Mature Follicles Research Articles

Transcriptome Dynamics and Cell Dialogs Between Oocytes and Granulosa Cells in Mouse Follicle Development.

The development and maturation of follicles is a sophisticated and multistage process. The dynamic gene expression of oocytes and their surrounding somatic cells and the dialogs between these cells are critical to this process. In this study, we accurately classified the oocyte and follicle development into nine stages and profiled the gene expression of mouse oocytes and their surrounding granulosa cells and cumulus cells. The clustering of the transcriptomes showed the trajectories of two distinct development courses of oocytes and their surrounding somatic cells. Gene expression changes precipitously increased at Type 4 stage and drastically dropped afterward within both oocytes and granulosa cells. Moreover, the number of differentially expressed genes between oocytes and granulosa cells dramatically increased at Type 4 stage, most of which persistently passed on to the later stages. Strikingly, cell communications within and between oocytes and granulosa cells became active from Type 4 stage onward. Cell dialogs connected oocytes and granulosa cells in both unidirectional and bidirectional manners. TGFB2/3, TGFBR2/3, INHBA/B, and ACVR1/1B/2B of TGF-β signaling pathway functioned in the follicle development. NOTCH signaling pathway regulated the development of granulosa cells. Additionally, many maternally DNA methylation- or H3K27me3-imprinted genes remained active in granulosa cells but silent in oocytes during oogenesis. Collectively, Type 4 stage is the key turning point when significant transcription changes diverge the fate of oocytes and granulosa cells, and the cell dialogs become active to assure follicle development. These findings shed new insights on the transcriptome dynamics and cell dialogs facilitating the development and maturation of oocytes and follicles.

Morphological assessment of ovaries during stimulation of ovulation in rab-bits with equine serum gonadotropin in different doses

Ovulatory stimulation of rabbits during artificial insemination requires the use of hormonal drugs to stimulate maturation of follicles, effective fertilization and full manifestation of female sexual function. For this purpose, gonadotropins or analogues of gonadotropin-releasing hormone are common in the practice of rabbit breeding. The latter have a selective effect on folliculogenesis and, under certain conditions, are not capable of inducing ovulation in rabbits of “zero” reproductive cycle, that is, those that have not given birth. Instead, gonadotropins, in particular serum gonadotropin obtained from foal mares (eCG), have a pronounced effect on folliculogenesis in animals. eCG is a glycoprotein hormone secreted by trophoblastic epithelial cells of fetal origin. In other species, except for horses, it is able to induce the release of both luteinizing and follicle-stimulating hormone. That is why eCG is widely used together with human chorionic gonadotropin to induce follicle growth and ovulation. However, there are data on complications and pathological conditions that may occur with long-term use of eCG, especially in genital tract. Therefore, the goal of our research was the morphological evaluation of the functional compartments of ovaries when using eCG to stimulate reproductive function of rabbits. Schemes of hormonal treatment of females in the conditions of a private rabbit farm, an experiment was conducted, which included the introduction of eCG in different doses (40 IU for animals of experimental group 1 and 25 IU for experimental group 2) during five reproductive cycles. Selection of material for histological studies, its fixation, production and analysis of tissue sections stained with hematoxylin and eosin were performed according to commonly used methods. The complex of morphofunctional changes in the ovaries of the rabbits of experimental groups corresponds to the pathomorphological picture of the syndrome of hyperstimulated ovaries. In our experiment, obtained changes were more pronounced at a dose of 40 IU, using which we additionally detected signs of increased immunoreactivity in body of rabbits, as evidenced by the presence of nodular lymphoid formations in outer theca of secondary and tertiary follicles. Thus, as a safe regimen for the use of eCG for ovulatory stimulation in rabbits, we recommend a dosage of 25 IU for 1–2 reproductive cycles.

#86 : Use of Triptorelin Versus Human Chorionic Gonadotropin as Ovulation Trigger

Background and Aims: Gonadotropin releasing hormone agonist (GnRH-a) trigger has been shown to be associated with significantly lower risk of moderate or severe ovarian hyperstimulation syndrome (OHSS) when compared among women at high risk of OHSS. However, trigger failure has been shown to occur in 2% of cycles using GnRH-a as trigger. This study aims to review the use of GnRH-a as ovulation trigger. Method: Retrospective review of records in antagonist cycles from an ART unit in a tertiary hospital from 2017-2021 was performed. Follicles were considered mature when they reach [Formula: see text] 15 mm. Ovulation triggered with urinary human chorionic gonadotropin (uhCG) (5000-10000 IU), recombinant hCG (rhCG) 250 mcg, or triptorelin 0.2 mg when there were [Formula: see text] 3 mature follicles. All embryos obtained from cycles using triptorelin were cryopreserved. Outcome including the number of oocytes retrieved, the follicle to oocyte index, ovarian hyperstimulation syndrome were reviewed. Results: 1378 cycles were included. 1089 cycles had uhCG or rhCG as trigger, while 284 cycles had triptorelin as trigger. Women in the triptorelin group were significantly younger with a lower basal follicle stimulating hormone level and a higher antral follicle count (Table 1). In the triptorelin group, the total number of mature sized follicles, estrogen level at trigger, oocytes retrieved, number of blastocysts were significantly higher compared to the hCG group (Table 2). Follicle to oocyte index was significantly higher in the triptorelin group but the proportion of mature oocytes were similar in both groups. There were no cases of trigger failure and OHSS in the triptorelin group, but 30 cases of OHSS, with18 cases early onset, 12 cases late onset, amongst them 10 cases (1%) were severe OHSS) in the hCG group. Conclusion: GnRH-a trigger is a good candidate as ovulatory trigger in women at risk of OHSS without hampering the laboratory outcome.

#285 : Amphiregulin Increases Pentraxin 3 Production via the EGFR-Mediated Erk1/2 Signaling in Human Granulosa Cells

Background and Aims: Pentraxin 3 (PTX3) plays a key role in the assembly of the extracellular matrix, which is critical for cumulus expansion, ovulation, and in vivo fertilization. As a member of the epidermal growth factor family, amphiregulin (AREG) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus expansion in mammalian follicles. The aim of this study was to investigate whether AREG regulates the expression and production of PTX3 in human granulosa cells and to examine its underlying molecular determinants. Method: Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein (hGL) cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression and accumulated levels of PTX3 were examined following cell incubation with AREG at different time points using the RT-qPCR and ELISA, respectively. Two kinase inhibitors (AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) were used to verify the specificity of the effects and to investigate the molecular mechanisms. The results were analyzed by one-way analysis of variance. Results: The results showed that AREG upregulated the expression and production of PTX3 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR receptor. Furthermore, we revealed that the Erk1/2 signaling is the downstream signaling pathway that mediates the increase in PTX expression and production in response to AREG treatment in hGL cells. Conclusion: AREG upregulated the expression of PTX3, leading to increased production of PTX3 in hGL cells. The AREG-induced cellular activities were most likely driven by the EGFR-mediated Erk1/2-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which AREG regulates the process of cumulus expansion and subsequent ovulation in humans.

Abstract B047: Tertiary lymphoid structures and B cells signatures determine clinically relevant T cell phenotype in ovarian cancer

Abstract Tertiary lymphoid structures (TLSs) are ectopic lymphoid formations that arise at sites with persistent inflammatory conditions, including tumors. TLSs are composed predominantly of B cells, T cells, and CD21+ CD23+ follicular dendritic cells and exhibit different levels of the organization, ranging from locally concentrated aggregates of immune cells to well-defined B cell follicles to mature follicles with germinal center formation. Supporting key relevance for TLSs in natural and immunotherapy-driven immunosurveillance, intratumoral TLS density has been associated with improved disease outcome and response to cancer immunotherapy, mainly check-point inhibitors (ICIs) in solid carcinomas. Here, we demonstrate that compared to the immunologically hot tumors like non-small cell lung carcinoma (NSCLC), human high-grade serous ovarian carcinoma (HGSOC) contains only a limited number of TLS with germinal center formation. In parallel, TLS frequency and maturation positively correlate with progenitor stem-like CD8+ T cell phenotype. In line with this notion, B cell depletion in HGSOC biopsies promoted terminally exhausted, poorly ICI-sensitive CD8+ T cells, suggesting a critical role of intratumoral B cells in maintaining T cell effector functions. These findings point to key numerical and functional differences between mature TLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative ICI-based immunotherapies for patients with HGSOC. Citation Format: Irena Kusova Moserova, Lenka Kasikova, Jana Rakova, Jana Tomankova, Michal Henser, Tereza Lanickova, Katerina Mojzisova, Jana Drozenova, Jan Laco, Lukas Rob, Michael Halaska, Ales Ryska, Robert Lischke, Jiri Vachtenheim, David Cibula, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Tertiary lymphoid structures and B cells signatures determine clinically relevant T cell phenotype in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B047.

#282 : Maximizing IVF Success Rates: Optimized Simulation Using a Novel Decision-Support Tool

Background and Aims: A mathematical procedure and a decision support tool, Opt-IVF, were developed to overcome the shortcomings of the current IVF practice of dosage profile determination by trial and error, which can provide a personalized model and optimized drug treatment. This paper describes the decision support tool for improving outcomes of IVF treatment for both antagonist and agonist protocols used in real practice. We present the results of two clinical trials. Method: Our earlier work developed models for agonist and antagonist protocols based on an analogy between superovulation and batch crystallization. This model was customized for each patient using two days of follicular data, and the optimal control method was applied to optimize the dosage profile. Validation of the model was conducted with retrospective data from 150 patients from India and 50 from the United States. Opt-IVF reduced total hormone dosage by 20 to 60%, reduced US testing by 50 to 75%, and provided a higher number of oocytes and high-quality embryos. Results: The primary outcome measures studied for two clinical trials with Opt-IVF include total hormonal dosage, % follicles retrieved, MII oocytes, Embryos, and Grade A Embryos. Results showed that Opt-IVF reduces drug dosage and testing and increases the number of embryos, Grade A embryos, and high-quality blastocysts significantly. Subgroup analysis showed improvements in outcomes were more significant for both Poor-responder and PCOS patients than for normal responders. Pregnancy rates were significantly higher with Opt-IVF patients than with standard protocol control patients. Conclusions: A mathematics-based approach to dosage profile determination results in optimal mature follicles, better embryos, and higher pregnancy with reduced dosage and the need for testing.

#336 : Improving Success of Intrauterine Insemination

Background and Aims: IUI is one of the most used methods to treat various forms of infertility. The aims were to compare the success rate of IUI with that of timed intercourse, review the methods used to time the IUI with ovulation, identify the factors to determine IUI outcome, compare the success rate of double IUI vs single IUI. Method: This is a cross-sectional prospective study conducted at a Private Fertility Centre, Chattogram, Bangladesh over a period of 40 months from 996 patients who underwent IUI. Variables such as age of women, cause of infertility, drugs used for COH, number of mature follicles, endometrial thickness post wash sperm count, type of IUI (single IUI and double IUI) were recorded and statistically analyzed. Results: The age for females and males were 18-41 and 23-52 respectively. The mean endometrial thickness was 8.9 mm ± 0.2 mm. Pregnancy rate for total motile sperm count less than 10 million was 7.2%. The successful conception rate for females were the age 28 was 9.6% compared to the whole group at 12%. Among 996 patients, 109 patients (10.94%) had successful conception. 209 patients (20.98%) underwent double IUI out of which 40 patients (19.14%) had a successful conception while 787 patients (79.01%) underwent single IUI out of which 69 patients (8.76%) had successful conception. Conclusion: IUI is currently a successful treatment modality for infertility especially as invasive treatment for couples with unexplained infertility, male factor infertility, anovulation, female less than 30 years of age. The numbers of motile sperms, appropriate endometrial thickness, have a remarkable impact on the outcome of IUI. Double IUI increases the success rate significantly. Acknowledgment: The authors are grateful to all who participated in the preparation of this manuscript.

#43 : Expression of miRNA-21-3p in Polycystic Ovary Syndrome and its Related Functions

Background and Aims: PCOS is an endocrine-related disease related to abnormal folliculogenesis and is a leading cause of infertility worldwide. Inhibition of GCs proliferation and increased GCs apoptosis have been identified as the major factors in aberrant follicle maturation. Recently, the roles of miRNAs were identified in polycystic ovary syndrome (PCOS). For instance, miR-21-3p was identified as an important miRNA regulating autophagy of BGCs. Besides, studies showed that AMSC-EXOs can rescue the polycystic phenotype and metabolic dysfunction in PCOS ovaries by transferring miR-21-5p to the livers of rats with PCOS, finally improve reproduction. However, there is no research clarified the association between miRNA-21-3p and GCs function to date. Method: In the present study, letrozole was used to establish the PCOS rat model. Separate the ovary and then miRNA-21-3p expression will be analyzed by RT-PCR. Primary rat ovarian GCs were isolated and incubated and transfected with mimic or inhibitor of miRNA-21-3p, CCK8 kit was used to detect cell viability, PR-PCR was used to detect cell proliferation and apoptosis related gene expression. Finally, flowcytometry was used to test apoptosis rate of Gcs. Results: The expression of miR-21-3p in the ovaries of PCOS rats was significantly increased compared with that in control rats. Primary rat ovarian GCs were isolated and incubated successfully, and then CCK-8 experiment showed that GCs viability was significantly decreased when miR-21-3p was overexpressed. Additionally, the miR-21-3p mimic significantly decreased the expression of PCNA, Bcl-2, cell cycle-related genes (CDK2, CCNE1) and increased the expression of Bax and caspase3 in primary rat ovarian GCs. By contrast, transfected primary rat ovarian GCs using the miR-21-3p inhibitor significantly upregulated the expression of PCNA, Bcl-2, cell cycle-regulated genes (CDK2, CCNE1) and reduced the expression of Bax and caspase3. Finally, flow cytometry analysis demonstrated that overexpression of miR-21-3p significantly increased the apoptosis rate of GCs. By contrast, the suppression of miR-21-3p resulted in a significant decrease in the apoptosis rate of GCs. Conclusion: In PCOS rat model, miRNA-21-3p was proved significantly increased compared with that in control rats. Which is participant in the development of PCOS by regulating the function of granular cells.

Insights into genetic diversity and phenotypic variations in domestic geese through comprehensive population and pan-genome analysis

BackgroundDomestic goose breeds are descended from either the Swan goose (Anser cygnoides) or the Greylag goose (Anser anser), exhibiting variations in body size, reproductive performance, egg production, feather color, and other phenotypic traits. Constructing a pan-genome facilitates a thorough identification of genetic variations, thereby deepening our comprehension of the molecular mechanisms underlying genetic diversity and phenotypic variability.ResultsTo comprehensively facilitate population genomic and pan-genomic analyses in geese, we embarked on the task of 659 geese whole genome resequencing data and compiling a database of 155 RNA-seq samples. By constructing the pan-genome for geese, we generated non-reference contigs totaling 612 Mb, unveiling a collection of 2,813 novel genes and pinpointing 15,567 core genes, 1,324 softcore genes, 2,734 shell genes, and 878 cloud genes in goose genomes. Furthermore, we detected an 81.97 Mb genomic region showing signs of genome selection, encompassing the TGFBR2 gene correlated with variations in body weight among geese. Genome-wide association studies utilizing single nucleotide polymorphisms (SNPs) and presence-absence variation revealed significant genomic associations with various goose meat quality, reproductive, and body composition traits. For instance, a gene encoding the SVEP1 protein was linked to carcass oblique length, and a distinct gene-CDS haplotype of the SVEP1 gene exhibited an association with carcass oblique length. Notably, the pan-genome analysis revealed enrichment of variable genes in the “hair follicle maturation” Gene Ontology term, potentially linked to the selection of feather-related traits in geese. A gene presence-absence variation analysis suggested a reduced frequency of genes associated with “regulation of heart contraction” in domesticated geese compared to their wild counterparts. Our study provided novel insights into gene expression features and functions by integrating gene expression patterns across multiple organs and tissues in geese and analyzing population variation.ConclusionThis accomplishment originates from the discernment of a multitude of selection signals and candidate genes associated with a wide array of traits, thereby markedly enhancing our understanding of the processes underlying domestication and breeding in geese. Moreover, assembling the pan-genome for geese has yielded a comprehensive apprehension of the goose genome, establishing it as an indispensable asset poised to offer innovative viewpoints and make substantial contributions to future geese breeding initiatives.

The effects of propolis-loaded chitosan nanoparticles and menstrual blood stem cells on LPS-induced ovarian inflammation in the murine ovary in vivo: An in vitro and in vivo study

Mammary glands infection via Gram-negative bacteria may cause infertility or reduced ovarian function. In the current study, a potential treatment for LPS-induced ovarian inflammation was developed. Propolis was loaded into chitosan nanoparticles and co-administered with menstrual blood stem cells (MenSCs) in mice infused with LPS. Various properties of propolis-loaded chitosan nanoparticles were evaluated using scanning electron microscopy, drug release assay, antibacterial assay, and radical scavenging assay. In vitro studies showed biocompatibility, anti-oxidative, and antibacterial properties of the developed propolis nanoformulation. In vivo study showed that mice treated with co-administration of propolis-loaded chitosan nanoparticles and MenSCs significantly increased the total ovarian follicle reserve in mice infused with LPS. Percentage of mature follicles in co-administration method was around 13.89 ± 1.72 %. Gene expression studies showed that the expression levels of inflammation related cytokines including IL6, IL8, IL-1β, and TNF-α were downregulated in this group compared with other groups. However, the expression levels of PTEN, AKT, FOXO3 did not show a significant difference between groups. The developed treatment may potentially considered as an approach for treating ovarian infection with gram-negative bacteria.

TNF-α and IFN-γ prestimulation enhances the therapeutic efficacy of human amniotic epithelial stem cells in chemotherapy-induced ovarian dysfunction

BackgroundExposure to a harsh ovarian microenvironment induced by chemotherapeutic agents seriously affects the remodeling of ovarian function and follicular development, leading to premature ovarian failure or insufficiency (POF/POI). For decades, the effectiveness of stem cell therapies in POI animal models has been intensively studied; however, strategies to enhance the therapeutic effect of stem cells remain challenging.MethodsIn this study, we first observed the pathological changes of the ovaries at different time points during chemotherapy, including the number of follicles, granulosa cell proliferation, oxidative stress damage, ovarian fibrosis, and inflammatory reaction. Moreover, we investigated whether activated hAECs stimulated by the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were more effective than native hAECs in repairing ovarian injury induced by chemotherapy.ResultsThe inhibitory effect of chemotherapy drugs on ovarian granulosa cells (GCs) in growing follicles mainly occurred on day 3 after chemotherapy in a mouse model. Then, continued ovarian injury, including oxidative damage and cell death cascades, resulted in the depletion of follicular reserves and inflammation-related ovarian fibrosis. Cytokine array demonstrated that activated hAECs secreted high levels of paracrine cytokines related to extracellular matrix (ECM) remodeling, angiogenesis, and immunomodulation. An in vivo study showed that the engraftment rate of activated hAECs in damaged ovaries was higher than that of native hAECs. Furthermore, activated hAECs in damaged ovaries had significantly upregulated expression of the antioxidant proteins thioredoxin1/2. In addition, activated hAECs had increased numbers of mature follicles and ameliorated the ovarian microenvironment by promoting angiogenesis and reducing ovarian fibrosis.ConclusionsThese results indicated that secondary ovarian damage induced by chemotherapy, including oxidative stress damage, chronic inflammatory response, and ovarian tissue fibrosis should be attended. Prestimulation with the proinflammatory factors TNF-α and IFN-γ could enhance the therapeutic efficacy of hAECs against chemotherapy-induced ovarian dysfunction, which may become a new feasible strategy to improve the therapeutic potential of hAECs in regenerative medicine.Graphical

Attenuated AKT signaling by miR-146a-5p interferes with chicken granulosa cell proliferation, lipid deposition and progesterone biosynthesis

Steroid hormones play a crucial role in the growth and maturation of poultry ovarian follicles, with progesterone secretion by granulosa cells (GC) being essential. According to our previous transcriptome analysis, it apparented that miR-146a-5p expressions were upregulated in the follicles undergoing atresia. In this study, we delved the depth to explore the underlying mechanisms by miR-146a-5p in the regulation of follicle functions in chicken. The study demonstrated that miR-146a-5p suppressed cell growth, lipids accumulation, and progesterone biosynthesis in chicken GC. Through targeting association validations, we identified delta 4-desaturase, sphingolipid 1 (DEGS1) as capable of interacting with miR-146a-5p. Co-transfection experiments further confirmed that DEGS1 reversed the impairment of GC functions by miR-146a-5p. Moreover, we discovered that miR-146a-5p suppressed AKT phosphorylation, while DEGS1 enhanced AKT phosphorylation. Phosphatidylinositol-3 kinase (PI3K) inhibitor (LY294002) studies showed that miR-146a-5p would inhibit AKT phosphorylation by governing the DEGS1/AKT pathway, which in turn regulates GC function. In summary, the findings revealed that miR-146a-5p suppressed cell growth, lipid deposition, and progesterone biosynthesis via the DEGS1/AKT pathway. These results may further enrich our understandings of how non-coding RNA regulates productive performance in chickens.

Association of Vitamin-D insufficiency and infertility: A Literature Review

Vitamin D receptors are present in both male and female central neural system and reproductive system hence Vitamin D sufficiency or insufficiency affects fertility. The present review aims to determine a correlation between Vitamin D insufficiency and infertility. Regarding female infertility, it was found that Vitamin D insufficiency is associated with increased issues during conception and higher miscarriage rate. Furthermore, Vitamin D insufficiency is associated with lower FSH levels affecting follicle formation, maturation and ovulation. Regarding male infertility, Vitamin D and its Receptor have been identified in the prostate, seminal vesicles, epididymis, germ cells, spermatogonia, spermatocytes and Sertoli cells. The prevalence of Vitamin D insufficiency is higher among men with lower sperm count and lower motility. However, the relation between Vitamin D and testosterone levels remain unclear. The present review stresses the effects of Vitamin D insufficiency in both male and female infertility.

Comparative analyses of laying performance and follicular development characteristics between fat and lean broiler lines

The deposition of high levels of fat in broiler breeder hens can have a profound impact on follicular development and laying performance. This study was formulated with the goal of comparing egg production and follicular development characteristics at different laying stages in the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The egg production was analyzed using the birds from the 19th to 24th generations of NEAUHLF; the follicular development characteristics were analyzed by hematoxylin-eosin staining and quantitative real-time polymerase chain reaction using the birds from the 24th generation of NEAUHLF. The results showed that the age at first egg of lean hens was significantly earlier than that of fat hens in this study. While no significant differences in total egg output from the first egg to 50 wk of age were noted when comparing these 2 chicken lines, lean hens laid more eggs from the first egg to 35 wk of age relative to fat hens, whereas fat hens laid more eggs from wk 36 to 42 and 43 to 50 relative to their lean counterparts. No differences in ovarian morphology and small yellow follicle (SYF) histological characteristics were noted when comparing these 2 chicken lines at 27 wk of age. At 35 and 52 wk of age, however, lean hens exhibited significantly lower ovarian weight, ovarian proportion values, numbers of hierarchical follicles, hierarchical follicle weight, and SYF granulosa layer thickness as compared to fat hens, together with a significant increase in the number of prehierarchical follicles relative to those in fat hens. Gene expression analyses suggested that follicle selection was impaired in the fat hens in the early laying stage, whereas both follicle selection and maturation were impaired in the lean hens in the middle and late laying stages. Overall, these data highlight that fat deposition in broiler hens can have a range of effects on follicular development and egg production that are laying stage-dependent.

Ovary of Zebrafish during Spawning Season: Ultrastructure and Immunohistochemical Profiles of Sox9 and Myostatin.

This study sought to examine the ovarian cellular and stromal components of the zebrafish (Danio rerio) throughout the spawning season using light and electron microscopic tools. The ovaries of zebrafish showed oocytes in all stages of follicular development and degeneration (atresia). Six stages of oogenesis were demonstrated: oogonia, early oocytes, late oocytes, vacuolated follicles, the yolk globule stage (vitellogenesis), and mature follicles. The SOX9 protein was expressed in the ooplasm of the primary and previtellogenic oocytes and the theca cell layer of the mature follicles. Myostatin was expressed in the granulosa and theca cells. Many stem cells in the ovarian stroma expressed myostatin and SOX9. During the spawning season, the EM results indicated that the zona radiata increased in thickness and was crossed perpendicularly by pore canals that contained processes from both oocytes and zona granulosa. The granulosa cells contained many mitochondria, rER, sER, and vesicles. Meanwhile, the thecal layer consisted of fibroblast-like cells. Atretic follicles could be demonstrated that involved both oocytes and their follicular walls. Several types of cells were distinguished in the ovarian stroma, including mast cells, telocytes, lymphocytes, fibroblasts, endocrine cells, macrophages, adipocytes, dendritic cells, and steroidogenic (stromal) cells. The ovary of the zebrafish serves as a model to investigate follicular development.

Gonadotropin Stimulation's Effects on Folliculogenesis in Mild and Severe Endometriosis

Infertility in endometriosis has been linked to a number of theories, including a disruption in the folliculogenesis process. Further research is needed to determine whether this issue is linked to the stage of endometriosis or gonadotropin stimulation. In a cross-sectional study design, the study comprised 28 women with moderate endometriosis and 25 women with severe endometriosis who had had in vitro fertilization. The data includes the total number of follicles, mature follicles, and ovarian responses. Bivariate and multivariate analysis were used to examine the data. In bivariate analysis, the t-test and chi-square were utilized. There was no significantly difference in number of follicles total between the two groups (15.2 vs. 10.6; p>0,05). In comparison to the severe endometriosis group, the moderate endometriosis group had considerably more mature follicles (2.891.62 vs. 1.761.17; p0,05).In the severe endometriosis group, insufficient ovarian response was more likely (RR 1.26 ; p 0.018). The number of follicles in total is the same in mild and severe endometriosis, but mild endometriosis has more developed follicles, and severe endometriosis has a higher rate of inadequate ovarian response

Association of Metabolic and Endocrine Disorders with Bovine Ovarian Follicular Cysts.

After estrus, when mature follicles fail to ovulate, they may further develop to form follicular cysts, affecting the normal function of ovaries, reducing the reproductive efficiency of dairy cows and causing economic losses to cattle farms. However, the key points of ovarian follicular cysts pathogenesis remain largely unclear. The purpose of the current research was to analyze the formation mechanism of ovarian follicular cysts from hormone and gene expression profiles. The concentrations of progesterone (P4), estradiol (E2), insulin, insulin-like growth factor 1 (IGF1), leptin, adrenocorticotropic hormone (ACTH) and ghrelin in follicle fluid from bovine follicular cysts and normal follicles were examined using enzyme-linked immunosorbent assay (ELISA) or 125I-labeled radioimmunoassay (RIA); the corresponding receptors' expression of theca interna cells was tested via quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the mRNA expression profiling was analyzed via RNA sequencing (RNA-seq). The results showed that the follicular cysts were characterized by significant lower E2, insulin, IGF1 and leptin levels but elevated ACTH and ghrelin levels compared with normal follicles (p < 0.05). The mRNA expressions of corresponding receptors, PGR, ESR1, ESR2, IGF1R, LEPR, IGFBP6 and GHSR, were similarly altered significantly (p < 0.05). RNA-seq identified 2514 differential expressed genes between normal follicles and follicular cysts. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis linked the ovarian steroidogenesis pathway, especially the STAR, 3β-HSD, CYP11A1 and CYP17A1 genes, to the formation of follicular cysts (p < 0.01). These results indicated that hormone metabolic disorders and abnormal expression levels of hormone synthesis pathway genes are associated with the formation of bovine ovarian follicular cysts.

Identification of Lipids and Cytokines in Plasma and Follicular Fluid before and after Follicle-Stimulating Hormone Stimulation as Potential Markers for Follicular Maturation in Cattle.

The process of follicle maturation leading to ovulation is a key milestone in female fertility. It is known that circulating lipids and cytokines play a role in the follicle's ability to go through follicular maturation and the ovulatory processes. However, the specific mechanisms are not well understood. We posit that dysregulation of granulosa cells influences the ovarian environment, which tries to adapt by changing released lipids and cytokines to achieve follicular maturation. Eleven non-lactating adult females underwent estrus synchronization with two injections of PGF2α 14 days apart. Daily blood samples were collected for 28 days to monitor steroid hormone production after the second injection. To understand the potential impacts of lipids and cytokines during ovulation, a low-dose FSH stimulation (FSHLow) was performed after resynchronization of cows, and daily blood samples were collected for 14 days to monitor steroid hormone production until ovariectomies. The lipidomic analysis demonstrated increased circulating diacylglycerides and triacylglycerides during the mid-luteal phase and after FSHLow treatment. Cholesteryl esters decreased in circulation but increased in follicular fluid (FF) after FSHLow. Increased circulating concentrations of TNFα and reduced CXCL9 were observed in response to FSHLow. Therefore, specific circulating lipids and cytokines may serve as markers of normal follicle maturation.

Unilateral lesion of the suprachiasmatic nucleus impairs estradiol feedback, follicular development, estrous cycle and ovulation.

Ovulation is timed by neural signals originating at the suprachiasmatic nucleus (SCN) that trigger ovulation when converge with high estradiol levels, which indicates the maturation of ovarian follicles. We have shown that the hypothalamic regulation of ovulation is asymmetrical and we hypothesized that the paired SCN could contribute to such symmetries. We unilaterally lesioned the SCN of rats at each stage of the estrous cycle and evaluated the acute effects on the progression of their estrous cycle, follicular development and ovulation. Lesions disrupted the estrous cycle when performed in estrus/metestrus but not in diestrus/proestrus. Abnormalities in follicular development were observed in the non-ovulating lesioned rats and this was independent of the side of the SCN destroyed and the stage of the cycle when surgery was performed. Groups of lesioned rats were then hormonally primed with GnRH or estradiol to assess the neuroendocrine pathway altered by the treatment. GnRH restored ovulation, suggesting that both SCN are needed for proper triggering of the preovulatory surge of GnRH and that unilateral lesion does not impair the sensitivity of the pituitary or the ovary to GnRH and gonadotropins, respectively. Estradiol was asymmetrically effective to restore ovulation in rats lesioned in estrous, partially effective on rats operated at diestrus and ineffective on metestrus. Our results indicate that the SCN regulates the activity of the hypothalamic-pituitary-ovarian axis not only by modulating the preovulatory surge of GnRH/gonadotropins, but also by promoting the hypothalamic integration of estrogenic signals from the ovaries in an asymmetric and stage-dependent fashion.

OR19-04 Hypoglycosylated FSH Enhances Ovarian Follicle Development and Gamete Quality

Abstract Disclosure: A. Converse: None. T. Kumar: None. F.E. Duncan: None. Gamete quality depends on the proper growth and maturation of the ovarian follicle, which consists of the oocyte and its supporting somatic cells. FSH is essential for follicle growth as female Fshb and Fshr null mice are infertile due to arrested follicle development. Macroheterogeneity in N-glycosylation on the FSHß-subunit confers differential bioactivities with hypoglycosylated FSH21 exhibiting higher FSHR binding affinity and bioactivity compared to fully glycosylated FSH24. In females, there is a shift from the highly bioactive FSH21 to FSH24 with increasing age which occurs coordinately with the age-dependent decline in female fertility. In this study, we used an encapsulated in vitro follicle growth assay to test the hypothesis that FSH glycoforms have differential abilities to promote follicle development and gamete quality. Early-stage (preantral) ovarian follicles were isolated from CD-1 mice, cultured in media containing 10 ng/ml of either FSH21 or FSH24 for up to 12 days, and then ex vivo ovulation was induced. While both glycoforms maintained &amp;gt;75% follicle survival through day 12, follicles treated with FSH21 were significantly larger than FSH24-treated follicles between days 4 and 12 of culture. Over the 12 day culture, FSH21-treated follicles grew from 110.9 ± 9.2 µm to 251.3 ± 24.4 µm, while FSH24-treated follicles grew from 109.3 ± 7.1 µm to 229.7 ± 15.4 µm. This difference in growth correlated with a 13.4 and 4.5-fold increase in estradiol secretion by FSH21-treated follicles compared to FSH24 treatment on days 8 and 12, respectively. After ex vivo maturation, FSH21-treated follicles produced a higher proportion (76.9%) of meiotically competent gametes that reached the metaphase II (MII)-arrested state compared to FSH24-treated follicles (62.5%). Furthermore, normal meiotic spindle configuration was observed in 76% of MII eggs from the FSH21 cohort but only 50% of the FSH24 cohort. These results indicate that FSH21 is more bioactive than FSH24 in promoting follicle development and high-quality oocytes. Mechanistically, this is likely due in part to the differential effects of FSH21 and FSH24 on the formation of transzonal projections (TZPs), which are cytoplasmic filaments essential for granulosa-oocyte communication. By 24 hrs, FSH21-treated follicles had more TZPs than FSH24-treated follicles, as demonstrated by increased actin staining (1.6x) and Myo10 foci (5.1x) in the zona pellucida region. Thus, the early establishment of TZPs and increased somatic-oocyte communication in FSH21-treated follicles ultimately results in a more developmentally competent gamete. In addition, we anticipate that ongoing transcriptomic analysis of 24 hr FSH glycoform-treated follicles will highlight other processes involved in early follicle development that contribute to the observed differential effects on oocyte quality. Presentation Date: Saturday, June 17, 2023