Xenobiotic effects on ovarian preantral follicles

Women are born with a finite population of ovarian follicles, which are slowly depleted during their reproductive years until reproductive failure (menopause) occurs. The rate of loss of primordial follicles is determined by genetic and environmental influences, but certain toxic exposures can accelerate this process. Ionizing radiation reduces preantral follicle numbers in rodents and humans in a dose-dependent manner. Cigarette smoking is linked to menopause occurring 1-4 yr earlier than with nonsmokers, and components of smoke, polycyclic aromatic hydrocarbons, can cause follicle depletion in rodents or in ovaries in vitro. Chemotherapeutic agents, such as alkylating drugs and cisplatin, also cause loss of preantral ovarian follicles. Effects depend on dose, type, and reactivity of the drug, and the age of the individual. Evidence suggests DNA damage may underlie follicle loss induced by one common alkylating drug, cyclophosphamide. Occupational exposures have also been linked to ovarian damage. In an industrial setting, 2-bromopropane caused infertility in men and women, and it can induce ovarian follicle depletion in rats. Solvents, such as butadiene, 4-vinylcyclohexene, and their diepoxides, can also cause specific preantral follicle depletion. The mechanism(s) underlying effects of the latter compound may involve alterations in apoptosis, survival factors such as KIT/Kit Ligand, and/or the cellular signaling that maintains primordial follicle dormancy. Estrogenic endocrine disruptors may alter follicle formation/development and impair fertility or normal development of offspring. Thus, specific exposures are known or suspected of detrimentally impacting preantral ovarian follicles, leading to early ovarian failure.     

Pathways coordinating oocyte attrition and abundance during mammalian ovarian reserve establishment

The mammalian ovarian reserve is comprised of a finite pool of primordial follicles, representing the lifetime reproductive capacity of females. In most mammals, the reserve is produced during embryonic and early postnatal development with oocyte numbers peaking during mid‐to‐late gestation, and then experiencing a dramatic decline continuing until shortly after birth. Oocytes remaining after the bulk of this attrition are subsequently surrounded by a layer of somatic pre‐granulosa cells with these units then referred to as “primordial follicles.” The complex and varied cell death mechanisms intrinsic to this process are not only characteristic of, but also essential for, the proper formation of this pool of follicles, and as a result must be immaculately balanced to ensure long‐term fertility and reproductive health. Too few follicles can lead to Primary Ovarian Insufficiency, resulting in fertility loss and other features of aging, such as an overall shorter lifespan. On the other hand, whereas an excess of follicles might extend reproductive lifespan, this might also be the underlying etiology of other ovarian pathologies. The last decade, in particular, has vastly expanded our understanding of oocyte attrition and determinants of ovarian reserve abundance. By continuing to decipher the intricacies underlying the cell death processes and development of the initial primordial follicle pool, we may be in a much better position to understand idiopathic cases of premature follicle depletion and improve ovarian health in reproductive‐age women.     

In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation

As cancer treatment improves, more young women of reproductive age are surviving, but they suffer from infertility as a consequence of the radiation and chemotherapy. Human ovarian tissue containing immature primordial follicles has been successfully cryopreserved. The ultimate aim of this technique is to induce ovarian function by re-plantation of ovarian tissue or, further into the future, by in vitro maturation (IVM) of the oocytes derived from the cryopreserved-thawed ovarian tissue, followed by routine in vitro fertilization. IVM of primordial follicles from young cancer survivors would avoid the risk of cancer re-transmission by the ovarian grafts. The present review discusses the current achievements in IVM of female germ cells and primordial ovarian follicles and the attempts to improve their development by adding various factors to the culture medium. The established methods for the evaluation of survival and growth in culture are also discussed: follicular counts, immunocytochemical methods, transmission electron microscopy, fluorescent viability markers and endocrine assays. Although the development of IVM systems is still in its infancy, researchers need to pursue their approach step-by-step, especially with regard to factors that might be involved in the activation of the ovarian follicles or female germ cells. The final measure of success will be the ability of the in vitro matured oocytes to fertilize and produce healthy offsprings. The availability of such treatment will probably lead to its demand not only by cancer patients but by other women as well.     

The acceleration of reproductive aging in Nrg1flox/flox;Cyp19‐Cre female mice

Irregular menstrual cycles, reduced responses to exogenous hormonal treatments, and altered endocrine profiles (high FSH/high LH/low AMH) are observed in women with increasing age before menopause. In this study, because the granulosa cell‐specific Nrg1 knockout mice (gcNrg1KO) presented ovarian and endocrine phenotypes similar to older women, we sought to understand the mechanisms of ovarian aging and to develop a new strategy for improving fertility in older women prior to menopause. In the ovary of 6‐month‐old gcNrg1KO mice, follicular development was blocked in bilayer secondary follicles and heterogeneous cells accumulated in ovarian stroma. The heterogeneous cells in ovarian stroma were distinguished as two different types: (i) the LH receptor‐positive endocrine cells and (ii) actin‐rich fibrotic cells expressing collagen. Both the endocrine and fibrotic cells disappeared following long‐term treatment with a GnRH antagonist, indicating that the high levels of serum LH induced the survival of both cell types and the abnormal endocrine profile to reduce fertility. Moreover, follicular development to the antral stages was observed with reduced LH and the disappearance of the abnormal stromal cells. Mice treated with the GnRH antagonist regained normal, recurrent estrous cycles and continuously delivered pups for at least for 3 months. We conclude that endocrine and matrix alternations occur within the ovarian stroma with increasing age and that abolishing these alternations resets the cyclical release of LH. Thus, GnRH antagonist treatments might provide a new, noninvasive strategy for improving fertility in a subset of aging women before menopause.     

Biological aging and the etiology of aneuploidy

A prevalent hypothesis concerning the cause of the rise in aneuploid conceptions with maternal age is that the changes that accompany normal ovarian aging increase the rate of meiotic errors in the oocyte. Biological aging of the ovary is accompanied by a decline in both the total oocyte pool and the number of antral follicles maturing per cycle, as well as changes in the levels of circulating reproductive hormones. The biological aging hypothesis predicts that aneuploidy rates should be higher in women with a prematurely reduced oocyte pool, and that women with trisomic conceptions should show signs of earlier ovarian aging than women of the same chronological age without trisomic conceptions. Comprehensive studies of aneuploidy in groups of women with known causes of premature ovarian failure remain to be done, though anecdotal evidence does suggest increased rates of pregnancy loss and aneuploidy. Smoking, which is a well-documented cause of earlier ovarian aging, is not associated with an increase in aneuploid conceptions. Evidence from women with unilateral ovariectomies is inconsistent. Support for the biological aging hypothesis was provided by one study showing that menopause occurred about a year earlier in women with a trisomic spontaneous abortion compared to women with chromosomally normal conceptions. Associations between high FSH and pregnancies with Down syndrome and chromosomally abnormal spontaneous abortions have also been reported. However, the most direct test of the hypothesis, which compared antral follicle counts and hormonal levels in women with trisomic pregnancies and those with chromosomally normal pregnancies, failed to find a difference in the expected direction. A prospective study of FSH levels in women with subfertility also failed to find an association with the rate of pregnancy loss. The bulk of evidence thus suggests that, if the processes of biological aging are indeed related to aneuploidy, they probably involve factors other than those measured by oocyte or antral follicle pool size and reproductive hormone levels.     

Insulin like growth factor 1 and regulation of ovarian function in mammals

Various growth factors have been proposed to play endocrine and/or paracrine role in mammalian ovarian follicular development. The insulin like growth factor 1 (IGF-1) is one such factor. More and more reports now support the existence of an intra-ovarian IGF system including receptors and binding proteins. The role of IGF-1 in ovary is to amplify gonadotropin hormone action in terms of increased steroidogenesis by ovarian granulosa cell and granulosa cell proliferation. The synthesis and proteolysis of insulin like growth factor binding proteins, under the control of follicle stimulating hormone, regulate the intra-follicular availability of IGF-1, which further determines the sensitivity of granulosa cells to gonadotropins. Besides gonadotropins, IGF-1 has been implicated in somatotropin hormone action in the ovarian function. Exact mechanism of IGF-1 action in the ovarian follicles needs to be worked out to elucidate whether or not IGF-1 is indispensable in addition to know endocrine factors like gonadotropic and ovarian steroid hormones. This will pave the way for better understanding of control(s) which ensure final development of dominant follicle(s) and atresia of other follicles of the cohort.     

Mechanisms of hormone-mediated carcinogenesis of the ovary in mice

Experimental ovarian carcinogenesis has been investigated in inbred and hybrid strains of mice and induced by a diversity of mechanisms including X-irradiation, oocytotoxic xenobiotic chemicals, ovarian grafting to ectopic or orthotopic sites, neonatal thymectomy, mutant genes reducing germ cell populations, and aging. The mechanisms are briefly reviewed whereby disruptions in the function of graafian follicles results in a spectrum of ovarian proliferative lesions including tumors. The findings in mutant mice support the concept of a secondary (hormonally-mediated) mechanism of ovarian carcinogenesis in mice associated with sterility. Multiple pathogenetic factors that either destroy or diminish the numbers of graafian follicles in the ovary result in decreased sex hormone secretion (especially estradiol-17β) leading to a compensatory over-production of pituitary gonadotrophins (particularly luteinizing hormone), which places the mouse ovary at an increased risk to develop tumors. The intense proliferation of ovarian surface epithelium and stromal (interstitial) cells with the development of unique tubular adenomas in response to sterility does not appear to have a counterpart in the ovaries of women.     

A review on the morphology of ovarian follicles in elasmobranchs: A case study in Rhizoprionodon taylori

The identification of the elasmobranch secondary ovarian follicles and their function can be challenging and the inconsistent use of terminology derived from other taxa is a matter of ongoing debate. In this study, the available information on the histology of the elasmobranch secondary ovarian follicles derived from atresia (preovulatory follicles) or ovulation (postovulatory follicles) is reviewed highlighting their morphology and steroidogenic capacity. Based on this literature review, the ovarian follicles of the Australian sharpnose shark Rhizoprionodon taylori were classified according to their preovulatory or postovulatory origin. Two types of secondary follicles originating from atresia of developing follicles (atretic previtellogenic follicles) and ripe follicles (atretic vitellogenic follicles), and one type of postovulatory follicle were identified throughout the reproductive year of this species. Morphological similarities of the elasmobranch secondary ovarian follicles and their variations in different species denote the difficulty to classify them. Given the multiple origins of ovarian follicles, their poorly understood functions and capacity to supply steroids, visual identification of these structures and the use of terminology derived from mammalian and other vertebrate studies (with the exception of the corpora lutea as a temporary endocrine gland) is not advisable. J. Morphol. 278:486–499, 2017. © 2017 Wiley Periodicals, Inc.     

Defining ovarian reserve to better understand ovarian aging

Though a widely utilized term and clinical concept, ovarian reserve (OR) has been only inadequately defined. Based on Medline and PubMed searches we here define OR in its various components, review genetic control of OR, with special emphasis on the FMR1 gene, and discuss whether diminished OR (DOR) is treatable. What is generally referred to as OR reflects only a small portion of total OR (TOR), a pool of growing (recruited) follicles (GFs) at different stages of maturation. Functional OR (FOR) depends on size of the follicle pool at menarche and the follicle recruitment rate. Both vary between individuals and, at least partially, are under genetic control. The FMR1 gene plays a role in defining FOR at all ages. Infertility treatments have in the past almost exclusively only centered on the last two weeks of folliculogenesis, the gonadotropin-sensitive phase. Expansions of treatments into earlier stages of maturation will offer opportunity to significantly improve ovarian stimulation protocols, especially in women with DOR. Dehydroepiandrosterone (DHEA) may represent a first such intervention. Data generated in DHEA-supplemented women, indeed, suggest a new ovarian aging concept, based on aging of ovarian environments and not, as currently is believed, aging oocytes.     

Imminent oocyte exhaustion and reduced follicular recruitment mark the transition to acyclicity in aging C57BL/6J mice

Quantitative cytological analyses of aging C57BL/6J mouse ovaries revealed that the populations of primordial and growing follicles were nearly exhausted by 13-14 months, the average age of ovulatory failure. Anovulatory animals of this age had, on the average, half the follicle number of their counterparts which were still ovulating. This result suggests that follicular depletion is a factor which contributes to the loss of ovarian cyclicity during aging. However, the considerable overlap of follicle number between the two groups suggests that other, possibly extraovarian, factors also influence the loss of ovulatory function. Although the numbers of follicles recruited for growth was much lower in ovaries from old cycling animals, the number of ova shed cyclically was generally within the range of younger individuals. The observed reduction in incidence of morphological atresia among medium-sized follicles may explain how ovulatory constancy is maintained virtually throughout the cyclical life of the ovary.     

The maternal recognition of pregnancy in mammals

Progesterone secreted by the corpora lutea stimulates uterine secretion of nutrients required for embryonic growth. But progesterone also blocks maturation of ovarian follicles and oestrous behaviour. When conception fails, therefore, the corpora lutea must lyse, in order to allow a second ovulation and oestrus, while in pregnancy the corpora lutea must be maintained. A number of different endocrine mechanisms exist to ensure the corpora lutea are controlled appropriately depending on pregnancy status; among the best studied of these are the secretion by the developing conceptus of interferons (in ungulates) or chorionic gonadotropins (in primates). These and other mechanisms are reviewed.     

Oogenesis from human somatic stem cells and a role of immune adaptation in premature ovarian failure

The central thesis is that, while embryonic oocytes originate from extra-ovarian sources, those generated during fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE). With the assistance of immune system-related cells, primitive granulosa and germ cells appear to originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly in order to be recognized as self and renewed later. With the onset of menarche, a periodical follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs are always available during the prime reproductive period. The periodical follicular renewal ceases between 35-40 years of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. Premature ovarian failure (POF) may result from premature termination of follicular renewal during adulthood, possibly due to the alteration of fetal follicular development during immune adaptation (idiopathic POF), or due to the alteration of the adult immune system by cytostatic chemotherapy. Factors responsible for the diminution of follicular renewal may be responsible for the aging of other tissues and the whole body in general. However, our recent research shows that OSE stem cells may produce new eggs in vitro, even when derived from ovaries lacking primary follicles. Consequently, their in vitro fertilization (IVF) and subsequent utilization of embryos for intrauterine implantation may represent a novel IVF approach for providing genetically related children to women with ovarian infertility, which is worthy of consideration and further exploration.     

Nonvitellogenic female sterile mutants and the regulation of vitellogenesis in Drosophila melanogaster.

The genetic and endocrine regulation of vitellogenesis was investigated by studying 18 female sterile mutations that disrupt the development of normal vitellogenic follicles. Applications of exogenous juvenile hormone analog and reciprocal ovarian transplants between flies of different genotypes were employed to accomplish our first two objectives: to find (1) whether the mutation blocked development of the ovary directly, and (2) whether the mutation altered the hormonal milieu. In 15 of the mutants the developmental defect was localized to the ovary, but in the other 3 the ovary was competent to respond to a permissive environment. The internal milieu of these three mutants (ap⁴, fs(3)A1, fs(2)A18) was unable to provoke normal development in wild-type ovaries, suggesting that these mutations cause endocrine defects. Our third objective was to find whether an endocrine organ was itself defective in any of these mutants. The corpus allatum from two of the mutants was unable to provoke vitellogenesis in isolated wild-type abdomens, but corpora allata from wild-type females or from other mutants were able to promote maturation of ovarian follicles in isolated abdomens. Our fourth objective was to find whether any of the mutants were able to produce yolk proteins. Immunoelectrophoresis of fly hemolymph demonstrated that in all mutants tested vitellogenins were found in the blood. These experiments permit four main conclusions. First, they identify the first Drosophila mutants in which an endocrine gland is shown to be intrinsically defective during adulthood. Second, they show that the production of morphologically normal late previtellogenic follicles is not required for the induction of vitellogenin synthesis and secretion. Third, they show that juvenile hormone can cause ovarian follicles to sequester yolk in mutant flies. And finally, they show that mutants with defective corpora allata still synthesize and secrete vitellogenin. Taken together, these conclusions suggest that in Drosophila melanogaster the uptake of vitellogenin into follicles depends upon the availability of juvenile hormone, but that the synthesis and secretion of vitellogenin are independent of both normal ovaries and totally normal corpora allata.     

Ovarian theca cell

Theca cells are the endocrine cells associated with ovarian follicles that play an essential role in fertility by producing the androgen substrate required for ovarian estrogen biosynthesis. Theca cells differentiate from the interfollicular stroma in response to proteins secreted from growing follicles. The most common endocrine cause of infertility is associated with excessive proliferation of theca cells and ovarian hyperandrogenism. Cell facts: -ovarian androgen-producing cells; -are associated only with developing follicles; -over-activity of theca cells causes infertility due to hyperandrogenism; -under-activity of theca cells causes infertility due to lack of estrogen. Theca cells: androgen-producing cells in the ovary.     

The sheep ovary: regulation of steroidogenic, haemodynamic and structural changes in the largest follicle and adjacent tissue before ovulation.

Important changes occur in the follicle that is destined to ovulate and also in the surrounding ovarian tissue of sheep during the 72 hr preceding ovulation. It is our purpose to describe the morphological and functional changes that take place in large follicles during the preovulatory period and to relate these to the local ovarian environment and general endocrine status prevailing at this time. A consideration of changes in the structure, blood supply and function of the ovaries in vivo, is followed by a discussion of the functional capacity of the individual ovarian components when isolated and studied in vitro. Against this background, the nature and sequence of events that occur in the preovulatory follicle and the mechanisms which regulate them will be discussed.     

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.     

Genetic markers of ovarian follicle number and menopause in women of multiple ethnicities

Oocyte loss has a significant impact on fertility and somatic health. Yet, we know little about factors that impact this process. We sought to identify genetic variants associated with ovarian reserve (oocyte number as measured by antral follicle count, AFC). Based on recently published genome-wide scans that identified loci associated with age of menopause, we also sought to test our hypothesis that follicle number and menopausal age share underlying genetic associations. We analyzed menopause-related variants for association with follicle number in an independent population of approximately 450 reproductive-aged women of European and African ancestry; these women were assessed for AFC, anthropometric, clinical, and lifestyle factors. One SNP strongly associated with later menopausal age in Caucasian women (+1.07?±?0.11?years) in previous work was also associated with higher follicle counts in Caucasians (+2.79?±?1.67 follicles) in our study. This variant is within the Minichromosome Maintenance Complex Component 8 (MCM8) gene, which we found was expressed within oocytes in follicles of the human ovary. In genome-wide scans of AFC, we also identified one marginally genome-wide and several nominally significant SNPs within several other genes associated with follicle number in both ethnic groups. Further, there were overlapping variants associated with multiple ovarian reserve markers (AFC, serum hormone levels, menopausal age). This study provides the first evidence for direct genetic associations underlying both follicle number and menopause and identifies novel candidate genes. Genetic variants associated with ovarian reserve may facilitate discovery of genetic markers to predict reproductive health and lifespan in women.     

Superovulatory responses of cattle

Non-histological examination of superovulated ovaries of cows does not allow one to distinguish between corpora lutea and luteinized follicles. A better estimation of ovulation rate could, therefore, be made from the number of embryos recovered or from the levels of E₂-17β in the plasma 60 hours after PMSG.For comparison of different treatments, it is necessary to characterize activities of the stimulatory agents used. Administration of an FSH - LH preparation twice a day at decreasing doses gives the best mean responses, but no treatment has been found which can clearly decrease the large variation between individuals in their responses.Numerical, kinetic and endocrine ovarian factors can partly explain the variability of ovarian responses to PMSG in the heifer. Individual differences in follicular populations at the time of treatment, or in E₂-17β levels after stimulation, could be related to differences in responses in ovulation rate. Normal follicles >1.7 mm diameter before treatment would usually ovulate following PMSG injection, whereas early atretic follicles of the same size mostly luteinize.     

Anti-mullerian hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can help to predict superovulatory responses in the cow

The major limitation to the development of embryo production in cattle is the strong between-animal variability in ovulatory response to FSH-induced superovulation, mainly due to differences in ovarian activity at the time of treatment. This study aimed to establish whether anti-Müllerian hormone (AMH) was an endocrine marker of follicular populations in the cow, as in human, and a possible predictor of the ovarian response to superovulation. Anti-Müllerian hormone concentrations in plasma varied 10-fold between cows before treatment and were found to be highly correlated with the numbers of 3- to 7-mm antral follicles detected by ovarian ultrasonography before treatment (r=0.79, P<0.001) and the numbers of ovulations after treatment (r=0.64, P<0.01). Between-animal differences in AMH concentrations were found to be unchanged after a 3-mo delay (r=0.87, P<0.01), indicating that AMH endocrine levels were characteristic of each animal on a long-term period. The population of healthy 3- to 7-mm follicles was the main target of superovulatory treatments, contained the highest AMH concentrations and AMH mRNA levels compared with larger follicles, and contributed importantly to AMH endocrine levels. In conclusion, AMH was found to be a reliable endocrine marker of the population of small antral gonadotropin-responsive follicles in the cow. Moreover, AMH concentrations in the plasma of individuals were indicative of their ability to respond to superovulatory treatments.