ETS transcription factors regulate precise matrix metalloproteinase expression and follicle rupture in Drosophila.

Drosophila matrix metalloproteinase 2 (MMP2) is specifically expressed in posterior follicle cells of stage-14 egg chambers (mature follicles) and is crucial for the breakdown of the follicular wall during ovulation, a process that is highly conserved from flies to mammals. The factors that regulate spatiotemporal expression of MMP2 in follicle cells remain unknown. Here, we demonstrate crucial roles for the ETS-family transcriptional activator Pointed (Pnt) and its endogenous repressor Yan in the regulation of MMP2 expression. We found that Pnt is expressed in posterior follicle cells and overlaps with MMP2 expression in mature follicles. Genetic analysis demonstrated that pnt is both required and sufficient for MMP2 expression in follicle cells. In addition, Yan was temporally upregulated in stage-13 follicle cells to fine-tune Pnt activity and MMP2 expression. Furthermore, we identified a 1.1 kb core enhancer that is responsible for the spatiotemporal expression of MMP2 and contains multiple pnt/yan binding motifs. Mutation of pnt/yan binding sites significantly impaired the Mmp2 enhancer activity. Our data reveal a mechanism of transcriptional regulation of Mmp2 expression in Drosophila ovulation, which could be conserved in other biological systems.

Immune and oxidative stress disorder in ovulation-dysfunction women revealed by single-cell transcriptome.

Introduction: Ovulation dysfunction is now a widespread cause of infertility around the world. Although the impact of immune cells in human reproduction has been widely investigated, systematic understanding of the changes of the immune atlas under female ovulation remain less understood. Methods: Here, we generated single cell transcriptomic profiles of 80,689 PBMCs in three representative statuses of ovulation dysfunction, i.e., polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI) and menopause (MENO), and identified totally 7 major cell types and 25 subsets of cells. Results: Our study revealed distinct cluster distributions of immune cells among individuals of ovulation disorders and health. In patients with ovulation dysfunction, we observed a significant reduction in populations of naïve CD8 T cells and effector memory CD4 T cells, whereas circulating NK cells and regulatory NK cells increased. Discussion: Our results highlight the significant contribution of cDC-mediated signaling pathways to the overall inflammatory response within ovulation disorders. Furthermore, our data demonstrated a significant upregulation of oxidative stress in patients with ovulation disorder. Overall, our study gave a deeper insight into the mechanism of PCOS, POI, and menopause, which may contribute to the better diagnosis and treatments of these ovulatory disorder.

Metagenomic analysis reveals a dynamic microbiome with diversified adaptive functions that respond to ovulation regulation in the mouse endometrium.

Understanding the microflora inhabiting the reproductive tract is important for a better understanding of female physiology and reproductive health. The endometrial fluid from mice in three reproductive stages (A: Unproductive mice; B: Postovulatory mice; C: Postpartum mice) was extracted for microbial DNA extraction and sequencing. Phenotypic and functional analyses of endometrial microbial enrichment was undertaken using LefSe. The results showed 95 genera and 134 species of microorganisms in the uteri of mice. There were differentially distributed genera, among which Lactobacillus, Enterococcus, and Streptococcus were more abundant in the endometrial fluid of mice in the unproductive group. That of mice in the postovulatory group was colonized with Salmonella enterica and Campylobacter and was mainly enriched in metabolic pathways and steroid biosynthesis. The presence of Chlamydia, Enterococcus, Pseudomonadales, Acinetobacter, and Clostridium in the endometrial fluid of postpartum mice, in addition to the enrichment of the endocrine system and the Apelin and FoxO signaling pathways, resulted in a higher number of pathogenic pathways than in the other two groups. The results showed that the microbial diversity characteristics in the endometrium of mice in different reproductive states differed and that they could be involved in the regulation of animal reproduction through metabolic pathways and steroid biosynthesis, suggesting that reproductive diseases induced by microbial diversity alterations in the regulation of animal reproduction cannot be ignored.

ADAMTS Proteases: Importance in Animal Reproduction.

Many reproductive physiological processes, such as folliculogenesis, ovulation, implantation, and fertilization, require the synthesis, remodeling, and degradation of the extracellular matrix (ECM). The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) family genes code for key metalloproteinases in the remodeling process of different ECM. Several genes of this family encode for proteins with important functions in reproductive processes; in particular, ADAMTS1, 4, 5 and 9 are genes that are differentially expressed in cell types and the physiological stages of reproductive tissues. ADAMTS enzymes degrade proteoglycans in the ECM of the follicles so that the oocytes can be released and regulate follicle development during folliculogenesis, favoring the action of essential growth factors, such as FGF-2, FGF-7 and GDF-9. The transcriptional regulation of ADAMTS1 and 9 in preovulatory follicles occurs because of the gonadotropin surge in preovulatory follicles, via the progesterone/progesterone receptor complex. In addition, in the case of ADAMTS1, pathways involving protein kinase A (PKA), extracellular signal regulated protein kinase (ERK1/2) and the epidermal growth factor receptor (EGFR) might contribute to ECM regulation. Different Omic studies indicate the importance of genes of the ADAMTS family from a reproductive aspect. ADAMTS genes could serve as biomarkers for genetic improvement and contribute to enhance fertility and animal reproduction; however, more research related to these genes, the synthesis of proteins encoded by these genes, and regulation in farm animals is needed.

Granulosa Cell Specific Loss of Adar in Mice Delays Ovulation, Oocyte Maturation and Leads to Infertility.

Adenosine deaminases acting on RNA-(ADAR) comprise one family of RNA editing enzymes that specifically catalyze adenosine to inosine (A-to-I) editing. A granulosa cell (GC) specific Adar depleted mouse model [Adar flox/flox:Cyp19a1-Cre/+ (gcAdarKO)] was used to evaluate the role of ADAR1 during the periovulatory period. Loss of Adar in GCs led to failure to ovulate at 16 h post-hCG, delayed oocyte germinal vesicle breakdown and severe infertility. RNAseq analysis of GC collected from gcAdarKO and littermate control mice at 0 and 4 h post-hCG following a super-ovulatory dose of eCG (48 h), revealed minimal differences after eCG treatment alone (0 h), consistent with normal folliculogenesis observed histologically and uterine estrogenic responses. In contrast, 300 differential expressed genes (DEGs; >1.5-fold change and FDRP < 0.1) were altered at 4 h post-hCG. Ingenuity pathway analysis identified many downstream targets of estrogen and progesterone pathways, while multiple genes involved in inflammatory responses were upregulated in the gcAdarKO GCs. Temporal expression analysis of GCs at 0, 4, 8, and 12 h post-hCG of Ifi44, Ifit1, Ifit3b, and Oas1g and Ovgp1 confirmed upregulation of these inflammatory and interferon genes and downregulation of Ovgp1 a glycoprotein involved in oocyte zona pellucida stability. Thus, loss of ADAR1 in GCs leads to increased expression of inflammatory and interferon response genes which are temporally linked to ovulation failure, alterations in oocyte developmental progression and infertility.

Steroidogenic Factor 1 Regulation of the Hypothalamic-Pituitary-Ovarian Axis of Adult Female Mice.

The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor-driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization.

Modulatory effects of R10 fraction of garlic (Allium sativum L.) on hormonal levels, T cell polarization, and fertility-related genes in mice model of polycystic ovarian syndrome.

Polycystic ovary syndrome (PCOS) is an inflammatory endocrine-metabolic disorder related to reproductive system characterized by polycystic ovarian morphology, androgen excess, and chronic anovulation. Current treatments haven't been very successful in PCOS treatment and the problem still remains as a challenge. Therefore, new approaches should be applied to overcome the disease. Previous studies demonstrated immunomodulatory effects of R10 fraction of garlic in the treatment of inflammatory conditions such as cancer. Considering previous studies suggesting immunomodulatory therapy for PCOS, therapeutic effects of R10 fraction was evaluated in a mouse model of PCOS. To do so, PCOS was developed by intramuscular injection of estradiol valerate. Treatment with R10 fraction, isolated from garlic, was performed and the alterations in hormonal levels (estradiol, progesterone, and testosterone), T cell polarization markers (IFN-γ, IL-4, and IL-17), and expression of fertility-related genes (Gpx3 and Ptx3) were evaluated. The results showed that hormonal levels were elevated in PCOS model comparing to normal animals but were markedly modulated after treatment with R10 fraction. Moreover, a severe disturbance in T cell polarization with a significant reduction of fertility-related genes expression were detected in PCOS-induced ovaries. Treatment with R10 fraction also represented modulatory effects on T cell polarization by increasing IL-4 and decreasing IL-17 and IFN-γ levels. Accordingly, fertility-related genes were also modulated following treatment with R10 fraction in PCOS. Our study elucidated that R10 fraction of garlic possess immunomodulatory effects alleviating PCOS symptoms. This approach could be adjusted to give rise the optimum therapeutic results and considered as a candidate therapeutic approach for PCOS.

Association of raised serum progesterone level with ovulation trigger and histology of endometrium in stimulated cycles

Abstract This was a forthcoming study of those patients, who undergo in-vitro fertilization (IVF) and freeze-all embryo, who acquiesce for the study. The number of participated patients (n=350) in this study, underwent for IVF. The blood sample was collected from patients to evaluate the level of serum progesterone in vacuum vials on the day of ovulation trigger. After 36 hrs of ovulation trigger, ovum picked up was done. Quantitative methods were used to estimate the level of serum progesterone through the electrochemiluminescence immunoassay and correlation of serum progesterone with embryo transfer (ET) outcomes. Main outcome of this current study was to evaluate the value of mean serum progesterone level i.e.0.868± 0.712 ng/ml and 0.88±0.723 ng/ml was found in case of pregnancy positive and negative respectively, at p=0.216 value. In antagonist (n=40) and agonist (n=310) cases, it was 8(20%) and 37(11.94%) PL occurrence was noted at p=0.143 respectively. An overall value of the premature lutenization (PL) occurrences was 13.63% and 15.25% observed in both positive and negative cases of pregnancy at p=0.216 respectively. This study concluded that 12.66% of PL occurrences were recorded in the case of IVF. Study results proved, there were no significant effect of PL on pregnancy outcomes.

Ovulatory upregulation of angiotensin-converting enzyme 2, a receptor for SARS-CoV-2, in dominant follicles of the human ovary.

OBJECTIVE: To determine the temporal expression of angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV-2, in dominant follicles throughout the periovulatory period in women and the regulatory mechanisms underlying ACE2 expression in human granulosa/lutein cells (hGLC). DESIGN: Experimental prospective clinical study and laboratory-based investigation. SETTING: University Medical Center and private in vitro fertilization center. PATIENT(S): Thirty premenopausal women undergoing surgery for tubal ligation and 16 premenopausal women undergoing in vitro fertilization. INTERVENTION(S): Administration of human chorionic gonadotropin (hCG) and harvesting of preovulatory/ovulatory follicles by timed laparoscopy, and collection of granulosa/lutein cells and cumulus cells at the time of oocyte retrieval. MAIN OUTCOME MEASURE(S): Expression and localization of ACE2 in granulosa cells and dominant follicles collected throughout the periovulatory period of the menstrual cycle and in hGLC using quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULT(S): ACE2 expression (mRNA and protein) is up-regulated in human ovulatory follicles after administration of hCG. ACE2 expression was higher in cumulus cells than in granulosa cells. hCG increased the expression of ACE2 in primary hGLC cultures; the increase was inhibited by RU486 (an antagonist for progesterone receptor and glucocorticoid receptor) and CORT125281 (a selective glucocorticoid receptor antagonist), but not by AG1478 (an EGF receptor tyrosine kinase inhibitor) or by dexamethasone. CONCLUSION(S): The hormone-regulated expression of ACE2 in granulosa cells suggests a potential role of ACE2 in the ovulatory process. These data also imply the possible impact of COVID-19 on a vital cyclic event of ovarian function and thus on women's overall reproductive health. However, SAR-CoV-2 infection in ovarian cells in vivo or in vitro has yet to be determined.

Expression Profile of New Gene Markers and Signaling Pathways Involved in Immunological Processes in Human Cumulus-Oophorus Cells.

The function of the immune system extends from defense against external pathogens to the recognition and elimination of mutated or dying cells, aiding elimination of malignant potential and/or maintaining homeostasis. The many cell types of the immune system secrete a broad range of factors to enable cellular signaling that is vital to physiological processes. Additionally, in the ovary, follicular selection and maturation, as well as ovulation, are directly regulated by the nearby immune cells. Additionally, ovulation and rupture of the follicle have been observed to resemble a local inflammatory response. Cells of the cumulus-oocyte complex (COC) show evolving gene expression profiles throughout the oocytes' lifespan, including genes associated with immunological processes. Analysis of these genes allows the identification of useful molecular markers, as well as highlighting gene functions and interactions in these cells. Cumulus cells were obtained from hormonally stimulated patients undergoing an in vitro fertilization procedure and studied under long-term culture conditions. The microarray technique made it possible to compare the level of CCs' gene expression on the 1st, 7th, 15th and 30th day of cultivation. Additionally, RNA microarray analysis was performed to map gene expression in these cells, associated with immunological processes and associated cytokine signaling. Subsequently, the use of DAVID software allowed us to identify the "defense response to other organism", "defense response", "defense response to virus", "cytokine secretion", "cytokine production" and "cytokine-mediated signaling pathway" GO BP terms, as well as allowing further analysis of the most differentially expressed genes associated with these processes. Of the 122 genes involved, 121 were upregulated and only one was downregulated. The seven most upregulated genes related to the abovementioned terms were ANXA3, IFIT1, HLA-DPA1, MX1, KRT8, HLA-DRA and KRT18. Therefore, genes involved in immunological defense processes are upregulated in CC cultures and could serve as useful molecular markers of growth and development in the COC, as well as the proliferation of granulosa and cumulus cells.

Nuclear progesterone receptor regulates ptger4b and PLA2G4A expression in zebrafish (Danio rerio) ovulation.

Previous studies have implicated the nuclear progesterone receptor (Pgr or nPR) as being critical to ovulation in fishes. This study investigated the expression of Pgr in zebrafish ovarian follicles throughout development as well as putative downstream targets of Pgr by searching the promoter regions of selected genes for specific DNA sequences to which Pgr binds and acts as a transcription factor. Expression of Pgr mRNA increases dramatically as follicles grow and mature. In silico analysis of selected genes linked to ovulation showed that the prostaglandin receptors ptger4a and ptger4b contained the progesterone responsive element (PRE) GRCCGGA in their promoter regions. Studies using full-grown follicles incubated in vitro revealed that ptger4b was upregulated in response to 17,20ß-P. Our studies also showed that the expression of phospholipase A2 (PLA2G4A) mRNA and protein, a key enzyme in prostaglandin synthesis, was upregulated in response to 17,20ß-P treatment. pla2g4a was not found to contain a PRE, indicating that it is regulated indirectly by 17,20ß-P or that it may contain an as-of-yet unidentified PRE in its promoter region. Collectively, these studies provide further evidence of the importance of Pgr during the periovulatory periods through its involvement in prostaglandin production and function by controlling expression of PLA2G4A and the receptor EP4b and that these genes appear to be regulated through the actions of 17,20ß-P.

The FOS/AP-1 Regulates Metabolic Changes and Cholesterol Synthesis in Human Periovulatory Granulosa Cells.

FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of FOS using primary human granulosa/lutein cells (hGLCs). Human chorionic gonadotropin (hCG) induced a biphasic increase in the expression of FOS, peaking at 1 to 3 hours and 12 hours. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Coimmunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated protein kinase A and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high-throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis, and cholesterol biosynthesis. Quantitative PCR analysis verified hCG-induced, T-5224-regulated expression of a selection of genes involved in these processes. Consistently, hCG-induced increases in metabolic activities and cholesterol levels were suppressed by T-5224. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 complex in metabolic changes and cholesterol biosynthesis in granulosa/lutein cells of human periovulatory follicles.

Morphology and Biochemistry of Ovulation Morfologia e bioquímica da ovulação

Abstract The process of ovulation involves multiple and iterrelated genetic, biochemical, and morphological events: cessation of the proliferation of granulosa cells, resumption of oocyte meiosis, expansion of cumulus cell-oocyte complexes, digestion of the follicle wall, and extrusion of the metaphase-II oocyte. The present narrative review examines these interrelated steps in detail. The combined or isolated roles of the folliclestimulating hormone (FSH) and luteinizing hormone (LH) are highlighted. Genes indiced by the FSH genes are relevant in the cumulus expansion, and LH-induced genes are critical for the resumption ofmeiosis and digestion of the follicle wall. A nonhuman model for follicle-wall digestion and oocyte release was provided.

Resumo O processo de ovulação envolve modificações genéticas, bioquímicas e morfológicas múltiplas e interrelacionadas: suspensão da proliferação das células da granulosa, reinício da meiose do oócito, expansão das células do complexo cumulus-oócito, digestão da parede folicular, e extrusão do oócito. Esta revisão narrativa examina em detalhes cada um desses eventos e os principais genes e proteínas envolvidos. Mais importante, a ação combinada ou isolada do hormônio folículo-estimulante (HFE) e do hormônio luteinizante (HL) é destacada. Detalha-se o papel do HFE na expansão do cumulus e do HL na digestão da parede folicular, permitindo a extrusão do oócito na superfície ovariana. Proveu-se um modelo não humano para explicar a digestão da parede folicular.

A central role for cAMP/EPAC/RAP/PI3K/AKT/CREB signaling in LH-induced follicular Pgr expression at medaka ovulation†.

Nuclear progestin receptor (PGR) is a ligand-activated transcription factor that has been identified as a pivotal mediator of many processes associated with ovarian and uterine function, and aberrant control of PGR activity causes infertility and disease including cancer. The essential role of PGR in vertebrate ovulation is well recognized, but the mechanisms by which PGR is rapidly and transiently induced in preovulatory follicles after the ovulatory LH surge are not known in lower vertebrates. To address this issue, we utilized the small freshwater teleost medaka Oryzias latipes, which serves as a good model system for studying vertebrate ovulation. In the in vitro ovulation system using preovulatory follicles dissected from the fish ovaries, we found that inhibitors of EPAC (brefeldin A), RAP (GGTI298), PI3K (Wortmannin), AKT (AKT inhibitor IV), and CREB (KG-501) inhibited LH-induced follicle ovulation, while the PKA inhibitor H-89 had no effect on follicle ovulation. The inhibitors capable of inhibiting follicle ovulation also inhibited follicular expression of Pgr and matrix metalloproteinase-15 (Mmp15), the latter of which was previously shown to not only be a downstream effector of Pgr but also a proteolytic enzyme indispensable for follicle rupture in medaka ovulation. Further detailed analysis revealed for the first time that the cAMP/EPAC/RAP/PI3K/AKT/CREB signaling pathway mediates the LH signal to induce Pgr expression in preovulatory follicles. Our data also showed that phosphorylated Creb1 is a transcription factor essential for pgr expression and that Creb1 phosphorylated by Akt1, rather than PKA, may be preferably used to induce pgr expression.

Oocyte competence is independent of the ovulation trigger adopted: a large observational study in a setting that entails vitrified-warmed single euploid blastocyst transfer.

PURPOSE: To assess whether the GnRH-agonist or urinary-hCG ovulation triggers affect oocyte competence in a setting entailing vitrified-warmed euploid blastocyst transfer. METHODS: Observational study (April 2013-July 2018) including 2104 patients (1015 and 1089 in the GnRH-a and u-hCG group, respectively) collecting ≥1 cumulus-oocyte-complex (COC) and undergoing ICSI with ejaculated sperm, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing, and vitrified-warmed transfers at a private clinic. The primary outcome measure was the euploid-blastocyst-rate per inseminated oocytes. The secondary outcome measure was the maturation-rate per COCs. Also, the live-birth-rate (LBR) per transfer and the cumulative-live-birth-delivery-rate (CLBdR) among completed cycles were investigated. All data were adjusted for confounders. RESULTS: The generalized-linear-model adjusted for maternal age highlighted no difference in the mean euploid-blastocyst-rate per inseminated oocytes in either group. The LBR per transfer was similar: 44% (n=403/915) and 46% (n=280/608) in GnRH-a and hCG, respectively. On the other hand, a difference was reported regarding the CLBdR per oocyte retrieval among completed cycles, with 42% (n=374/898) and 25% (n=258/1034) in the GnRh-a and u-hCG groups, respectively. Nevertheless, this variance was due to a lower maternal age and higher number of inseminated oocytes in the GnRH-a group, and not imputable to the ovulation trigger itself (multivariate-OR=1.3, 95%CI: 0.9-1.6, adjusted p-value=0.1). CONCLUSION: GnRH-a trigger is a valid alternative to u-hCG in freeze-all cycles, not only for patients at high risk for OHSS. Such strategy might increase the safety and flexibility of controlled-ovarian-stimulation with no impact on oocyte competence and IVF efficacy.

Upsurge in autophagy, associated with mifepristone-treated polycystic ovarian condition, is reversed upon thymoquinone treatment.

Polycystic ovarian syndrome (PCOS) is a multi-factorial gynecological endocrine disorder. It affects fertility in women and also predisposes to insulin resistance, type 2 diabetes mellitus, obesity etc. Earlier, significance of autophagy has been explored in PCOS-related metabolic disorders and during normal folliculogenesis. Increasing evidences reveal connection of autophagy with chronic inflammatory behaviour, an associated phenomena in polycystic ovaries. However, understanding of the association of autophagy with PCOS is still obscure. This study reveals that increased autophagy in mifepristone (RU486) treated KK-1 cells and in vivo PCO rat model is characterized by upregulated Androgen Receptor (AR) expression and downregulated PCO biomarker aromatase. The prevalence of autophagy has been observed to be concomitant with increased expression of two autophagic markers Beclin1 and MAP-LC3-II while the autophagy substrate p62/SQSTM1 was downregulated. Immunohistochemical staining revealed increased localization of MAP-LC3 in the compacted granulosa layers of the follicular cysts in the PCO model. The PCO rat models also demonstrated augmented levels of p65, the active subunit of NF-κB, which acts as a transcriptional regulator of several pro-inflammatory factors. NF-κB repressor and anti-inflammatory herbal drug thymoquinone, known to alleviate PCO condition, downregulated autophagy modules substantially. Pre-treatment with thymoquinone upregulated aromatase, reduced AR levels and decreased autophagic markers as well as p65 levels, simulating super-ovulated condition. In conclusion, the anti-inflammatory phytochemical thymoquinone alleviated PCO condition.

The impact of timing modified natural cycle frozen embryo transfer based on spontaneous luteinizing hormone surge.

PURPOSE: To evaluate whether adjusting timing of modified natural cycle frozen embryo transfer (mNC-FET) 1 day earlier in the setting of a spontaneous LH surge has an impact on pregnancy outcomes. METHODS: This retrospective cohort study evaluated all mNC-FET with euploid blastocysts from May 1, 2016 to March 30, 2019, at a single academic institution. Standard protocol for mNC-FET included ultrasound monitoring and hCG trigger when the dominant follicle and endometrial lining were appropriately developed. Patients had serum LH, estradiol, and progesterone checked on day of trigger. If LH was ≥ 20 mIU/mL, trigger was given that day and FET was performed 6 days after surge (LH/HCG+6), with the intent of transferring 5 days after ovulation. If LH was < 20 mIU/mL, FET was performed 7 days after trigger (hCG+7). Primary outcomes included clinical pregnancy and live birth rates. To account for correlation between cycles, a generalized estimating equation (GEE) method for multivariable logistic regression was used. RESULTS: Four hundred fifty-three mNC-FET cycles met inclusion criteria, of which 205 were in the LH/HCG+6 group and 248 were in the HCG+7 group. The overall clinical pregnancy rate was 64% and clinical miscarriage rate was 4.8%, with similar rates between the two groups. The overall live birth rate was 60.9% (61.0% in LH/HCG+6 group and 60.9% in HCG+7 group). After implementing GEE, the odds of CP (aOR 0.97, 95% CI [0.65-1.45], p = 0.88) and LB (aOR 0.98, 95% CI [0.67-1.45], p = 0.93) were similar in both groups. CONCLUSIONS: In our study cohort, mNC-FET based on LH/HCG+6 versus HCG+7 had similar pregnancy outcomes.

The mediation of pigeon egg production by regulating the steroid hormone biosynthesis of pigeon ovarian granulosa cells.

The aim of this study was to determine the molecular mechanism of miR-205b targeting 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1) on the apoptosis and proliferation of granulosa cells (GC) of pigeons. Our previous studies suggested that HSD11B1 was the target gene of miR-205b and played a key role in steroid hormone biosynthesis and GC development. The adenovirus-miR-205b recombinant virus and adenovirus-cli-miR-205b-sh recombinant virus were generated, verified, and their characteristics determined. The recombinant viruses were used to infect the GC of pigeons, with real time quantitative PCR used to examine the expressions of HSD11B1 and related genes. The HSD11B1 antibody was obtained and verified, and Western blotting was used to detect the protein level of HSD11B1. The Cell Counting Kit-8 assay kit was used to detect cell viability, and the Annexin V-FITC/PI kit was used for the apoptosis assays. The expression of HSD11B1 was significantly lower in the overexpression (OE) than in OE negative control (OE-NC) treatments and significantly higher in short hairpin (SH) than in SH negative control (SH-NC) treatments. The expression levels of cytochrome P4503A5 was significantly higher in SH and lower in OE treatments, and the rhythms of cytochrome P450 aromatase mRNA levels were similar. The mRNA level of cytochrome P450scc in OE was lower than in OE-NC treatments and higher in SH than in SH-NC treatments. The protein expressions of HSD11B1 were decreased in the GC of OE, whereas increased in the SH group. The Cell Counting Kit-8 assay revealed that overexpression of miR-205b significantly suppressed proliferation of the GC of pigeons, whereas interference of miR-205b significantly induced the proliferation of the GC. The overexpression and the interference of miR-205b did not have a significant effect on cell cycle. The overexpression of miR-205b significantly increased the number of apoptotic cells, whereas the interference of miR-205b decreased the number of apoptotic cells. These findings indicated that miR-205b mediated pigeon egg production by regulating the steroid hormone biosynthesis of the pigeon ovarian GC by targeting HSD11B1, which may be useful in increasing pigeon egg production.

Novel PGK1 determines SKP2-dependent AR stability and reprograms granular cell glucose metabolism facilitating ovulation dysfunction.

BACKGROUND: Disordered folliculogenesis is a core characteristic of polycystic ovary syndrome (PCOS) and androgen receptors (ARs) are closely associated with hyperandrogenism and abnormalities in folliculogenesis in PCOS. However, whether the new AR binding partner phosphoglycerate kinase 1 (PGK1) in granulosa cells (GCs) plays a key role in the pathogenesis of PCOS remains unclear. METHODS: We identified the new AR binding partner PGK1 by co-IP (co-immunoprecipitation) in luteinized GCs, and reconfirmed by co-IP, co-localization and GST pull down assay, and checked PGK1 expression levels with qRT-PCR and western blotting. Pharmaceuticals rescue assays in mice, and metabolism assay, AR protein stability and RNA-seq of PGK1 targets in cells proved the function in PCOS. FINDINGS: PGK1 and AR are highly expressed in PCOS luteinized GCs and PCOS-like mouse ovarian tissues. PGK1 regulated glucose metabolism and deteriorated PCOS-like mouse metabolic disorder, and paclitaxel rescued the phenotype of PCOS-like mice and reduced ovarian PGK1 and AR protein levels. PGK1 inhibited AR ubiquitination levels and increased AR stability in an E3 ligase SKP2-dependent manner. Additionally, PGK1 promoted AR nuclear translocation, and RNA-seq data showed that critical ovulation-related genes were regulated by the PGK1-AR axis. INTERPRETATION: PGK1 regulated GCs metabolism and interacted with AR to regulate the expression of key ovulation genes, and also mediated cell proliferation and apoptosis, which resulted in the etiology of PCOS. This work highlights the pathogenic mechanism and represents a novel therapeutic target for PCOS. FUNDING: National Key Research and Development Program of China; National Natural Science Foundation of China grant.

Practical application of an impractical bovine genotype: creating bilateral twin pregnancies in Trio allele carriers.

Bovine twin birth is associated with detriments, including increased embryo/fetal losses, malpresentation, and dystocia. Incidence of these is lessened in bilateral compared with unilateral twin pregnancy. This study was undertaken to assess the use of follicular ablation by aspiration to create bilateral twin pregnancies in females with genetic potential for ~3.5 ovulations per cycle (Trio allele carriers). In experiment 1, carriers (n = 30) and noncarriers (n = 10) were synchronized for ovulation and timed artificial insemination (TAI). Follicles (>5 mm) in excess of one per ovary were aspirated ~16 h preceding TAI. Follicle count for females with follicles on only one ovary was reduced to two. Blood was sampled 2 wk post-TAI to assess progesterone (P4) concentrations; embryo count was determined by ultrasound 6 wk post-TAI. Circulating P4 concentration post-TAI was significantly (P < 0.001) associated with both genotype and subsequent pregnancy status (pregnant noncarriers: 7.06 ± 0.68 ng/mL; pregnant carriers: 5.54 ± 0.55 ng/mL; nonpregnant noncarriers: 5.22 ± 1.05 ng/mL; nonpregnant carriers: 3.13 ± 0.42 ng/mL). Experiment 2 was undertaken to offset the negative effects of follicular aspiration on subsequent P4 concentration observed in experiment 1. Carriers (n = 38) and noncarriers (n = 32) were submitted to TAI and follicle ablation as described for experiment 1. Additionally, accessory corpora lutea (CL) were induced in carriers by the administration of human chorionic gonadotropin (carriers) at day 6 post-TAI. Consequently, P4 concentration post-TAI was significantly (P < 0.05) associated with subsequent pregnancy status (pregnant: 8.48 ± 0.61 ng/mL; nonpregnant: 6.70 ± 0.63 ng/mL) but not with genotype (carrier: 8.01 ± 0.59 ng/mL; noncarrier: 7.17 ± 0.64 ng/mL). Embryo number was greater in carriers (exp. 1: 1.64 ± 0.81; exp 2: 1.45 ± 0.09) vs. noncarriers (1.00 ± 0.00, both experiments). Single, twin, and triplet pregnancies occurred in carriers in experiment 1, whereas multiples in experiment 2 were limited to twin pregnancies. Genotype effects on pregnancy rate were not significant (P > 0.10) in either experiment. Results suggest that follicular ablation to create bilateral twin pregnancies in Trio carriers is feasible but requires the induction of accessory CL to offset the negative effects of follicular aspiration on subsequent P4 concentration and associated fertility outcomes.