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In females, reproduction is a key facet of women’s health, livestock production, and conservation of endangered species. The ovary is the gonad of the female and is a central component of female reproductive physiology as it houses the germ cells (i.e., oocytes) within follicles and produces hormones to coordinate folliculogenesis. The ovary is a target organ for developing novel assisted reproductive technologies (ART) such as safeguarding fertility via cryopreserving ovarian tissue containing follicles and developing strategies to grow early staged preantral follicles in vitro for oocyte production. Moreover, a better understanding of cellular and molecular events constituting early gonad development would be widely valuable to further advance ART that capitalizes on components of the ovary. In this work, we describe three aims using the bovine as a model and collectively expand our current knowledge in 1) ovarian tissue cryopreservation (OTC), 2) in vitro bovine preantral follicle development, and 3) in vitro differentiation of bovine embryonic stem cells (bESCs) into progenitors of bipotential gonad-like cells. In Aim 1, we found that, overall, slow freezing outperformed vitrification when bovine ovarian fragments were thawed and cultured as indicated by higher rates of normal follicle morphology, lower rates of apoptosis in stromal cells, and proper expression of the gap junction protein connexin 37. In addition to the use of OTC and culturing ovarian tissue fragments to rescue folliculogenesis, in Aim 2 we explored the use of bioengineered and proteolytically degradable (poly)ethylene glycol (PEG) hydrogels for in vitro culture of isolated bovine preantral follicles. We also tested co-encapsulation of mesoderm-like cells (MeLCs) from bESCs or native bovine ovarian cells (BOCs) with follicles to determine their propensity to become theca-like cells and improve folliculogenesis in vitro. After customizing a PEG hydrogel to complement bovine preantral follicle extracellular matrix (ECM) enzyme expression, we found that smaller follicles grew better in PEG hydrogels compared to two-dimensional control over ten days. However, MeLCs did not survive PEG hydrogel encapsulation process. Although BOCs could survive in PEG hydrogels throughout the culture period, BOCs did not better support follicle survival and development when co-encapsulated compared to PEG-only control. Moreover, the culture conditions did not maintain expression of selected genes essential for theca cell differentiation. Finally, in Aim 3, we tested the ability of bESCs to be driven towards the intermediate mesoderm (IM), early coelomic epithelium (eCE), and steroidogenic state during in vitro culture using several protocols. Our data indicated that inclusion of the steroidogenic factor-1 (SF1) agonist, RJW100, did not upregulate steroidogenic genes during the differentiation. Similarly, the cytokines basic fibroblast growth factor (bFGF) and bone morphogenic protein-4 (BMP4) were dispensable for inducing IM and eCE gene expression, and WNT activation was a predominant driver of eCE and IM gene expression, high concentrations of BMP4 led to upregulation of lateral plate mesoderm gene expression, and paraxial mesoderm gene expression remained unchanged across all regimens. Collectively, this dissertation work sheds light on several facets of ovarian biology including a better understanding of the impacts of OTC, in vitro preantral folliculogenesis, and use of embryonic stem cells to recapitulate bipotential gonad somatic cell differentiation. Nevertheless, additional research is required to elucidate mechanisms that are pivotal to both early folliculogenesis and early embryonic gonad formation in the bovine model.

MiR-202-5p is identified as the maker microRNA (miRNA) of germplasm in fish. However, the role of miR-202-5p in gonadal development is still unclear. Here we investigated the expression patterns of miR-202-5p in Japanese flounder (Paralichthys olivaceus). Real-time PCR displayed that miR-202-5p was specifically expressed in bisexual gonads, and its transcription in the testis was relatively abundant than that in the ovary. Moreover, it was gradually increased in testis from stage II to stage IV. Fluorescent in situ hybridization (FISH) illustrated that miR-202-5p was co-localized with vasa (a marker gene of germ cells) in testis, and it was predominately expressed in spermatogonia and spermatocytes, implying that miR-202-5p might participate in spermatogenesis. Considering the important role of retinoic acid (RA) on regulating meiosis during spermatogenesis, we treated Japanese flounders by injecting all-trans retinoic acid (ATRA). MiR-202-5p in testis was shown to be up-regulated, and ccnd1, a key cell cycle regulator, was found to be down-regulated after ATRA treatment. Furthermore, ccnd1 was demonstrated to be a direct regulatory target of miR-202-5p using Dual-luciferase assay. In the primary testis cell from Japanese flounder, we further confirmed that ccnd1 was down-regulated by miR-202-5p. The results suggest that miR-202-5p exerts a crucial role in gonadal development by inhibiting ccnd1 in Japanese flounder.

Reproduction is a critical component of an animal’s fitness, but it is also energetically intensive. In order to maximize reproductive fitness, organisms modulate their reproductive investment or timing in response to environmental cues that provide information about resource availability or other indicators of reproductive success. Physiologic systems that monitor these cues then influence both reproductive readiness and more scaled measures of investment, including number of offspring or offspring provisioning effort. This kind of physiologic modulation is especially salient to mammals, for whom reproduction (gestation and lactation) is exceptionally intensive, and there is extensive documentation of variation in reproductive effort and success under specific environmental and physiological challenges. Still, the underlying mechanisms that coordinate these shifts in investment remain poorly defined and understood. This dissertation identifies novel endocrine and paracrine mechanisms that regulate ovarian and uterine function in the context of reproductive success. Chapter 1 summarizes our current understanding of female reproductive function and modulation, ultimately identifying 4 key areas of modulation for which the molecular mechanisms are poorly understood or defined: (1) evaluating autonomic nervous system regulation of reproductive organs in ecophysiological contexts, (2) identifying new signals regulating ovary and uterine function; (3) determining the extent to which reproductive stage (e.g., pregnancy) alters regulatory networks and sensitivity; and (4) scaling function of specific cell types or organs to organism-level reproductive outcomes. Chapters 2-5 address areas 2-4 by identifying new mechanisms of mammalian reproductive system control in the ovary and uterus and by evaluating the function of established signaling networks in other reproductive states. Chapter 2 interrogates the direct sensitivity of the ovary to physiological cues using mouse ovarian explants. By using pharmacologic inhibition of glucose metabolism, these experiments identify novel glucose sensitivity in the ovary. Chapter 3 examines the effect of neuropeptide gonadotropin inhibitory hormone (GnIH) on ovarian follicle growth, survival, and steroidogenesis in the domestic cat. As has been shown in other mammals to-date, GnIH promotes follicle degradation in vitro. These findings support a conserved role for GnIH in vertebrate ovarian folliculogenesis. Together, these chapters provide new paracrine and endocrine mechanisms of ovarian regulation that adds to our molecular understanding of how reproductive effort and investment are modulated.Chapter 4 explores the sensitivity of feline uterine endometrial epithelial cells to sex steroids. We demonstrate that sex steroids stimulate morphological changes to endometrial epithelial cell 3D growth in vitro, and that morphological changes are associated with functional changes in gene expression. Though often manipulated as part of normal fertility treatment, we do not have a good understanding of how these hormones facilitate uterine preparedness for implantation and pregnancy in felines. Chapter 4 thus identifies specific mechanisms of reproductive modulation in the feline uterus and also beings to flesh out molecular mechanisms underlying reproductive failure at the organism level. Our results have direct application to endangered felid breeding programs, which utilize in vitro follicle maturation and in vivo hormones treatments to promote breeding success of genetically-important individuals. Finally, chapter 5 uses a classic stress physiology paradigm to identify the mechanism(s) by which psychological stress inhibits ovarian steroid synthesis during pregnancy in mice. These experiments demonstrate that stress-dependent inhibition of ovarian function does not occur through classical, hypothalamic inhibition, emphasizing the dynamic shifts in endocrine function across mammalian pregnancy. These studies add to our comparative understanding of reproductive function, which is critical because of its fundamental connection to organismal fitness. Taken together, these studies also highlight the utility of in vitro approaches for experimentally approaching the challenge of connecting molecular variation to organismal function. We summarize the results and their ultimate importance to female reproductive physiology research in Chapter 6.

The gonad specific expression of insulin-like growth factor 3 (igf3) has suggested an important role of igf3 in fish reproduction. In this study, medaka igf3 was isolated and its expression patterns were compared with igf1, and germ cell gene vasa in adult medaka ovary. Molecular cloning and sequencing showed that the open reading frame (ORF) of medaka igf3 was comprised of 501 nucleotides and encoded 166 amino acid residues. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that igf3 RNA was specifically expressed in developing embryos and adult gonads of both sexes. Real time quantitative PCR (q-PCR) indicated that igf3 expression was gradually increased during oogenesis, and reached its highest level at stage V. Using Chromogenic and fluorescent in situ hybridization, igf3 was shown to be present in the germ cells of oogonia and in oocytes at stages I-III, as well as abundant in granulosa cells and theca cells of oocytes at stages IV–V. In addition, igf3 and igf1 were expressed mutually in the outer theca cells of stage V oocytes in ovary. Collectively, we demonstrated that gonad specific igf3 could mark medaka ovarian somatic cells and germ cells. These findings highlight the importance of igf3 during ovarian development.

Heat stress not only affects the physical condition but also affects reproductive performance in sheep. A thorough understanding of the molecular and physiological mechanisms underlying heat stress would certainly improve livestock productivity and provide genetic evaluation ways for heat resistant breeds selection. In this study, 85 Turpan Black sheep, a breed exhibited excellent heat resistance from long-term artificial selection, and 85 heat sensitive Kazakh sheep in Turpan basin were tested for physiological and reproductive performance from July to August in summer. The results showed that the estrus rate was significantly higher in Turpan Black sheep (P 0.05), while the heart rate and respiratory rate of Turpan Black sheep are significantly lower than that of Kazakh sheep (P 0.05). Furthermore, to clarify genes participated in heat stress response, the pituitary, ovarian and hepatic tissues from three Turpan Black sheep and three Kazakh sheep were subjected to RNA-seq. The results indicated that 32, 49 and 69 genes were up-regulated, and 39, 60 and 145 genes were down-regulated in pituitary, ovarian and hepatic tissues in Turpan Black sheep compared with that of the Kazakh sheep, respectively. KEGG and gene set enrichment analysis showed that the differentially expressed genes were mainly involved in signal transduction pathways. In particular, the differentially expressed genes in hepar were enriched in the energy metabolism pathway, while the differentially expressed genes in ovarian tissue were enriched in the ovarium steroidogenesis pathway. In conclusion, our results implied that the pituitary-ovary axis might include hepar as downstream targeted organism in heat resistant regulation. Under heat stress, the signals released from pituitary would impact steroidogenesis in ovary, and further alter energy metabolism in hepar. As we know, this is the first comparative study to investigate the gene expression in multi-tissue in sheep under heat stress.

Dwarf mice are characterized by extremely long lifespan, delayed ovarian ageing, altered metabolism, lower age-related oxidative damage and cancer incidence rate. Snell dwarf, Ames dwarf and growth hormone receptor knockout mice are three commonly used models. Despite studies focusing on ageing and metabolism, the reproductive features of female dwarf mice have also attracted interest over the last decade. Female Snell and Ames dwarf mice have regular oestrous cycles and ovulation rates, as in normal mice, but with a larger ovarian reserve and delayed ovarian ageing. The primordial follicle reserve in dwarf mice is greater than in normal littermates. Anti-Müllerian hormone (AMH) concentration is seven times higher in Ames dwarf mice than in their normal siblings, and ovarian transcriptomic profiling showed distinctive patterns in older Ames dwarf mice, especially enriched in inflammatory and immune response-related pathways. In addition, microRNA profiles also showed distinctive differences in Ames dwarf mice compared with normal control littermates. This review aims to summarize research progress on dwarf mice as models in the reproductive ageing field. Investigations focusing on the mechanisms of their reserved reproductive ability are much needed and are expected to provide additional molecular biological bases for the clinical practice of reproductive medicine in women.

5 fluorouracil (5FU), an antineoplastic drug, is often utilized in the therapeutic regimen for several types of cancer, including the hepatoblastoma in children. The effects of 5FU on the population of ovarian preantral follicles, which is the largest oocyte reservoir, is still poorly understood. The integrity of the ovarian preantral follicle pool is important for lifelong fertility. The better understanding of such effects may favor intervention strategies to protect fertility in 5FU-treated children and women coping with cancer. To analyze the effects of 5FU on isolated murine secondary follicles in vitro, ovaries were collected from young mice (28-30 days old), and secondary follicles were isolated and cultured for 12 days in basic culture medium, with or without 5FU at concentrations of 0.3 mM, 1 mM, 3 mM, 10 mM, and 30 mM. In the in vitro study, we analyzed the percentage of morphologically normal follicles, antrum formation, follicular diameter, and hormone production. On day 12, oocytes were recovered for in vitro maturation. 5FU treatment did not alter the percentage of morphologically normal follicles. On day 12, only 1, 10, and 30 mM 5FU significantly reduced the percentage of antrum. From day 4 onwards, 5FU treatments significantly reduced follicle diameter. The meiosis resumption rate was significantly lower in all 5FU treatments. 5FU concentrations ≥3 mM reduced estradiol levels. In conclusion, 5FU does not affect follicular morphology. However, 5FU deleteriously affects follicular growth, estradiol production, and oocyte maturation in isolated ovarian follicles.

Fibrillin-1 (FBN1) functions as a structural protein in the ovary, while the role of its protein product asprosin remains unknown. Both proteins are encoded by the FBN1 gene and when it is cleaved at the C-terminal end, asprosin is produced. Asprosin is associated with various metabolic parameters and sex-related hormones in women. One goal of this research was to quantify FBN1 and the presumed asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1) mRNA in water buffalo granulosa cells and correlate them to aromatase (CYP19A1) gene expression. A second goal was to determine the effect of asprosin on follicular growth in vivo. In Exp. 1, ovaries were collected from a local slaughterhouse, follicular fluid and granulosa cells from small (6 mm) and large (6-13 mm) follicles were aspirated, cellular RNA extracted for gene expression analysis, data analyzed using ANOVA, and Pearson correlation coefficients were calculated among FBN1, OR4M1, and CYP19A1 gene expression. In Exp. 2, an intra-follicular injection of asprosin (600 ng of asprosin/194 μL of PBS) or vehicle (200 μL of PBS; Controls) was given via the theca layer of the dominant follicle of synchronized cows (n = 5/group) 1 day after injection of PGF2α, follicle sizes were measured daily via transrectal ultrasonography for 3 days, a two-way repeated measures ANOVA was used to determine the effect of asprosin on growth rate of follicles from day 0-2, and Chi-square analysis for the percentage of cows ovulated 2 days following asprosin injections. In Exp. 1, FBN1 mRNA abundance was 1.9-fold greater in cells of follicular aspirates from small than large follicles (P 0.05), but abundance of OR4M1 and CYP19A1 mRNA did not differ (P 0.10) between the two sizes of follicles. Abundance of FBN1 mRNA was positively correlated with CYP19A1 (r = 0.55, P 0.05) and OR4M1 mRNA (r = 0.50, P 0.06) across follicle sizes. In Exp. 2, cows treated with asprosin revealed a greater follicle growth rate from day 0-2 (63.4% increase in diameter) than placebo cows (36.8% increase in diameter) post-injection, and more follicles from asprosin treatment vs. control group (100% vs. 20%; P 0.05) ovulated within 2 days. These findings suggest that FBN1 may be developmentally regulated in follicular cells, and that asprosin may induce follicular growth in buffaloes, but further studies will be required to determine if asprosin directly regulates estradiol production during follicle development.

Recently, we have demonstrated that neonatal exposure to androgen and estrogen agonists or antagonists influenced the number of ovarian follicles in piglets. Since the FOXL2 transcription factor is required for proper ovarian follicle formation and activation, the objective of the study was to examine effects of exposure of the neonatal porcine ovary to testosterone propionate (TP; an androgen), flutamide (FLU; an antiandrogen), 4-tert-octylphenol (OP; compound with estrogenic activity), ICI 182,780 (ICI; an antiestrogen), and methoxychlor (MXC; compound with estrogenic, antiestrogenic and antiandrogenic properties) on FOXL2 expression and expression of its target genes, AMH and CYP19A1. Piglets were injected subcutaneously with TP, FLU, OP, ICI, MXC, or corn oil (control) between postnatal days 1 and 10 (n = 4/each group). Ovaries were excised from the 11-day-old piglets and the expression of FOXL2, AMH and CYP19A1 was examined using immunohistochemistry and/or real-time PCR and Western blot. FOXL2 was localized in stroma cells surrounding egg nests and in granulosa cells. TP, OP and MXC increased both FOXL2 and AMH mRNAs, while FLU and ICI decreased CYP19A1 mRNA. The increased FOXL2 protein abundance was found in all examined groups. In addition, TP, OP, ICI and MXC increased AMH protein abundance, while TP, FLU and OP decreased CYP19A1 protein abundance. In conclusion, neonatal exposure to sex steroid receptor agonists and antagonists increased FOXL2 expression at mRNA and/or protein levels and affected FOXL2 target genes in the ovaries of 11-day-old piglets. Therefore, it seems that impaired ovarian folliculogenesis induced by altered steroid milieu during the neonatal development period in pigs may, at least in part, involve FOXL2.

ATP-binding cassette (ABC) transporters perform multiple functions in reproductive tissues. During ovarian tissue vitrification, the plasma membrane has important functions in the influx or efflux of water, and substances such as cryoprotectants and channel proteins that are required in this process. Thus, the present study aimed to verify the relative abundance of mRNA transcript of ABC transporters ABCB1, ABCG2, and MRP2 after vitrification and in vitro culture (IVC) of ovine ovarian tissue. For this study, the ovarian cortex fragments were proportioned into four groups as fresh control, vitrified control, fresh culture, and vitrified culture groups. After vitrification and in vitro culture, the ovarian tissue was evaluated using morphological procedures. Further, relative abundance of ABCB1, ABCG2, and MRP2 transporter mRNA transcripts in the ovarian cortex subjected to aforementioned treatment conditions were evaluated using qPCR. Our results showed a negative association between degenerated follicles and mRNA transcript abundances of ABCB1 and ABCG2. In addition, the percentage of growing follicles in the ovine ovarian cortex after vitrification was similar to that of the fresh control tissue without in vitro culture. The in vitro culture of fresh and vitrified tissue however, showed a significant decrease in the percentage of growing follicles. To the best of our knowledge, we believe that our data for the first time has studied the relative abundances of ABCB1 and ABCG2 mRNA transcripts in the ovine ovarian cortex. In addition, alterations of these protein channels may be indicative of a deleterious effect of osmotic stress on follicular survival during vitrification. Furthermore, these effects were detectable only after the IVC of the ovarian tissues. Nonetheless, further studies are required to investigate the functions of ABC transporters in ovine folliculogenesis, especially after in vitro culture of ovarian tissue.

Immunotherapy has emerged at the forefront of cancer treatment. Checkpoint inhibitor pembrolizumab (KEYTRUDA), a chimeric antibody which targets programmed cell death protein 1 (PD-1), has been approved by the Food and Drug Administration (FDA) for use in pediatric patients with relapsed or refractory classical Hodgkin’s lymphoma. However, there is currently no published data regarding the effects of pembrolizumab on the ovary of female pediatric patients. In this study, prepubertal immunocompetent and immunodeficient female mice were injected with pembrolizumab or anti-mouse PD-1 antibody. The number of primordial follicles significantly decreased post-injection of both pembrolizumab and anti-mouse PD-1 antibody in immunocompetent mice. However, no changes in follicle numbers were observed in immunodeficient nude mice. Superovulation test and vaginal opening experiments suggest that there is no difference in the number of cumulus–oocyte complexes (COCs) and the timing of puberty onset between the control and anti-mouse PD-1 antibody treatment groups, indicating that there is no effect on short-term fertility. Elevation of pro-inflammatory cytokine TNF-α following COX-2 upregulation was observed in the ovary. CD3+ T-cell infiltration was detected within some ovarian follicles and between stromal cells of the ovaries in mice following treatment with anti-mouse PD-1 antibody. Thus, PD-1 immune checkpoint blockade affects the ovarian reserve through a mechanism possibly involving inflammation following CD3+ T-cell infiltration.

The knowledge of the testicular and ovarian morphology of a particular fish species is of paramount importance. Such analyze enables the development of studies and techniques aiming the improvement of their reproduction, management, commercialization and even their conservation. This study performed the ovarian and testicular characterization of the ornamental Amazon fish Serrapinnus kriegi. A total of three males and three females had their gonads analyzed by optical microscopy. Females present ovaries filled with oocytes in asynchronous development, indicating partial spawning in the species. Moreover, the micropyle and micropilar cell formation was observed in primary growing oocytes, representing a precocious oocyte development; and the zona radiata in the final vitellogenic oocytes is thicker than other related species, evidencing the development of a better protection to the embryos in function of the waters’ turbulence that characterize it spawning sites in the Amazonian streams. The male specimens’ present anastomosed tubular testes with unrestricted spermatogonia spread along the entire seminiferous tubules. The present data elucidate the dynamic of spermatogenesis and oogenesis of an ornamental Amazonian species, through the description of the male and female germ cells development. Resumo O conhecimento da morfologia testicular e ovariana de uma determinada espécie de peixe é de suma importância, pois através destas análises é possível o desenvolvimento de estudos e técnicas visando o melhoramento de sua reprodução, manejo e comercialização e até mesmo auxiliar em sua conservação. Este estudo realizou a caracterização ovariana e testicular do peixe Amazônico ornamental Serrapinnus kriegi. Um total de três machos e três fêmeas tiveram suas gônadas analisadas através de microscopia óptica. As fêmeas apresentam ovários preenchidos por oócitos em desenvolvimento assincrônico, indicando desova parcelada da espécie. Além disso, observou-se a formação de micrópila e célula micropilar em oócitos em crescimento primário, representando o desenvolvimento precoce do oócito; a zona radiata nos oócitos vitelogênicos finais é mais espessa em comparação a outras espécies relacionadas, evidenciando o desenvolvimento de uma melhor proteção aos embriões, em função das águas turbulentas que caracterizam seu local de desova nos córregos amazônicos. Os machos apresentam testículos do tipo tubular anastomosado com espermatogônias irrestritas, espalhadas por todo o túbulo seminífero. Os dados apresentados elucidam a dinâmica da espermatogênese e oogênese de uma espécie de peixe ornamental amazônica, por meio da descrição das células germinativas masculinas e femininas.

Turkey bird is one of the popular poultry species which is reared primarily for meat production and considered as one of the major sources of animal protein. With such importance of this species, this study was designed to investigate the gross and histomorphology of the ovary and oviduct with especial emphasis on sperm-host glands of the turkey hen involving ten mature female turkeys (Meleagris gallopavo). The present study highlighted the distribution pattern of sperm-host glands (SHGs) in the oviduct of turkey hen that has a potential role in producing a fertile egg in poultry industries. The oviduct of turkey consists of the infundibulum, magnum, isthmus, uterus, and vagina which are sole distributors for making nutrition enriched egg. The tissue samples were collected from the ovary, different segments of the oviduct and especially uterovaginal junction (UVJ) and infundiomagnal junction of the oviduct. The ovaries and the oviducts were dissected and fixed in Bouins solution and processed for a light microscopic study. Histologically, the left ovary of turkey consisted of an outer cortex and inner medulla, with different stages of follicles. In all areas of the oviduct except the infundibulum and vagina, the tunica mucosa epithelium was lined with ciliated pseudo stratified columnar epithelium, and the lamina propria-submucosa contained branched tubular glands. Sperm-storage tubules were observed in the uterovaginal junction and infundibulo-magnum junction. These tubules were mostly branched, slightly coiled and extended into the lamina propria from the bases of the mucosal folds. These glands had proximal and distal parts; the proximal part was lined by pseudostratified columnar epithelium and distal part by non-ciliated simple columnar epithelium. The number of sperm host glands was more at uterovaginal junction than infundibulomagnal junction. The sperm-host glands might play a functional role in the storage and release of spermatozoa from the SHGs in response to oviposition or ovulation. The results would help poultry scientists and farmers in developing effective disease control and growth strategies.

The Notch signaling pathway is conserved among mammalian species and controls proliferation, differentiation, and cell death in many organs throughout the body including the reproductive tract. Notch signaling plays critical roles in the development and function of both the male and female reproductive systems. Specifically, within the female reproductive tract, Notch signaling is hormone regulated and mediates key reproductive events important for ovarian and uterine function. In this review, we highlight the tissues that express Notch receptors, ligands, and downstream effectors and distinguish how these molecules regulate reproductive function in male and female mice, non-human primates, and humans. Finally, we describe some of the aberrations in Notch signaling in female reproductive pathologies and identify opportunities for future investigation.

Phthalates are a family of chemicals that can be found in plastic and personal care products used by consumers every day and they are known endocrine disrupting chemicals that can disrupt female reproduction. In previous studies, an environmentally relevant phthalate mixture was shown to affect female reproduction in a transgenerational manner. However, limited information was available on the effect of phthalate mixtures on ovarian steroidogenesis and folliculogenesis. Ovarian steroidogenesis is important for producing hormones needed for reproduction and ovarian regulation, and folliculogenesis is essential for the development of ovarian follicles and successful fertility. Thus, this study tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture adversely affects ovarian steroidogenesis and folliculogenesis in the F1 generation of adult female mice. Pregnant dams (F0 generation) were orally dosed with vehicle control or a phthalate mixture (20 μg/kg/day-500 mg/kg/day) daily from gestational day 10 to birth, and the adult F1 females were the offspring of the dosed dams. The ovaries of the F1 generation were collected at postnatal day 60. One ovary was used for histological examination of the numbers and percent of different follicle types. The other ovary was used to measure expression of theca and granulosa cell enzymes. Additionally, sera were collected for measuring hormone levels. The results indicate that prenatal exposure to the phthalate mixture decreases hormone levels and gene expression, alters the transitioning of follicle types, and leads to a higher incidence of atresia in the F1 generation offspring.

Mitotically active cellular fibroma (MACF) of the ovary is a relatively newly recognized histological disease entity which explains its scarce reporting in literature and unknown prognosis [1]. In this tumor type, high mitotic activity is noticed without severe atypia. These tumors are categorized as benign ovarian tumors with late recurrence, if any. However, we herein report an unusual case of an ovarian mitotically active atypical fibrous tumor case which presented as recurrence within six months of being operated thereby raising suspicion of an element of malignancy. On presentation, the patient suffered from abdominal pain and abdomino-pelvic mass. After imaging confirmed an ovarian neoplastic lesion patient underwent bilateral salpingo-oophorectomy and hysterectomy. Histopathology defined the lesion as “ovarian mitotically active atypical fibrous tumor”. Six months later, abdominal pain recurred for which imagining was done and confirmed a newly developed well defined irregular complex solid cystic mass causing intestinal obstruction giving an impression of a malignant neoplasm, probably ovarian carcinoma. However, histopathology for the new lesion remained the same as was for the previous mass, i.e. mitotically active ovarian fibroma, but with lung and mesenteric lymph nodes metastases. Patient couldn’t be operated on due to her co-morbid conditions, and the mass was vascular. She was given supportive care with consideration to start hormonal therapy and during this period died due to sepsis. This case highlights the importance of having a high suspicion of the possibility of malignancy when mitotically active cellular fibroma manifests as a recurrence of mass as this tumor may tend to have an uncertain malignant potential. Thereby, stressing the importance of long term follow up even after the primary tumor is surgically treated without any surgical difficulty or tumor rupture [2]. Such tumors need to be followed and reported more often to help understanding the way they behave and their management.

In vitro systems capable of reconstituting the process of mouse oogenesis are now being established to help develop further understanding of the mechanisms underlying oocyte/follicle development and differentiation. These systems could also help increase the production of useful livestock or genetically modified animals, and aid in identifying the causes of infertility in humans. Recently, we revealed, using an in vitro system for recapitulating oogenesis, that the activation of the estrogen signaling pathway induces abnormal follicle formation, that blocking estrogen-induced expression of anti-Müllerian hormone is crucial for normal follicle formation, and that the production of α-fetoprotein in fetal liver tissue is involved in normal in vivo follicle formation. In mouse fetuses, follicle formation is not carried out by factors within the ovaries but is instead orchestrated by distal endocrine factors. This review outlines findings from genetics, endocrinology, and in vitro studies regarding the factors that can affect the formation of primordial follicles in mammals.

Tramadol at the dose levels of 1mg and 3mg/100g body weight was administered to normal cycling rats for 20 days through intraperitoneal routes. At autopsy on 21st day significant reduction in the ovarian, uterine and body weight was observed. Histological observations showed decrease in the number and size of Graafian follicles, corpora lutea and increase in the atretic follicles in the ovary. The uterus showed absences of endometrial glands, decrease in the height of myometrium, endometrium and its epithelial cells. The total protein and glycogen content of the ovary and uterus is decreased whereas the cholesterol content is increased. The hypothalamo-hypophyseal gonadal axis is prominent regulator of reproductive activities in animals through neuro-endocrine regulation. In this study action of tramadol on ovary and uterine parameters is discussed.

International audience; Although puberty can occur as early as 14-15 months of age, depending on breed and use, the reproductive career of mares may continue to advanced ages. Once mares are used as broodmares, they will usually produce foals once a year until they become unfertile, and their productivity can be enhanced and/or prolonged through embryo technologies. There is a general consensus that old mares are less fertile, but maternal age and parity are confounding factors because nulliparous mares are usually younger and older mares are multiparous in most studies. This review shows that age critically affects cyclicity, folliculogenesis, oocyte and embryo quality as well as presence of oviductal masses and uterine tract function. Maternal parity has a nonlinear effect. Primiparity has a major influence on placental and foal development, with smaller foals at the first gestation that remain smaller postnatally. After the first gestation, endometrial quality and uterine clearance capacities decline progressively with increasing parity and age, whilst placental and foal birthweight and milk production increase. These combined effects should be carefully balanced when breeding mares, in particular when choosing and caring for recipients and their foals.

Fibroblast growth factors (FGFs) are polypeptides involved in the regulation of oogenesis and folliculogenesis by inducing ovarian mitogenic, homeostatic and angiogenic activity. This study was aimed at determining the localisation of FGF ligands (FGF1 and FGF2) and FGF receptor 2 (FGFR2) in the rat ovary by immunohistochemical analyses, at pregnancy and the postpartum period. During pregnancy and the postpartum period, positive FGF1 immunoreactions were observed in the nucleus and cytoplasm of germinative epithelial cells, granulosa cells of follicles in different developmental stages, theca interna cells, interstitial cells, luteal cells and atretic follicles. FGF2 immunoreactivity was strong in the cytoplasm of the endothelial cells and smooth muscle cells of the ovarian blood vessels and in the smooth muscle cells of the ovarian cortex and medulla. Strong FGFR2 immunoreactivity was observed in the stromal cells surrounding the blood vessels and rete ovarii. Immunoreaction intensity of the FGF1, FGF2 and FGFR2 had relatively similar abundances between the periods examined. Considering that FGFs act as local regulators in oogenesis, folliculogenesis, follicular atresia, ovulation, corpus luteum formation and regression and angiogenesis, this study supports the idea that FGFs may also be involved in these physiological functions in rat ovaries during pregnancy and postpartum period.

Atypical protein serine kinase RIOK3 is involved in cellular invasion and survival. The spatiotemporal expression pattern and regulatory mechanisms controlling expression of Riok3 were investigated in the rat ovary during the periovulatory period. Immature female rats (22–23 days old) were treated with pregnant mare’s serum gonadotropin (PMSG) to stimulate follicular development, followed 48 h later by injection with human chorionic gonadotrophin (hCG). Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration. Both real-time polymerase chain reaction (PCR) and in situ hybridisation analysis revealed that Riok3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. Riok3 expression was induced in theca-interstitial cells at 4 h after hCG. However, the expression of Riok3 mRNA was stimulated in granulosa cells at 8 h. Both protein kinase C inhibitor (GF109203) and the protein kinase A inhibitor (H89) could block the stimulation of Riok3 mRNA by hCG. Furthermore, Riok3 induction is dependent on new protein synthesis. Inhibition of prostaglandin synthesis or progesterone action did not alter Riok3 mRNA expression, whereas inhibition of the epidermal growth factor (EGF) pathway downregulated Riok3 expression. In conclusion, our findings suggest that the induction of the RIOK3 may be important for ovulation and luteinisation.

Context First-degree relatives of women with polycystic ovary syndrome (PCOS) present hormonal and metabolic alterations compared to girls unrelated to PCOS. It is unknown whether glucose intolerance in the PCOS proband confers a more severe metabolic predisposition on their first-degree relatives. Objective To determine whether glucose tolerance status in women with PCOS is associated with worsened glucose metabolism and sex hormone levels in their peripubertal daughters or sisters. Design Cross-sectional study. Setting Seven academic centers in North America, South America, and Europe. Patients Sixty-four pairs of women with PCOS and their daughters or younger sisters aged between 8 and 14 years were recruited. Twenty-five mothers or older sisters with PCOS were glucose intolerant (GI) and 39 were normal glucose tolerant (NGT). Main Outcome Measures Beta-cell function estimated by the insulin secretion-sensitivity index-2 (ISSI-2) during an oral glucose tolerance test and by the disposition index during a frequently sampled IV glucose tolerance test. Free testosterone and 17-hydroxyprogesterone (17-OHP) levels. Results Being related to a GI PCOS proband was associated with a lower ISSI-2 (P-value = 0.032) after adjusting for ethnicity, body mass index z-score, and pubertal stage. They also had higher free testosterone (P-value = 0.011) and 17-OHP levels compared to girls with an NGT proband, the latter becoming significant after adjusting for confounders (P-value = 0.040). Conclusions Compared to first-degree female relatives of women with PCOS and NGT, first-degree relatives of women with PCOS and GI display lower beta-cell function and hyperandrogenemia, putting them at higher risk of GI and PCOS development.

Although it is well known that the ovulation occurs during a period of time after LH surge in dogs, there are few reports of observing the entire process of development, ovulation and luteinization of each follicle. This study aimed to detect the ovulation kinetics by ultrasonography in combination with progesterone monitoring and therefore identify the time-range of the ovulation process in a dog. Daily transabdominal ultrasonography and progesterone monitoring were conducted for 24 natural oestrus cycles of Labrador Retrievers. Ovarian follicles were observed as anechoic structure with contours before ovulation. Ovulation (follicular collapse) was defined as when follicles became cloudy and contours obscure by transabdominal ultrasonography. Ultrasound imaging was capable of identifying the day of ovulation for 94.7% (178/188) of the follicles through the appearance of collapsed follicle or corpus luteum. Ovulation was observed between LH 0 (the day of LH surge) and LH 5, with 48.0%, 33.5% and 15.0% for LH 2, LH 3 and LH 1, respectively. The total number of ovulations on LH 2 and LH 3 accounted for 81.5% (141/173) of the total ovulation in 24 cycles examined. Ovulation occurred in 12 cycles for 2 d and for 3 d in 12 cycles. Seventeen cycles (70.8%) with multiple days of ovulation showed the largest number of ovulations on LH 2. The average follicle diameter 3 d before the LH surge was less than 5 mm, then exceeded 5 mm 2 d before the LH surge. The average follicle diameter at the time of ovulation (follicular collapse) was 6.1 ± 1.0 mm (n = 118). On the day before ovulation, the average diameters of the follicles ovulated on LH 1, LH 2 and LH 3 were 5.0 ± 0.7 mm, 5.8 ± 1.2 mm and 6.2 ± 1.3 mm, respectively. There was a significant difference in the follicle diameter between LH1 and LH2 (p

STUDY QUESTION Can serum anti-Müllerian hormone (AMH) replace polycystic ovary morphology (PCOM) determined by ultrasound as a diagnostic component of polycystic ovary syndrome (PCOS)? SUMMARY ANSWER Despite good correlations between serum AMH and PCOM, the use of a high serum AMH as a proxy for PCOM resulted in the reclassification of PCOS in 5% of study participants, with the main effect being more women identified, although some women previously classified as having PCOS were no longer classified as such. WHAT IS KNOWN ALREADY AMH has been proposed as an alternative to PCOM as a diagnostic component of PCOS. Previous studies are limited by poorly defining PCOS, use of infertile women as comparators, measurement of hormones by immunoassay that lack precision in the female range, low-resolution ovarian ultrasound and inconsistent handling and storage of serum samples. STUDY DESIGN, SIZE, DURATION This is an Australian cross-sectional study of 163 non-healthcare-seeking women. PARTICIPANTS/MATERIALS, SETTING, METHODS Serum AMH was measured by both the Ansh picoAMH assay and the Beckman Coulter Access 2 (BA2) assay, in parallel with androgens measured by liquid chromatography–tandem mass spectrometry, in blood samples of women, not pregnant, breast feeding or using systemic steroids, who also underwent high-resolution ovarian ultrasound. PCOS was determined by the Rotterdam criteria with PCOM defined by the Androgen Excess-PCOS Taskforce recommendation of ≥25 follicles in at least one ovary. Cut-off serum concentrations that best identified women as having PCOM were identified by receiver operator characteristic (ROC) curves. MAIN RESULTS AND THE ROLE OF CHANCE A total of 163 women, mean (SD) age 32.5 (5.5) years, who provided a blood sample and had both ovaries visualized on ultrasound were included in the analysis. Women with isolated PCOM had higher median (range) Ansh AMH and BA2 AMH concentrations than those with no PCOS characteristics [56.9 pmol/l (34.6, 104.2) versus 18.7 (3.2, 50.9), P = 0.002 and 38.5 pmol/l (22.2, 100.2) versus 16.7 (3.5, 38.9), P = 0.002, respectively]. An AMH ≥ 44.0 pmol/l, suggested by the ROC curve, identified 80.6% of women with PCOM, falsely identified 15.2% of women without PCOM as having PCOS and had a positive predictive value of 55.6%. The negative predictive value was 94.9%. An AMH BA2 assay cut-off of ≥33.2 pmol/l provided a sensitivity of 80.6%, a specificity of 79.5% and a positive predictive value for PCOM of 48.1%. The negative predictive value was 94.6% for PCOM. When serum AMH was used in the place of PCOM as a diagnostic criterion for PCOS, the Ansh assay resulted in an additional seven women classified as having PCOS and no longer classified one woman as having PCOS. For the BA2 assay, eight additional and two fewer women were classified as having PCOS. Overall, both assays resulted in six more women being classified as having PCOS. LIMITATIONS, REASONS FOR CAUTION Women with functional hypogonadotrophic hypogonadism were not excluded and may have been misclassified as having an oligo-amenorrhoea-PCOM phenotype. As study participants were predominantly Caucasian/White, our findings cannot be generalized to women of other ethnicities. WIDER IMPLICATIONS OF THE FINDINGS Although serum AMH reflects the number of developing ovarian follicles, the absolute values vary between assays and specific reference ranges for individual assays are required. Irrespective of the assay used, replacing PCOM with serum AMH to diagnose PCOS in a community-based sample altered the number of women classified as having or not having PCOS. Consequently, although overall the risk of women being identified as having PCOS would be increased, some women would no longer be classified as having this condition. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by the Norman Beischer Research Foundation and the Grollo-Ruzzene Foundation. S.R.D. is an NHMRC Senior Principal Research Fellow (Grant No. 1135843). S.R.D. reports unrelated support that includes grants from the NHMRC Australia, personal fees for educational activities from Besins Healthcare, Abbott Chile, BioFemme and Pfizer Australia, personal Advisory Board/consultancy fees from Theramex, Abbott Laboratories, Astellas, Mayne Pharmaceuticals, Roche Diagnostics, Lawley Pharmaceuticals and Que Oncology and has received institutional grant funding from Que Oncology and Ovoca research. The other authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Mulberry-leaf flavonoids (MF), extracted from mulberry leaves, exert antioxidant and hypolipidemic effects. The purpose of this experimental study was to investigate the effects of dietary MF on the ovarian function and liver lipid metabolism of aged breeder hens. We used 270 (60-weeks-old) Qiling breeder hens randomly assigned in 3 treatments with supplemental dietary MF doses (0, 30, 60 mg/kg). The results showed that dietary MF significantly improved the egg-laying rate, followed by the reduced feed conversion rate (FCR) (p 0.05). However, there is no obvious difference in hatchability and fertilised eggs hatchability among the three groups (p 0.05). The level of T-CHO, LDL-C and AKP in serum was reduced, and the HDL-C concentrations were increased by dietary MF (p 0.05). MF treatment also improved the antioxidant capacity and reduced the apoptotic index of the ovary (p 0.05). Additionally, dietary MF significantly increased the serum estradiol (E2) levels (p 0.05) and the transcription level of CYP19A1 and LHR in the ovary (p 0.05). Dietary MF enhanced fatty acid β-oxidation in the liver via up-regulating the mRNA expressions of PPARα and CPT-I (p 0.05). Moreover, the HMF group significantly decreased mRNA expressions of SREBP-1c (p 0.05) and increased mRNA expressions of ERα, VTG-Ⅱ and ApoB in the liver (p 0.05). In conclusion, dietary MF could improve the reproduction performance of aged breeder hens through improving ovary function and hepatic lipid metabolism.

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and that vascular endothelial growth factor A would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later-stage follicles compared to controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, anti-Mullerian hormone, and A4 secretion compared to controls. In trial 2, we tested if vascular endothelial growth factor A isoforms could rescue the High A4 phenotype. High A4 (n = 5) and control (n = 5) ovarian cortex was cultured with (1) PBS, (2) VEGFA165 (50 ng/mL), (3) VEGFA165B (50 ng/mL), or (4) VEGFA165 + VEGFA165B (50 ng/mL each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids and had greater indicators of inflammation compared to controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. The VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFβ secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and may promote folliculogenesis.

Objective The oxidative stress status and changes of chicken ovary tissue after shading were studied, to determine the mechanism of the effect of shading on follicular development. Methods Twenty healthy laying hens (40 weeks old) with uniform body weight and the same laying rate were randomly divided into two groups (the shading group and normal light group). In the shading group, the cage was covered to reduce the light intensity inside the cage to 0 without affecting ventilation or food intake. The normal lighting group received no additional treatment. After 7 days of shading, oxidative stress related indicators and gene expression were detected. Results Analysis of paraffin and ultrathin sections showed that apoptosis of ovarian granulosa cells increased significantly after light shading. ELISA (enzyme linked immunosorbent assay) results revealed that the levels of T-AOC (total antioxidant capacity), MDA (malondialdehyde), SOD (superoxide dismutase), GSH (glutathione), CAT (catalase) and other substances in the sera, livers, ovaries, and follicular granulosa cells of laying hens increased significantly after shading for 7 days; and ROS (reactive oxygen species) levels in the livers of laying hens also increased significantly. ROS in the serum, ovarian and granulosa cell (GCs)also increased. After shading for 7 days, the levels of 8-OHdG (8-hydroxy-2 deoxyguanosine) in the sera and ovarian tissues of laying hens increased significantly. CCK-8 (cell counting kit-8) detection showed that the proliferation activity of GCs in layer follicles decreased after shading for 7 days; the expression level of the anti-apoptotic gene BCL2 (B-cell lymphoma-2) in ovarian tissue and follicular GCs was significantly reduced, and the expression levels of pro-apoptotic casp3 (caspase 3) and SOD, GPX2 (glutathione peroxidase 2) and CAT were all significantly increased. Conclusion Oxidative stress induced by shading light has a serious inhibitory effect on follicular development during reproduction in laying hens.

Background Insulin resistance and hormonal imbalances are key features in the pathophysiology of polycystic ovarian syndrome (PCOS). We have previously shown that Ficus deltoidea var. deltoidea Jack (Moraceae) can improve insulin sensitivity and hormonal profile in PCOS female rats. However, biological characteristics underpinning the therapeutic effects of F. deltoidea for treating PCOS remain to be clarified. This study aims to investigate the biochemical, hormonal, and histomorphometric changes in letrozole (LTZ)-induced PCOS female rats following treatment with F. deltoidea. Methods PCOS was induced in rats except for normal control by administering LTZ at 1 mg/kg/day for 21 days. Methanolic extract of F. deltoidea leaf was then orally administered to the PCOS rats at the dose of 250, 500, or 1000 mg/kg/day, respectively for 15 consecutive days. Lipid profile was measured enzymatically in serum. The circulating concentrations of reproductive hormone and antioxidant enzymes were determined by ELISA assays. Ovarian and uterus histomorphometric changes were further observed by hematoxylin and eosin (H&E) staining. Results The results showed that treatment with F. deltoidea at the dose of 500 and 1000 mg/kg/day reduced insulin resistance, obesity indices, total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL), malondialdehyde (MDA), testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) to near-normal levels in PCOS rats. The levels of high-density lipoprotein cholesterol (HDL), estrogen, and superoxide dismutase (SOD) are also similar to those observed in normal control rats. Histomorphometric measurements confirmed that F. deltoidea increased the corpus luteum number and the endometrial thickness. Conclusions F. deltoidea can reverse PCOS symptoms in female rats by improving insulin sensitivity, antioxidant activities, hormonal imbalance, and histological changes. These findings suggest the potential use of F. deltoidea as an adjuvant agent in the treatment program of PCOS.

Cryopreservation of ovarian tissue followed by transplantation represents a strategy to restore ovarian function and fertility. Stress from cryopreservation-thawing processes can lead to alterations and/or damage to mitochondrial structure and functionality. High resolution respirometry and histological analysis were used to evaluate the effect of cryopreservation and transplantation on ovarian tissue. Four different conditions were performed: Fresh non-transplanted tissue, Fresh transplanted tissue, Cryopreserved non-transplanted tissue and Cryopreserved transplanted tissue. All groups were able to respond to the substrates-uncoupler-inhibitor protocol. We found a dramatic decrease in general oxygen consumption in hemi-ovaries submitted to cryopreservation and/or transplantation. The effect of cryopreservation on mitochondrial metabolism was less intense than effect of transplantation, since the transplantation affected all of the mitochondrial states. A total of 2644 follicles were analyzed. Of these, 2198 were classified as morphologically normal. The percentage of morphologically normal follicles was significantly lower in the Cryopreserved transplanted group when compared to the Cryopreserved non-transplanted group and the Fresh transplanted group (p-value

Red palm weevil [Rhynchophorus ferrugineus (Olivier)], is native to South Asia and expanding its distribution range globally. Recent invasions of red palm weevil around the world, including Saudi Arabia, has become a global constraint for the production of palm species. Although, several control measures have been tested, none of them seemed successful against this invasive species. Therefore, we focused on silencing the reproduction control gene vitellogenin (Vg) based on RNA interference (RNAi) strategy for its possible application in the management of R. ferrugineus. The Vg is a major yolk protein precursor critical for oogenesis. To do this, fat body transcriptome of R. ferrugineus female adults was sequenced, which provided partial Vg gene transcript (FPKM 5731.60). A complete RfVg gene transcript of 5504 bp encoding 1787 amino acids was then sequenced using RCAE-PCR strategy and characterized. Phylogenetic analysis suggested that RfVg has closer ancestry to the coleopteran insects. The RfVg-based RNAi significantly suppressed the expressions of Vg gene. The 15, 20 and 25 days post-injection periods suppressed Vg expressions by 95, 96.6 and 99%, respectively. The suppressed Vg expressions resulted in the dramatic failure of Vg protein expression, which caused atrophied ovaries or no oogenesis and ultimately eggs were not hatched. These results suggest that knockdown of Vg gene involved in R. ferrugineus reproduction is a promising target for RNAi-based management of R. ferrugineus.

Cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (BMPR-IB), follicle-stimulating hormone (FSHR), and inhibin α (INHA), were determined in granulosa cells. Results showed that CTSD was expressed in corpus luteum, follicle, and granulosa cells. Notably, CTSD expression in the monotocous group was significantly higher than that in the polytocous group. In addition, CTSD knockdown could improve granulosa cell proliferation, inhibit cell apoptosis, and significantly elevate the expression of proliferating cell nuclear antigen (PCNA) and B cell lymphoma 2 (Bcl-2), but it lowered the expression of Bcl-2-associated X (Bax) and caspase-3. Furthermore, CTSD knockdown significantly reduced the ratios of cells in the G0/G1 and G2/M phases but substantially increased the ratio of cells in the S phase. The expression levels of cyclin D2 and cyclin E were elevated followed by the obvious decline of cyclin A1 expression. However, the expression levels of BMPR-IB, FSHR, and INHA clearly increased as a result of CTSD knockdown. Hence, our findings demonstrate that CTSD is an important factor affecting the litter size trait in goats by regulating the granulosa cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific trait.

The high rate of SARS-CoV-2 infection poses a serious threat to public health. Previous studies have suggested that SARS-CoV-2 can infect human ovary, the core organ of the female reproductive system. However, it remains unclear which type of ovarian cells are easily infected by SARS-CoV-2 and whether ovarian infectivity differs from puberty to menopause. In this study, public datasets containing bulk and single-cell RNA-Seq data derived from ovarian tissues were analyzed to demonstrate the mRNA expression and protein distribution of the two key entry receptors for SARS-CoV-2—angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2). Furthermore, an immunohistochemical study of ACE2 and TMPRSS2 in human ovaries of different ages was conducted. Differentially expressed gene (DEG) analysis of ovaries of different ages and with varying ovarian reserves was conducted to explore the potential functions of ACE2 and TMPRSS2 in the ovary. The analysis of the public datasets indicated that the co-expression of ACE2 and TMPRSS2 was observed mostly in oocytes and partially in granulosa cells. However, no marked difference was observed in ACE2 or TMPRSS2 expression between young and old ovaries and ovaries with low and high reserves. Correspondingly, ACE2 and TMPRSS2 were detected in the human ovarian cortex and medulla, especially in oocytes of different stages, with no observed variations in their expression level in ovaries of different ages, which was consistent with the results of bioinformatic analyses. Remarkably, DEG analysis showed that a series of viral infection-related pathways were more enriched in ACE2-positive ovarian cells than in ACE2-negative ovarian cells, suggesting that SARS-CoV-2 may potentially target specific ovarian cells and affect ovarian function. However, further fundamental and clinical research is still needed to monitor the process of SARS-CoV-2 entry into ovarian cells and the long-term effects of SARS-CoV-2 infection on the ovarian function in recovered females.

Excessive autophagy-induced follicular atresia of ovarian granulosa cells might be one of the pathogenesis of Premature Ovarian Insufficiency (POI), and melatonin (MT) exerted many beneficial effects on mitochondria. However, there was little report regarding the beneficial effects of MT on excessive autophagy-induced mitochondrial and ovarian reserve function deficiency, and the mechanisms have not been clearly identified. Autophagy played a protective role in cells survival, however, high level of autophagy could lead to cell death. In this report, firstly, Chinese hamster ovary cell damage model stably expressing EGFP-LC3 was established. Next, we systematically investigated the protective effects of MT on mitochondrial and ovarian reserve function and molecular mechanisms using this cell damage model. Our results revealed that 10-9 M MT not only protected against the decline of anti-mullerian hormone (AMH) expression induced by excessive autophagy, but also rescued excessive autophagy-induced impairment of mitochondrial expression and mitochondrial membrane potential. Furthermore, MT protected against excessive autophagy-induced decrease of nucleus-encoded proteins including SDHA and mitofilin, and mitochondrial dynamic-related proteins including OPA1, MFN2, and DRP1. MT also decreased mitochondrial oxidative stress, increased antioxidant enzyme superoxide dismutase 2 (SOD2) expression and ameliorated the G2/M cell cycle arrest induced by excessive autophagy. Finally, MT inhibited excessive autophagy-induced activation of extracellular signal regulated kinase (ERK) signaling pathway. In conclusion, our study showed that MT rescued impairment of mitochondrial and ovarian reserve function, and production of mitochondrial ROS and cell cycle arrest induced by excessive autophagy through down-regulated ERK pathway, implying the potential therapeutic drug target for POI.

The ovaries play a critical role in female reproductive health because they are the site of oocyte maturation and sex steroid hormone production. The unique cellular processes that take place within the ovary make it a susceptible target for chemical mixtures. Herein, we review the available data regarding the effects of chemical mixtures on the ovary, focusing on development, folliculogenesis, and steroidogenesis. The chemical mixtures discussed include those to which women are exposed to environmentally, occupationally, and medically. Following a brief introduction to chemical mixture components, we describe the effects of chemical mixtures on ovarian development, folliculogenesis, and steroidogenesis. Further, we discuss the effects of chemical mixtures on corpora lutea and transgenerational outcomes. Identifying the effects of chemical mixtures on the ovaries is paramount to preventing and treating mixture-inducing toxicity of the ovary that has long-term consequences such as infertility and ovarian disease.

Research question What is the impact of radiation exposure on oocyte quality and female fertility? Design Prepubertal mice underwent whole-body irradiation with a single dose (0.02, 0.1, 0.5, 2, 8 Gy) of gamma- or X-rays. Oocytes were quantified in irradiated (n = 36) and sham-treated (n = 8) mice. After a single exposure to 2 Gy, formation of DNA double-strand breaks (n = 10), activation of checkpoint kinase (Chk2) (n = 10) and dynamics of follicular growth (n = 18) were analysed. Fertility assessment was performed in adult irradiated mice and controls from the number of pups per mouse (n = 28) and the fetal abortion rate (n = 24). Ploidy of mature oocytes (n = 20) was analysed after CREST immunostaining, and uterine sections were examined. Results Radiation exposure induced a massive loss of primordial follicles with LD50 below 50 mGy for both gamma and X-rays. Growing follicles survived doses up to 8 Gy. This difference in radiosensitivity was not due to a different amount of radio-induced DNA damage, and Chk2 was activated in all oocytes. Exposure to a 2 Gy dose abolished the long-term fertility of females due to depletion of the ovarian reserve. Detailed analysis indicates that surviving oocytes were able to complete folliculogenesis and could be fertilized. This transient fertility allowed irradiated females to produce a single litter albeit with a high rate of fetal abortion (23%, P = 0.0096), related to altered ploidy in the surviving oocytes (25.5%, P = 0.0035). Conclusions The effects of radiation on surviving oocyte quality question natural conception as a first-line approach in cancer survivors. Together, the data emphasize the need for fertility preservation before radiation exposure and call for reassessment of the use of cryopreserved oocytes.

There is a worldwide trend for women to have their first pregnancy later in life. However, as oocyte quality declines with maternal aging, this trend leads to an increase in subfertility. The cellular mechanisms underlying this decline in oocyte competence are poorly understood. Oocyte mitochondria are the subcellular organelles that supply the energy that drives early embryogenesis, and thus their quality is critical for successful conception. Mitochondria contain their own DNA (mtDNA) and mutations in mtDNA cause mitochondrial diseases with severe symptoms, such as neurodegeneration and heart disease. Since mitochondrial function declines in tissues as humans age accompanied by an accumulation of mtDNA mutations, mtDNA is implicated as a cause of declining oocyte quality in older mothers. While this mutation load could be caused by declining accuracy of the mitochondrial replisome, age-related decline in mitochondrial quality control likely contributes, however knowledge is lacking. Mitophagy, a cellular process which specifically targets and recycles damaged mitochondria may be involved, but studies are scarce. And although assisted reproductive technologies can help older mothers, how these techniques affect the mechanisms that regulate mitochondrial and oocyte quality have not been studied. With the long-term goal of understanding the molecular mechanisms that control mitochondrial quality in the oocyte, model systems including Drosophila and mouse as well as human oocytes have been used. In this review, we explore the contribution of mitophagy to oocyte quality and the need for further systematic investigation in oocytes during maternal aging using different systems. Lay summary Mitochondria are small parts of cells called organelles that generate the chemical energy needed for life. Hundreds of thousands of mitochondria in the developing eggs of the mother support the initial growth and development of the fertilized egg. However, due to increasingly diminished function over time, mitochondria generate less energy as we age, posing real problems for older women considering pregnancy. It is possible that this declining energy could be responsible for declining fertility as women age. Energy may decline because mitochondria fail and the cell’s way of keeping them healthy become less efficient as we age. This review summarizes what is known about mitochondrial quality control in developing eggs as they age. In the future, understanding how the best mitochondria are selected and maintained in the egg, and hence the future baby, may enable older women with or without mitochondrial problems, to have healthy children.

Background The ewe lamb nutritional and physiological state interfere with the ovarian environment and fertility. The lack or excess of circulating nutrients reaching the ovary can change its gene expression. A protein deficiency in the blood caused by an Haemonchus contortus abomasal infection is detrimental to the organism’s development during puberty. The peripubertal period is a time of intensive growth that requires a high level of nutrients. An essential feature controlling pubertal arousal and female reproductive potential is ovarian follicle growth activation. Protein supplementation improves the sheep’s immune response to helminthic infections. We aimed to determine if supplementing protein in infected ewe lambs’ diet would impact the ovarian environment leading to earlier ovarian follicle activation than in infected not supplemented animals. Methods We fed 18 Santa Ines ewe lambs (Ovis aries) - bred by the same ram - with either 12% protein (Control groups) or 19% protein (Supplemented groups) in their diets. After 35 days of the diet, they were each artificially infected or not with 10,000 Haemonchus contortus L3 larvae. Following 77 days of the diet and 42 days of infection, we surgically collected their left ovaries and examined their genes expression through RNA sequencing. Results We found that protein supplementation in infected animals led to an up-regulation of genes (FDR p-values Conclusion Diet’s protein supplementation of peripubertal infected animals allowed them to express genes related to a more mature ovarian follicle stage than their half-sisters that were not supplemented. These results could be modelling potential effects of the interaction between environmental factors, nutrition and infection on reproductive health. When ovarian activation is achieved in a timely fashion, the ewe may generate more lambs during its reproductive life, increasing sheep breeders’ productivity.

SUMMARY OBJECTIVE: This study evaluates the effects of a ketogenic diet on morphology and follicle reserve. METHOD: Sixteen Sprague-Dawley rats were randomized into two groups: standard diet group (n=8) and ketogenic diet group (n=8). Rats were time mated. Dams were permitted to deliver spontaneously. The animals were monitored for the onset of puberty. All the rats were weighed and anesthetized, serum anti-Müllerian hormone level was measured, and the oviducts were removed. The morphological characteristics of follicles were determined and total ovarian volumes were calculated. RESULTS: The mean ovarian volume was statistically significantly lower in the ketogenic diet group compared to the standard diet group (14.41±0.99 mm3 versus 18.89±1.28 mm3) (p=0.000). The mean number of antral follicles was 13.63±1.80 in the standard diet group and 4.462±0.760 in the ketogenic diet group. The mean ovarian weight of the ketogenic diet group was significantly lower than that of the standard diet group (0.42±0.06 g versus 0.815±107 g). The mean anti-Müllerian hormone levels were significantly higher in the standard diet group compared to the ketogenic diet group (1.023±4.75 ng/mL versus 0.69±0.07 ng/mL) (p=0.000). The mean percentage of staining of Ki-67 was 35.28±4.75 in the standard diet group and 16.98±3.33 in the ketogenic diet group (p=0.000). CONCLUSION: Maternal ketogenic diet reduces ovarian follicular reserve in female offspring and has important implications for maintaining reproductive potential at a population level.

Germ cells of many organisms exhibit a unique perinuclear cytoplasmic cloud-like structure called the chromatoid body (CB). Specific components of the CB, such as vasa, are crucial for germline development. Herein we identify another component of the CB, the Oryzias latipes gcnf homolog (Olgcnf). By reverse transcription polymerase chain reaction (RT-PCR), Olgcnf transcript was restricted to gonads in the adult tissues and was maternally provided and persisted throughout embryogenesis. By in situ hybridization on gonadal sections, Olgcnf transcript was restricted to germ cells. In the ovary, Olgcnf was predominantly expressed in the early stages of oocytes. In the testis, Olgcnf showed dynamic expression in round spermatids and sperm, accumulated in the CB during spermatid morphogenesis and concentrated in elongated spermatids of acrosome cap region and flagellar manchette. Moreover, Olgcnf was colocalized with vasa, which is the best-characterized component of the CB. Therefore, Olgcnf is specific to germ cells of both sexes and is a component of the CB during spermiogenesis.