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Cotton rats (Sigmodon hispidus, CRs) are commonly used as animal models in biomedical research. However, the reproductive characteristics and ovarian development in the CRs has not been widely investigated. We have previously shown that female CRs, in particular, show several unique phenotypes associated with the urogenital system, such as chronic kidney disease and pyometra. Our investigation revealed unique morphologies in CR ovaries, particularly in oocytes. Cotton rat ovaries at 6–8 weeks of age were obtained from the Hokkaido Institute of Public Health, and their sections analyzed by light microscopy and transmission electron microscopy. Although the general histology and folliculogenesis of CR ovaries were similar to those of other experimental rodents, multi-oocyte follicles (MOFs) and double nucleated oocytes (DNOs) were also observed. Although MOFs were found at all stages of follicular development, a greater frequency of MOFs was observed in the primary and secondary stages. However, DNOs tended to be frequently observed in primordial follicles. Almost all MOF oocytes and a few DNOs possessed a clear zona pellucida, expressed DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 and Forkhead box protein 2, a representative marker of oocytes and follicular epithelial cells. Thus, our investigations revealed the unique phenotypes of the CR ovary. As MOFs and DNOs are occasionally observed in human patients with infertility, the CR would be a useful animal model to study for gaining a better understanding of folliculogenesis and oocytogenesis, as well as their abnormalities in humans and other animals.

Abstract Granulosa cells (GCs) are somatic cells surrounding oocytes within follicles and are essential for folliculogenesis. Pathological changes in GCs are found in several ovarian disorders. Recent reports have indicated that long non-coding RNAs (lncRNAs), which modulate gene expression via multiple mechanisms, are key regulators of the normal development of GCs, follicles, and ovaries. In addition, accumulating evidence has suggested that lncRNAs can be utilized as biomarkers for the diagnosis and prognosis of GC-related diseases, such as polycystic ovary syndrome (PCOS) and premature ovarian insufficiency (POI). Therefore, lncRNAs not only play a role in GCs that are involved in normal folliculogenesis, but they may also be considered as potential candidate biomarkers and therapeutic targets in GCs under pathological conditions. In the future, a detailed investigation of the in vivo delivery or targeting of lncRNAs and large-cohort-validation of the clinical applicability of lncRNAs is required.

Gametogenesis, the production of eggs and sperm, is a fundamental process in sexually reproducing animals. Following gametogenesis commitment and sexual fate decision, germ cells undergo several developmental processes to halve their genomic size and acquire sex-specific characteristics of gametes, including cellular size, motility, and cell polarity. However, it remains unclear how different gametogenesis processes are initially integrated. With the advantages of the teleost fish medaka (Oryzias latipes), in which germline stem cells continuously produce eggs and sperm in mature gonads and a sexual switch gene in germ cells is identified, we found that distinct pathways initiate gametogenesis cooperatively after commitment to gametogenesis. This evokes the concept of functional modules, in which functionally interlocked genes are grouped to yield distinct gamete characteristics. The various combinations of modules may allow us to explain the evolution of diverse reproductive systems, such as parthenogenesis and hermaphroditism.

Oocyte maturation is a complex and dynamic process regulated by the coordination of ovarian cells and numerous extraovarian signals. From mammal studies, it is learnt that lipid metabolism provides sufficient energy for morphological and cellular events during folliculogenesis, and numerous lipid metabolites, including cholesterol, lipoproteins, and 14-demethyl-14-dehydrolanosterol, act as steroid hormone precursors and meiotic resumption regulators. Endogenous and exogenous signals, such as gonadotropins, insulin, and cortisol, are the upstream regulators in follicular lipid metabolic homeostasis, forming a complex and dynamic network in which the key factor or pathway that plays the central role is still a mystery. Though lipid metabolites are indispensable, long-term exposure to a high-fat environment will induce irreversible damage to follicular cells and oocyte meiosis. This review specifically describes the transcriptional expression patterns of several lipid metabolism–related genes in human oocytes and granulosa cells during folliculogenesis, illustrating the spatiotemporal lipid metabolic changes in follicles and the role of lipid metabolism in female reproductive capacity. This study aims to elaborate the impact of lipid metabolism on folliculogenesis, thus providing guidance for improving the fertility of obese women and the clinical outcome of assisted reproduction.

Polycystic ovary syndrome (PCOS) is a complex reproductive and endocrine disorder affecting 5-10% of women of reproductive age, but the pathophysiology of PCOS still remains unknown. Here, the aim of our study was to analyze the effects of rapamycin treatment that may regulate impaired hormonal levels and folliculogenesis in dehydroepiandrosterone (DHEA)-treated PCOS mouse. We hypothesized that rapamycin may ameliorate the negative effects of PCOS in DHEA-induced PCOS mouse model. The target of rapamycin (TOR) gene product is a serine/threonine kinase that has been implicated in the control of cell growth, proliferation and autophagy, and rapamycin is a potent inhibitor of mTORC1 pathway. In this study, for the first time, mTORC1 and activation products are presented at protein and mRNA levels after rapamycin treatment in DHEA-induced PCOS mouse ovary. We showed that rapamycin treatment may regulate follicular development, hormonal levels and provide ovulation in DHEA-induced PCOS mouse. Additionally, we assessed decreased primordial follicle reserve, increased number of primary and secondary follicles, corpus luteum structure forms again after 10 days of rapamycin treatment. This study presented here suggests rapamycin treatment regulates hormonal phenotype and folliculogenesis in the ovary and also mTOR signalling pathway in granulosa cells of DHEA-induced PCOS mouse ovary which may have potential to attenuate understanding the mechanism of dominant follicle selection and anovulatory infertility.

The preceding decades have seen an extensive emergence of the harmful effects of tobacco smoke on systemic health. Among the various compounds of tobacco, nicotine is one of the principal, potentially hazardous, and toxic components which is an oxidant agent that can affect both men's and women's fertility. Nicotine exerts its effect by modulating the expression of transmembrane ligand-gated ion channels called nicotinic acetylcholine receptors. The activities of female reproduction might be disrupted by exposure to nicotine at various sites, such as the ovary or reproductive tract. It's been demonstrated that nicotine might cause oxidative stress, apoptosis, hormonal imbalance, abnormalities in chromosomal segregation, impact oocyte development, and disruption in ovarian morphology and functions. This review paper summarizes the findings and provides an updated overview of the evidence on the harmful effects of nicotine use on women's reproductive health and the resulting detrimental impacts on the body. Additionally, it provides the detailed possible mechanisms involved in impairing reproductive processes like folliculogenesis, oocyte maturation, steroidogenesis, and pregnancy in different animal species., (© 2024 John Wiley & Sons Ltd.)

Folliculogenesis is controlled by intimate communications between oocytes and surrounding follicle cells. Epidermal growth factor (EGF/Egf) is an important paracrine/autocrine factor in vertebrate ovary, and it is well known for its stimulation of oocyte maturation. However, the role of EGF signaling through its receptor (EGFR/Egfr) in ovarian folliculogenesis is poorly understood, especially at early stages of follicle development. In this study, we created zebrafish mutants for Egf (egf−/−) and Egfr (egfra−/− and egfrb−/−) by CRISPR/Cas9 technique. Surprisingly, these mutants all survived well with little abnormality in growth and development. Spermatogenesis and folliculogenesis were both normal in egf−/− males and females. Their fecundity was comparable to that of the wildtype fish at 4 months post-fertilization (mpf); however, the fertilization rate of mutant eggs (egf−/−) decreased significantly at 7 mpf. Interestingly, disruption of egfra (egfra−/−) led to failed follicle activation with folliculogenesis being blocked at primary–secondary growth transition (PG-SG transition), leading to female infertility, whereas the mutant males remained fertile. The mutant ovary (egfra−/−) showed abnormal expression of a substantial number of genes involved in oxidative metabolism, gene transcription, cytomembrane transport, steroid hormone biosynthesis, and immune response. The stunted PG oocytes in egfra−/− ovary eventually underwent degeneration after 6 months followed by sex reversal to males with functional testes. No abnormal phenotypes were found in the mutant of truncated form of EGFR (egfrb). In summary, our data revealed critical roles for EGFR signaling in early folliculogenesis, especially at the PG-SG transition or follicle activation.

The occurrence of two antral follicle recruitment waves in a single inter-ovulatory interval has been detected in ovaries of normal women. This data supports the claim that a double ovarian stimulation in the same cycle may benefit poor responder patients with an increased recovery of mature oocytes and good quality embryos per single cycle. The double stimulation protocol was the object of several published studies in which, surprisingly, the mechanism and the safety of the double stimulation in the same cycle were poorly addressed. We propose that in the double stimulation protocol, the first stimulation impacts more committed oocytes progenitors ready to differentiate into mature oocytes. Conversely, the protracted exposure of developmentally earlier less-committed ovarian stem cells to FSH, which occurs in the double stimulation protocol, impacts the less differentiated stem cells which take longer to differentiate into oocytes. The proposed mechanism has broad implications for the safety of the double stimulation strategy. [ABSTRACT FROM AUTHOR]

Apoptosis is the cellular mechanism of ovarian follicular atresia in mammals; the aim of this study was to examine the apoptosis‐related cyclic changes in follicular cells of different‐sized antral follicles throughout the oestrous cycle in canines. Ovaries were collected from 26 adult female dogs (1–4 years) following routine ovariohysterectomy. Antral follicles were classified as small, medium or large antral or preovulatory. The percentage of apoptotic cells was determined flow cytometrically using the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate (dUTP) DNA nick end labelling (TUNEL) assay. Apoptosis rate was quantified as the percentage of TUNEL‐positive cells on a logarithmic scale. Percentages of TUNEL‐positive cells obtained in the flow cytometric assay were compared among oestrous phases and follicular sizes using analysis of variance. Apoptotic follicles were observed in all types of canine follicles in different cycle phases and stages of development, possibly corresponding to the physiological process of the oestrous cycle. Both the oestrous phase and follicular size significantly influenced the apoptosis rate (p <.05). Apoptosis rate increased significantly (p <.05) as follicular development progressed. Apoptosis rate was the highest in large follicles during the oestrous phase (9.2%; p <.05) and the lowest in small follicles during the anestrus period (1.8%; p <.05). In conclusion, our results demonstrate significant differences in the apoptosis rate during the oestrous cycle related to follicle development in the canine ovary. Furthermore, flow cytometry using the TUNEL assay was found to be an effective method for detecting apoptosis in canine follicles. [ABSTRACT FROM AUTHOR]

It is well known that oocyte quality is essential in the determination of the developmental potential of the fertilized oocytes. The oocyte developmental potential decreases in all species with increasing age. Use of serum and follicular fluid concentration of GDF9 as biomarkers of oocyte and embryo quality and their relation to pregnancy rate. Eighty-eight women were included in this study are selected from those undergoing intra-cytoplasmic sperm injection. Positive pregnancy was achieved by 14 women. The difference in mean serum and follicular GDF9 between pregnant and non-pregnant women was not significant. MI oocyte count was not significantly correlated to serum and follicular GDF9 (p > 0.05). MII oocyte count showed a nonsignificant correlation to serum and follicular GDF9 (p > 0.05). Grade 1 embryo count showed a non-significant correlation to serum and follicular GDF9. Grade 2 embryo count showed a non-significant correlation to serum and follicular GDF9. Also, grade 3 embryo count showed a non-significant correlation to serum and follicular GDF9. The current study revealed that serum and follicular GDF9 could not be used as indicators for oocyte maturity, embryo quality, or pregnancy rate. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The molecular mechanisms regulating follicular development and ensuring primordial follicle activation remain undefined. To help elucidate these mechanisms, this proteomic study of bovine ovarian tissue identified the differential molecular profiles of preantral follicles together with the spatial distribution of the most abundant molecular components in the tissue. Isolated primordial, primary and secondary follicles were individually placed on a MALDI target plate for mass spectral acquisitions, with detection of different m/z ranges. Ovarian tissue was sectioned and analysed in the m/z 400–2,000 range. Results of the first analysis indicated a similarity pattern in the molecular protein profile among different follicular classes in the m/z ranges of 100–1000 and 25,000–200,000, but in the m/z ranges of 800–4000, 4000–20,000 and 15,000–70,000, primary and secondary follicles shared similar clustering profiles which were different from primordial follicles (p <.05). In the second analysis, it was possible to correlate some intense molecular components in the tissue from global mass spectrum with the ions detected in the first analysis. Molecular components at m/z 11,325 (±230) were also detected in primary and secondary follicles in the experiment with isolated follicles, in addition to ions at m/z 4,029 (±120), 13,799 (±70), 5,547 (±9), 15,313 (±200), 7,018 (±40) and 7,663 (±90) which were also intensely detected in primary and secondary follicles. The present proteomic approaches evaluated different mass ranges of preantral follicles in bovine ovarian tissue and also indicated the spatial distribution of the most abundant molecular components. This study hopes to pave the way for future research identifying and characterizing specific proteins involved in follicle activation in bovine follicles, in order to better understand folliculogenesis and potentially improve mammalian follicle culture systems. [ABSTRACT FROM AUTHOR]

Follicular thyroid tissue originates from progenitors derived from a midline endodermal primordium. Current understanding infers that folliculogenesis in the embryonic thyroid designates the latest morphogenetic event taking place after the final anatomical shape and position of the gland is established. However, this concept does not consider the fact that the thyroid isthmus develops chronologically before the lobes and also contains all progenitors required for lobulation. To elucidate whether cells committed to a thyroid fate might be triggered to differentiate asynchronously related to maturation and developmental stage, mouse embryonic thyroid tissues from E12.5-17.5 were subjected to immunofluorescent labeling of biomarkers (progenitors: NKX2-1; differentiation: thyroglobulin/TG); folliculogenesis: E-cadherin/CDH1; luminogenesis: mucin 1/MUC1; apical polarity: pericentrin/PCNT; basement membrane: laminin; growth: Ki67), quantitative RT-PCR analysis (Nkx2.1 , Tg , Muc1) and transmission electron microscopy. Tg expression was detectable as early as E12.5 and gradually increased >1000-fold until E17.5. Muc1 and Nkx2.1 transcript levels increased in the same time interval. Prior to lobulation (E12.5-13.5), MUC1 and TG distinguished pre-follicular from progenitor cells in the developing isthmus characterized by intense cell proliferation. Luminogenesis comprised redistribution of MUC1+ vesicles or vacuoles, transiently associated with PCNT, to the apical cytoplasm and the subsequent formation of MUC1+ nascent lumens. Apical polarization of pre-follicular cells and lumen initiation involved submembraneous vesicular traffic, reorganization of adherens junctions and ciliogenesis. MUC1 did not co-localize with TG until a lumen with a MUC1+ apical membrane was established. MUC1 delineated the lumen of all newly formed follicles encountered in the developing lobes at E15.5-17.5. Folliculogenesis started before establishment of a complete follicular basal lamina. These observations indicate that embryonic thyroid differentiation is an asynchronous process consistent with the idea that progenitors attaining a stationary position in the connecting isthmus portion undergo apical polarization and generate follicles already at a primordial stage of thyroid development, i.e. foregoing growth of the lobes. Although the thyroid isthmus eventually comprises minute amounts of the total thyroid volume and contributes little to the overall hormone production, it is of principal interest that local cues related to the residence status of cells – independently of a prevailing high multiplication rate – govern the thyroid differentiation program. [ABSTRACT FROM AUTHOR]

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Follicular thyroid tissue originates from progenitors derived from a midline endodermal primordium. Current understanding infers that folliculogenesis in the embryonic thyroid designates the latest morphogenetic event taking place after the final anatomical shape and position of the gland is established. However, this concept does not consider the fact that the thyroid isthmus develops chronologically before the lobes and also contains all progenitors required for lobulation. To elucidate whether cells committed to a thyroid fate might be triggered to differentiate asynchronously related to maturation and developmental stage, mouse embryonic thyroid tissues from E12.5-17.5 were subjected to immunofluorescent labeling of biomarkers (progenitors: NKX2-1; differentiation: thyroglobulin/TG); folliculogenesis: E-cadherin/CDH1; luminogenesis: mucin 1/MUC1; apical polarity: pericentrin/PCNT; basement membrane: laminin; growth: Ki67), quantitative RT-PCR analysis (Nkx2.1, Tg, Muc1) and transmission electron microscopy. Tg expression was detectable as early as E12.5 and gradually increased >1000-fold until E17.5. Muc1 and Nkx2.1 transcript levels increased in the same time interval. Prior to lobulation (E12.5-13.5), MUC1 and TG distinguished pre-follicular from progenitor cells in the developing isthmus characterized by intense cell proliferation. Luminogenesis comprised redistribution of MUC1+ vesicles or vacuoles, transiently associated with PCNT, to the apical cytoplasm and the subsequent formation of MUC1+ nascent lumens. Apical polarization of pre-follicular cells and lumen initiation involved submembraneous vesicular traffic, reorganization of adherens junctions and ciliogenesis. MUC1 did not co-localize with TG until a lumen with a MUC1+ apical membrane was established. MUC1 delineated the lumen of all newly formed follicles encountered in the developing lobes at E15.5-17.5. Folliculogenesis started before establishment of a complete follicular basal lamina. These observations indicate that embryonic thyroid differentiation is an asynchronous process consistent with the idea that progenitors attaining a stationary position in the connecting isthmus portion undergo apical polarization and generate follicles already at a primordial stage of thyroid development, i.e. foregoing growth of the lobes. Although the thyroid isthmus eventually comprises minute amounts of the total thyroid volume and contributes little to the overall hormone production, it is of principal interest that local cues related to the residence status of cells – independently of a prevailing high multiplication rate – govern the thyroid differentiation program.

Previous studies have shown that nicotine could impair the germ cell cyst breakdown and the primordial follicle assembly by autophagy. In this paper, we discovered that luteinizing hormone (LH) and follicle-stimulating hormone (FSH) could counteract the damage caused by nicotine of mouse germ cell cyst breakdown. The neonatal mice were separately intraperitoneally injected with nicotine, nicotine plus LH, nicotine plus FSH, and saline (control) for 4 days. Compared with the nicotine group, the quality of oocytes and the number of follicles were remarkably increased in the nicotine plus LH group or nicotine plus FSH group. LH and FSH could alleviate nicotine-induced oocyte autophagy by different pathways. LH reduced the nicotine-induced autophagy by restoring the phosphorylation level of adenosine 5′-monophosphate-activated protein kinase α-1, while FSH by downregulating the phosphorylation level of Forkhead box class O 1. In addition, in a subsequent study of 6-week mice in different treated groups, we found that LH and FSH supplementation significantly improved normal maturation rates, fertilization rates, and embryo’s developmental potential of oocytes in oocytes exposed to nicotine. Taken together, these results suggested that LH and FSH could counteract the damage caused by nicotine and finally ensure normal germ cell cyst breakdown and early embryo development.

Objective: To document associations between anti‐Müllerian hormone (AMH) and circulating androgens in nonhealthcare‐seeking premenopausal women. Design: Community‐based, cross‐sectional study. Setting: Eastern states of Australia. Participants: Women aged 18–39 years not using systemic hormones, not pregnant or breastfeeding within 3 months, and not postmenopausal. Measurements: AMH, measured by the Beckman Access 2, 2 site immunometric assay from fresh samples, and testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 11‐oxygenated C19 steroids, measured by liquid chromatography–tandem mass spectrometry. Results: Data were available for 794 women, median age of 33 years (range: 18–39). 76.1% were of European ancestry and 48.2% were parous. Serum AMH was positively associated with testosterone (rho =.29, p <.001) androstenedione (rho =.39, p <.001) and DHEA (rho =.10, p =.005) but not 11‐ketoandrostenedione or 11‐ketotestosterone. When adjusted for age, body mass index and smoking, using quantile regression, independent positive associations remained between AMH and testosterone (β coefficient: 20.90, 95% confidence interval [CI]: 13.79–28.03; p <.001) and androstenedione (β coefficient: 5.90, 95% CI: 3.76–8.03; p <.001). The serum concentration of testosterone was greater at the top AMH quintile than other quintiles (0.56 nmol/L [range: 0.21–1.90] vs. 0.36 nmol/L [range: 0.13–0.87]; p =.001) in women with self‐reported polycystic ovary syndrome. Conclusions: The positive associations between serum testosterone and androstenedione and AMH in premenopausal women is consistent with androgens directly or indirectly influencing AMH production during follicular development. As the highest AMH concentrations are most likely to be seen in women with multifollicular ovaries, it would be expected that women with multifollicular ovaries would have higher serum testosterone. Therefore, whether hyperandrogenemia and multifollicular ovaries should be considered independent characteristics of polycystic ovary syndrome warrants review. [ABSTRACT FROM AUTHOR]

Background: Congenital hypothyroidism is often caused by genetic mutations that impair thyroid hormone (TH) production, resulting in growth and development defects. XB130 (actin filament associated protein 1 like 2) is an adaptor/scaffold protein that plays important roles in cell proliferation, migration, intracellular signal transduction, and tumorigenesis. It is highly expressed in thyrocytes, however, its function in the thyroid remains largely unexplored. Methods:Xb130−/− mice and their littermates were studied. Postnatal growth and growth hormone levels were measured, and responses to low or high-iodine diet, and levothyroxine treatment were examined. TH and thyrotropin in the serum and TH in the thyroid glands were quantified. Structure and function of thyrocytes in embryos and postnatal life were studied with histology, immunohistochemistry, immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction. Results:Xb130−/− mice exhibited transient growth retardation postnatally, due to congenital hypothyroidism with reduced TH synthesis and secretion, which could be rescued by exogenous thyroxine supplementation. The thyroid glands of Xb130−/− mice displayed diminished thyroglobulin iodination and release at both embryonic and early postnatal stages. XB130 was found mainly on the apical membrane of thyroid follicles. Thyroid glands of embryonic and postnatal Xb130−/− mice exhibited disorganized apical membrane structure, delayed folliculogenesis, and abnormal formation of thyroid follicle lumina. Conclusion: XB130 critically regulates folliculogenesis by maintaining apical membrane structure and function of thyrocytes, and its deficiency leads to congenital hypothyroidism. [ABSTRACT FROM AUTHOR]

The objective of the study was to investigate the effect of dietary supplementation with nettle or fenugreek on folliculogenesis and steroidogenesis in the juvenile rabbit ovary. To gain insight into the mechanism of action of these herbs, we examined follicle formation, ovarian cell proliferation and apoptosis, steroidogenic enzyme abundance and steroid concentrations in ovarian tissue and plasma. Animals were fed with control, 1% nettle- or 1% fenugreek-supplemented pellets from 5 to 12 weeks of age (n = 10 per each group), when animals were slaughtered for ovary and blood collection. The addition of nettle decreased the numbers of primordial (P = 0.015) and early antral (P = 0.02) follicles and increased the number of primary (P = 0.04) ones when compared with the control group. Following fenugreek supplementation, the numbers of primary (P = 0.008) and antral (P = 0.027) follicles were greater, while the number of early antral (P = 0.003) follicles was lower in comparison with the control group. Nettle revealed apoptotic activity through activation of caspases 9 (P = 0.047), 8 (P = 0.022) and 3 (P = 0.004), whereas fenugreek increased (P = 0.042) follicular cell proliferation marked by PCNA protein abundance. Furthermore, only fenugreek targeted steroidogenic enzymes, decreasing CYP17A1 (P = 0.043) and increasing CYP19A1 (P = 0.048) protein abundances that resulted in enhanced estradiol biosynthesis and its elevated (P = 0.006) plasma concentration. In conclusion, both herbs affected follicle development in the rabbit ovary in a stage specific manner. Additionally, fenugreek altered ovarian steroidogenesis in a way that might affect sexual maturation in rabbits. • Nettle and fenugreek affect follicle development in a stage specific manner. • Nettle induces apoptosis of follicular cells. • Fenugreek increases follicular cell proliferation. • Fenugreek increases ovarian estradiol biosynthesis. [ABSTRACT FROM AUTHOR]

The optimal folliculogenesis process describes a good condition of the female reproductive system. One of the main growth factors in the folliculogenesis is IGF-II which is important for proliferation and differentiation of granulosa cells and also steroidogenesis. Chronic stress can cause ovulation failure and folliculogenesis disorders. Increased corticosterone levels due to chronic stress will cause disruption in the GnRH pulse, causing a decrease in the gonadotropin and lead to folliculogenesis disorders, and decrease IGF-II production. This study aims to prove the effect of increased corticosterone levels due to chronic stress on IGF-II expression in the ovaries of Rattus norvegicus. The research sample consisted of 34 Rattus norvegicus which were divided into 2 groups. The treatment group was given Chronic Unpredictable Mild Stress (CUMS) method for 20 days. Corticosterone levels were detected by the ELISA method as a marker of chronic stress and IGF-II expression was evaluated by immunohistochemical methods. The results of statistical tests showed that there were significant differences in corticosterone levels (p = 0.000) and IGF-II expression (p = 0.042) in the two groups (p <0.05). So the conclusion is increased corticosterone levels due to chronic stress can reduce IGF-II expression in the ovaries of Rattus norvegicus. [ABSTRACT FROM AUTHOR]

We evaluated the effect of quercetin on the in vitro culture of bovine ovarian fragments in relation to morphology, development, and oxidative stress. Ovaries (n = 12) from Nelore heifers (n = 6) were used. Each pair of ovaries was divided into nine fragments, and one fragment from each animal was fixed in Bouin solution for 24 h (histology control) or frozen (− 80°C; control for oxidative stress). Other ovarian fragments (n = 8) were distributed into concentrations of 0, 10, 25, and 50 μg/mL of quercetin added to the culture medium for 5 or 10 d. Data were analyzed by chi-square test or ANOVA followed by Tukey's test (P < 0.05). Treatment with 25 μg/mL quercetin resulted in the highest proportion of total intact follicles for 5 (67.3%) and 10 d (57.1%); the concentration of 25 μg/mL also presented the best proportion of developing follicles for 5 d (68.7%) and 10 d (62.8%). Treatment with 25 μg/mL quercetin resulted in significant ferric reduction for 10 d of culture, but not for 5 d. No difference (P > 0.1) was observed in the production of reactive oxygen species or in the oxidative degradation of lipids between treatments and non-cultivated controls. Treatment with 25 μg/mL quercetin preserved the morphological integrity of the developing follicles for 5 and 10 d of culture, in addition to promoting the best antioxidant potential after 10 d of culture in bovine ovarian fragments. [ABSTRACT FROM AUTHOR]

Objective: In humans, polycystic ovary syndrome (PCOS) is an androgen-dependent ovarian disorder. Aberrant gene expression in folliculogenesis can arrest the transition of preantral to antral follicles, leading to PCOS. We explored the possible role of altered gene expression in preantral follicles of estradiol valerate (EV) induced polycystic ovaries (PCO) in a mouse model. Methods: Twenty female balb/c mice (8 weeks, 20.0±1.5 g) were grouped into control and PCO groups. PCO was induced by intramuscular EV injection. After 8 weeks, the animals were killed by cervical dislocation. Blood serum (for hormonal assessments using the enzyme-linked immunosorbent assay technique) was aspirated, and ovaries (the right ovary for histological examinations and the left for quantitative real-time polymerase) were dissected. Results: Compared to the control group, the PCO group showed significantly lower values for the mean body weight, number of preantral and antral follicles, serum levels of estradiol, luteinizing hormone, testosterone, and follicle-stimulating hormone, and gene expression of TGFB1, GDF9 and BMPR2 (p<0.05). Serum progesterone levels were significantly higher in the PCO animals than in the control group (p<0.05). No significant between-group differences (p>0.05) were found in BMP6 or BMP15 expression. Conclusion: In animals with EV-induced PCO, the preantral follicles did not develop into antral follicles. In this mouse model, the gene expression of TGFB1, GDF9, and BMPR2 was lower in preantral follicles, which is probably related to the pathologic conditions of PCO. Hypoandrogenism was also detected in this EV-induced murine PCO model. [ABSTRACT FROM AUTHOR]

What is the involvement of pigment epithelium-derived factor (PEDF), expressed in granulosa cells, in folliculogenesis? mRNA expression of PEDF and other key factors [ Cyp19 , anti-Müllerian hormone receptor (AMHR) and vascular endothelial growth factor (VEGF)] in mice follicles was examined in order to typify the expression of PEDF in growing follicles and in human primary granulosa cells (hpGC), and to follow the interplay between PEDF and the other main players in folliculogenesis: FSH and AMH. mRNA expression of PEDF increased through folliculogenesis, although the pattern differed from that of the other examined genes, affecting the follicular angiogenic and oxidative balance. In hpGC, prolonged exposure to FSH stimulated the up-regulation of PEDF mRNA. Furthermore, a negative correlation between AMH and PEDF was observed: AMH stimulation reduced the expression of PEDF mRNA and PEDF stimulation reduced the expression of AMHR mRNA. Folliculogenesis, an intricate process that requires close dialogue between the oocyte and its supporting granulosa cells, is mediated by various endocrine and paracrine factors. The current findings suggest that PEDF, expressed in granulosa cells, is a pro-folliculogenesis player that interacts with FSH and AMH in the process of follicular growth. However, the mechanism of this process is yet to be determined. [Display omitted] [ABSTRACT FROM AUTHOR]

Obesity in adult females impairs fertility by altering oxidative stress, DNA repair and chemical biotransformation. Whether prepubertal obesity results in similar ovarian impacts is under-explored. The objective of this study was to induce obesity in prepubertal female mice and assess puberty onset, follicle number, and abundance of oxidative stress, DNA repair and chemical biotransformation proteins basally and in response to 7,12-dimethylbenz(a)anthracene (DMBA) exposure. DMBA is a polycyclic aromatic hydrocarbon that has been shown to be ovotoxic. Lactating dams (C57BL6J) were fed either a normal rodent containing 3.5% kCal from fat (lean), or a high fat diet comprised of 60% kCal from fat, and 9% kCal from sucrose. The offspring were weaned onto the diet of their dam and exposed at postnatal day 35 to either corn oil or DMBA (1 mg/kg) for 7 d via intraperitoneal injection. Mice on the HFD had reduced (P < 0.05) age at puberty onset as measured by vaginal opening but DMBA did not impact puberty onset. Heart, spleen, kidney, uterus and ovary weight were increased (P < 0.05) by obesity and liver weight was increased (P < 0.05) by DMBA exposure in obese mice. Follicle number was largely unaffected by obesity or DMBA exposure, with the exception of primary follicle number, which were higher (P < 0.05) in lean DMBA exposed and obese control relative to lean control mice. There were also greater numbers (P < 0.05) of corpora lutea in obese relative to lean mice. In lean mice, DMBA exposure reduced (P < 0.05) the level of CYP2E1, EPHX1, GSTP1, BRCA1, and CAT but this DMBA-induced reduction was absent in obese mice. Basally, obesity reduced (P < 0.05) the abundance of CYP2E1, EPHX1, GSTP1, BRCA1, SOD1 and CAT. There was greater (P < 0.05) fibrotic staining in obese DMBA-exposed ovaries and PPP2CA was decreased (P < 0.05) in growing follicles by both obesity and DMBA exposure. Thus, prepubertal obesity alters the capacity of the ovary to respond to DNA damage, ovotoxicant exposure and oxidative stress. • Pre-pubertal obesity altered ovarian and uterine weight. • Chemical biotransformation was reduced by pre-pubertal obesity. • Ovarian DNA repair was compromised in pre-pubertal obese females. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the development of ovarian follicles in female albino rats following vitamin D3 supplementation. Methods: Eighteen prepubertal female albino rats, aged 3-4 weeks, weighing (70.25±9.16) g, were assigned to three groups (n=6 in each group). Group A was treated with 5.00 mL/kg of distilled water and served as the control group, group B was treated with 0.025 mg/kg of vitamin D3 dissolved in distilled water, and group C was treated with 0.125 mg/kg of vitamin D3 dissolved in distilled water. All treatments were administered orally, twice weekly for 28 days. Blood and ovaries were harvested under anaesthesia. Serum vitamin D3 levels were determined by using spectrophotometric method. Ovaries were processed for histology and every10th hematoxylin and eosin stained-section was selected for histomorphometry. The number of follicles at each developmental stage was estimated. Results: Both 0.025 mg/kg and 0.125 mg/kg of vitamin D3 significantly increased serum concentrations of vitamin D3 and calcium (P0.05). The control group had fewer growing follicles (primary, secondary and antral follicles) and more non-growing follicles (primordial and atretic follicles) when compared with the vitamin D3-supplemented groups (P

Objectives: Polycystic ovary syndrome (PCOS) is an endocrine disorder with still unknown pathophysiology. Insulin resistance is one of major factors that affect the pathogenesis of PCOS (69%). Green tea has compounds that can be used to improve the condition of insulin resistance, so that folliculogenesis may recur in the case of PCOS. Materials and Methods: The subjects in this study were rats which were divided into 5 groups, consisting of 2 groups serving as a control group (negative and positive) and 3 groups as the treatment group. Each group consisted of 7 experimental animals. Negative control group was given with distilled water for 14 days. Positive control group was given with injections of testosterone propionate 1 mg/100 g BW intramuscularly in the thigh for 28 days to obtain a model of insulin-resistant PCOS and the distilled water was used as therapy. The treatment groups were given wth injections of testosterone propionate 1 mg/100gBW intra-muscularly for 28 days and then treated with green tea extract in doses of each 200 mg/kg, 400 mg/kg, and 800 mg/kg for 14 days. Rats were dissected on day 42 for harvesting the right and left ovary and heart blood sample was taken for examining insulin levels. Results: The results showed that administration of green tea extract on K5 can reduce insulin levels and increase folliculogenesis significantly (significance p

Abstract Background The complex regulatory mechanism involved in ovarian follicular development is not completely understood. Neuronal neuropeptide Y (NPY) is involved in the regulation of feeding behavior, energy homeostasis, and reproduction behavior, while its function in ovarian follicular development is not clear. The objective of this study was to investigate if and how NPY regulates follicle development in the ovary. Methods All experiments were performed using Sprague Dawley rats. To understand NPY expression pattern at different stages of follicular development, NPY content was assessed using immunohistochemistry in individual follicles. NPY and its receptors expression pattern were evaluated in granulosa cells isolated from preantral (PA), early antral (EA) and late antral follicles (LAF). The influence of NPY on granulosa cell proliferation and apoptosis were further assessed in vitro, using Ki67- and TUNEL-positivity assays. To investigate whether NPY induced-proliferation in EA granulosa cells is mediated through the activation of NPY receptor Y5 (NPY5R) and Mitogen-activated protein kinase (MEK) signal pathway, EA granulosa cells were treated with NPY5R antagonist (CGP71683) and MEK inhibitors (PD98059 and U0126), and Ki67-positive cells were assessed. Results NPY protein expression was follicular stage-dependent and cell type-specific. NPY signal intensity in EA was higher than those in PA and LAF. Antral granulosa cells showed the highest signal intensity compared to mural granulosa cells, cumulus cells and theca cells. Granulosa cells NPY protein content and mRNA abundance were higher in EA than in LAF. NPY receptor contents in granulosa cells were follicular stage-dependent. While NPY reduced apoptosis of EA granulosa cells, it increased the proliferation through NPY5R and MEK pathway. In contrast, in LAF granulosa cells, NPY reduced proliferation and increased the number of apoptotic cells, with no significant effects on PA granulosa cells. Conclusion This study is the first to evaluate the intraovarian role of NPY in granulosa cells at various stage of follicular development. These results indicate that NPY regulates granulosa cells proliferation and apoptosis in a follicular stage-dependent and autocrine manner. NPY may play a role in pathogenesis of ovarian follicular disorders.

Abstract Background Ovarian follicular fluid influences follicle and oocyte growth, but the fluctuation of its protein content during folliculogenesis has not been comprehensively analyzed. Here we used a shotgun approach and bioinformatics analyses to investigate and compare the proteomes of porcine follicular fluid (pFF) obtained from small ( 6–12 mm) follicles. Results Follicular fluid samples containing highest estrogen levels were selected as non-atretic from small (SNA: 26.1 ± 15 ng/mL), medium (MNA: 162 ± 54 ng/mL), and large (LNA: 290 ± 37 ng/mL) follicles for proteomic analyses. We detected 1627, 1699, and 1756 proteins in SNA, MNA, and LNA samples, respectively. Nearly 60–63% of total proteins were specific to each sample, 11–13% were shared in pairwise comparisons, and 247 proteins were shared among all samples. Functional categorization indicated comparable gene ontology (GO) terms distribution per cellular component, molecular function, and biological process categories across samples; however, the ranking of highly significantly enriched GO terms per category revealed differences between samples. The patterns of protein-to-protein interactions varied throughout follicle development, and proteins such as serine protease inhibitor, clade E (SERPINE); plasminogen activator, urokinase (PLAU); and plasminogen activator, urokinase receptor (PLAUR) appeared stage-specific to SNA, MNA, and LNA, respectively. The “complement and coagulation cascades” was the common major pathway. Besides, properdin and fibulin-1 were abundant proteins that appeared absent in LNA samples. Conclusion This study provides extensive and functional analyses of the pFF proteome changes during folliculogenesis and offers the potential for novel biomarker discovery in pFF for oocyte quality assessment.

Objective: Polycystic ovarian syndrome (PCOS) is characterized by hormonal imbalance, oxidative stress and chronic anovulation. The present study was designed to assess ameliorative effect of auto-locating platelet-rich plasma (PRP), as a novel method, for inhibiting PCOS-induced pathogenesis in experimentally-induced hyperandrogenic PCOS. Materials and Methods: In this experimental study, 30 immature (21 days old) female rats were assigned into five groups, including control (sampled after 30 days with no treatment), 15 and 30 days PCOS-sole-induced as well as 15 and 30 days PRP auto-located PCOS-induced groups. Serum levels of estrogen, progesterone, androstenedione, testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), ovarian total antioxidant capacity (TAC), malondialdehyde (MDA), glutathione peroxidase (GSH-px) and superoxide dismutase (SOD) were evaluated. Expression of estrogen receptor α (Erα), β (Erβ) and c-Myc were assessed. Finally, the numbers of intact follicles per ovary and mRNA damage ratio were analyzed. Results: PRP groups significantly (P

Eggs are female germ cells that are required for producing offspring through sexual reproduction. In mammals, eggs are produced in the ovary and ovulated into the oviduct. It is well known that over 99% of eggs are degenerated without ovulation, so that many studies have attempted in vitro folliculogenesis to produce many eggs in different species for a few decades. Although many methods have been developed, a success of in vitro egg production with the resultant live birth of offspring has been limited, especially in livestock animals. More recently, we have succeeded in producing live pups derived from in vitro/ex vivo egg production in mice. This review aims to introduce our recent findings with a brief history of in vitro/ex vivo culture systems for follicles and ovaries.

Abstract Background The fibrinolytic system and its inhibitors play a number of roles, apart from their function in blood haemostasis and thrombosis, namely in ovarian folliculogenesis and in ovulation. Plasminogen is converted to active plasmin at the time of follicular rupture through a decrease in plasminogen activator inhibitor-1 (PAI-1) and an increase in plasminogen activators. Oligo−/anovulation and follicle arrest are key characteristics of PCOS, but studies evaluating fibrinolytic/proteolytic markers within human or animal PCOS ovaries are lacking. We aimed to investigate and compare the expression and distribution of the plasminogen system markers in PCOS and control ovaries. Methods A hyperandrogenised PCOS mouse model was used that mimics the ovarian, endocrine and metabolic features of the human condition. Immunohistochemistry and digital image analysis were used to investigate and compare fibrinolytic/proteolytic markers plasminogen, plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator and inhibitor PAI-1 in PCOS and control ovaries. Student’s t-test was used to compare data sets for normally distributed data and Wilcoxon-Mann Whitney test for non-normally distributed data. Results We noted differences in the ovarian distribution of PAI-1 that was expressed throughout the PCOS ovary, unlike the peripheral distribution observed in control ovaries. Plasminogen was present in small follicles only in PCOS ovaries but not in small follicles of control ovaries. When we assessed and compared PAI-1 expression within follicles of different developmental stages we also noted significant differences for both the PCOS and control ovaries. While we noted differences in distribution and expression within specific ovarian structures, no differences were noted in the overall ovarian expression of markers assessed between acyclical PCOS mice and control mice at the diestrus stage of the estrous cycle. Conclusions Our novel study, that comprehensively assessed the fibrinolytic/proteolytic system in the mouse ovary, showed the expression, differential localisation and a potential role for the plasminogen system in the physiological mouse ovary and in PCOS. Androgens may be involved in regulating expression of the ovarian plasminogen system. Further studies evaluating these markers at different time-points of ovulation may help to further clarify both physiological and potential pathological actions these markers play in ovulatory processes distorted in PCOS.

Background and Objectives: To test the long-term ability of human ovarian cortex cells to develop in unconventional culture conditions. Materials and Methods. Ovarian cortex cells from fetuses aged 23 to 39 weeks gestation were cultured for 90 days in hollow chitosan hydrogel micro-bioreactors and concurrently in traditional wells. Various cell-type counts were considered. Results: With intact follicles as a denominator, the percentage of growing intact follicles at Day 0 varied widely between ovaries (0 to 31.7%). This percentage tended to increase or stay relatively constant in bioreactor as in control cultures; it tended more toward an increase over time in bioreactor vs. control cultures. Modeled percentages showed differences (though not significant) in favor of bioreactor cultures (16.12% difference at D50 but only 0.12% difference at D90). With all follicles present as a denominator, the percentage of growing primary and secondary follicles at D0 varied widely between ovaries (0 to 29.3%). This percentage tended to increase over time in bioreactor cultures but to decrease in control cultures. Modeled percentages showed significant differences in favor of bioreactor cultures (8.9% difference at D50 and 11.1% difference at D90). At D50 and D90, there were only few and sparse apoptotic cells in bioreactor cultures vs. no apoptotic cells in control cultures. Conclusions: Over three months, bioreactor folliculogenesis outperformed slightly traditional culture. This is an interesting perspective for follicle preservation and long-term toxicological studies.

Introduction: Obtaining in vitro mature oocytes from ovarian tissue to preserve women’s fertility is still a challenge. At present, there is a therapeutic deadlock for girls and women who need emergency fertility preservation in case of a high risk of ovary invasion by malignant cells. In such a case, ovarian tissue cannot be engrafted; an alternative could be in vitro folliculogenesis. Methods: This review focuses on the progress of in vitro folliculogenesis in humans. PubMed and Embase databases were used to search for original English-language articles. Results: The first phase of in vitro folliculogenesis is carried out in the original ovarian tissue. The addition of one (or more) initiation activator(s) is not essential but allows better yields and the use of a 3D culture system at this stage provides no added value. The second stage requires a mechanical and/or enzymatic isolation of the secondary follicles. The use of an activator and/or a 3D culture system is then necessary. Conclusion: The current results are promising but there is still a long way to go. Obtaining live births in large animals is an essential step in validating this in vitro folliculogenesis technique.

The ovary is an indispensable unit of female reproduction and health. However, the study of ovarian function in mammals is hindered by unique challenges, which include the desynchronized development of oocytes, irregular distribution and vast size discrepancy of follicles, and dynamic tissue remodeling during each hormonal cycle. Overcoming the limitations of traditional histology, recent advances in optical tissue clearing and three-dimensional (3D) visualization offer an advanced platform to explore the architecture of intact organs at a single cell level and reveal new relationships and levels of organization. Here we summarize the development and function of ovarian compartments that have been delineated by conventional two-dimensional (2D) methods and the limits of what can be learned by these approaches. We compare types of optical tissue clearing, 3D analysis technologies, and their application to the mammalian ovary. We discuss how 3D modeling of the ovary has extended our knowledge and propose future directions to unravel ovarian structure toward therapeutic applications for ovarian disease and extending female reproductive lifespan.

This is isobaric tags for a relative and absolute quantification (iTRAQ)-Based Proteomic Data on bovine plasma (PL) and follicular fluid (FF) containing high and low pre-ovulatory circulating concentration of estradiol (E2). The PL and FF were collected from nine beef cows that were identified to initiate a new follicular wave on day -4 during synchronization. Follicular dynamics and ovulatory response were monitored using transrectal ultrasonography. Blood samples were collected at slaughter and FF was aspirated from dominant follicles (DF; >10 mm). Estradiol concentrations in PL and FF were measured by radioimmunoassays. Plasma and FF were labeled as containing high E2 (PL HE2 and FF HE2) or low E2 (PL LE2 and FF LE2). Abundant proteins (albumin, IgG, IgA, and alpha-1-antitrypsin) were depleted from the four PL and FF samples. Peptides were labeled with iTRAQ reagents and analyzed using 2-dimentional liquid chromatography ESI-based mass spectrometry. Proteins were identified and quantified using SEQUESTTM search engine embedded in Proteome Discoverer. The proteins matched with at least one unique peptide at minimum 95% confidence were considered positive identifications. Protein expression levels were determined by assigned fold change of >2.0 or

• Bisphenol A decreased the number and diameter of preovulatory follicles. • Bisphenol A and bisphenol S altered the expression of genes related to oogenesis, immune function, and ovarian cancer. • Bisphenol S produced similar ovarian toxicities as bisphenol A by inducing inflammation. Both bisphenol A (BPA) and its analog bisphenol S (BPS) are industrial chemicals that have been used to make certain plastic products applied in chicken farms, including food and water containers. They are endocrine disrupting chemicals (EDCs) with xenoestrogenic activities and affect reproductive success in many ways. It was hypothesized that BPA and BPS could adversely affect the folliculogenesis in chickens due to their disruption of the estrogen responses, using either genomic or non-genomic mechanisms. This study investigated the deleterious effects of BPA and BPS on the ovaries when adult layer chickens were orally treated with these EDCs at 50 μg/kg body weight, the reference dose for chronic oral exposure of BPA established by the U.S. EPA. The chickens in both BPA and BPS-treated groups showed a decreased number of the preovulatory follicles. BPA-treated chickens showed a significant decrease in the diameter of F1. Additionally, both BPA and BPS treatments increased the infiltrations of lymphocytes and plasma cells in ovaries. Moreover, it was found that the ovaries of BPS-treated chickens weighed the most among the groups. RNA sequencing and subsequent pathway enrichment analysis of differentially expressed genes revealed that both BPA- and BPS-treatment groups showed significant changes in gene expression and pathways related to reproduction, immune function and carcinogenesis. Taken together, both BPA and BPS are potentially carcinogenic and have deleterious effects on the fertility of laying chickens by inducing inflammation, suggesting that BPS may not be a safe replacement for BPA. [ABSTRACT FROM AUTHOR]

Follicular granulosa cells (GCs) form an important association with follicle's survival and health that determines ovulation. Apoptosis induction in GCs leads to follicular atresia and infertility problems. The present study aims at assessing the ultrastructural toxicity of methoxychlor (MXC), an organochlorine insecticide and reproductive toxin, on structural aspects of GCs owing to its potential for inducing apoptosis and the ameliorating potential of N-acetyl-L-cysteine (NAC) in this toxicity. The ultrastructural morphology revealed MXC induced degenerative changes in GCs like loss of cellular junction complexes and membrane integrity; nuclear abnormalities like occurrence of condensed and marginated chromatin, crescent shaped or blebbed nucleus; presence of disrupted mitochondria with disrupted cristae, vacuolization, empty spaces, large number of homogenous lipid droplets and extensive network of rough endoplasmic reticulum and several cytoplasmic processes at various doses of MXC with maximum degeneration observed at 100 pg/ml. NAC supplementation reduced the observed apoptotic characteristics of GCs with most significant changes at 5- and 10 mM concentration. Thus, it is evident that MXC acts as an apoptotic inducer in GCs that influences the quality of antral follicles in mammals; however, NAC, with its anti-oxidative and anti-inflammatory properties, turns out to be a potential therapeutic and anti-apoptotic agent against MXC toxicity. [ABSTRACT FROM AUTHOR]

Knowledge of follicle development during pregnancy under experimental conditions could be a key factor to understanding maternal ovarian activity. Thus, this study evaluated the effects of maternal protein restriction before and during pregnancy on folliculogenesis. Swiss outbred female mice were allocated to either a control (CC; 20% protein) or treated (TT; 8%protein) group. Pregnant females were killed either on Gestational day (GD) 7.5 or GD17.5 and the ovaries were evaluated using histomorphometric and immunohistochemical methods. TT females showed higher feed and energy intakes, but lower bodyweight gain at GD17.5 (P<0.05). They also had lower number of secondary follicles at GD7.5 and a higher proportion of primordial follicles at GD17.5 (P<0.05). In addition, the areas of the secondary follicles and their granulosa layer were smaller in the TT group on GD7.5, whereas the areas of the oocyte and granulosa layer from atretic follicles were larger (P<0.05). Notwithstanding the slight increase in the insulin-like growth factor 1 (IGF1) receptor expression on GD7.5 in the TT group, there was a marked reduction in IGF1 expression detected in secondary follicles on GD17.5 (P<0.05). Collectively, these results demonstrate that protein restriction during pregnancy negatively affects follicle quality by reducing the size and activation capacity, which is more severe in late pregnancy. [ABSTRACT FROM AUTHOR]

Background: Wnt signaling pathway plays critical role in ovarian follicle development. Therefore, the aim of this study was to evaluate the effects of vitrification on the expression of Wnt pathway related genes in preantral follicles (PFs). Methods: Isolated PFs (n=982) of 14-16 day old female mice (n=45: 15 for each group) were divided into fresh (n=265), toxicity (n=272), and vitrified (n=265). The mRNA levels of Wnt2, Wnt4, Lrp5 and Fzd3 were evaluated by real-time PCR on the 2nd and 6th days of culture period. One-way ANOVA was conducted to analyze the data. Post hoc Tukey's HSD was used for multiple comparisons and p-value less than 0.05 was considered statistically significant. Results: The developmental parameters of fresh PFs were significantly higher than those of vitrified (p<0.001). There were no differences between fresh and vitrified PFs on the 2nd day of culture (p<0.001). Wnt4 expression levels decreased significantly in vitrified groups compared with fresh ones (p<0.001). Fzd3 and Lrp expression levels increased significantly in vitrified groups compared with those in the fresh group on the 2nd day (p<0.001). On the 6th day of culture period, the expression levels of Wnt2 and Fzd3 increased significantly in vitrified group compared to those of fresh group (p<0.001). Moreover, the expression levels of Wnt4 and Lrp increased significantly in toxicity groups compared to those of the control group (p<0.001). Conclusion: Vitrification increase the expression levels of Wnt2, Lrp and Fzd3 genes of PFs during in vitro culture. [ABSTRACT FROM AUTHOR]

Evolutionary and comparative approaches can yield novel insights into human adaptation and disease. Endometriosis and polycystic ovary syndrome (PCOS) each affect up to 10% of women and significantly reduce the health, fertility, and quality of life of those affected. PCOS and endometriosis have yet to be considered as related to one another, although both conditions involve alterations to prenatal testosterone levels and atypical functioning of the hypothalamic–pituitary–gonadal (HPG) axis. Here, we propose and evaluate the novel hypothesis that endometriosis and PCOS represent extreme and diametric (opposite) outcomes of variation in HPG axis development and activity, with endometriosis mediated in notable part by low prenatal and postnatal testosterone, while PCOS is mediated by high prenatal testosterone. This diametric disorder hypothesis predicts that, for characteristics shaped by the HPG axis, including hormonal profiles, reproductive physiology, life‐history traits, and body morphology, women with PCOS and women with endometriosis will manifest opposite phenotypes. To evaluate these predictions, we review and synthesize existing evidence from developmental biology, endocrinology, physiology, life history, and epidemiology. The hypothesis of diametric phenotypes between endometriosis and PCOS is strongly supported across these diverse fields of research. Furthermore, the contrasts between endometriosis and PCOS in humans parallel differences among nonhuman animals in effects of low versus high prenatal testosterone on female reproductive traits. These findings suggest that PCOS and endometriosis represent maladaptive extremes of both female life‐history variation and expression of sexually dimorphic female reproductive traits. The diametric disorder hypothesis for endometriosis and PCOS provides novel, unifying, proximate, and evolutionary explanations for endometriosis risk, synthesizes diverse lines of research concerning the two most common female reproductive disorders, and generates future avenues of research for improving the quality of life and health of women. [ABSTRACT FROM AUTHOR]

Significance: To improve the outcomes of in vitro culture of human oocytes and embryos, the dynamic balance and roles of reactive oxygen species (ROS) in folliculogenesis and embryo development merit further consideration. Recent Advances: ROS have been demonstrated to participate in various signaling processes and act as mediators in various physiological events in germ cells. An imbalance between pro-oxidants and antioxidants seems to explain the high failure rate of assisted reproduction. Critical Issues: Oxidative stress induced by excessive ROS or insufficient antioxidant protection can cause detrimental effects on both male and female reproduction. In this study, oxidative stress in folliculogenesis and embryo development are summarized and the multiple modifiable factors of in vitro culture systems in relation to ROS are discussed. Future Directions: More studies are needed to establish an optimal redox state in in vitro culture systems for human oocytes and embryos. [ABSTRACT FROM AUTHOR]

The development of culture systems capable of maintaining follicular growth since the preantral stage has been the target of investigations. Mesenchymal stem cells (MSC) present potential for use in a wide range of applications, including research aimed at preserving fertility. Therefore, this study investigated the use of caprine Wharton's Jelly Mesenchymal Stem Cells (WJMSC) on the survival and in vitro development of goat preantral follicles enclosed in ovarian fragments cultured for 1 or 7 days. Fragments of the ovarian cortex were immediately fixed (non-cultured control) or distributed in four treatments: ovarian tissue cultured in control medium (α-MEM+); ovarian tissue cultured in α-MEM+ supplemented with FBS (α-MEM+ + FBS); ovarian tissue co-cultured with stem cells in α-MEM+ (α-MEM+ + SC); and ovarian tissue co-cultured with stem cell in α-MEM+ + FBS (α-MEM+ + SC + FBS). The rates of cell proliferation, follicular survival, and activation, as well as follicular diameter, were evaluated. After 7 days, the treatment co-cultured with stem cells showed a higher (P < 0.05) percentage of morphologically normal preantral follicles compared to the other treatments, as well as a higher (P < 0.05) activation rate compared to cultured control. Moreover, the follicular diameter was higher (P < 0.05) in the treatment co-cultured with stem cells compared to co-cultured with stem cells plus FBS. This study demonstrates for the first time that in vitro co-culture of caprine WJMSC with preantral follicles enclosed in goat ovarian tissue improves activation and early follicular development. [ABSTRACT FROM AUTHOR]

This study measured the sequelae of cholecalciferol (VD3) therapy on ovarian functions in adult VD-replete rats (n = 48). The animals were distributed into the control and VD groups following estrous cycle synchronisation. The VD group received VD3 injections for 4 weeks (600 IU/Kg; 3 times/week). Vaginal cytology and cycle durations were recorded throughout the study. Serum VD (25-OH VD), Ca2+, gonadotrophins (FSH & LH) and sex steroids (E2 & progesterone) were measured following euthanasia. Follicles and corpora lutea were counted in ovarian tissue sections. VD receptor, binding protein, Ca2+-sensing receptor and retinoid X receptor-α genes and proteins were measured by quantitative RT-PCR and immunohistochemistry. Serum VD, LH, E2 and progesterone levels were significantly higher, whereas FSH declined, in the VD group than controls. VD3 therapy was also associated with markedly higher rates alongside shorter durations of estrous cycles than controls. While serum Ca2+ levels were equal between the study groups, they correlated directly with serum 25-OH VD. The numbers of small and medium size ovarian follicles were equal in both study groups, whereas large follicles and corpora lutea counts were significantly higher in the VD group. The mRNAs and proteins of targeted molecules also increased substantially in the VD group than controls. In conclusion, treating VD-sufficient female rats with supraphysiological VD3 supplements was not associated with hypercalcaemia, and could contribute to ovarian functions by regulating the hypothalamic-pituitary-ovarian hormones and ovarian VD-related molecules. However, further studies are still needed to illustrate the clinical significance of VD3 in female reproduction. [ABSTRACT FROM AUTHOR]

Granulosa cells (GCs) play important roles in the regulation of ovarian functions, and in vitro culture is a relevant model for the study of steroidogenesis in ovarian follicles. Thus, growth factors secreted by the oocyte, like Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), play an important part in the luteinization of granulosa cells. The aim of this work was to express GDF9 and BMP15 genes in bovine GCs in vitro and evaluate their effects on the luteinization process. Samples of culture medium and GCs transfected with GDF9 and BMP15 were obtained for 21 consecutive days to analyse the steroidogenic hormones' concentration (progesterone (P4) and estradiol (E2)) and the expression of STAR, GDF9 and BMP15 and their respective receptors. The results demonstrated an inhibitory effect of GDF9 and BMPF15 on P4 secretion in bovine GCs cultured in vitro. Moreover, our study demonstrated the entire expression of their respective receptors (TGFBR1, BMPR1B and BMPR2) and the inhibition of the steroidogenic marker, STAR gene. This work sheds light on a novel biological function of BMP15 and GDF9 in bovine GCs physiology, which could elucidate a non‐described biological role for GDF9 and BMP15 in bovine granulosa cells' metabolism. [ABSTRACT FROM AUTHOR]

• Adriamycin causes damage of histoarchitecture of testis. • It affects steroidogenesis and spermatogenesis. • It affects the DNA methylation in spermatocytes. • It affects ovarian architecture and folliculogenesis. • Interrupts the reproductive cyclicity. The anticancer drug doxorubicin has been associated with several adverse side-effects including reproductive toxicity in both genders. The current review has complied the mechanisms of doxorubicin induced reproductive toxicity. The articles cited in the review were searched using Google Scholar, PubMed, Scopus, Science Direct. Doxorubicin treatment has been found to cause a decrease in testicular mass along with histopathological deformities, oligospermia and abnormalities in sperm morphology. Apart from severely affecting the normal physiological role of both Leydig cells and Sertoli cells, doxorubicin also causes chromosome abnormalities and affects DNA methylase enzyme. Testicular lipid metabolism has been found to be negatively affected by doxorubicin treatment resulting in altered profile of sphingolipids glycerophospholipids and neutral lipids. Dysregulation of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β- hydroxysteroid dehydrogenase (17β-HSD) are strongly linked to testicular exposure to doxorubicin. Further, oxidative stress along with endoplasmic reticulum stress are also found to aggravate the male reproductive functioning in doxorubicin treated conditions. Several antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase (GPx) are downregulated by doxorubicin. It also disturbs the hormones of the hypothalamic-pituitary-gonadal (HPG)-axis including testosterone, luteinizing hormone, follicle stimulating hormone etc. In females, the drug disturbs folliculogenesis and oogenesis leading to failure of ovulation and uterine cycle. In rodent model the drug shortens pro-estrous and estrous phases. It was also found that doxorubicin causes mitochondrial dysfunction in oocytes with impaired calcium signaling along with ER stress. The goal of the present review is to comprehends various pathways due to which doxorubicin treatment promotes toxicity in male and female reproductive system. [ABSTRACT FROM AUTHOR]

Objectives: Salvia officinalis (sage) is widely sold in Saudi Arabia to make a popular herbal tea that women with reproductive disorders can drink daily. Despite the wide use of sage in traditional medicine, to our knowledge, little attention has been given to its effect on female reproduction. Our purpose was to understand the effects of sage extract on ovarian function to potentially shed light on its use in treating female infertility. Methods: Forty female Wistar virgin female rats were randomly assigned to four groups, with 10 animals in each group. The control group received distilled water, and the three treatment groups received different concentrations of sage extract: 3.5, 15 or 60 mg/kg during 14 days. Ovarian tissues were cut into 5-μm sections and stained with hematoxylin and eosin. Plasma levels of reproductive steroids were measured using ELISA, and gene expression was examined via RT-PCR. Results: Our results showed that low doses of sage (3.5 mg/kg and 15 mg/kg) have no effects on serum concentration levels of steroid hormone, the number of growing follicles, and on the transcripts of the steroidogenesis and folliculogenesis genes. In contrast, a high dose of the extract (60 mg/kg) significantly increased estradiol concentration levels. As a result, the number of growing follicles increased, while the number of abnormal follicles significantly decreased. Interestingly, sage extract also affected mRNA levels of genes involved in folliculogenesis and steroidogenesis processes. Two autophagy related genes, LC3 and ATG12, were not affected, but all of the investigated genes, INSL3, CYP17a, LHR, CCND2, IGF1, ESR1, ESR2, ACTB, GDF, and ATG5, significantly increased with the highest dose of sage. Conclusion: As a remedy, sage extract is best used at a high dose when treating female infertility disorders, as sage extract may promote ovarian function by stimulating folliculogenesis and steroidogenesis.