Endocrine alterations and signaling changes associated with declining ovarian function and advanced biological aging in follicle-stimulating hormone receptor haploinsufficient mice

Reproductive aging in female mammals is characterized by a progressive decline in fertility due to loss of follicles and reduced ovarian steroidogenesis. In this study we examined some of the endocrine and signaling parameters that might contribute to a decrease in ovulation and reproductive performance of mice with haploinsufficiency of the FSH receptor (FSH-R). For this purpose we compared ovarian changes and hormone levels in FSH-R heterozygous (+/-) and wild-type mice of different ages (3, 7, and 12 mo). Hormone-induced ovulations in immature and 3-mo-old +/- mice were consistently lower. The number of corpora lutea (CL) were lower at 3 and 7 mo, and none were present in 1-yr-old +/- females. The plasma steroid and gonadotropin levels exhibited changes associated with typical ovarian aging. Plasma FSH and LH levels were higher in 7-mo-old +/- mice, but FSH levels continued to rise in both genotypes by 1 yr. Serum estradiol and progesterone were lower in +/- mice at all ages, and testosterone was several-fold higher in 7-mo-old and 1-yr-old +/- mice. Inhibin alpha (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.     

Changes in germ cell population in young and adult female mice from two inbred strains: CBA/Kw and KE

Female mice from two inbred strains CBA/Kw and KE differ markedly in fertility. The gametes of females from KE strain are of poorer quality than those of CBA/Kw. We analyzed the number of oocytes per ovary in KE and CBA/Kw mice aged 5, 25, 90, 180 and 360 days. The ovaries were dissected and processed according to the routine histological methods. In case of five-day-old females we used a modified distributed point counting method while in order to examine the gonads of older females, the nucleoli counting method was applied. In general, we observed gradual decrease in germ cell number throughout the whole life of females from both strains. The noticeable wave of oocyte loss occurs between 5th and 25th days of life. The mice from KE inbred strain on day 25th (1650 +/- 322 vs. 1140 +/- 210) and 90(th) (1040 +/- 211 vs. 692 +/- 89) days have significantly (p<0.005) more germ cells than the females from CBA/Kw strain. In older females the differences were not statistically significant. Interestingly, CBA/Kw females were found to have more rapid loss of primordial follicles throughout their lives. This can explain their shortened reproductive lifespan which was observed earlier.     

Defects in reproductive functions in PACAP-deficient female mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide and widely expressed in both brain and peripheral tissues, including several reproductive organs (e.g., testis and ovary). PACAP stimulates syntheses of several sexual hormones and steroids, suggesting it has possible roles in reproductive function. In this study, the role of PACAP in female reproductive functions such as fertility, mating behavior and maternal behaviors were investigated by using mice lacking PACAP (PACAP(-/-)). PACAP(-/-) females showed reduced fertility (the number of parturitions relative to the number of pairings). Mating experiments using vasectomized males revealed that mating frequency and its intervals in some PACAP(-/-) females were quite different (zero to eight times/4 weeks), whereas the frequency was relatively constant (two to three times/4 weeks) in wild-type females. In PACAP(-/-) females, maternal crouching behavior tended to decrease compared to wild-type females, although the influence of litter size on maternal behavior needs to be considered. These data suggest a role for endogenous PACAP in female reproductive processes.     

In vitro fertilization and microinsemination with round spermatids for propagation of nephrotic genes in mice

The ICGN is an inbred strain of mice with a hereditary nephrotic syndrome of an unknown cause. The disease progresses to renal failure, resulting in the deterioration of the systemic condition and in a drastic reduction of reproductive capacity. The present study was undertaken to determine if in vitro fertilization (IVF) and microinsemination using round spermatids could ameliorate the reduced fertility of ICGN mice. Mature oocytes (9.4 +/- 1.1 per mouse) were obtained from PMSG/hCG-primed females 2 to 5 mo of age. When spermatozoa from males aged 3 to 4 mo was used for IVF, a high fertilization rate (82.6%) was achieved and a high rate of embryos developed into blastocysts (92.6%). When spermatozoa from males aged 5 to 7 mo was used, the rates of fertilization and development to blastocysts were significantly lower (63.9 and 47.1%, respectively; P < 0.001). However, the production rate of offspring after embryo transfer was satisfactory irrespective of the age of males (59.1%). When males older than 9 mo were used, no fertilization was achieved due to the very poor motility of the spermatozoa. Microinsemination techniques with round spermatids (electrofusion and intracytoplasmic injection) resulted in the production of normal offspring from the older males, including one azoospermic case. These findings indicate that a conventional IVF technique and microinsemination using round spermatids can be used for propagating mutant genes which cause poor reproductive ability in mice.     

Moderate caloric restriction initiated in rodents during adulthood sustains function of the female reproductive axis into advanced chronological age

Age-related ovarian failure in women heralds the transition into postmenopausal life, which is characterized by a loss of fertility and increased risk for cardiovascular disease, osteoporosis and cognitive dysfunction. Unfortunately, there are no options available for delaying loss of ovarian function with age in humans. Rodent studies have shown that caloric restriction (CR) can extend female fertile lifespan; however, much of this work initiated CR at weaning, which causes stunted adolescent growth and a delayed onset of sexual maturation. Herein we tested in mice if CR initiated in adulthood could delay reproductive aging. After 4 months of CR, the ovarian follicle reserve was doubled compared to ad libitum (AL)-fed age-matched controls, which in mating trials exhibited a loss of fertility by 15.5 months of age. In CR females returned to AL feeding at 15.5 months of age, approximately one-half remained fertile for 6 additional months and one-third continued to deliver offspring through 23 months of age. Notably, fecundity of CR-then-AL-fed females and postnatal offspring survival rates were dramatically improved compared with aging AL-fed controls. For example, between 10 and 23 months of age, only 22% of the 54 offspring delivered by AL-fed females survived. In contrast, over 73% of the 94 pups delivered by 15.5- to 23-month-old CR-then-AL-fed mice survived without any overt complications. These data indicate that in mice adult-onset CR maintains function of the female reproductive axis into advanced age and dramatically improves postnatal survival of offspring delivered by aged females.     

Evaluation of Follicular Synchronization Caused by Estrogen Administration and Its Reproductive Outcome

To evaluate multiple follicular development synchronization after estrogen stimulation in prepubertal mice, follicular responsiveness to gonadotropin superovulation, the prospective reproductive potential and ovarian polycystic ovary syndrome (PCOS)-like symptoms at adulthood, prepubertal mice were intraperitoneally injected with estrogen to establish an animal model with solvent as control. When synchronized tertiary follicles in ovaries, in vitro oocyte maturation and fertilization rates, blastocyst formation rate, developmental potential into offspring by embryo transfer, adult fertility and PCOS-like symptoms, and involved molecular mechanisms were focused, it was found that estrogen stimulation (10μg/gBW) leads to follicular development synchronization at the early tertiary stage in prepubertal mice; reproduction from oocytes to offspring could be realized by means of the artificial reproductive technology though the model mice lost their natural fertility when they were reared to adulthood; and typical symptoms of PCOS, except changes in inflammatory pathways, were not remained up to adulthood. So in conclusion, estrogen can lead to synchronization in follicular development in prepubertal mice, but does not affect reproductive outcome of oocytes, and no typical symptoms of PCOS remained at adulthood despite changes related to inflammation.     

Short day lengths delay reproductive aging

Caloric restriction and hormone treatment delay reproductive senescence in female mammals, but a natural model of decelerated reproductive aging does not presently exist. In addition to describing such a model, this study shows that an abiotic signal (photoperiod) can induce physiological changes that slow senescence. Relative to animals born in April, rodents born in September delay their first reproductive effort by up to 7 mo, at which age reduced fertility is expected. We tested the hypothesis that the shorter day lengths experienced by late-born Siberian hamsters ameliorate the reproductive decline associated with advancing age. Short-day females (10L:14D) achieved puberty at a much later age than long-day animals (14L:10D) and had twice as many ovarian primordial follicles. At 10 mo of age, 86% of females previously maintained in short day lengths produced litters, compared with 58% of their long day counterparts. Changes in pineal gland production of melatonin appear to mediate the effects of day length on reproductive aging; only 30% of pinealectomized females housed in short days produced litters. Exposure to short days induces substantial decreases in voluntary food intake and body mass, reduced ovarian estradiol secretion, and enhanced production of melatonin. One or more of these changes may account for the protective effect of short day lengths on female reproduction. In delaying reproductive senescence, the decrease in day length after the summer solstice is of presumed adaptive significance for offspring born late in the breeding season that first breed at an advanced chronological age.     

Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in rats

The effect of age and melatonin on the activity of the neuroendocrine reproductive system was studied in young cyclic (3-5 months-old), and old acyclic (23-25 month-old) female rats. Pituitary responsiveness to a bolus of GnRH (50 ng per 100 g body weight) was assessed at both reproductive stages in control and melatonin-treated (150 micrograms melatonin per 100 g body weight each day for 1 month) groups. After this experiment, female rats were treated for another month to study the influence of ageing and melatonin on the reproductive axis. Plasma LH, FSH, prolactin, oestradiol and progesterone were measured. A positive LH response to GnRH was observed in both control groups (cyclic and acyclic). However, a response of greater magnitude was observed in old acyclic rats. Melatonin treatment reduced this increased response in acyclic rats and produced a pituitary responsiveness similar to that of young cyclic rats. FSH secretion was independent of GnRH administration in all groups, indicating desynchronization between LH and FSH secretion in response to GnRH in young animals and during senescence. No effect on prolactin was observed. Significantly higher LH (3009.11 +/- 1275.08 pg ml(-1); P < 0.05) and FSH concentrations (5879.28 +/- 1631.68 pg ml(-1); P < 0.01) were seen in acyclic control rats. After melatonin treatment, LH (811.11 +/- 89.71 pg ml(-1)) and FSH concentrations (2070 +/- 301.62 pg ml(-1)) decreased to amounts similar to those observed in young cyclic rats. However, plasma concentrations of oestradiol and progesterone were not reduced. In conclusion, the results of the present study indicate that, during ageing, the effect of melatonin is exerted primarily at the hypothalamo-pituitary axis rather than on the ovary. Melatonin restored the basal concentrations of pituitary hormones and pituitary responsiveness to similar values to those observed in young rats.     

Effect of dietary restriction on estrous cyclicity and follicular reserves in aging C57BL/6J mice

Restricting the food intake of female mice by alternating days of feeding and fasting delayed the age-related loss of estrous cycling potential and retarded the rate of follicular depletion, as determined after reinstatement of ad libitum (AL) feeding. During the period of food restriction (FR; 3.5-10.5 mo), food intake and body weight were about 80% of AL values. Mice were acyclic and predominantly in a state of diestrus during FR, but after reinstatement of an AL diet at 10.5 mo all FR mice resumed cycling regularly. By contrast, 80% of AL controls had become acyclic by this age, and the cycles of the remaining mice were significantly longer than those of the reinstated FR mice. Follicular reserves of 12.5-mo-old FR mice were twice those of age-matched AL controls. Cycling performance of reinstated FR mice, measured by cycle length and the proportion of mice still cycling, was equivalent to that of AL mice when the latter were 2-5 mo younger. Ovarian age, measured by the size of the follicular reserve, was similarly retarded in FR mice. Based on these data and previous evidence that follicular depletion plays a major role in the cessation of cyclicity in this strain, we hypothesize that the delayed loss of estrous cyclicity in aging FR mice is mediated at least in part by the retarding effect of dietary restriction on the rate of follicular depletion.     

A delay in age at first mating results in the loss of future reproductive potential via apoptosis

Summary Women who delay childbearing risk subfertility. However, this loss of fertility is not a simple function of aging. Women who have had children early in life tend to maintain fertility longer, measured as a later age at menopause. But why should otherwise healthy women lose reproductive capacity? Loss of fertility independent of senescence, menopause, has been approached from two perspectives: evolution and development. Evolutionary biologists focus on how natural selection favors survival after reproductive ability has ceased, whereas reproductive biologists examine mechanisms by which women lose fertility with age and factors that influence the rate of reproductive aging. Combining mechanistic studies with evolutionary theory should allow us to define principles of the evolution of postembryonic development of ovaries, including the role of reproductive timing relative to sexual maturation. Achieving this will require identifying appropriate, and more experimentally tractable, taxa in which to study how early reproductive events influence lifetime fertility. We work with an invertebrate species, the cockroach Nauphoeta cinerea, with a complex reproductive biology in which females experience reproductive cycles, give live birth, and show age‐related decline in fertility. Thus, N. cinerea provides an opportunity to use an experimental approach to examine mechanisms by which females lose reproductive potential as they delay reproduction. Our results demonstrate that the loss of both oocytes ready for fertilization and future oocytes in females that delay mating is because of apoptosis. We suggest that loss of fertility because of delayed mating may originate in a nonadaptive response in control of apoptosis through mistiming of reproduction.     

Developmental and molecular aberrations associated with deterioration of oogenesis during complete or partial follicle-stimulating hormone receptor deficiency in mice

Targeted disruption of the mouse FSH receptor gene (FSH-R) that mediates the action of the FSH results in a gene dose-related ovarian phenotype in the developing as well as the adult animal. While null females (FORKO) are sterile, the haplo-insufficient mice experience early reproductive senescence. The purpose of this study was to first record changes in oocyte development in the null FORKO and haplo-insufficient mice. Oocyte growth is significantly retarded in the null mutants with thinner zona pellucida in preantral follicles, but thicker zona pellucida in secondary follicles. This morphometric change indicates developmental aberrations in coordination of the germ cell (oocyte) and the somatic granulosa cell (GC) compartments. Markers for primordial germ cell proliferation and oocyte growth, such as the c-Kit/Kit-ligand and bone morphogenetic protein-15 (BMP-15) were downregulated in both null and +/- ovaries, suggesting disrupted communication between oocyte and GCs. Extensive changes in the expression of other oocyte-specific gene products like the zona pellucida glycoproteins (zona pellucida A, B, and C) indicate major alteration in the extracellular matrix surrounding the germ cells. This led to leaky germ cells that allowed infiltration of somatic cells. These results show that the loss of FSH-R signaling alters the follicular environment, where oocyte-granulosa interactions are perturbed, creating an out-of-phase germ cell and somatic cell development. We believe that these data provide an experimental paradigm to explore the mechanisms responsible for preserving the structural integrity and quality of oocytes at different ages.     

The endocrinology of puberty and reproductive functioning in female cotton-top tamarins (Saguinus oedipus) under varying social conditions

Sexual maturation and fertility were assessed in fourteen cotton-top tamarin (Saguinus oedipus) females under various social conditions. Six tamarin females (20-28 mo of age) showed a suppression of fertility while living with their families. Hormonal profiles demonstrated low, acyclic levels of urinary luteinizing hormone (LH) and estrone-conjugates (E1C). A rapid onset of ovarian and pituitary cyclicity occurred when four of the six females were removed from their families and paired with an unrelated male. In one female, an ovulatory LH peak occurred as early as eight days after pairing and resulted in conception and full-term pregnancy. Two of the six females were housed in total isolation for 30 days following their removal from the family and prior to pairing. Gradual increases in hormone concentrations occurred during isolation; however, there was no ovarian cyclicity until each female was paired with an unrelated male. In all six females, conception occurred before or as a result of the third ovulatory cycle. Partial isolation of a 36-mo-old female resulted in elevated LH and E1C levels, but cyclicity was not observed until the female was paired with an unrelated male. These findings indicate that removal of a female from the family alone does not initiate ovarian cycling. Sexual maturation, or puberty, occurs in female tamarins living with their families between 15 and 17 mo of age when mean LH and E1C levels began to increase. However, when a female is removed and paired at 9 mo of age with an unrelated male, elevated levels of LH and E1C may be seen by 10 and 11 mo of age. Our findings indicate that a suppression of fertility occurs in cotton-top tamarins living with their families, but that reproductive suppression does not affect the process of sexual maturation. Both removal from the family environment and stimulation by an unrelated male tamarin were necessary to induce normal reproductive activity. An acceleration of puberty occurred when a female tamarin was removed from her family early in development and paired with a male.     

Immunocytochemical detection of estrogen and progesterone receptors in the rabbit submandibular gland.

Rabbit submandibular glands produce secretions involved in olfactory communication. The histology of these glands and their secretory activity are: sexually dimorphic; vary across the female reproductive cycle; and are modified by gonadectomy. This suggests that gonadal steroids regulate the structure and function of such glands. To further support this idea we assessed by immunocytochemistry the presence of estrogen and progesterone receptors in male and female rabbit submandibular glands. Immunoreactivity was detected only in the nucleus of acini cells. The number of estrogen receptor-immunoreactive cells/field varied among estrus (26 +/- 6; mean +/- S.E.), ovariectomized (19 +/- 2), and ovariectomized-estrogen-treated animals (13 +/- 3). Intact males showed a significantly smaller number of estrogen receptor-immunoreactive cells/field (12 +/- 1) than estrous females. Interestingly, progesterone receptor-immunoreactive cells were more abundant in estrous (32 +/- 7) than in ovariectomized animals (7 +/- 1). Estradiol benzoate (5 micrograms daily for 5 days) increased the number of progesterone receptor-immunoreactive cells/field in ovariectomized females (17 +/- 1). Intact males showed fewer progesterone receptor-immunoreactive cells/field (16 +/- 2) than estrous females. Results show that the rabbit submandibular gland is a target for estrogen and progesterone and support the idea that these hormones participate in regulating the physiology of this gland.     

Astrocytes from acyclic female rats exhibit lowered capacity for neuronal differentiation

Astrocytes comprise a large proportion of the central nervous system support cells and play a critical role in neural injury and repair. The present study examined the impact of ovarian aging using an ex vivo model system, where astrocytes were derived from the olfactory bulb of young, reproductively competent females and reproductive senescent females. Cellular morphology and the spatial pattern of laminin deposition was altered in astrocyte cultures derived from reproductive senescent females. Young adult astrocytes had a flattened polygonal shape with actin bundles at the cell edges, while reproductive senescent astrocytes had a contractile appearance with thick stress fibers visible throughout the cell. Moreover, in reproductive senescent astrocytes, BDNF was elevated with a concomitant reduction in expression of the BDNF receptor, TrkB. To examine the ability of astrocytes derived from young adult and reproductive senescent females to promote neuronal differentiation, neural progenitor cells (NPCs) were co-cultured with astrocytes derived from these groups. At day 4 in vitro, MAP-2(+) NPCs were located in smaller clusters when co-cultured with young adult astrocytes and in large clusters when co-cultured with older astrocytes. At days 6 and 10, neuronal differentiation was significantly reduced in reproductive senescent astrocyte-NPC co-cultures, as determined by NeuN(+) cell numbers and MAP-2(+) process lengths. Furthermore, estrogen only enhanced neuronal differentiation in young adult-NPC co-cultures. The ovarian age-related astrocyte phenotype thus limits the ability of this cell to promote neuronal differentiation in NPC populations and suggests that the astrocyte-mediated microenvironment in older acyclic females is less conducive to repair following neurovascular injury.     

Loss of abnormal spindle-like,microcephaly-associated (Aspm) disrupts female folliculogenesis in mice during maturation and aging

The abnormal spindle-like, microcephaly-associated (ASPM) gene is a causative gene of autosomal recessive primary microcephaly (MCPH) 5 in humans, which is characterized by a reduction in brain volume. It was previously reported that truncated Aspm proteins in transgenic mice caused major defects in the germline, a severe reduction in ovary weight and the number of follicles accompanied by reduced fertility. However; it remains unknown whether a loss of Aspm induces abnormal ovarian function, resulting in female infertility. In order to assess the ovary function, we examined vaginal smear cytology from the age of 7 weeks to 100 weeks in CAG-mediated Cre-loxP conditional Aspm^-/- knockout mice and control female mice. In addition, we evaluated the ovarian size, fibrosis ratio and the number of follicles (primordial, primary, secondary, antral and atretic follicles) in mice from 15 weeks to 100 weeks old by image analyses. Mann-Whitney U-test was used for statistical analysis. The size of the ovary was significantly reduced in Aspm knockout mice at 15–20 weeks, 40–50 weeks and 70–80 weeks old compared with the control mice. Furthermore, at all stages, we found a severe decrease in the number of developing follicles at 10–15 weeks, 40–50 weeks and 70–80 weeks old, accompanied by disrupted cyclic changes of vaginal cytology and an aberrant upregulation of Foxo3, Kitl, and Lhcgr in Aspm knockout female. These results suggested that Aspm might play an important role in the folliculogenesis and estrous cyclicity of the postnatal ovary during maturation and aging.     

Bovine model for the study of reproductive aging in women: follicular, luteal, and endocrine characteristics

At present, there is no well-characterized animal model to study the effects of aging on fertility in women. The objectives of the study were to characterize age-related changes in ovarian and endocrine functions in old cows and to investigate the validity of a bovine model for the study of human reproductive aging. We tested the hypotheses that aging in cattle is associated with 1) elevated concentrations of gonadotropins and reduced concentrations of steroid hormones in systemic circulation and 2) increased recruitment of ovarian follicles during wave emergence. Daily ultrasonography was performed in 13- to 14-yr-old cows (n = 10) and their 1- to 4-yr-old daughters (n = 9) for one interovulatory interval to study ovarian function. Plasma samples were obtained every 12 h for determination of FSH, LH, progesterone, and estradiol concentrations. Circulating FSH concentrations were higher (P = 0.009) during follicular waves in old cows than in their daughters, but the number of 4- to 5-mm follicles recruited into a wave was lower (P = 0.04) in old cows. Plasma LH concentrations did not differ between groups (P = 0.4), but the ovulatory follicle in two-wave cycles was smaller in old cows (P = 0.04). Plasma estradiol concentrations were higher (P = 0.01) in old cows, and luteal phase progesterone tended to be lower (P = 0.1). We conclude that these changes are consistent with those reported for women approaching menopause transition. Therefore, our results validate the use of the bovine model to study reproductive aging in women.