Ovarian volume and antral follicle counts as indicators of menopausal status.

OBJECTIVE: Recent studies suggest that ovarian volume and antral follicle numbers may be sensitive, specific, and early indicators of menopausal status. The accuracy of these markers, however, has not been compared directly to more traditional markers [age and follicle-stimulating hormone (FSH) levels]. Thus, the purpose of this study was to test whether ovarian volume and antral follicle counts are more sensitive and specific markers of menopausal status than age or FSH levels. DESIGN: Premenopausal (n = 34) and postmenopausal (n = 25) women between 40 and 54 years old received a transvaginal ultrasound for determination of ovarian volume and antral follicle numbers, provided blood for measurement of FSH levels, and completed a questionnaire. FSH levels, age, ovarian volume, and antral follicle numbers were compared using t tests. Receiver operating characteristic curves were generated to evaluate the sensitivity and specificity of each marker. RESULTS: Postmenopausal women had significantly higher FSH levels (p < or = 0.0001), smaller ovarian volumes (p < or = 0.002), and fewer antral follicles (p < or = 0.002) than premenopausal women. Ovarian volume and antral follicle numbers had similar sensitivity (27.3-100%) and specificity (3.4-92.9%) in indicating postmenopausal status as FSH levels and age. CONCLUSION: These data suggest that ovarian volume and antral follicle numbers may be useful indicators of menopausal status.

Nerve growth factor is required for early follicular development in the mammalian ovary.

Nerve growth factor (NGF) epitomizes a family of proteins known as the neurotrophins (NTs), which are required for the survival and differentiation of neurons within both the central and peripheral nervous system. Synthesis of NGF in tissues innervated by the peripheral nervous system is consistent with its function as a target-derived trophic factor. However, the presence of low- and high-affinity NGF receptors in the gonads suggests another function for the NTs within the reproductive endocrine system. We now report that NGF is required for the growth of primordial ovarian follicles, a process known to occur independently of pituitary gonadotropins. Both the NT receptor p75(NTR) and the NGF tyrosine kinase receptor trkA were found to be expressed in the ovaries of infantile normal mice and mice carrying a null mutation of the NGF gene. The ovaries from homozygote NGF-null (-/-) mutant animals, analyzed after completion of ovarian histogenesis, exhibited a markedly reduced population of primary and secondary follicles in the presence of normal serum gonadotropin levels, and an increased number of oocytes that failed to be incorporated into a follicular structure. Assessment of mitogenic activity using two complementary proliferation markers revealed a conspicuous reduction in somatic cell proliferation in the ovaries of NGF-deficient mice. These results suggest that the delay in follicular growth observed in NGF(-/-) mice may be related to the loss of a proliferative signal provided by NGF to the nonneural endocrine component of the ovary.

Effect of bcl-2 on the primordial follicle endowment in the mouse ovary.

Little is known about the embryonic factors that regulate the size of the primordial follicle endowment at birth. A few studies suggest that members of the B-cell lymphoma/leukemia-2 (bcl-2) family of protooncogenes may be important determinants. Thus, the purpose of this study was to test whether bcl-2 regulates the size of the primordial follicle pool at birth. To test this hypothesis, three lines of transgenic mice (c-kit/bcl-2 mice) were generated that overexpress human bcl-2 in an effort to reduce prenatal oocyte loss. The overexpression was targeted to the ovary and appropriate embryonic time period with the use of a 4.8-kilobase c-kit promoter. This promoter provided two to three times more expression of bcl-2 in the ovaries with minimal or no overexpression in most nongonadal tissues. On Postnatal Days 8-60, ovaries were collected from homozygous c-kit/bcl-2 and nontransgenic littermates (controls) and processed for histological evaluation of follicle numbers. All lines of c-kit/bcl-2 mice were born with significantly more primordial follicles than control mice (P < or = 0.05). By Postnatal Days 30-60, however, there were no significant differences in follicle numbers between c-kit/bcl-2 and control mice. These results indicate that bcl-2 overexpression increases the number of primordial follicles at birth, but that the surfeit of primordial follicles is not maintained in postnatal life. These data suggest that it is possible that the ovary may contain a census mechanism by which excess numbers of primordial follicles at birth are detected and removed from the ovary by adulthood.

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental defects in the rat vagina.

At puberty, female rats exposed in utero to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit a persistent thread of mesenchymal tissue surrounded by keratinized epithelium that partially occludes the vaginal opening. Our objective was to determine the earliest time during fetal development that morphological signs of this vaginal canal malformation could be detected and to obtain greater insight into mechanisms involved in this effect. Pregnant rats were administered a single dose of vehicle (control) or TCDD (1.0 microg/kg, po) on gestation day (GD) 15 and were sacrificed on GD 18, 19, 20, and 21 for histological evaluation of female. Gestational exposure to TCDD affected vaginal morphogenesis as early as GD 19, 4 days after exposure of pregnant dams. In exposed fetuses, the thickness of mesenchymal tissue between the caudal Mullerian ducts was increased, which resulted in a failure of the Mullerian ducts to fuse, a process normally completed prior to parturition. In addition, TCDD exposure appeared to inhibit the regression of Wolffian ducts. Thus, TCDD interferes with vaginal development by impairing regression of the Wolffian ducts, by increasing the size of interductal mesenchyme, and by preventing fusion of the Mullerian ducts. Taken together, these effects appear to cause the persistent vaginal thread defect observed in rats following in utero and lactational TCDD exposure.

Workshop to identify critical windows of exposure for children's health: reproductive health in children and adolescents work group summary.

This work group report addresses the central question: What are the critical windows during development (preconception through puberty) when exposure to xenobiotics may have the greatest adverse impact on subsequent reproductive health? The reproductive system develops in stages, with sex-specific organogenesis occurring prenatally and further maturational events occurring in the perinatal period and at puberty. Complex endocrine signals as well as other regulatory factors (genetics, growth factors) are involved at all stages. Evidence from animal models and human studies indicates that many specific events can be perturbed by a variety of toxicants, with endocrine-mediated mechanisms being the more widely studied. Prioritized research needs include basic studies on the cellular-molecular and endocrine regulation of sexual differentiation and development; increased efforts regarding potential adverse effects on development in females, including breast development; expanded animal studies on different classes of chemicals, comparing responses during development (prenatal and postnatal) with responses in adults; and, more extensive explorations regarding the reproductive biology and toxicology of puberty in humans.

Ovarian volume and menopausal status.

OBJECTIVE: The purposes of this study were to (1) examine whether ovarian volume differs by age and menopausal status in healthy women; (2) evaluate whether ovarian volume could be a sensitive and specific predictor of menopausal status; and (3) assess whether ovarian volume is affected by cigarette smoke, oral contraceptives (OCs), and hormone replacement therapy (HRT). DESIGN: Each participant (527 women) completed an extensive in-home interview that assessed age, menopausal status, smoking history, OC use, and HRT use. Each participant also received a transvaginal ultrasound that measured ovarian volume. Geometric means for ovarian volume were compared between premenopausal and postmenopausal women using t tests. Tests for trends were conducted using linear regression analyses. RESULTS: Ovarian volume declined with age (p < or = 0.0001) and also differed by menopausal status; postmenopausal women had smaller ovarian volumes than premenopausal women of the same age (p < or = 0.0001). Ovarian volume was not associated with smoking history or HRT use. However, it was significantly smaller in current users of OCs compared with past users of or those who never used OCs (p < or = 0.0001). Ovarian volume was a sensitive and specific predictor of postmenopausal status. CONCLUSIONS: The data suggest that age, menopausal status, and OC use may be determinants of ovarian volume. They also suggest that ovarian volume may be useful for predicting menopausal status in women.

90-day feeding and one-generation reproduction study in Crl:CD BR rats with 17 beta-estradiol.

Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic toxicity/oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic/biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P1 generation, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17 beta-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17 beta-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17 beta-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminization of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary; hypertrophy of the endometrium and endometrial glands in the uterus; degeneration of seminiferous epithelium; and atrophy of the testes and the accessory sex glands. In the reproduction portion of this study, rats fed 10 or 50 ppm 17 beta-estradiol did not produce litters. While there was no evidence that the 50 ppm treated rats mated, 33.3% of the rats fed 10 ppm mated but did not produce litters. No effects on mating and fertility indices were observed in rats fed 0.05 and 2.5 ppm 17 beta-estradiol. Pup weights at birth were statistically decreased relative to control in the groups fed 0.05 and 2.5 ppm 17 beta-estradiol. Weights of the rats in the 0.05 ppm group recovered by postnatal day 4 and remained similar to control throughout the remainder of the study. The mean gestation length of the 0.05 ppm group was slightly, albeit not statistically significantly, shorter (0.5 days) than that of the control group, which may have contributed to the decrease in birth weight of the 0.05 ppm group. In contrast, the weights of the F1 generation rats fed 2.5 ppm 17 beta-estradiol remained decreased relative to the control group throughout the study. Parental administration of 17 beta-estradiol did not alter anogenital distance in male or female pups. The onset of sexual maturation, as measured by day of preputial separation in males and day of vaginal opening in females, was delayed in male rats fed 2.5 ppm (by 8.2 days) and was hastened in female rats fed 0.05 and 2.5 ppm (by 1.6 and 8.8 days, respectively). The age at vaginal opening ranged from 26 to 37, 26 to 35, and 21 to 25 days for rats fed 0, 0.05, and 2.5 ppm 17 beta-estradiol, respectively. Hence, the range of age at vaginal opening was similar between the control and 0.05 ppm group. The organ weight and pathologic alterations observed in the adult F1 generation rats were similar to those observed in the P1 generation rats. (ABSTRACT TRUNCATED)

Chronically elevated luteinizing hormone depletes primordial follicles in the mouse ovary.

A few years before reproductive senescence, primordial follicles are depleted from the ovary at a dramatically accelerated rate. It has been proposed that this depletion is due to transient increases in gonadotropin levels. To test this hypothesis, we used mice that produce chronically elevated levels of serum LH via expression of an LHbeta subunit transgene. Ovaries were collected from transgenic and control mice, and complete serial sections were prepared for histological examination. Each section was scanned for morphological abnormalities, and every fifth section was sampled to estimate the total number of primordial, primary, and large preantral follicles per ovary. Until 3 wk postpartum, ovaries from transgenic and control mice were morphologically similar. By 5 wk, control ovaries contained many healthy primordial, primary, and large preantral follicles as well as atretic follicles. Transgenic ovaries contained blood-filled cysts, misshapen granulosa cells, luteinized cells, and approximately 45% fewer primordial follicles than controls. By 3 mo, transgenic ovaries had about 68% fewer primordial follicles and 53% fewer primary follicles than controls. These results suggest that, in addition to having profound effects on growing follicles, chronically elevated LH levels deplete the primordial follicle pool and thus may hasten the onset of reproductive senescence.

Elevated luteinizing hormone in prepubertal transgenic mice causes hyperandrogenemia, precocious puberty, and substantial ovarian pathology.

In women, chronically elevated androgens have been associated with polycystic ovarian syndrome and infertility. Recently, we described transgenic mice with elevated serum LH secondary to targeted expression of a transgene encoding a chimeric LH beta-subunit. Mature transgenic females exhibit elevated androgens, anovulation, and a range of ovarian phenotypes including cysts, widespread luteinization, and tumors. In the present study we have examined serum levels of LH and testosterone and the concurrent development of the reproductive system in prepubertal mice. Serum LH in prepubertal females was elevated despite increased serum testosterone and estradiol, indicating a relative insensitivity to steroid negative feedback. Elevated serum LH and hyperandrogenemia resulted in accelerated vaginal opening and ovarian follicular development in transgenic females. Precocious antral follicle formation and conspicuous hypertrophy of the theca-interstitium preceded the development of large cysts with marked hemorrhage. Based on these studies we conclude that chronic prepubertal elevation of serum LH results in gonadotropin-dependent hyperandrogenemia, leading to abnormal sexual development and significant ovarian pathology.

Overview of ovarian follicular development: considerations for the toxicologist.

Folliculogenesis is the lengthy process that results in the production of a species-specific, highly consistent number of follicles, which ripen during each reproductive cycle at precisely the appropriate time for ovulation. Certain features of folliculogenesis may have special implications for toxicologists studying effects of environmental mutagens on oocytes. Such features include the constantly changing geometry of the ovarian follicle, the great excess of developing follicles (most of which will degenerate rather than ovulate), the exponential nature of follicular growth, the acceleration of cell proliferation as follicular size increases, and the location of the principal feedback regulatory step at the penultimate stage of the developmental process. Because the ovary can respond quickly and completely to loss of homeostasis over the short term, damage from toxic insult may not be readily apparent. However, long-range fertility may nevertheless be impaired. The finite size of the follicular pool and the absence of feedback regulatory steps during the early stages of follicular growth render the ovary incapable of restoring the status quo among small and medium-sized follicles. This will eventually result in loss of fine control over the number of follicles that ripen and the regularity of the reproductive cycles and could reduce the overall duration of the fertile life span.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces genital dysmorphogenesis in the female rat.

Recently, Gray and Ostby (Toxicol. Appl. Pharmacol. 133, 285-294, 1995) reported that in utero and lactational TCDD exposure causes striking abnormalities in the rat female reproductive system, including reduced fecundity and vaginal threads. The mechanism by which TCDD induces such abnormalities is unknown. Thus, we sought to determine: (1) whether TCDD reduced fecundity by destroying ovarian follicles and (2) whether the vaginal threads resulted from a TCDD-induced developmental defect during embryogenesis or abnormal vaginal opening at puberty. Pregnant Holtzman rats were treated with 1.0 microgram TCDD/kg or vehicle by a single oral dose on gestation day (GD) 11, 15, or 18. Female offspring were monitored for vaginal opening and terminated on postnatal days 2, 21, and 42. The reproductive tract was removed and evaluated for structural abnormalities. The number of primordial follicles also was determined for each ovary. TCDD exposure on GD 11, 15, or 18 did not change the day of vaginal opening, affect ovarian morphology, or reduce the number of primordial follicles. However, this exposure induced the cleft clitoris and vaginal thread originally described by Gray and Ostby (1995) in approximately 55-96% and 36-44% of the litters in our study, respectively. Histologically the thread presented as a thick cord of mesenchyme surrounded by epithelial cells. This defect was clearly visible in histological sections at birth and was noted in the closed vaginas of prepubertal animals. These data suggest that in utero and lactational exposure to TCDD does not reduce the size of the primordial follicle pool; however, it induces developmental abnormalities in the vaginal canal.

Patterns of ovarian cell proliferation in rats during the embryonic period and the first three weeks postpartum.

The purpose of this study was to characterize the patterns of ovarian cell proliferation during the earliest stages of folliculogenesis, which occur in the embryonic period and the first weeks postpartum in rats. Rats were given continuous infusions of [3H]thymidine (3H-TdR) or bromodeoxyuridine (BrdU), and cells that were synthesizing DNA were visualized by autoradiography or immunohistochemistry. There were dramatic changes in the patterns of cell proliferation during the period studied. Mesenchymal cells proliferated extensively in the embryonic and neonatal ovary, but their growth fraction declined precipitously as follicles formed. Epithelial cells in the medulla of the ovary left the cell cycle at about embryonic Day 12, then resumed proliferation as soon as they were incorporated into follicles just after birth. Epithelial cells towards the cortex of the organ continued to proliferate until late in the embryonic period; they apparently became quiescent around the time of birth, and incorporation into follicles did not release them from their quiescent state. After the follicles had formed, patterns of cell proliferation continued to change. At 5 days postpartum, approximately 36% of the smallest follicles (1-8 granulosa cells in cross section) had at least 1 granulosa cell that was labeled following a 24-h infusion of 3H-TdR; by Day 20 only 14% of these follicles were labeled, and by Day 30 only 4.4% were labeled.

Interleukin-1 beta-converting enzyme-related proteases (IRPs) and mammalian cell death: dissociation of IRP-induced oligonucleosomal endonuclease activity from morphological apoptosis in granulosa cells of the ovarian follicle.

The Caenorhabditis elegans death susceptibility gene, ced-3, has a number of homologs in vertebrate species, including interleukin-1 beta (IL-1 beta)-converting enzyme (ICE), Ich-1long, and CPP32. These genes, which encode a family of related proteases, have been shown to induce apoptosis when transfected into eukaryotic cells. However, it remains to be determined whether these proteases are involved in apoptotic cell death under physiological conditions. The purpose of these studies was to examine the role of ICE-related proteases (IRPs) in apoptosis using a physiologically relevant model system, the ovarian follicle. Somatic granulosa cells within ovarian follicles undergo apoptosis during follicular atresia, a process responsible for the depletion of greater than 95% of the follicles established in the postnatal ovary. To accomplish these studies, we cloned partial rat complementary DNAs encoding ICE, Ich-1, and CPP32 and used these complementary DNAs to examine the gonadotropin regulation of ICE, Ich-1, and CPP32 gene expression in the immature rat ovary. We also examined levels of ICE activity in healthy and atretic rat follicles by monitoring the conversion of exogenous pro-IL-1 beta to the active cytokine, and then evaluated the actions of recombinant IL-1 beta on apoptosis in follicles incubated in vitro. Finally, we tested the requirement for IRP activity in granulosa cell apoptosis and follicular atresia by incubating follicles without and with IRP inhibitors. Northern blot analysis of total RNA samples indicated that gonadotropin-promoted follicular survival was associated with reduced ovarian expression of messenger RNAs encoding Ich-1 and CPP32. In contrast, ICE messenger RNA levels were extremely low and were not affected by gonadotropin treatment. We were also unable to detect ICE activity in proteins extracted from either healthy or atretic rat follicles, collectively suggesting that ICE per se may not function in granulosa cell death. As another approach to determine whether ICE is involved in atresia, healthy antral follicles were isolated from ovaries of gonadotropin-primed immature rats and incubated for 24 h in the absence or presence of 100 ng/ml transforming growth factor-alpha (TGF alpha) without and with 100 ng/ml IL-1 beta. Granulosa cells within follicles incubated in medium alone exhibited extensive levels of apoptosis, and this onset of apoptosis was prevented by the inclusion of TGF alpha. Addition of IL-1 beta did not alter basal levels of apoptosis nor did the cytokine antagonize TGF-alpha-promoted follicle survival, providing additional evidence that ICE activity is not required for atresia to occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Vasoactive intestinal peptide-mediated suppression of apoptosis in the ovary: potential mechanisms of action and evidence of a conserved antiatretogenic role through evolution.

Vasoactive intestinal peptide (VIP)-containing nerve fibers are present in ovarian follicles at all stages of development, and VIP, acting primarily via the cAMP pathway, has been reported to modulate many aspects of granulosa cell function. Herein we examined the effects of VIP and its potential mechanisms of action on apoptosis in antral follicles isolated from ovaries of gonadotropin-primed immature rats and incubated in vitro under serum-free conditions. Additionally, the effects of VIP on apoptosis in isolated avian granulosa cells incubated in vitro were used as a comparative model system to determine whether the ability of VIP to modulate apoptosis in the ovary has been conserved through evolution. Genomic DNA extracted from incubated rat antral follicles exhibited extensive levels of internucleosomal DNA cleavage characteristic of cell death via apoptosis. Treatment of follicles with VIP (1-1000 nM) caused a dose-dependent reduction in the extent of apoptotic DNA breakdown, with a maximal effect achieved with 100 nM VIP. Provision of the adenylyl cyclase activator, forskolin (10 microM), mimicked the inhibitory effect of VIP on apoptosis and concomitantly increased intrafollicular cAMP accumulation, suggesting a role for the cAMP pathway in mediating the immediate actions of VIP on follicular cell survival. Moreover, treatment of rat antral follicles with insulin-like growth factor-binding protein 3 (3 micrograms/ml) partially antagonized the ability of VIP (100 nM) to suppress apoptosis, suggesting involvement of endogenous insulin-like growth factor I in mediating the downstream actions of VIP in incubated rat antral follicles. To further confirm that VIP and activation of the cAMP pathway prevented atresia, individual rat antral follicles incubated for 24 h in the absence or presence of VIP (100 nM) or forskolin (10 microM) were fixed, embedded, and sectioned for morphological analysis. Follicles fixed immediately after isolation from equine CG-primed rat ovaries were classified as morphologically healthy, consistent with the absence of biochemical evidence for apoptosis (e.g. oligonucleosomes) in this pool of follicles. Follicles incubated for 24 h in the absence of tropic support displayed extensive granulosa cell pyknosis and disorganization characteristic of follicles at a moderate stage of atresia. Inclusion of VIP or forskolin maintained the morphological health status of incubated follicles at that resembling healthy follicles fixed immediately after isolation from ovaries of equine CG-primed rats. Lastly, extensive levels of internucleosomal DNA cleavage were also detected in avian granulosa cells incubated for 6 h under serum-free conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated: changes at the time of folliculogenesis.

An emerging body of evidence suggests that neurotrophins not only promote neuronal survival and differentiation, but can also target nonneuronal cells for their actions. Neurotrophins initiate their biological effects by binding to cell membrane tyrosine kinase receptors of the trk protooncogene family. In addition, all neurotrophins recognize with similar affinity a different receptor molecule known as p75 nerve growth factor receptor (p75 NGFR) or low affinity NGFR, which appears to interact with the trk receptors to potentiate their response to neurotrophins. The mature mammalian ovary has been shown to synthesize several neurotrophins, including nerve growth factor (NGF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5). The ovary also expresses some of the neurotrophin receptors, including p75 NGFR, trkB [the receptor for NT-4/5 and brain-derived neurotropic factor (BDNF)], and trkA (the NGF receptor). The present experiments were undertaken to determine whether neurotrophins and their receptors are expressed at the time of definitive ovarian histogenesis, and whether any of them exhibit a developmental pattern of expression related to the completion of folliculogenesis. Immunohistochemical identification of p75 NGFR in rat embryonic ovaries revealed that the receptor is predominantly expressed in mesenchymal cells. By gestational day 18, these cells have formed pockets that enclose presumptive pregranulosa cells and groups of oocytes into ovigerous cords. Immediately after birth, the ovigerous cords are subdivided, resulting in the abrupt formation of primordial follicles between 24-48 h after birth. Consistent with these observations, the p75 NGFR messenger RNA (mRNA) content increased after birth and remained elevated at the time of follicular assembly. The NGF and trkA genes showed a different pattern of expression, as the ovarian content of both NGF and trkA mRNA decreased at the time of folliculogenesis. In contrast to the drop in NGF and trkA mRNA expression, NT-4 mRNA levels increased at the time of follicular assembly, coinciding with the abrupt appearance of trkB mRNA. In situ hybridization showed that the increase in NT-4 mRNA expression occurred in a subpopulation of oocytes between 24-48 h after birth, and that the trkB gene became predominantly expressed at this time in epithelial pregranulosa cells. Substantial, but unchanging, levels of NT-3 mRNA and the mRNA encoding trkC, the preferred NT-3 receptor, were detected throughout the perinatal period examined. Very low and invariable levels of BDNF were also detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship between the supply of primordial follicles and the onset of follicular growth in rats.

Primordial follicles enter a state of suspended animation when they are formed and constitute a "stockpile" from which all growing follicles are derived. The factors that release individual follicles from their quiescent state are unknown. Many investigators believe that the number of follicles in the primordial stockpile is a major factor in determining the rate at which follicles begin to grow. However, the relationship between the size of the stockpile of primordial follicles and the rate at which follicles move into the growing pool is unclear. The purpose of the present study was to attempt to clarify this relationship. The initial size of the stockpile of primordial follicles was experimentally reduced by exposing rats to various doses of busulphan (BUS) in utero. Ovaries were collected at various ages postpartum and prepared for histological analysis. A computer-controlled image analyzer was used to perform size/frequency analysis of oocytes in control and treated ovaries; onset of follicular growth was recognized by enlargement of the oocyte. There was an inverse correlation between the number of primordial follicles in the ovary at birth and the rate at which they moved into the growing pool. In rats most severely affected by the BUS, all of the remaining follicles began to grow very early in life. By the time these severely affected rats reached adulthood, the stockpile of primordial follicles had been nearly exhausted. Nevertheless, the number of large antral follicles remained at normal levels until the follicular reserve was completely depleted.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of cell populations that contribute to the formation of primordial follicles in rats.

To study the events that lead to the formation of primordial follicles, pregnant rats were given continuous infusions of [3H]thymidine (3H-TdR) beginning on Days 14-19 of pregnancy (e14-e19) and continuing for 48-120 h. Ovaries from the pups were collected and plastic-embedded histological sections were prepared for autoradiography. The autoradiographs revealed that within the core of the developing ovary were a large number of cells that remained mitotically inactive (failed to incorporate label) from e14 through the day of birth. These unlabeled cells gave rise to the granulosa cells of the first follicles that formed, were located in the medulla of the ovary, and were the first to begin growth. The unlabeled cells did not appear to contribute to the formation of the follicles that formed later in the cortical region of the ovary. When 3H-TdR infusion was begun during late pregnancy, a small subset of the germ cells incorporated label, although the vast majority did not. The labeled germ cells are presumed to represent those that were lagging in their development (had not yet entered meiosis). After ovarian histogenesis was completed during the first week postpartum, the unlabeled ocytes were found concentrated in the core of the ovary, enclosed in the earliest growing follicles; labeled oocytes were found exclusively in the cortex of the ovary, within tiny, quiescent primordial follicles. These observations provide some empirical support for long-held, but heretofore untested, hypotheses concerning early folliculogenesis: that the first follicles that begin to grow are qualitatively different from the remaining follicles in the ovary and that primordial follicles begin to grow in the order in which they were first formed.

The changing architecture of the neonatal rat ovary during histogenesis.

The purpose of this study was to describe the changing histological organization of the rat ovary during postpartum days one through three (p1-p3). A PC-based image-combining microscope system was used to reconstruct the ovary in three dimensions. On p1, cyclindrical pocket-like structures radiated from the core of the ovary that were open toward the surface epithelium. The walls of the pockets contained connective tissue cells and capillaries (stroma). By p2, these pockets had completely closed; each pocket enclosed a small nest of oocytes and a few presumptive granulosa cells. By p3, the pocket-like organization had disappeared. On p1, only one or two primordial follicle-like structures were observed in the core and toward the periphery of the ovary; most oocytes were not enclosed in follicles. By p3, very few naked oocytes remained; primordial follicles predominated in all the regions of the ovary and some of the follicles had multiple layers of granulosa cells. There were changes in location, area, and volume of the rete tubules during these postnatal days. The extraovarian rete was visible on all 3 days but changed its orientation relative to the ovary. The connecting rete was found beneath the epithelial layer of the ovary on all 3 days and showed dramatic increase in area on p2. The wide lumen of the intraovarian rete was in direct contact with some of the oocytes near by on all 3 days, but these "communication points" were most abundant on p2. Based on our observations of different cell-cell associations during this time period, we hypothesize (1) that the mesenchymal-presumptive granulosa cell association is essential for the completion of folliculogenesis, and (2) the rete ovarii may have an inductive role in follicle assembly. These observations suggest that the first 3 days postpartum are critically important for studying the heterogeneous cell interactions that lead to the assembly of primordial follicles. The regional differences in tissue organization during this formative period may have significant implications on later aspects of follicular development.

Correlation of age-associated increases in follicle stimulating hormone secretion with decreases in antral follicles: failure of progesterone-induced acyclicity to prevent these changes.

It is well known that the number of follicles in the mammalian ovary decreases with age. In light of previous data from this laboratory showing age-related alterations in the secretion and production of follicle-stimulating hormone (FSH) in rats by 5-6 months of age, one objective of the present study was to determine if alterations in FSH secretion were accompanied by changes in the number of antral follicles. A second objective of this study was to determine whether or not interruption of cyclic activity by continuous progesterone (P) treatment could decelerate age-associated changes in FSH secretion possibly by retarding the depletion of follicles through ovulation. For this study, one group of 4-day cycling, 7-week-old rats received one empty Silastic implant while another group received 3-40 mm implants containing 30 mm crystalline P. Implants were replaced every 2 weeks until the animals were 5 months old. Progesterone-implanted rats were acyclic during treatment exhibiting predominantly leukocytic vaginal smears. Regular 4-day cycles resumed when P implants were withdrawn (rats approximately 5-6-months-old). A group of 2-3-month-old untreated rats were used for comparison. As expected from our previous results, serum FSH levels at 1600 h on estrus were significantly higher in 5-6-month-old rats receiving empty capsules than in younger rats. Serum FSH concentrations measured in P-treated rats at this time also were significantly higher than levels of this gonadotropin measured in younger rats. Ovaries of older control and P-treated rats contained significantly fewer medium and large antral follicles (greater than 250 microns) than the ovaries of younger rats despite the curtailment of estrous cyclicity and ovulation by continuous P treatment. Interestingly, P treatment prevented the age-associated decrease in thymus weight. Taken together, the present observations suggest that a decrease in the number of growing follicles may be a factor contributing to early age-related alterations in FSH secretion. Furthermore, the prevention (at least temporarily) of age-related thymic involution by P treatment may be indicative of an interrelationship between thymic and reproductive aging.