Role In Ovarian Development Research Articles

Molecular characterization of miR-31 for regulating egg production in female Schistosoma japonicum

Schistosomiasis is caused by Schistosoma infection and affects more than 200 million people worldwide. A large number of eggs produced by adult Schistosoma play central the role in host pathology and subsequent disease dissemination. However, the underlying mechanisms of egg production in Schistosoma still need to be further elucidated. Previously, we found that miR-31 was highly enriched in the female reproductive organs of Schistosoma japonicum (S. japonicum), which was shown to be associated with ovarian development. In the present study, we analyzed the potential targets of miR-31 including mRNA and long noncoding RNAs (lncRNAs) in S. japonicum by RNA seq combined with bioinformatics. Then, six putative targets of miR-31 including three mRNAs such as EWB00_000918, EWB00_004242, and EWB00_009323 and three lncRNAs such as LncSJG_010465, LncSJG_015374 and LncSJG_013128 were further analyzed their expressions in the parasites treated with miR-31 inhibitor by qPCR to confirm their potential regulations. Whole mount in suit hybridization (WISH) analysis of some miR-31 targets were carried out to determine their colocalizations with miR-31. Furthermore, we selected EWB00_009323, which is an eggshell synthetic protein and also a target of miR-31, to inhibit its functions by small interfering RNA. The results indicated that inhibition of EB00_009323 led to decreased oviposition and defective ovarian morphology. Overall, the potential targets of miR-31 including mRNA and lncRNAs were identified in female S. japonicum and the results indicated that miR-31 coordinates with its targets, at least EWB00_009323, play an important role in ovarian development and egg production.

Smad4 and FoxH1 potentially interact to regulate cyp19a1a promoter in the ovary of ricefield eel (Monopterus albus)

BackgroundCyp19a1a is a key enzyme in the pathway that converts androgens into estrogen and is regulated by TGF-β signaling. Smad4 and FoxH1 are downstream effectors of TGF-β signaling and may play important roles in ovarian development in M. albus.MethodsWe investigated the expression pattern of the Smad4 and FoxH1 using qRT‒PCR and immunofluorescence, then tested the changes of smad4 and foxh1 by qRT‒PCR after ovary incubation with FSH in vitro, and analysed the regulation of cyp19a1a transcription by Smad4 and FoxH1 by dual-luciferase reporter assays.ResultsWe found that Smad4 encoded a putative protein of 449 amino acids and harbored the three conserved domains typical of this protein family. Smad4 and foxh1 exhibited similar expression patterns during ovarian development and after FSH incubation, with Pearson’s coefficients of 0.873 and 0.63–0.81, respectively. Furthermore, Smad4, FoxH1 and Cyp19a1a colocalized in the granulosa cells and theca cells of ovaries during the mid-to-late vitellogenic stage. Smad4 repressed cyp19a1a activity via SBE1 (− 1372/−1364) and SBE2 (− 415/−407) in the cyp19a1a promoter, whereas mutating SBE1 or SBE2 restored cyp19a1a promoter activity. Co-overexpression of Smad4 and FoxH1 significantly reduced cyp19a1a promoter activity.ConclusionsThis study provides new insights into the potential functions of transcription factors Smad4 and FoxH1 in ovarian development and the transcriptional regulation mechanism of cyp19a1a in M. albus, which will reveal Smad4/FoxH1-mediated TGF-β signaling in reproduction and the regulation of the cyp19a1a.Plain English summaryAromatase, encoded by cyp19a1a, is involved in ovarian development and plays an important role in the quality of eggs, as well the sex ratio, of the teleost fish, M. albus. The research on the transcriptional regulation of cyp19a1a has contributed to the understanding of its role in ovarian development. In previous study, it was shown that FoxH1 inhibits cyp19a1a transcription. In the present study, Smad4 was confirmed as a cyp19a1a transcriptional repressor and Smad4 may also coordinate with FoxH1 to repress cyp19a1a transcription. At present, we provide a new perspective for the transcriptional regulation of cyp19a1a by transcription factors Smad4 and FoxH1 in teleost fish ovary. In the future, the regulatory networks of Smad4 and FoxH1 will be further studied and the gene editing technology will be applied to screen specific regulatory factors of cyp191a1a gene, so as to alter the female cycle and modulate the sex ratio of the eggs production.

Molecular characterization of a short neuropeptide F signaling system in the swimming crab, Portunus trituberculatus, and its role in ovarian development

Molecular characterization of a short neuropeptide F signaling system in the swimming crab, Portunus trituberculatus, and its role in ovarian development

Identification of ETH receptor and its possible roles in the mud crab Scylla paramamosain

Ecdysis-triggering hormone (ETH) is a neuropeptide hormone characterized by a conserved KxxKxxPRx amide structure widely identified in arthropods. While its involvement in the regulation of molting and reproduction in insects is well-established, its role in crustaceans has been overlooked. This study aimed to de-orphanise a receptor for ETH in the mud crab Scylla paramamosain and explore its potential impact on ovarian development. A 513-amino-acid G protein-coupled receptor for ETH (SpETHR) was identified in S. paramamosain, exhibiting a dose-dependent activation by SpETH with an EC50 value of 75.18 nM. Tissue distribution analysis revealed SpETH was in the cerebral ganglion and thoracic ganglion, while SpETHR was specifically expressed in the ovary, hepatopancreas, and Y-organ of female crabs. In vitro experiments demonstrated that synthetic SpETH (at a concentration of 10−8 M) significantly increased the expression of SpVgR in the ovary and induced ecdysone biosynthesis in the Y-organ. In vivo experiments showed a significant upregulation of SpEcR in the ovary and Disembodied and Shadow in the Y-organ after 12 h of SpETH injection. Furthermore, a 16-day administration of SpETH significantly increased 20E titers in hemolymph, gonadosomatic index (GSI) and oocyte size of S. paramamosain. In conclusion, our findings suggest that SpETH may play stimulatory roles in ovarian development and ecdysone biosynthesis by the Y-organ.

Histological and Transcriptomic Insights into the Ovary Development of Hemibarbus labeo Injected with Spawn-Inducing Hormones.

Naturally, the ovaries of many farmed fish can only develop to stage IV (mainly including stage IV oocytes, known as full-grown postvitellogenic oocytes). Therefore, spawn-inducing hormone injections are used to promote ovary development and oocyte maturation, facilitating reproduction in the aquaculture industry. The study of spawn-inducing hormones and their underlying neuroendocrine mechanisms has been a recent focus in fish reproductive biology. However, the intra-ovarian regulatory mechanisms of ovary development and oocyte maturation after hormone injection require further investigation. In this study, we explored the histological and transcriptomic map of the ovary of Hemibarbus labeo after hormone injection to reveal changes in the ovary. The gonad index significantly increased after hormone injection for 5.5h, after which no significant change was observed. Histological analysis showed that the nuclei had moved to one side of the oocytes at 5.5h after hormone injection. Moreover, the volume of the oocytes increased and their yolk membranes thickened. Oocytes then underwent their first meiotic division at 5.5-11h after hormone injection. Subsequently, the follicular membrane was ruptured, and ovulation was completed at 11-16.5h after hormone injection. In addition, we identified 3189 differentially expressed genes (DEGs) on comparing the transcriptomes at different time points after hormone injection. These DEGs were significantly enriched in the GO terms of nervous system process, molecular transducer activity, and extracellular region, and the KEGG pathways of TNF signaling and cytokine-cytokine receptor interaction; these may play important roles in ovary development and oocyte maturation. Within these pathways, genes such as apoe, creb3, jun, junb, il11, and il8 may play important roles in steroid hormone synthesis and ovulation. Conclusively, our results show detailed sequential dynamics of oocyte development and provide new insights into the intra-ovarian regulatory mechanisms of ovarian development and oocyte maturation in H. labeo. These findings may be important for research on improving egg quality and reproduction in aquaculture.

Effect of TSH on aromatase expression of ovarian granulosa cells in obese mice.

Aromatase plays an important role in ovarian development, the normal progress of the menstrual cycle, and fertility status. Elevated aromatase activity is linked to obesity. There is a bidirectional relationship between obesity and thyroid function. Few studies have investigated the relationship between TSH and ovarian aromatase in obesity. Our aim was to investigate the effect of TSH on aromatase expression of ovarian granulosa cells in obese mice. Female mice pups were divided into an obesity group and a control group. Obese parameters and the time of pubertal onset were recorded. At the age of 5weeks, blood and tissues were obtained. Serum aromatase and hormone concentrations were measured using ELISA. The granulosa cells were isolated and exposed to variable concentrations (0μM, 1μM, 10μM, 100μM) of TSH. The expression of CYP19A1 mRNA and protein were assessed via RT-qPCR and western blot. In female mice, body weight, Lee's obesity index, and serum levels of E2, aromatase, and TSH were significantly higher in the obesity group compared to the control group, whereas the time of pubertal onset and serum T3 and T4 concentrations were significantly lower (all P < 0.001). In granulosa cells, the expression of CYP19A1 mRNA in the obesity group was lower than that in the control group at 1μM and 100μM concentrations of TSH (both P < 0.001). The expression of CYP19A1 protein in the obesity group was higher than that in the control group after TSH stimulation (P = 0.014, P < 0.001, and P = 0.004, respectively). With the increase of TSH concentrations, the expression of CYP19A1 mRNA and protein in the two groups significantly increased (all P < 0.001). Early puberty and elevated serum aromatase and TSH levels were found in obese female mice. In the granulosa cells of obese mice, TSH directly regulates aromatase expression in a dose-dependent manner.

Changes in ovarian tissue structure and distribution of oestrogen receptors in Huanghuai goats at different ages.

To observe developmental changes in the ovarian tissue structure and distribution characteristics of oestrogen receptors (ERs) in the ovaries of Huanghuai goats at different ages, we selected healthy Huanghuai goats ewes and divided them into five groups (i.e. 3-, 30-, 60-, 90- and 120-day-old groups), with 10 animals in each group. The serum was separated after blood collection through the jugular vein, and the contents of oestrogen (E) and progesterone (P) in the serum of Huanghuai goats at each age were determined. Three goats were randomly selected from each group and sacrificed after anaesthesia, and the ovarian tissue was quickly obtained and placed in 4% paraformaldehyde fixative to prepare the tissue sections. Using HE, oestrogen receptors were immunohistochemically stained and observed. These results showed many primordial follicles and occasional secondary follicles in the ovaries of 3-day-old Huanghuai goats. Ovarian reticular structures were observed in 30-day-old ovarian medulla, with occasional near-mature growing follicles. Mature follicles and corpus luteum were occasionally detected in 60-day-old ovarian cortex. The 90-120-day-old ovarian cortices contained growing and mature follicles, and the number of mature follicles and corpora lutea increased, implying a significant luteal involution period. The E and P contents in the 120-day-old group were significantly higher than those in the 3-, 30-, 60- and 90-day-old groups. The levels of ERα and ERβ in the 3- and 30-day-old groups were mainly distributed in the granulosa cells of ovarian reproductive epithelial cells, primordial follicles, atretic follicles, and primary and secondary follicles. The ERα and ERβ levels of the 60-, 90- and 120-day-old groups were also distributed in the granulosa cells and luteal cells of mature follicles, especially in the 120-day-old endometrial cells of mature follicles, where ERβ was distributed significantly. The overall expression of ERβ in the ovary was higher than that of ERα. The results of this study provide basic data on the ovarian development and the specific expression of ERs and PRs in the ovaries of Huanghuai white goats, which play an important role in ovarian development and precocity.

Bulk and single-cell transcriptome datasets of the mouse fetal and adult rete ovarii and surrounding tissues

The rete ovarii (RO) is an epithelial structure that arises during development in close proximity to the ovary and persists throughout adulthood. However, the functional significance of the RO remains elusive, and it is absent from recent discussions of female reproductive anatomy. The RO comprises three regions: the intraovarian rete within the ovary, the extraovarian rete in the periovarian tissue, and the connecting rete linking the two. We hypothesize that the RO plays a pivotal role in ovarian homeostasis and responses to physiological changes. To begin to uncover the nature and function of RO cells, we conducted transcriptomic profiling of the RO. This study presents three datasets, and reports our analysis and quality control approaches for bulk, single-cell, and nucleus-level transcriptomics of the fetal and adult RO tissues using the Pax8-rtTA; Tre-H2B-GFP mouse line, where all RO regions express nuclear GFP. The integration and rigorous validation of these datasets will advance our understanding of the RO’s roles in ovarian development, female maturation, and adult female fertility.

Genome-Wide Identification of Vitellogenin Gene Family and Comparative Analysis of Their Involvement in Ovarian Maturation in Exopalaemon carinicauda.

Vitellogenin (Vtg) is a precursor of yolk proteins in egg-laying vertebrates and invertebrates and plays an important role in vitellogenesis and embryonic development. However, the Vtg family remains poorly characterized in Exopalaemon carinicauda, a major commercial mariculture species found along the coasts of the Yellow and Bohai Seas. In this study, 10 Vtg genes from the genomes of E. carinicauda were identified and characterized. Phylogenetic analyses showed that the Vtg genes in crustaceans could be classified into four groups: Astacidea, Brachyra, Penaeidae, and Palaemonidae. EcVtg genes were unevenly distributed on the chromosomes of E. carinicauda, and a molecular evolutionary analysis showed that the EcVtg genes were primarily constrained by purifying selection during evolution. All putative EcVtg proteins were characterized by the presence of three conserved functional domains: a lipoprotein N-terminal domain (LPD_N), a domain of unknown function (DUF1943), and a von Willebrand factor type D domain (vWD). All EcVtg genes exhibited higher expression in the female hepatopancreas than in other tissues, and EcVtg gene expression during ovarian development suggested that the hepatopancreas is the main synthesis site in E. carinicauda. EcVtg1a, EcVtg2, and EcVtg3 play major roles in exogenous vitellogenesis, and EcVtg3 also plays a major role in endogenous vitellogenesis. Bilateral ablation of the eyestalk significantly upregulates EcVtg mRNA expression in the female hepatopancreas, indicating that the X-organ/sinus gland complex plays an important role in ovarian development, mostly by inducing Vtg synthesis. These results could improve our understanding of the function of multiple Vtg genes in crustaceans and aid future studies on the function of EcVtg genes during ovarian development in E. carinicauda.

Effects of light spectra on the ovarian maturation and maternal immunity in the little yellow croaker (Larimichthys polyactis)

Before spawning, fish ovaries undergo multiple events to prepare for embryogenesis. Although light plays a vital role in ovarian development and neuroendocrine activities, it is unclear how it affects ovarian maturation in the little yellow croaker (Larimichthys polyactis). This study aimed to investigate the influence of different light spectra [i.e., red, blue, dark, green, white, and ambient] on the gonadosomatic index (GSI) and hepatosomatic index (HSI); histology of the ovary; vitellogenin (VTG) levels in hepatic cells; plasma 17β-estradiol and growth hormone (GH) levels; total length; body weight; and innate/adaptive immune factors in captive-bred L. polyactis. The plasma 17β-estradiol levels gradually decreased from Jan 8 to Apr 9, and were higher in the red, green, and blue groups than in the white, dark, and control groups. By the end of the experiment, the VTG levels were significantly higher in the red group than in the control, white, and dark groups, while those in the blue group were the lowest among the treatments. The HSI fluctuated among the groups and was higher in the control, white, and green groups on Apr 9. The proportion of oocytes at stage IV was highest in the red group, which also exhibited the best synchronization of oocyte maturation. Maternal immunoglobulin M levels were significantly higher in the red and green groups than in the control, blue, dark, and white groups at the end of the experiment, while there was no significant difference in the complement component 3 (C3) levels. Collectively, these findings demonstrate that different light spectra affect 17β-estradiol secretion, VTG production, follicle synchronization and maternal immunity during ovarian maturation in L. polyactis.

Impact of IGF3 stimulation on spotted scat (Scatophagus argus) liver: Implications for a role in ovarian development

Insulin-like growth factor 3 (IGF3) plays an essential role in the reproductive system and influences the liver of teleosts. However, the mechanism of IGF3 action on the liver remains unknown. In this study, we analyzed the transcriptome patterns and enzymatic activities in the liver of Scatophagus argus following IGF3 injection. We identified 155 differentially expressed genes (DEGs), which were enriched in terms ‘retinol metabolism’, ‘steroid hormone biosynthesis’, ‘epidermal growth factor receptor tyrosine kinase inhibitor resistance’, and ‘one carbon pool by folate’ in Kyoto Encyclopedia of Genes and Genomes pathway analysis. Notable DEGs included inhbb, shmt2, slc2a5, cyp1a1, got1, mid1ip1, sstr5, hnf6, slc39a14, and hspa1a. The activities of amylase, lipase, malic enzyme, fatty acid synthase, and lipoprotein lipase increased significantly, whereas those of phospholipid hydroperoxide glutathione peroxidase and pyruvate kinase decreased significantly. The combined analysis of transcriptome patterns and enzyme activities suggests IGF3 may promote energy storage and protein synthesis by the liver. These findings indicate IGF3 may support the liver to promote ovarian development and maturation in S. argus.

SEMA4G targeted by miR‐363‐5p regulates the proliferation of granulosa cells in Yunshang black goats

AbstractProliferation of granular cells (GCs) plays an important role in ovary development, providing energy and a microenvironment for oocyte ovulation. In this study, we explored the spatiotemporal expression of SEMA4G and its effects on the growth and development of goat GCs using primary GCs cultured in vitro as a model. The results showed that the expression level of SEMA4G was significantly higher in the ovaries of high‐fertility goats than in those of low‐fertility goats (p &lt; 0.05). The mRNA and protein expression levels of the cell proliferation markers of GCs were significantly increased after the overexpression of SEMA4G in goat primary GCs. The EdU and CCK8 results showed that cell viability was elevated in goat GCs and that proliferation was promoted by an increase in the number of proliferating cells. The proliferation of goat GCs was significantly inhibited by SEMA4G inhibition (p &lt; 0.05). The results of online miRNA and target gene prediction software and dual luciferase activity analysis confirmed that SEMA4G could bind to mi‐363‐5p and was one of its target genes. The RT‒qPCR results showed that the expression level of miR‐363‐5p was significantly lower in the ovaries of high‐fertility goats than in those of low‐fertility goats in contrast to the expression level of SEMA4G (p &lt; 0.05). After the overexpression of miR‐363‐5p in goat GCs, the expression of SEMA4G was significantly suppressed (p &lt; 0.05). Collectively, the results of this study could lay the foundation for exploring the molecular mechanisms by which SEMA4G and miR‐363‐5p regulate the growth and development of goat GCs and provide targets for breeding high‐fertility goats.

Function Analysis of Cholesterol 7-Desaturase in Ovarian Maturation and Molting in Macrobrachium nipponense: Providing Evidence for Reproductive Molting Progress.

The Cholesterol 7-desaturase gene plays an important role in insect ecdysone synthesis, but its role in ovarian development has not been reported. In this study, characteristics and the phylogenetic relationship of Cholesterol 7-desaturase were identified by bioinformatics. qPCR showed that the Mn-CH7D gene was highly expressed in the ovary, which was much higher than that in other tissues, and the expression level of Mn-CH7D reached the highest level at the third stage of the ovarian development stage (O-III). During embryonic development, the Mn-CH7D gene expression was highest in the zoea stage. The function of the Mn-CH7D gene was explored by RNA interference. The experimental group was injected with Mn-CH7D dsRNA through the pericardial cavity of M. nipponense, while the control group was injected with the same volume of dsGFP. Statistical analysis of gonadal development and GSI calculation showed that the silencing of Mn-CH7D resulted in the suppression of gonadal development. In addition, the molting frequency of the experimental group was significantly lower than that of the control group during the second molting cycle after silencing Mn-CH7D. On the seventh day after silencing, ecdysone content in the experimental group was significantly reduced. These results demonstrated that the Mn-CH7D gene played a dual role in ovarian maturation and molting of M. nipponense.

Lipophorin receptor is required for the accumulations of cuticular hydrocarbons and ovarian neutral lipids in Locusta migratoria

Lipophorin is the most abundant lipoprotein particle in insect hemolymph. Lipophorin receptor (LPR) is a glycoprotein that binds to the lipophorin and mediates cellular uptake and metabolism of lipids by endocytosis. However, the roles of LPR in uptake of lipids in the integument and ovary remain unknown in the migratory locust (Locusta migratoria). In present study, we characterized the molecular properties and biological roles of LmLPR in L. migratoria. The LmLPR transcript level was high in the first 2 days of the adults after eclosion, then gradually declined. LmLPR was predominately expressed in fat body, ovary and integument. Using immuno-detection methods, we revealed that LmLPR was mainly localized in the membrane of oenocytes, epidermal cells, fat body cells and follicular cells. RNAi-mediated silencing of LmLPR led to a slight decrease of the cuticle hydrocarbon contents but with little effect on the cuticular permeability. However, the neutral lipid content was significantly decreased in the ovary after RNAi against LmLPR, which led to a retarded ovarian development. Taken together, our results indicated that LmLPR is involved in the uptake and accumulation of lipids in the ovary and plays a crucial role in ovarian development in L. migratoria. Therefore, LmLPR could be a promising RNAi target for insect pest management by disrupting insect ovarian development.

Identification of the cyclooxygenase (COX) gene and its role in ovarian development and ovulation of the oriental river prawn Macrobrachium nipponense

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs), which are involved in ovarian development and ovulation in crustaceans. In this study, complementary DNA (cDNA) of COX in Macrobrachium nipponense (MnCOX) was identified, and its function in ovarian development and ovulation was investigated by quantitative real-time PCR (qPCR), fluorescence in situ hybridization (FISH), RNA interference (RNAi) and arachidonic acid (AA), dexamethasone (DEX) and DEX+ prostaglandin E2 (PGE2) administration. The results showed that the complete cDNA of MnCOX is 3238 bp, including an 1848 bp open reading frame (ORF) that encodes 615 amino acids. The MnCOX protein conservatively possesses a membrane-binding domain, an epidermal growth factor-like (EGF-like) domain, an animal haem peroxide domain, three N-glycosylation sites and seven essential catalytic residues. The richest MnCOX transcript of nine tissues was found in the gill, followed by the ovary and intestine. In six ovarian development stages, the MnCOX transcript was the richest in the stage Ⅴ ovary. FISH showed that the MnCOX transcript was present in the cytoplasm of oocytes. MnCOX knockdown led to decreases in PGE2 content, expression levels of the vitellogenin (Vg) and vitellogenin receptor (Vgr) genes and the gonadsomatic index (GSI) of prawns. Moreover, oocyte development was delayed, and the oviposition rate and the number of eggs ovulated were reduced after MnCOX knockdown. AA administration increased the expression of MnCOX, PGE2 content and the oviposition rate of prawns. However, DEX administration decreased the expression of MnCOX, PGE2 content, oviposition rate, and the number of eggs ovulated by prawns. The addition of PGE2 to DEX injection rescued the negative effect of DEX on the ovulation of M. nipponense. This investigation illustrates that MnCOX plays an important role in regulating ovarian development and ovulation in M. nipponense by affecting the synthesis of PGs.

Regulation of CHH family in eyestalk by A-type allatostatin in the mud crab Scylla paramamosain

The eyestalk ganglion is a principal neuroendocrine organ in crustaceans for the synthesis, storage and release of the crustacean hyperglycemic hormone neuropeptide (CHH) family. A-type allatostatin (AST-A) is involved in a variety of physiological functions, although its function in regulating the CHH family in the eyestalk ganglion has not been reported. The present study identified AST-A in the mud crab Scylla paramamosain. AST-A was highly expressed in the ganglia as shown by reverse transcription PCR, while AST-A receptor (AST-AR) was expressed in multiple tissues, such as the eyestalk ganglion. Both in vitro and in vivo experiments revealed that AST-A mature peptide significantly increased mRNA expression of AST-AR, vitellogenesis-inhibiting hormone (VIH), molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH). In contrast, RNA interference by injection of AST-AR-dsRNA into crabs significantly reduced the expressions of VIH, MIH, and CHH in the eyestalk ganglion. This study reports for the first time that AST-A might play roles in ovarian development, molting and glucose metabolism in S. paramamosain via regulating CHH family neuropeptides in the eyestalk ganglion.

Sumoylation regulates functional properties of the oocyte transcription factors SOHLH1 and NOBOX.

SOHLH1 and NOBOX are oocyte-expressed transcription factors with critical roles in ovary development and fertility. In mice, Sohlh1 and Nobox are essential for fertility through their regulation of the oocyte transcriptional network and cross-talk to somatic cells. Sumoylation is a posttranslational modification that regulates transcription factor function, and we previously showed that mouse oocytes deficient for sumoylation had an altered transcriptional landscape that included significant changes in NOBOX target genes. Here, we show that mouse SOHLH1 is modified by SUMO2/3 at lysine 345 and mutation of this residue alters SOHLH1 nuclear to cytoplasmic localization. In NOBOX, we identify a non-consensus SUMO site, K97, that eliminates NOBOX mono-SUMO2/3 conjugation, while a point mutation at K125 had no effect on NOBOX sumoylation. However, NOBOXK97R/K125R double mutants showed loss of mono-SUMO2/3 and altered higher molecular weight modifications, suggesting cooperation between these lysine's. NOBOXK97R and NOBOXK97R/K125R differentially regulated NOBOX promoter targets, with increased activity on the Gdf9 promoter, but no effect on the Pou5f1 promoter. These data implicate sumoylation as a novel regulatory mechanism for SOHLH1 and NOBOX, which may prove useful in refining their roles during oogenesis as well as their function during reprogramming to generate de novo germ cells.

Activin Stimulates Follicle Formation and Activation and Modulates Steroidogenesis in Fetal Bovine Ovarian Tissue in Vitro 1

The earliest stages of ovarian follicular development, follicle formation and development of the capacity to activate, are critical to the size of the ovarian reserve. In primates and most domestic animals, these stages occur during fetal life and thus, are difficult to study. Our laboratory has used cattle, an excellent model for human ovarian development, to elucidate the regulation of these early stages in fetal bovine ovarian tissue in vitro. The experiments described here were designed to determine if fetal bovine ovaries have activin A and its receptors and if activin A affects follicular formation and/or activation to begin growth. During the second trimester, when follicles first form, activin increased the total number follicles and the number of primary (activated) follicles in cultured ovarian cortical pieces, compared to control medium; its effects were similar to the positive control, insulin. During the second trimester, activin decreased primordial follicle numbers and increased primary and secondary follicles, again mimicking the positive control. Our previous experiments showed that fetal ovaries produce ovarian steroids, particularly during the second trimester, and that estradiol and progesterone inhibit follicle formation and activation. In control and gonadotropin-treated ovarian pieces, activin A increased progesterone, but decreased androstenedione and estradiol accumulation in the medium. Messenger RNA for activin and its type II receptors was detected in fetal bovine ovaries, with ACVR2A receptor mRNA more abundant than ACVR2B. Immunohistochemistry localized mRNA for activin A and ACVR2A to germ cells of all stages, granulosa cells, and theca cells of early secondary follicles. Staining for ACVR2B was weaker and mostly confined to germ calls. Taken together, these results strongly suggest that activin A of fetal ovarian origin 3 plays a role in ovarian development by promoting follicle formation and activation and modulating ovarian steroidogenesis.

Pivotal role of High-Mobility Group Box 2 in ovarian folliculogenesis and fertility

BackgroundHigh-Mobility Group Box 1 (HMGB1) and HMGB2 are chromatin-associated proteins that belong to the HMG protein family, and are involved in the regulation of DNA transcription during cell differentiation, proliferation and regeneration in various tissues. However, the role of HMGB2 in ovarian folliculogenesis is largely unknown.MethodsWe investigated the functional role of HMGB1 and HMGB2 in ovarian folliculogenesis and fertilization using C57BL/6 wild type (WT) and HMGB2-knockout (KO) mice. Ovarian tissues were obtained from WT and HMGB2-KO mice at postnatal days 0, 3, 7, and 2, 6 months of age, then performed immunohistochemistry, qPCR and Western blotting analyses. Oocyte fertilization capability was examined by natural breeding and in vitro fertilization experiments.ResultsIn HMGB2-KO mice, ovary weight was decreased due to reduced numbers of oocytes and follicles. Natural breeding and in vitro fertilization results indicated that HMGB2-KO mice are subfertile, but not sterile. Immunohistochemistry showed that oocytes expressed HMGB2, but not HMGB1, in neonatal and adult WT ovaries. Interestingly, in HMGB2-KO ovaries, a compensatory increase in HMGB1 was found in oocyte nuclei of neonatal and 2-month-old mice; however, this was lost at 6 months of age.ConclusionsThe depletion of HMGB2 led to alterations in ovarian morphology and function, suggesting that HMGB2 plays an essential role in ovarian development, folliculogenesis and fertilization.

The forkhead factor Foxl2 participates in the ovarian differentiation of Chinese soft-shelled turtle Pelodiscus sinensis

The forkhead transcription factor Foxl2 plays a major role in ovarian development and function in mice and fish, and acts as a female sex-determining gene in goat. Its functional role in the sex determination and gonadal differentiation has not yet been investigated in reptiles. Here, we characterized Foxl2 gene in Chinese soft-shelled turtle Pelodiscus sinensis, exhibiting ZZ/ZW sex chromosomes. Foxl2 exhibited a female-specific embryonic expression pattern throughout the critical sex determination periods in P. sinensis. The expression of Foxl2 was induced at early stage in ZZ embryonic gonads that were feminized by estrogen treatment. Most importantly, Foxl2 knockdown in ZW embryos by RNA interference resulted in female-to-male sex reversal, characterized by obvious masculinization of gonads, significant up-regulation of testicular markers Dmrt1 and Sox9, and remarkable down-regulation of ovarian regulator Cyp19a1. Conversely, gain-of-function study showed that overexpression of Foxl2 in ZZ embryos led to largely feminized genetic males, production of Cyp19a1, and a decline in Dmrt1 and Sox9. These findings demonstrate that Foxl2 is both necessary and sufficient to initiate ovarian differentiation in P. sinensis, thereby acting as a key upstream regulator of the female pathway in a reptilian species.