Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.

Zinc fluctuations regulate key steps in late oocyte and preimplantation embryo development; however, roles for zinc in preceding stages in early ovarian follicle development, when cooperative interactions exist between the oocyte and somatic cells, are unknown. To understand the roles of zinc during early follicle development, we applied single cell X-ray fluorescence microscopy, a radioactive zinc tracer, and a labile zinc probe to measure zinc in individual mouse oocytes and associated somatic cells within early follicles. Here, we report a significant stage-specific increase and compartmental redistribution in oocyte zinc content upon the initiation of early follicle growth. The increase in zinc correlates with the increased expression of specific zinc transporters, including two that are essential in oocyte maturation. While oocytes in follicles exhibit high tolerance to pronounced changes in zinc availability, somatic survival and proliferation are significantly more sensitive to zinc chelation or supplementation. Finally, transcriptomic, proteomic, and zinc loading analyses reveal enrichment of zinc targets in the ubiquitination pathway. Overall, these results demonstrate that distinct cell type-specific zinc regulations are required for follicle growth and indicate that physiological fluctuation in the localization and availability of this inorganic cofactor has fundamental functions in early gamete development.

The mammalian ovarian follicle is the complex reproductive unit comprising germ cell, somatic cells (Cumulus and Granulosa cells), and follicular fluid (FF): paracrine communication among the different cell types through FF ensures the development of a mature oocyte ready for fertilization. This paper is focused on non-coding RNAs in ovarian follicles and their predicted role in the pathways involved in oocyte growth and maturation. We determined the expression profiles of microRNAs in human oocytes and FF by high-throughput analysis and identified 267 microRNAs in FF and 176 in oocytes. Most of these were FF microRNAs, while 9 were oocyte specific. By bioinformatic analysis, independently performed on FF and oocyte microRNAs, we identified the most significant Biological Processes and the pathways regulated by their validated targets. We found many pathways shared between the two compartments and some specific for oocyte microRNAs. Moreover, we found 41 long non-coding RNAs able to interact with oocyte microRNAs and potentially involved in the regulation of folliculogenesis. These data are important in basic reproductive research and could also be useful for clinical applications. In fact, the characterization of non-coding RNAs in ovarian follicles could improve reproductive disease diagnosis, provide biomarkers of oocyte quality in Assisted Reproductive Treatment, and allow the development of therapies for infertility disorders.

Ovarian primordial follicles are critical for female reproduction and comprise a finite pool of gametes arrested in development. A systems biology approach was used to identify regulatory gene networks essential for primordial follicle development. Transcriptional responses to eight different growth factors known to influence primordial follicles were used to construct a bionetwork of regulatory genes involved in rat primordial follicle development. Over 1,500 genes were found to be regulated by the various growth factors and a network analysis identified critical gene modules involved in a number of signaling pathways and cellular processes. A set of 55 genes was identified as potential critical regulators of these gene modules, and a sub-network associated with development was determined. Within the network two previously identified regulatory genes were confirmed (i.e., Pdgfa and Fgfr2) and a new factor was identified, connective tissue growth factor (CTGF). CTGF was tested in ovarian organ cultures and found to stimulate primordial follicle development. Therefore, the relevant gene network associated with primordial follicle development was validated and the critical genes and pathways involved in this process were identified. This is one of the first applications of network analysis to a normal developmental process. These observations provide insights into potential therapeutic targets for preventing ovarian disease and promoting female reproduction.

Reelin plays an important role in cerebral cortex development and synaptogenesis. In the hippocampus, the neurosteroid estrogen affects reelin expression. In this study we tested a potential crosstalk between estradiol and reelin, thus the possibility of a reelin-induced activation of the estradiol synthesizing enzyme aromatase. As a model system, we used ovaries, which express reelin and are a major source of estradiol. We found that in wild-type mice, reelin and aromatase are expressed in granulosa cells of growing follicles. The expression of reelin varies with the estrus cycle and is highest shortly before ovulation, when estradiol serum levels are at their maximum. In ovaries of reelin-deficient reeler mice, aromatase mRNA and protein are significantly reduced, as evidenced by real-time PCR, western blot analysis, and quantitative immunohistochemistry in granulosa cells of preovulatory follicles. In line with reduced estradiol synthesis, ovarian estrus cycle length is prolonged in reeler mice. Most importantly, treating cultured granulosa cells with recombinant reelin results in significant upregulation of aromatase mRNA and protein and increased secretion of estradiol into the supernatant. Our data provide evidence of a local increase of aromatase expression by reelin. Regarding reproduction, this crosstalk may contribute to follicular stability and counteract luteinization in ovaries.

Silent information regulator 2 related enzyme 1 (SIRT1) is one of the key factors in the mechanism of calorie restriction (CR) extending lifespan of animals. The aim of the study is to investigate if CR prolongs ovarian lifespan in mice through activating SIRT1 signaling.

Ovarian follicle development is a complex and well-orchestrated biological process of great economic significance for poultry production. Specifically, understanding the molecular mechanisms underlying follicular development is essential for high-efficiency follicular development can benefit the entire industry. In addition, domestic egg-laying hens often spontaneously develop ovarian cancer, providing an opportunity to study the genetic, biochemical, and environmental risk factors associated with the development of this cancer. Here, we provide high-quality RNA sequencing data for chicken follicular granulosa cells across 10 developmental stages, which resulted in a total of 204.57 Gb of clean sequencing data (6.82 Gb on average per sample). We also performed gene expression, time-series, and functional enrichment analyses across the 10 developmental stages. Our study revealed that SWF (small while follicle), F1 (F1 hierarchical follicles), and POFs (postovulatory follicles) best represent the transcriptional changes associated with the prehierarchical, preovulatory, and postovulatory stages, respectively. We found that the preovulatory stage F1 showed the greatest divergence in gene expression from the POF stage. Our research lays a foundation for further elucidation of egg-laying performance of chicken and human ovarian disease.

Primordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a female's reproductive life.

The endocrinology regulating ovulation of the desired number of oocytes in the ovarian cycle is well described, particularly in mono-ovulatory species. Less is known about the characteristics that make one follicle suitable for ovulation while most other follicles die by atresia. Bromodeoxyuridine (BrdU) injection was used to characterize granulosa cell proliferation rates in developing ovarian follicles in the estrous cycle of mice. This methodology allowed identification of follicle diameters of secondary (80-130 μm), follicle-stimulating hormone (FSH)-sensitive (130-170 μm), FSH-dependent (170-350 μm), and preovulatory (>350 μm) follicles. Few preovulatory-sized follicles were present in the ovaries of mice at estrus, the beginning of the cycle. Progressive increases were seen at metestrus and diestrus, when full accumulation of the preovulatory cohort (~10 follicles) occurred. BrdU pulse-chase studies determined granulosa cell proliferation rates in the 24-48 h before the follicle reached the preovulatory stage. This showed that slow-growing follicles were not able to survive to the preovulatory stage. Mathematical modeling of follicle growth rates determined that the largest follicles at the beginning of the cycle had the greatest chance of becoming preovulatory. However, smaller follicles could enter the preovulatory follicle pool if low numbers of large antral follicles were present at the beginning of the cycle. In this instance, rapidly growing follicles had a clear selection advantage. The developing follicle pool displays heterogeneity in granulosa cell proliferation rates, even among follicles at the same stage of development. This parameter appears to influence whether a follicle can ovulate or become atretic.

Microfluidic chips have been proved effective in mimicking different organs of human body. Simulating human ovarian follicles by microfluidic device will be useful in exploring the mechanism of folliculogenesis and related diseases. In this paper, a microfluidic chip was designed to culture a single human pre-antral follicle. Ovarian follicles were first encapsulated in 3D calcium alginate hydrogel beads and then cultured on chip and in dish under same conditions. The diameters of cultured ovarian follicles were measured, and the same amount of medium was collected from microfluidic device or dish per two days for measuring the estradiol and androgen concentrations. The results confirmed the successful growth of ovarian follicles on chip with their hormonal trends and diameters increase, which were similar to ovarian follicles cultured in dish. It is concluded that this microfluidic chip can be used to culture a single human ovarian follicle, which provides a useful tool to explore the hormonal changes and their interactions during folliculogenesis.

Protein arginine methyltransferase 5 (Prmt5) is the major type II enzyme responsible for symmetric dimethylation of arginine. Here, we found that PRMT5 was expressed at high level in ovarian granulosa cells of growing follicles. Inactivation of Prmt5 in granulosa cells resulted in aberrant follicle development and female infertility. In Prmt5-knockout mice, follicle development was arrested with disorganized granulosa cells in which WT1 expression was dramatically reduced and the expression of steroidogenesis-related genes was significantly increased. The premature differentiated granulosa cells were detached from oocytes and follicle structure was disrupted. Mechanism studies revealed that Wt1 expression was regulated by PRMT5 at the protein level. PRMT5 facilitated IRES-dependent translation of Wt1 mRNA by methylating HnRNPA1. Moreover, the upregulation of steroidogenic genes in Prmt5-deficient granulosa cells was repressed by Wt1 overexpression. These results demonstrate that PRMT5 participates in granulosa cell lineage maintenance by inducing Wt1 expression. Our study uncovers a new role of post-translational arginine methylation in granulosa cell differentiation and follicle development.

Introduction: Ovarian follicle growth is a key step in the success of assisted reproductive treatment, but limited data exists to directly relate follicle growth to recombinant FSH (rFSH) dose. In this study, we aim to evaluate FSH requirements for follicular growth during controlled ovarian stimulation. Method: Single center retrospective cohort study of 1,034 IVF cycles conducted between January 2012-January 2016 at Hammersmith Hospital IVF unit, London, UK. Median follicle size after 5 days of stimulation with rFSH and the proportion of antral follicles recruited were analyzed in women treated with rFSH alone to induce follicular growth during IVF treatment. Results: Starting rFSH dose adjusted for body weight (iU/kg) predicted serum FSH level after 5 days of rFSH (r 2 = 0.352, p < 0.0001), median follicle size after 5 days of rFSH, and the proportion of antral follicles recruited by the end of stimulation. Day 5 median follicle size predicted median follicle size on subsequent ultrasound scans (r 2 = 0.58-0.62; p < 0.0001), and hence time to oocyte maturation trigger (r 2 = 0.22, P < 0.0001). Insufficient rFSH starting dose that required >5% dose-increase was associated with increased variability in follicle size on the day of oocyte maturation trigger, and negatively impacted the number of mature oocytes retrieved. Conclusion: Weight-adjusted rFSH dose correlates with follicular growth during ovarian stimulation. Early recruitment of follicles using a sufficient dose of rFSH from the start of stimulation was associated with reduced variability in follicle size at time of oocyte maturation trigger and an increased number of mature oocytes retrieved.

Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles in vitro?

Introduction Adalimumab is used in the treatment of many chronic inflammatory diseases, especially rheumatoid arthritis. The aim of this experimental study is to determine the histological and biochemical effects of adalimumab on rat ovary. Methods Wistar albino female rats were randomly divided into three groups prior to the experiment: a healthy control group, a 2 mg/kg adalimumab group, and a 5 mg/kg adalimumab group. Then, histopathological findings and biochemical examinations were made in the ovaries of the rats. Hematoxylin-eosin staining, morphometric examination, and Masson trichrome staining were performed. Antimullerian hormone (AMH) levels were measured in the biochemical examination. Results Ovarian follicle count and AMH level were significantly higher in the groups given low-dose adalimumab and high-dose adalimumab (p <0.001). In addition, fibrosis decreased in proportion to the dose of adalimumab (p <0.001). Conclusion Adalimumab is an important biological agent that contributes to the preservation of ovarian function by increasing ovarian follicle reserve and has shown that it can help preserve ovarian reserve in women of reproductive age suffering from chronic inflammatory diseases.

Isoliquiritigenin is a botanical estrogen used as a dietary supplement. Previous studies show that other botanical estrogens affect ovarian estradiol synthesis, but isoliquiritigenin's effects on the ovary are unknown. Thus, this study tested the hypothesis that isoliquiritigenin inhibits ovarian antral follicle growth and steroidogenesis. Antral follicles from CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or isoliquiritigenin (0.6μM, 6 μM, 36 μM, and 100 μM) for 48-96h. During culture, follicle diameters were measured daily to assess follicle growth. After culture, media were collected for hormone assays and follicles were collected for gene expression analysis of steroidogenic enzymes. Isoliquiritigenin inhibited antral follicle growth and altered estradiol, testosterone, and progesterone levels. Additionally, isoliquiritigenin altered the mRNA levels of cytochrome P450 steroid 17-α-hydroxylase 1, aromatase, 17β-hydroxysteroid dehydrogenase 1, and steroidogenic acute regulatory protein. These data indicate that exposure to isoliquiritigenin inhibits growth and disrupts steroid production in antral follicles.

We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed.

In the present investigation, we examined whether a change in whole body energy fluxes could affect ovarian follicular development, employing mice ectopically expressing uncoupling protein 1 in skeletal muscle (UCP1-TG). Female UCP1-TG and wild-type (WT) mice were dissected at the age of 12 weeks. Energy intake and expenditure, activity, body weight and length, and body composition were measured. Plasma insulin, glucose, leptin, plasma fibroblast growth factor 21 (FGF21) and plasma insulin-like growth factor 1 (IGF1) levels were analyzed and ovarian follicle and corpus luteum numbers were counted. IGF1 signaling was analyzed by immunohistochemical staining for the activation of insulin receptor substrate 1/2 (IRS1/2) and AKT. UCP1-TG female mice had increased energy expenditure, reduced body size, maintained adiposity, and decreased IGF1 concentrations compared to their WT littermates, while preantral and antral follicle numbers were reduced by 40% and 60%, respectively. Corpora lutea were absent in 40% of the ovaries of UCP1-TG mice. Phospho-IRS1, phospho-AKT -Ser473 and -Thr308 immunostaining was present in the granulosa cells of antral follicles in WT ovaries, but faint to absent in the antral follicles of UCP1-TG mice. In conclusion, the reduction in circulating IGF1 levels due to the ectopic expression of UCP1 is associated with reduced immunostaining of the IRS1-PI3/AKT pathway, which may negatively affect ovarian follicle development and ovulation.

Aberrant signaling between germ cells and somatic cells can lead to reproductive disease and depends on diffusible signals, including transforming growth factor-beta (TGFB) -family proteins. The TGFB-family protein Gsdf (gonadal soma derived factor) controls sex determination in some fish and is a candidate for mediating germ cell/soma signaling.

Regulation of human ovarian steroidogenesis differs from other species and precise knowledge on how human small antral follicles (hSAF) develop and acquire competence for continued growth and steroid output is still incomplete. The present study has characterized almost 1,000 normal hSAF collected in connection with cryopreservation of ovarian tissue for fertility preservation. The antral follicles (ranging from 3 to 13 mm) were generally aspirated from one ovary surgically removed during the natural cycle, and the follicular fluid (FF) and the granulosa cells (GC) were isolated and snap-frozen. In FF, the following hormones were measured: inhibin-B, inhibin-A, AMH, follistatin, PAPP-A, estradiol, progesterone, testosterone, and androstenedione. In GC, mRNA gene expressions using q-PCR were measured for the following genes: FSHR, AMH, CYP19, and AR. All samples in which one of the abovementioned parameters was measured were included, but typically multiple parameters were measured. Highly significant differences in concentration and follicular content in relation to follicular diameter were found for all measured hormones despite massive variability in-between follicles for any given diameter. The results demonstrate that profound changes take place in the hormonal microenvironment around follicular diameters of 8-11 mm corresponding to when follicular selection occurs. At this point, inhibin-B and inhibin-A showed distinct peaks concomitant with a significant reduction in both AMH protein and mRNA expression. Concentrations of inhibins, androgens, FSHR, and AR were intimately associated, and it is suggested that inhibin-B in combination with PAPP-A and thereby IGF2 activity exerts important paracrine signaling at follicular selection. At the same time upregulation of estradiol synthesis and CYP19 mRNA expression increased steroid output profoundly. Furthermore, the highly significant association between FSHR and AR mRNA gene expression enforces important functions of androgens in follicular development. Collectively, these data reintroduce the understanding of the follicular phase as two parted in which regulation of steroidogenesis differs. The profound changes taking place around follicular selection highlight important paracrine actions of TGF-β family members and IGFs for securing dominance of the selected follicle.

Liver Kinase b1 (LKB1/STK11)is a tumor suppressor responsible for the Peutz-Jeghers syndrome, an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. Besides, the C allele of a SNP in the Lkb1 gene impedes the likelihood of ovulation in polycystic ovary syndrome (PCOS) in women treated with metformin, a known LKB1-AMPK activator. It is very likely that LKB1 plays roles in female fertility. To identify the physiological functions of LKB1 in the mouse ovary, we selectively disrupted LKB1 in oocytes by the Cre-LoxP conditional knockout system and found that Lkb1fl/fl; Gdf9-Cre mice were severely subfertile with significantly enlarged ovaries compared to Lkb1fl/fl mice. Interestingly, without Lkb1 expression in oocytes from the primordial follicle stage, the entire primordial follicle pool was activated but failed to mature and ovulate, subsequently causing premature ovarian failure (POF). Further investigation demonstrated that elevated mTOR signaling regulated by an AKT-independent LKB1-AMPK pathway was responsible for the excessive follicle activation and growth. Our findings reveal the role of LKB1 as an indispensable gatekeeper for the primordial follicle pool, offer new functional understanding for the tumor suppressor genes in reproductive organs, and might also provide valuable information for understanding POF and infertility.