarian function relies upon the perfect interaction between FSH and its own receptor (FSH-R) localized exclusively on granulosa cells in the ovary [18]. In women and experimental animals, deficiency of FSH [19, 20] or FSH-R [21–25] is associated with various disturbances of reproductive development and adult reproductive function. Some investigators have suggested that perhaps the last remaining follicles become refractory to gonadotropin stimulation [26] in postmenopausal women and women with premature ovarian failure also have impaired ovarian responsiveness [27]. To our knowledge, there was no study that questioned or linked the quantitative status/aspects of ovarian FSH-R signaling in aging.
In the preceding communication we reported that female mice with haploinsufficiency of the FSH-R undergo accelerated reproductive senescence and biological aging by losing follicles and oocytes and ceasing their reproductive life much earlier than wild-type mice [28]. The availability of this model and a previous report of reduced fertility and fecundity in the heterozygous mice [25] prompted us to examine the hypothesis that the numbers of FSH-R play a key role in the triggering of ovarian cessation.
In the present study we show that FSH-R haploinsufficiency in mice leads to a decrease in ovulation, altered ovarian steroidogenesis, and neuroendocrine impairments resulting in early reproductive senescence. We are not aware of any animal model displaying such an important gene dosage effect leading to profound alterations in ovarian function that impact on aging processes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments were conducted with the approval of our institutional ethics committee. Details of the +/- FSH-R mutant mice and handling conditions are given in the preceding article [28]. For characterization of the hormonal profiles, blood samples were collected at different times from a total of 87 wild-type and 92 heterozygous mice. Samples were collected from all animals by the intracardiac method under ether anesthesia on the morning of proestrus and placed in centrifuge tubes containing 0.05% EDTA. As 12-mo-old +/- mice lose cyclicity, sampling was performed in the morning without reference to stage. Plasma was obtained by centrifugation and stored at -20°C. In the ovulation-induction experiment (see below), immature mice were also included.
Hormone Assays
FSH and LH were determined in the plasma and pituitary glands of the mice following extraction with an aqueous buffer [29]. Rat FSH and LH radioimmunoassays (RIA) were performed according to suggested instructions using kits from the National Hormone and Pituitary Distribution Program (courtesy of Dr. A.F. Parlow, University of California-Los Angeles, Torrance, CA). All samples for each hormone were measured in a single assay, with intraassay coefficients of variation of 9% and 8% for FSH and LH, respectively. The sensitivity of the assays was 0.04 ng/ml for both hormones. The total number of mice for gonadotropin measurements was for wild-type, n = 79, and for heterozygous, n = 108. Plasma concentrations of estradiol, progesterone, and testosterone [25] were determined in the samples using Coat-a-count kits (Diagnostic Products Corp., Los Angeles, CA) following the manufacturer's instructions.
Assessment of Corpora Lutea
The total number of corpora lutea (CL) present in the ovary was determined by histological examination of serial sections (each section = 5 µm) of the ovary without knowledge of the genotypes. As CL have a diameter of about 175 µm [30], every 35th section was checked for CL to determine the total number in the ovary.
Ovarian Responsiveness to eCG and hCG or Ovulation Rate
Immature 21-day-old (n = 4 +/+; n = 9 +/-) and 3-mo-old (n = 8 +/+; n = 11 +/-) female mice were injected with 5 IU/mouse, i.p. equine chorionic gonadotropin eCG (Pregnecol serum gonadotrophin injection, Horizon Technology Pty, Sydney, NSW, Australia) [31] followed by an injection with hCG (10 IU/mouse, i.p.) 48 h later (A.P.L. chorionic gonadotropin for injection USP, Wyeth-Ayerst Inc., Montreal, PQ, Canada). About 17 h afterward, mice were killed, and the morphological appearance of both ovaries was noted. Each ovary was then weighed and fixed for histology. The oviducts were squeezed between 2 slides for examining eggs under light microscope.
Western Blotting
Western blotting of desired proteins from pooled ovaries of each mouse was performed on the same day. Fresh tissues were extracted with a lysis buffer containing detergent and a protease inhibitor cocktail (50 mM Tris-HCl, pH 7.2, 1% NP-40, 50 mM glycerophosphate, 5 mM DTT, 1 mM sodium vanadate, 0.05 mM NaF, 0.1 mM PMSF, and 5 µg/ml leupeptin). Fifty micrograms of protein was run on SDS-PAGE gels and transferred to nitrocellulose for reaction with the following antibodies at appropriate dilutions. Each experiment was performed 2–3 times with different extracts. An antibody [29] to the N-terminal peptide of inhibin subunit (1:500) was given to us by Dr. B.D. Schanbacher (formerly of USDA, Clay Center, NE). LH receptor polyclonal antibody (1:500) [32] was obtained from Dr. N.R. Moudgal from the Indian Institute of Science in Bangalore, India. SF-1 antibody to mouse antigen (DNA binding domain) purchased from Upstate Biotechnology (Lake Placid, NY) was used at 1:10 000 dilution. Cyclin D2 antibody to carboxyl terminus of mouse protein from Santa Cruz Biotechnology (Santa Cruz, CA) was employed at 1:500 dilution. Antibody to ß-actin (Sigma Chemicals, St. Louis, MO) was used at 1:500 for verifying equivalent protein loading. After treatment of the blots with 1:2000 of a corresponding second antibody (Santa Cruz, CA), bands were finally detected by the Amersham-ECL kit (Buckinghamshire, U.K.) and compared with the reported values for molecular weight.
Immunohistochemistry
Ovaries were immediately removed from animals and placed in 10% formalin at 4°C overnight. After fixation, the ovaries were processed and embedded in paraffin for cutting sections (5 µm). To expose epitopes, sections were placed in a microwave oven for 15 min at 100°C in sodium citrate buffer (0.01 M, pH 5.7). Sections were then incubated overnight at 4°C with polyclonal antibodies against inhibin (1:800) and LH-R (1:500). Binding sites of antibodies were visualized by the Immunosystem kit (Santa Cruz, CA) as described previously [29].
LH-R and FSH-R Binding Assays
Ovaries from 3-, 7-, and 12-mo-old heterozygous and wild-type mice were collected, weighed, and placed in liquid nitrogen. Total of 3–4 samples for each mean value was used for these assays. The level of LH-R binding in mouse ovarian membrane preparation was determined using purified, radiolabeled ovine LH prepared in our laboratory [33]. The dissected ovaries from each animal was homogenized in buffer A (25 mM Tris-HCl, 0.3 M sucrose, pH = 7.2) and centrifuged at high speed (15 000 x g) for 15 min to collect the membrane pellet. The pellet was dispersed in another buffer (25 mM Tris-HCl pH 7.2, containing 10 mM MgCl2 and 1 mg/ml BSA) containing 50 000 cpm of labeled ovine LH. The tubes were incubated overnight at room temperature. Specific binding was calculated by subtracting the count obtained from the binding to the ovarian membrane in the presence of excess oLH (1 µg/tube) from the binding seen in the absence of unlabeled oLH. A similar method was used to assess the level of FSH binding in the mouse ovarian membrane receptor employing labeled human FSH. In this case, unlabeled oFSH was used to determine nonspecific binding. In both series of experiments the results were calculated to express receptor quantity as femtomole of LH or FSH bound per milligram of ovary. We have previously shown that these heterologous hormones bind with high affinity to the mouse gonadal LH and FSH-R, respectively [33].
Statistics
Data are presented as the mean ± SEM and analyzed by Student t-test or ANOVA with a Fischer least significant difference (LSD) post hoc test. A value of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In 3-mo-old heterozygous and wild-type females the basal FSH levels were similar, 3.3 ± 0.4 ng/ml and 3.6 +/- 0.7 ng/ml, respectively (Fig. 1). However, in the 7-mo-old +/- females the plasma FSH level was significantly higher (6.4 ± 0.7 ng/ml) than wild-type and heterozygote females of the same age (3.3–3.9 ng/ml; Fig. 1A). The plasma concentration of FSH increased even more in 12-mo-old +/- mice (7.6 ± 0.6 ng/ml). This was statistically different from values in +/+ females of the same age (5.7 ± 0.4 ng/ml) as well as in +/- females of 7 mo of age (P < 0.05).
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No statistically significant difference was found in plasma LH values in 3-mo-old females of the 2 genotypes (Fig. 1B). In contrast, LH level was significantly increased in 7-mo-old +/- mice (3.5 ± 1.3 ng/ml) compared to both 7-mo-old +/+ and 3-mo-old +/- (1.6 ± 1.2 ng/ml) values. Interestingly, the levels of LH in 1-yr-old heterozygous females decreased more than 50% (1.62 ± 0.2 ng/ml) in comparison to +/- females of 7 mo of age. However, the hormone was significantly elevated compared with 12-mo-old wild-type litter mates.
Pituitary levels of FSH and LH for all ages examined are also presented in Figure 1, C and D. There appeared to be a gradual accumulation of gonadotropin contents in the pituitary with increasing age in both genotypes. However, only the 1-yr-old +/- pituitary contained significantly higher levels of LH compared with wild-type litter mates (Fig. 1D).
Plasma Steroid Levels
The level of estradiol in +/- FSH-R females on the morning of proestrus tended to be decreased at all ages compared with wild-type mice at the same stage of the cycle (Fig. 2A). However, the reduction attained significance only in +/- females at 7 and 12 mo of age. There was a significant increase in the secretion of testosterone from the +/- ovaries at 7 and 12 mo compared with that in wild-type siblings (Fig. 2B). As the ratio of these 2 steroid hormones may impact ovarian function, a comparison was made at different ages. There was an elevation of the testosterone:estradiol ratio in 7-mo-old +/- mice (3.5:1) compared with that in wild-type litter mates (1:2.5). At 12 mo of age the ratios were 7:1 and 3:1 in +/- and wild-type animals, respectively. The plasma progesterone level in 3- and 7-mo-old +/- females was reduced significantly (P < 0.05) compared with wild-type controls (Fig. 2C). Continuing the steady decline, there was even less progesterone in 12-mo-old FSH-R +/- mice (30%) compared with that in wild-type, age-matched females.
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Corpora Lutea
In previous studies we had initially examined representative sections of the 3-mo heterozygous ovary [25] and concluded that there were no major changes. However, more detailed and quantitative studies examining serial sections by an established method [30] now show that the +/- ovaries contained decreased numbers of corpora lutea (CL), indicating some problems with ovulation. At 3 and 7 mo of age there were diminished numbers of CL in the +/- ovaries (mean of 13.6 in +/+ vs. 7.5 in +/- at 3 mo and 9.6 vs. 3.5 at 7 mo, respectively). These reductions were statistically significant (P < 0.05). By 12 mo, no identifiable CL was present in any +/- ovary, whereas the +/+ ovary had an average of 7.1 ± 3.2 CL.
Superovulation
To assess responsiveness of the ovary we performed hormonal stimulation using established eCG-hCG treatment at 2 different ages [32]. Immature 21-day-old heterozygous and wild-type female mice were stimulated with gonadotropins to examine ovulated eggs from their oviducts. Whereas all normal +/+ mice responded to hormonal stimulation, 2 +/- females were refractory. The calculated average ovulation rate among immature heterozygous mice (13.6 ova/mouse) was lower than that in wild-type siblings (26 ova/mouse; Table 1). At 21 days there was no significant difference in the number of ova shed between right and left ovaries in both +/- and +/+ mice. The mean weight of the right and left ovary was similar (5.3 vs. 5.5 mg), respectively. The uterus appeared to be stimulated to the same extent in both genotypes (not shown).
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The mean number of ova released in hormone-treated 3-mo-old +/- females was reduced by 57% in comparison to wild-type litter mates (P < 0.001). Interestingly at this age, the ovarian weight of the +/- right ovary (11.9 ± 0.6 mg) as well as number of eggs ovulated by the right ovary (7.4 ova/mouse) was significantly different from the left (weight, 9.4 ± 0.6 mg, P < 0.05; number of eggs, 4 ± 2.4 ova/mouse, P < 0.05).
Histological analysis revealed some defects in ovarian response to gonadotropin treatment in the 3-mo-old +/- mice. Ovaries isolated from wild-type females contained numerous CL (arrows) and the absence of mature preovulatory follicles, features that are typical of a recently ovulated ovary (Fig. 3, A and C). Interestingly, the FSH-R +/- ovaries also had CL plus a few mature "preovulatory" follicles or unruptured follicles characterized by the presence of entrapped oocyte and granulosa cell cumulus expansion occurring just before ovulation (Fig. 3, B and D).
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Inhibin
As FSH stimulates the expression and secretion of inhibin from granulosa cells [34], we examined inhibin subunit expression in the ovary of wild-type and +/- mice at 3, 7, and 12 mo of age. Using peptide antibodies that were available to us, Western blot revealed a gradual decrease in the expression of inhibin with age in both genotypes (Fig. 4A). However, the reduction of this protein in the FSH-R +/- ovary appeared to be greater, and by the age of 12 mo it had declined drastically to about 25% of the wild-type value (Fig. 4A, lane 6 and densitometry scan). Since the protein band with a higher Mr did not appear to change in both genotypes at different ages, we have assumed that it might represent a nonspecific reaction.
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Immunohistochemistry with antibodies against a inhibin subunit also revealed diminished expression of protein in +/- compared with wild-type ovaries at 3 and 7 mo (data not shown). By 12 mo of age the +/- ovaries contained only a few cells positive for inhibin (Fig. 5, B and D), whereas a +/+ ovary was highly stained with inhibin peptide antibody (Fig. 5, A and C).
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Status of Gonadotropin Receptors in the Ovary
One of the major markers of the FSH-induced differentiation of granulosa cells is the expression of LH-R [35] that is also essential for mediating the response to LH during induction of ovulation. To examine this we performed an LH-R binding assay. The binding of LH-R in 3-mo-old whole ovaries was not different between the 2 genotypes (wild-type 26 fmol and heterozygous 25 fmol/mg protein; Fig. 6A). By the age of 1 yr, there was an almost 2-fold increase of LH-R binding in the +/- ovary (60.6 ± 7 fmol/mg protein) over the values in wild-type litter mates (31.8 ± 2.6 fmol/mg protein; P < 0.05). However, Western blot of ovarian extracts revealed no changes between wild-type and +/- mice in LH-R expression at all ages studied (Fig. 3B), indicating that small differences are not detected by this method. When we examined the expression of LH-R in different compartments of the ovary by IHC, no major changes were evident in 3-mo-old females between 2 genotypes (data not shown), yet the 3-mo +/- ovary had responded poorly to ovulation induction (Table 1). However, 12-mo-old +/- ovaries contained abundant LH-R positive cells within the stroma, which presented large islands of these polygonal steroidogenic cells widespread all around the ovary (Fig. 5E). The localization of LH-R in normal ovaries of this age was confined to thecal cells, granulosa cells in mature follicles, and CL (Fig. 5G). Granulosa cells in the very few follicles remaining in the 1-yr-old +/- ovary did not express LH-R at any stage of follicle development, and LH-R was expressed only in thecal and interstitial cells (Fig. 5, E and F).
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FSH-R binding assays revealed a 40% reduction in the hormone binding of a 3-mo-old +/- ovary (4.4 ± 0.2 fmol/mg protein) compared with wild-type litter mates (7.1 ± 0.1 fmol/mg protein; P < 0.004; Fig. 6B). By 7 mo of age FSH binding was reduced by about 50% in the +/- ovary (3.7 vs. 6.9 fmol/mg protein) compared with wild-type, age-matched ovaries (P < 0.02). There was a constant decrease with age of ovarian FSH binding in both genotypes. Wild-type mice had a 25% reduction in receptor binding assay from age 3 to 12 mo of age, whereas +/- females lost more than 50% of the receptor from the values that were already low at 3 mo. Whereas there was no big change in FSH binding between 3- and 7-mo-old wild-type mice, the 7-mo-old heterozygous females showed about a 15% loss of the receptor, which reflects the lower number of follicles.
Selected Signaling/Regulatory Proteins
To get an idea of how ovarian structural changes may be reflected in functional alterations, we monitored 2 important and selected regulatory proteins, namely SF-1 and Cyclin D2, that mediate various pathways. As shown by Western blots in Figure 4, C and D, both were reduced in the 1-yr-old +/- extracts in comparison to the content in wild-type ovaries of the same age. One of the 2 (top) bands detected in the wild-type ovary by the cyclin D2 antibody was absent in the 12-mo-old +/- ovary.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FSH in humans, as well as in experimental animals, increases with increasing age. This age-related increase in FSH is associated with a decrease in inhibin, steroids, and a loss of follicles. In contrast, ovarian aging is attenuated in hpg female mice, which lack both gonadotropins [15]. In adult mice, hypophysectomy retards the loss of follicles [16], suggesting that the absence of both pituitary hormones play an anti-aging role. In contrast to this, experimental genetic ablation of FSH-R signaling appears to exert aging effects on the ovary in mice. The follicle-stimulating hormone receptor knockout (FORKO; null) mice that produce high FSH but lack the FSH-R repertoire show early symptoms of aging (high level of gonadotropins, low estradiol, skeletal deformity, and obesity), which is in sharp contrast to their normal litter mates [25]. These null females, as well as the aging heterozygotes, also go on to develop ovarian tumors accompanied by cachexia [29].
As reported recently, the manifestation of reproductive deficits that intensify with age in FSH-R haploinsufficient mice are striking and surprising [28]. The total number of follicles in the FSH-R +/- females at 7 mo of age decreased by more than 75% compared with the younger 3-mo-old +/- ovary. The increase in the number of growing follicle pools in 7-mo-old +/- ovaries was coincident with a rise in plasma FSH and LH levels, suggesting that high levels of gonadotropins recruit more follicles to develop. Our data on elevated LH levels at 7 mo (Fig. 1B) together with the observations in LH transgenic mice suggest that LH might indeed accelerate growth of primordial follicles into the primary and large preantral stage [36]. It was interesting to note that at 7 mo of age when the total number of follicles decreased severely in +/- mice [28], the levels of estradiol were still maintained slightly below the 3-mo level (Fig. 2). The sustained, albeit lower, levels of estradiol in +/- animals could be explained by an increase in the number of growing follicles capable of secreting estradiol and elevated levels of LH providing higher testosterone as a substrate for aromatization. The changes in estradiol, LH, and FSH in 7-mo-old +/- FSH-R female mice are strikingly parallel with trends reported in middle-aged rats [37] and postmenopausal women [38].
Our previous study showed that the 12-mo-old +/- ovary was comprised of very few follicles (2% of values in 3-mo-old +/- mice) and no CL at all (Table 1). As expected, aging changes also occur in wild-type ovaries, but these changes appear to be accelerated in FSH-R haploinsufficient mice. We have noted that the wild-type ovaries lost 70% of oocytes, and by 12 mo of age they had only 30% of the 3-mo-old oocyte pool with active folliculogenesis and hundreds of different types of follicles and CL [28]. The loss of follicles in both genotypes was accompanied by decreased values in FSH-R binding assay in the present study (Fig. 6B). However, the heterozygous females lost 98% of the entire oocyte pool and 50% of FSH-R. Therefore, acceleration of oocyte loss in the +/- ovary apparently corresponded to the greater reduction in the numbers of FSH-R in the follicle and initiated much earlier.
The high levels of both LH and FSH stored in the pituitaries of the 12-mo-old heterozygous females confirm the absence of any significant negative ovarian feedback in the form of estradiol and inhibin. However, the significant increase in plasma FSH and LH in the 7-mo-old heterozygous mice demonstrates differences in the sensitivity of feedback. Interestingly, the higher circulating level of testosterone by itself appears unable to exert a negative feedback influence on the hypothalamic-pituitary axis. Additional investigations would be required to understand the mechanisms involved in this upregulation of synthesis and/or release.
Increased testosterone secretion in the +/- ovary from 7-mo onward (Fig. 2B) was coupled with elevated plasma LH levels (Fig. 1B), ovarian stromal hyperplasia, and a high expression of LH-R in the ovary (Fig. 5, E and F). Our findings of high levels of LH and androgen might contribute to enhanced atresia in the +/- ovary [25], as it is known that high levels of these hormones bring about widespread atresia in transgenic mice [36].
According to the existing information, age-associated increases in circulating FSH levels at estrus may be linked to decreases in inhibin secretion in rats [4] and postmenopausal women [7]. The age-associated decrease in ovarian inhibin secretion in +/- FSH-R mice as determined indirectly in our study by Western blot and immunohistochemistry (Figs. 4A and 5, B and D) was coincident with elevated serum FSH levels in older mice. Our data in mice appear to support the findings of previous studies that showed high levels of FSH and low levels of inhibin B in postmenopausal women [7] and in middle-aged rats [4]. The low levels of inhibin in 7- and 12-mo-old FSH-R +/- females parallel the dramatic decrease in the number of healthy early antral follicles that usually secrete this protein. It is now known that decreased levels of inhibin B in older ovulatory women reflect a diminished follicular pool, typical of ovarian aging [5].
Although we have not yet examined all aspects of oocyte status in our +/- mice, certain observations derived from the superovulation experiments of the current study are worthy of note. Despite conflicting data in the literature on the age-related number of ovulated oocytes in normal mice [39, 40], the declining patterns of induced ovulation noted in the present study from the immature state to 3-mo in the FSH-R +/- mice are quite remarkable (Table 1). We assume that a decrease in sensitivity to gonadotropins and/or a decline in follicles that are ready to be stimulated might cause the poor response of +/- ovaries to exogenous hormones. The reduced binding of 125I FSH in the +/- ovaries compared with wild-type litter mates at all ages studied (Fig. 6B) indicates that stimulation with exogenous eCG was insufficient for the ovary to release the equivalent (to wild-type values) number of ova shed. Subtle inadequacy of LH receptor induction in granulosa cells of the maturing follicle could cause a poor response in hormone-induced ovulation. In addition, as LH surge is known to stimulate transcription of the progesterone receptor (PR) gene in cultured rat granulosa cells [41] and females that lack this receptor are anovulatory [42], it is not unreasonable to speculate that the PR gene status in the ovary may be inadequate in the FSH-R +/- mice.
The observations of the present study allow us to draw 2 important conclusions: 1) full expression of FSH-R is required for the normal appearance of LH-R in granulosa cells, but it is not obligatory for the constitutive expression of LH-R in thecal cells and interstitial tissue; and 2) since granulosa cells from the FSH-R +/- preovulatory follicle lack LH receptors partially or completely, they cannot respond appropriately and rapidly to an LH surge. As a consequence they might be unable to undergo luteinization (ovulation) through activation of cell cycle inhibitors and other associated events [43]. The decreases that we have observed in the orphan receptor SF-1 that modulates inhibin promoter activity [44] and cyclin D2 that is required for granulosa cell proliferation [43] are consistent with the decline in ovarian function in the FSH-R haploinsufficient mice. Although we did not examine the progesterone receptor in the +/- ovary during aging, it is possible that its expression/function may also be suboptimal. Thus all these events might contribute to a decrease in the number of oocytes released in the +/- FSH-R mice. The presence of unruptured follicles with improper expansion of cumulus cells in FSH-R +/- ovaries after priming by gonadotropins (Fig. 3, B and D) also suggests potential defects in these cells that surround the ovum. We therefore predict that communication between the oocyte and granulosa cells is disrupted under these conditions. Perturbations in the progesterone receptor A and B forms that we had previously detected in the uterus of +/- FSH-R mice [25] at 3 mo together with potential defects of this system in the ovary could contribute to the initial reduction in fertility and its eventual disappearance with advancing age. However, because these events occur within a short span of about 3–4 mo [28], we believe there is a good opportunity to examine new experimental paradigms in the FSH-R haploinsufficient females.
Poor ovarian response in the field of assisted reproduction or empty follicles syndrome (EFS), a condition in which no oocytes are retrieved in an IVF treatment cycle in women, is a significant clinical problem. Although many hypotheses ranging from dysfunctional folliculogenesis to a drug-related impairment have been put forward, the mechanisms responsible for this condition remain obscure. Ovarian aging, through altered folliculogenesis, might be implicated in the etiology of EFS and its recurrence [45]. Based on our data ([28] and this report), accelerated ovarian dysfunction and biological aging due to partial deficiency of the FSH-R might play a significant role in reducing the effects of gonadotropin treatments.
In summary, our data suggest that age-related changes in neuroendocrine and ovarian functions associated with haploinsufficiency of the FSH-R in females reflect diminished follicular reserves. We believe that these studies provide experimental evidence that declining FSH-R and a rise in FSH levels with age reflects increasing ovarian resistance to follicular development. Perhaps the last remaining follicles become refractory to gonadotropin stimulation because of an absence or the improper functioning of FSH-R. Therefore, the role of FSH-R in triggering the reproductive events appears to be paramount in quantitative as well as qualitative terms and in determining ovarian aging. A mouse model of biologically advanced reproductive aging provides an important opportunity for investigating various mechanisms of senescence and enhancing the prognostic value of potential biomarkers of the aging process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Canadian Institutes of Health Research. N.D. and W.X. are the holders of a CIHR doctoral fellowship. 
2 Correspondence: M. Ram Sairam, Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Ave. West, Montreal, QC, Canada H2W 1R7. FAX: 514 987 5585; sairamm{at}ircm.qc.ca
Accepted: February 28, 2002.
Received: December 7, 2001.
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N. Danilovich, I. Roy, and M. R. Sairam Emergence of Uterine Pathology during Accelerated Biological Aging in FSH Receptor-Haploinsufficient Mice Endocrinology, September 1, 2002; 143(9): 3618 - 3627. [Abstract] [Full Text] [PDF]
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N. Danilovich and M. R. Sairam Haploinsufficiency of the Follicle-Stimulating Hormone Receptor Accelerates Oocyte Loss Inducing Early Reproductive Senescence and Biological Aging in Mice Biol Reprod, August 1, 2002; 67(2): 361 - 369. [Abstract] [Full Text] [PDF]
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