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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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aging, apoptosis, follicle-stimulating hormone receptor, follicular development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been well documented that FSH decreases atresia in granulosa cells obtained from hypophysectomized rats [5], prevents apoptotic changes of cultured preovulatory follicles, and serves as the major survival factor in early antral follicles [6]. At this developmental stage, also called the "penultimate" stage, the follicles are most susceptible to atresia, and adequate exposure to FSH appears to be the most critical survival signal for their rescue and further development [7]. These conclusions are directly supported by recent gene disruption studies of both the hormone ß subunit [8] and the FSH receptor (FSH-R) [9, 10]. However, the quantitative aspects of the role of FSH-R signaling in events leading up to ovarian failure and senescence have not been fully understood in any species.
In the ovary, FSH binds selectively to its own receptor exclusively localized on the granulosa cells, resulting in activation of cAMP-dependent and other signaling pathways [11]. To analyze the role of FSH-R signaling on reproductive functions, we recently generated mice that lack FSH-R [9]. Our initial findings in 3-mo-old mice [9], followed by more investigations that are detailed, showed that the null follitropin receptor knockout (FORKO) (-/-) female mice are acyclic and infertile, showing many important outward signs of estrogen deficiency [10]. An additional and important finding was that the haploinsufficient female mice with partial (+/-) disruption of the receptor gene also exhibited recognizable phenotypes that are clearly age-dependent. Although young FORKO +/- mice at 3–4 mo of age appear to be otherwise healthy, they show consistent reduction in fertility and fecundity [10] and begin to exhibit a number of characteristics that are consistent with an accelerated aging process. These include premature reproductive senescence by about 7 mo of age, skeletal deformity, and late onset of obesity [10]. These characteristics prompted a closer examination of events that are related to ovarian development in FSH-R haploinsufficient mice. The aim of the present study was to understand events that cause reduced fertility and fecundity, resulting in earlier cessation of reproductive life. We hypothesized that suppressed reproductive performance in the FSH-R heterozygous mice occurs because of accelerated oocyte loss due to increased cell death in the ovary. Parts of this study have been presented at a recent meeting [12]. Interestingly, this stage of reproductive failure in +/- FSH-R mice corresponds approximately to middle age in women, a time of declining fertility that often has negative health consequences.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The generation of mice with targeted disruption of the FSH-R has been recently described [9]. All experiments were performed according to accepted standards and with the approval of an institutional ethics committee. For all the experiments, virgin heterozygous and wild-type mice in the SV129 background were used at 3, 7, and 12 mo of age. The primers and amplification conditions used for the multiplex PCR to identify the genotypes have been described in detail elsewhere [10]. The mice were housed (5 mice per cage) under controlled lighting (12D:12L) and were provided with standard commercial food pellets and tap water ad libitum. No particular attention was paid to the presence of ingredients in the diet with potential estrogenic activity. Unless specified otherwise the number of mice used in each group of the experiment is given in the tables or figure legends.
Age of Puberty
To study age of pubertal onset as 1 of the potential reproductive markers of aging [13], we examined daily vaginal introitus in females starting 1 wk before the expected age of vaginal opening. This usually occurs in wild-type mice around Day 30 postpartum [14]. For this experiment, we examined pups from heterozygous crosses, since this type of breeding allowed us to have all 3 different genotypes (+/+, +/-, and -/-) and perform accurate comparisons.
Estrous Cycle
Virgin +/- and wild-type females at different ages (3, 7, and 12 mo of age) were used for this experiment. After 1 wk of adaptation, the mice were examined for vaginal patency, and smears were taken daily by lavage for at least 5 cycles to establish the length of the estrous cycle. To avoid problems such as acyclicity while examining the estrous cycle of females housed together, all females were exposed to male pheromones daily by placing them in cages soiled with male urine.
Effects of Age on Fertility and Fecundity
Virgin heterozygous and wild-type females at 3, 7, and 12 mo of age were mated to wild-type proven males. Around the expected date, all females were examined each morning for delivery to note the litter size. The number of pups surviving on Day 21 after birth was also recorded as an index of weaning success.
Ovarian Histology and Oocyte Counting
The mice were killed on the morning of proestrus. The right ovaries were removed and fixed overnight in 10% formalin. Serial paraffin histological sections of 5-µm thickness was cut for staining with hematoxylin and eosin. The number of oocytes were estimated and classified according to the appearance of the granulosa cell (GC) layer [15] with the following descriptors: stage I or primordial follicle (PF) with 1 layer of flattened GCs; stage II or primary follicle (PY) with 1 layer of cuboidal GCs; stage III or secondary (2ry) with 2 layers of GCs; stage IV or tertiary (3ry) with 3 layers of GCs; stage V or quaternary (4+ry) with 
4 layers, no antrum; and stage VI or antral (A; antrum present). Every seventh section was examined under a microscope at 400x magnification to count the number of follicles. The total number of follicles was obtained by multiplying the sum of the counted numbers by 7 to account for the fact that every seventh section was used in the analysis and applying a suggested correction factor [16]. The total numbers of primordial and primary follicles were estimated on the basis of morphometric studies (including diameter of the germinal vesicle) using the correction factor described in studies of the human ovary [17]. Only those follicles in which at least 50% of the oocyte nucleus was visible were recorded to avoid overestimation.
Oocyte and Follicle Diameters
Ovaries from mice aged 3, 7, and 12 mo were examined (n = 3–4 ovaries per age and per genotype). In ovaries from mice aged 3 mo, 38 and 54 oocyte-follicle pairs were measured from wild-type and heterozygous ovaries, respectively. In ovaries from mice aged 7 mo, 48 and 56 oocyte-follicle pairs were measured in wild-type and heterozygous ovaries, respectively. In ovaries from mice aged 12 mo, 30 and 20 oocyte-follicle pairs were measured from wild-type and heterozygous ovaries, respectively. Follicle and oocyte diameters were measured in the section where the nucleolus of the oocyte was visible.
Follicular Population and Atresia
To count atretic follicles in the ovaries we examined preantral, early antral, and preovulatory follicles in wild-type and heterozygous mice of different ages [18]. Counts and assessments of atresia in 3-mo-old mice were done without knowledge of genotype. The following classification was used: preantral (2 or more layers of GCs, no antrum); the number of early antral follicles with a confluent antrum; and the number of preovulatory follicles (with a bigger antral cavity and displacement of the oocyte to an eccentric position) [19]. To evaluate atretic follicles, several histological characteristics such as the presence of 2 or more pyknotic nuclei in the GCs, disruption of the GC layer, and/or degeneration of the nucleus of the oocyte were used [20]. The results were expressed as the percentages of preantral, early antral, and preovulatory follicles of total counted follicles (sum of preantral, early antral, and preovulatory) [19].
TUNEL Analysis
TUNEL staining was performed on ovarian sections for apoptotic DNA fragmentation (Oncor, Gaithersburg, MD) as described elsewhere [19]. Ovaries removed from animals were immediately placed in 10% formalin at 4°C overnight. After fixation, the ovaries were processed and embedded in paraffin. Sections (5 µm) were cut and mounted on slides.
Statistics
Data are presented as the mean ± SEM. Body weights, litter sizes, number of follicles per ovary, and oocyte diameters were normally distributed and analyzed by Student t-test or ANOVA with a Fischer least square difference (LSD) post-hoc test using P < 0.05 as the level of significance. For numbers of females delivering (reproductive success) litters as well as numbers of atretic follicles in the ovary, chi-square tests were used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first outward sign of FSH-R disruption in females was evident by a change in their body weights. The body weight of the 3-mo-old +/- female mice was 9.6% higher compared to their wild-type siblings, but this increase was not significantly different. However, by 7 mo of age, the body weight of +/- mice averaged 28.3 ± 2 g and differed significantly from the +/+ females of the same age (Fig. 1). Signs of obesity were clearly apparent in older (12-mo) females. They were pear-shaped with body weight increasing by 
25% compared with wild-type, age-matched mice. Another clear sign of aging in these mice is the development of ovarian tumors in >12-mo-old +/- mice [21].
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Age of Puberty
The animals (22 wild-type and 33 heterozygous animals) were checked daily for establishment of vaginal opening starting from Day 21 postpartum. The age of pubertal onset was slightly but significantly advanced in FORKO +/- females and occurring 1.8 days earlier than in their wild-type siblings (26.9 ± 0.37 days vs. 28.7 ± 0.31 days, P < 0.005). At weaning the mean body weight of 21-day-old immature heterozygous females (9.7 ± 0.3 g, n = 22) was reduced by 9% in comparison with wild-type siblings (10.7 ± 0.2 g, n = 15, P < 0.05).
Breeding Performance
In the present study, we bred wild-type proven males with virgin +/- (n = 92) and +/+ (n = 88) females of different ages to determine reproductive success over a period of time. The percentage of mated (as indicated by presence of vaginal plugs) 3-mo-old +/- females that delivered litters was 85% as compared with wild-type counterparts (success rate 96%; Fig. 2A). Increases in age resulted in even lower fertility rates for the +/- females. By the age of 7 mo, the percentage of mated +/- females that delivered pups was reduced to 49% in comparison to +/+ controls that showed 83% success. Further increases in maternal age (12 mo) diminished the percentage of +/- females that gave pups to only 12%. At this time, the majority of 1-yr-old wild-type controls (73%) continued to produce live offspring.
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Other changes were also apparent in the FSH-R haploinsufficient mice. Review of breeding performance showed that young 3-mo-old +/- females had significantly smaller litter sizes (6.3 ± 1) than wild-type litter mates (9.4 ± 1.1 pups, P < 0.05; Fig. 2B). The litter size in 7-mo-old +/- females was further reduced (54% less). By the age of 12 mo, +/- females delivered an average of only 0.5 ± 0.3 offspring that died soon after birth. In contrast, although the litter size was lower (4.5 ± 2 pups), all the offspring of 12-mo-old wild-type females were viable and successfully reared to weaning. In addition, another dramatic change noticeable in the +/- mice was that the interval between the establishing of the vaginal plug and the delivery in 12-mo-old +/- females ranged between 45 and 60 days. Thus not even a single +/- 1-yr-old female was able to produce a litter within the normal duration after mating. At the same time, 1-yr-old wild-type mice gave healthy pups within 24–28 days.
The success at first weaning in the crosses between +/- female and wild-type male mice was reduced by 25% compared with values in the 3-mo-old wild-type females. When we analyzed the index of weaning success in 7-mo-old females, we found that in both genotypes there was a decrease in the number of surviving pups by 21 days after birth. The index of weaning success dropped to 43 ± 2.5% in +/- mice compared with 71% ± 3.1% in wild-type, age-matched animals (P < 0.005).
Estrous Cycle
To determine the length and regularity of the estrous cycle, vaginal smears were taken daily for a period of 40 days in wild-type and heterozygous female mice at 3, 7, and 12 mo of age. All 3-mo-old +/- females (n = 25) had prolonged cycles (6.2 ± 0.4 days) due to extended diestrus (P < 0.02) as compared with 4.9 ± 0.2 day cycles in wild-type females (n = 25; Table 1). At 7 mo, the mean length (±SEM) of the estrous cycle for the 75 +/- animals studied was 11.6 ± 1.4 days with a range of 4–15 days. Only 8 +/- females showed the appearance of changes compatible with some cyclicity; the rest were either acyclic with only leukocytes in the smears or with smears containing only cornified cells (persistent vaginal cornification). In contrast, 7-mo-old wild-type females (n = 45) had 5.6 ± 0.2 day cycles (P < 0.001) with a range of 4–7 days. At the age of 12 mo, all 65 +/- mice examined were acyclic with leukocyte-containing smears. Twelve-month-old wild-type females (n = 50) had 6.7 ± 0.6 day cycles. Age changes in cycle length paralleled those of cycle frequency. Cycle frequency declined steadily with age in +/- mice from 4.5 cycles per mo in 3-mo-old females and 2.5 cycles per mo in 7-mo-old females (Table 1), which disappeared altogether by 1 yr.
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Although the number of cycles with extended cornification (greater then 2 days) increased with advancing age in both genotypes, the +/- mice showed a higher incidence of this phenomenon at a much earlier age. The percentage of virgin +/- mice cycling at the age of 3 mo (84%) dropped dramatically to 49% in 7-mo-old females, and to 0 in 12-mo-old animals (Table 1). In contrast, these values for virgin wild-type controls were 92%, 78%, and 62%, respectively. Interestingly, the long cycles in 7-mo-old +/- female mice were interspersed with short ones, and intermittent ovulatory cycles were intermingled with periods that were hormonally indistinct from the acyclic state.
Ovarian Histology
On gross inspection, the ovaries of 3-mo-old heterozygous females were not generally distinguishable from wild-type litter mates and contained structures in all stages of development including corpora lutea (CL; Fig. 3, A and B). However, their histological appearance was characterized by the presence of large numbers of atretic follicles that indicated disruption of the cellular layers and the presence of pyknotic nuclei, which is typical of atretic granulosa cells. Pyknotic nuclei and cellular debris were present in the antrum of some large follicles, particularly at 7 mo (Fig. 3G). By histological appearance, many antral follicles were clearly abnormal. Some 7-mo-old +/- ovaries contained follicles with strange-looking or double oocytes (Fig. 3H). In 7-mo-old wild-type animals both fresh and old CL were evident, indicating an active estrous cycle, whereas the presence of corpus luteum in +/- ovaries was sparse (Fig. 3D). Figure 2E illustrates a 12-mo-old +/+ ovary with various follicles as well as numerous corpora lutea, whereas a 12-mo-old +/- mouse ovary was already on its path of advanced regression containing very few follicles and no corpus luteum at all (Fig. 3F). At this later age, the main part of the +/- ovary consisted of mostly fibroid interstitial tissue and cystic follicles (CyF; Fig. 3F). In some cases, the cysts (Cy) formed a greater part of the ovarian space (Fig. 3F).
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Number of Follicle and Oocyte Counts
To test the hypothesis that FSH-R haploinsufficiency might play a role in oocyte depletion, the entire resting (primordial and primary) and growing (stages III–VI) follicle population in virgin 3-, 7-, and 12-mo-old mice was compared with corresponding elements in the wild-type mice (Fig. 4). Although the total number of follicles seemed to be decreased in 3-mo-old +/- females compared with age-matched controls, the difference was not significant (Fig. 4A, inset). However, there was a marked reduction in oocyte numbers in the 7-mo-old heterozygous ovaries (Fig. 4B, inset). In fact, at 7 mo of age the total number of follicles dropped by 75% in heterozygotes compared with their 3-mo-old values (Fig. 4B, inset). Interestingly, the entire oocyte pool was also diminished in 7-mo-old wild-type females; however, the total number of follicles was reduced only by 40% (Fig. 4B, inset). By 12 mo of age, the +/- ovaries contained very few oocytes and had <2% of the total oocyte population present in 3-mo-old mice (Fig. 4C, inset). In contrast, age-matched wild-type females had lost two thirds of the oocyte pool of the 3-mo-old values and still had 35% of follicles (Fig. 4C, inset). As anticipated, primordial and primary follicle numbers were always in excess of numbers of growing follicles regardless of age in wild-type mice. The 7- and 12-mo-old +/- ovaries had an exhausted resting pool with larger numbers of growing follicles (Fig. 4, B and C). In 3-mo-old +/- ovaries, the growing follicle population (stages III–VI) was significantly reduced compared with age-matched, wild-type mice (Fig. 4A), suggesting inadequate delivery of hormonal signaling via its receptor. However, by 7 mo of age there was an accelerated rate of follicle recruitment from the resting pool characterized by increased numbers of developing or growing follicles in +/- ovaries (Fig. 4B). Although the absolute number of the entire oocyte population was severely reduced in the 7-mo-old heterozygous females, the fraction of growing follicles constituted a greater proportion of the reserve pool of follicles. At 12 mo of age there were very few follicles left in the heterozygous ovaries (Fig. 4C). In contrast, wild-type ovaries had numerous follicles, including those at primordial and primary stages.
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Oocyte and Follicular Diameters
The oocyte diameters within PF, PY, 2ry, and A follicles of +/- ovaries at 3 mo of age were greater than those found in the corresponding wild-type follicles (Fig. 5A). Interestingly, in the 7- and 12-mo-old +/- ovaries, oocyte diameters within all types of follicles were reduced compared with wild-type values (Fig. 5, B and C). However, the difference attained significance only within antral follicles of 7-mo-old ovaries and within primordial follicles of 12-mo-old ovaries.
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Follicular Atresia
To understand the basis underlying such a dramatic oocyte loss in the FORKO +/- ovary, we counted the numbers of atretic follicles applying standard histological characteristics (see Materials and Methods). The percentage of atretic follicles (including preantral, early antral, and preovulatory in the ovaries from the +/- and wild-type mice was 70% and 47%, respectively (P < 0.05). The percentage of preantral follicles with elements of attrition was significantly higher in the +/- ovaries at all ages studied (Fig. 6). In contrast, only a small percentage of preantral atretic follicles were observed in the wild-type ovaries (<6%). More early antral follicles underwent degeneration and constituted the vast majority of atretic follicles in both genotypes. However, the percentage of attrition in the early antral follicles was much higher in the +/- ovaries at all ages studied. The degeneration of preovulatory follicles was more evident in the +/- mice at any age, but the differences were not significant since the values displayed high ranges (Fig. 6).
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To validate the presence of follicular atresia, we performed TUNEL staining. Granulosa cell apoptosis, as determined by TUNEL, occurred in the ovary of both genotypes at all ages studied (Fig. 7, A–H). However, in wild-type ovaries apoptosis was detected most notably in some of the granulosa cells of large, antral-size follicles (Fig. 7, A and C), whereas in the FSH-R +/- ovaries apoptotic cells were mostly present in preantral medium-sized follicles (Fig. 7, B and D). It is interesting to note that in the 12-mo-old +/- ovary apoptosis was lower and scattered (Fig. 7, F and H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The earlier age of pubertal onset in +/- females, indicated by precocious vaginal opening, suggests that an altered hormonal milieu has already taken effect at this young age. It is known that this parameter is positively correlated with the reproductive life span and longevity [13]. The heterozygous females started showing reduction in litter size (by 33%) from 12 wk of age (Fig. 2B). That this becomes even more profound with age is in agreement with a recognized concept that in litter-bearing mammals a decrease in litter size is a function of age [1]. The decline in litter size with age occurred in both genotypes, but in heterozygous females it was more dramatic. Thus it is reasonable to suggest that for a given chronological age the +/- females had undergone more advanced reproductive aging. Another conclusion that can be drawn from these observations is that there was premature ovarian failure in +/- FSH-R mice. We are not aware of any animal model with partial disruption of a gene that would have such important changes leading to an apparent phenotype of profound physiological consequence for fertility in the female. For instance, in mice with targeted disruption of the aromatase gene [22] or in the FSH-ß disrupted mouse [8] there are no significant differences in litter sizes between wild-type and heterozygous animals. Because we used wild-type males of proven fertility in our evaluations, the decrease in fertility can be attributed directly to the haploinsufficiency of FSH-R.
Impairment in ovarian function could account for the fact that 40% of the offspring from 7-mo-old +/- females and all pups (if any) delivered by 12-mo-old +/- females died within 18–48 h after birth, apparently due to some embryopathy. As autopsy of the few newborns revealed milk in their stomach, serious maternal problems may be unlikely but cannot be excluded altogether. Our postulation of embryopathies in +/- mice that have a prolonged cycle is consistent with previous observation of similar occurrences that are more common in rats with extended reproductive cycles [23]. In addition, it may be noted that the probability of aneuploidy in mice and conceiving a trisomic fetus (Down syndrome) in middle-aged women appears to be determined mainly by the size of the remaining oocyte store and attributed to biological aging [24]. Thus further investigations in this regard in our FSH-R haploinsufficient model might be very informative. By the age 7 mo, the +/- ovaries had lost 75% of the oocyte pool, and by the age of 12 mo the +/- ovary was virtually depleted of all oocytes, as in the aging human female [25].
Age-related changes of estrous cycles in rodents are characterized by using several parameters such as estrous frequency, the fraction of mice cycling at any age, and cycle lengths, as well as the total number of estrous cycles [26]. Our observations of a decline of these events in FORKO +/- females at different ages, and terminating altogether in 12-mo-old animals (Table 1), all point to the phenomenon that can be collectively characterized as early reproductive senescence. One of the explanations for lengthened cycles in the +/- females might be the delayed preovulatory rise of estradiol shown in aging mice [27]. Additionally, diminished or absent LH surges on proestrus in rats leads to prolonged estrous cycles or the complete cessation of cyclicity [28]. Thus the depleted follicle pool in the +/- FORKO ovary might have an impact on the magnitude of preovulatory LH surges.
Our findings that the total number of ovarian follicles in young (3-mo-old) ovaries are higher than that of older (7- and 12-mo-old) ovaries in both genotypes is in agreement with a previous report on C57BL/6J mice [4]. However, the rate of decline differed thereafter, becoming steeper in +/- mice (<75% of the 3-mo stock by 7 mo). Eventually all +/- ovaries were barren by the time they reached 12 mo (Fig. 4C). Age-matched, wild-type ovaries still had 30% of values in 3-mo-old mice with active folliculogenesis and hundreds of different types of follicles and CL. The ability to sire viable offspring at this age proves the fertility status of wild-type females.
It has been suggested that acyclicity results when a critical threshold number of late secretory follicles, required for maintaining the positive feedback mechanism for pituitary gonadotropin release, is absent [24]. This might have contributed to early biological aging in FSH-R +/- females. The presence of clearly recognizable phenotypic effects in FSH-R haploinsufficient mice allows an evaluation of the dynamics of follicular development. The primordial follicle endowment in the mouse is determined at birth and is constantly depleted subsequently as a result of cellular death and recruitment into growing stages [4]. As follicular reserves decline, the probability of acyclicity increases, thereby advancing ovulatory failure in the mouse [24]. In the present study, we observed that with increasing age, the population of primordial follicles progressively declined in both genotypes (Fig. 4). The age-related depletion of the resting pool has been well documented in the human and rodent ovaries [4, 16, 29, 30]. However, the rate at which nongrowing follicles disappeared was hastened in the +/- ovaries apparently because of increased atresia (Fig. 6) and recruitment into the growing pool at 7 mo of age (Fig. 4B). Although the precise mechanisms underlying this accelerated demise of primordial follicles in the 7-mo-old +/- ovaries are not known yet, higher circulating LH levels may be implicated. There is evidence that chronic LH-induced reduction of the primordial follicle pool occurs in transgenic mice expressing high levels of LH [31].
Our findings of a 50% reduction in the number of primary follicles as well as a significant decrease in the numbers of growing follicles in +/- ovaries (Fig. 4A) supports the hypothesis that FSH/FSH-R signaling is involved in the transformation of granulosa cells from a flattened (primordial follicles) to a cuboidal shape (primary follicles). This change permits these follicles to enter the growth phase [32]. In addition, FSH action via control of steel factor production localized in the granulosa cells can stimulate c-kit receptors in the oocyte, leading to the resumption of follicular growth [32].
It is well known that FSH is a major survival factor for early antral follicles [7], and only those follicles able to respond to rising FSH levels continue to develop, whereas the majority of early antral follicles become atretic [33]. Our findings of increased apoptosis in +/- ovaries correspond to the fact that high levels of LH and androgen bring about widespread atresia [31], and both these hormones are elevated in 7-mo-old +/- mice. Interestingly, even in the 3-mo-old +/- ovary, TUNEL staining revealed not only antral but also many preantral follicles undergoing apoptosis (Fig. 7, B and D), and the percentage of degenerating preantral follicles was significantly higher in the FORKO +/- females compared with wild-type litter mates (15% ± 1.2% vs. 4.3% ± 0.5%, respectively; Fig. 6). An understanding of endocrine alterations with respect to aging in FSH-R haploinsufficient mice might shed light on the degenerative changes in the ovary.
In conclusion, this study for the first time reports the occurrence of premature ovarian failure in haploinsufficient FSH-R mice. As various phenomena related to this condition, which prevails in many women of reproductive age or middle age, are reproducible in these genetically altered female mice, we suggest that they might be useful in extending our knowledge in this important area of reproductive aging and women's health. Some of the endocrine alterations that impact on these events are presented in the accompanying article [34].
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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 (CIHR). N.D. is the holder 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 5, 2001.
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