Male Sexual Differentiation Research Articles

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5′-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

Chicken ovalbumin upstream promoter transcription factor type II interacts and functionally cooperates with GATA4 to regulate anti-Müllerian hormone receptor type 2 transcription in mouse MA-10 Leydig cells.

Leydig cells produce testosterone and insulin-like 3, two hormones essential for male sex differentiation and reproductive function. The orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor type II (COUP-TFII), and the zinc finger factor GATA4 are two transcription factors involved in Leydig cell differentiation, gene expression, and function. Several Leydig cell gene promoters contain binding motifs for both GATA factors and nuclear receptors. The goal of the present study is to determine whether GATA4 and COUP-TFII cooperate to regulate gene expression in Leydig cells. The transcriptomes from GATA4- and COUP-TFII-depleted MA-10 Leydig cells were analyzed using bioinformatic tools. Functional cooperation between GATA4 and COUP-TFII, and other related family members, was assessed by transient transfections in Leydig (MA-10 and MLTC-1) and fibroblast (CV-1) cell lines on several gene promoters. Recruitment of GATA4 and COUP-TFII to gene promoters was investigated by chromatin immunoprecipitation. Co-immunoprecipitation was used to determine whether GATA4 and COUP-TFII interact in MA-10 Leydig cells. Transcriptomic analyses of GATA4- and COUP-TFII-depleted MA-10 Leydig cells revealed 44 commonly regulated genes including the anti-Müllerian hormone receptor type (Amhr2) gene. GATA4 and COUP-TFII independently activated the Amhr2 promoter, and their combination led to a stronger activation. A GC-rich element, located in the proximal Amhr2 promoter was found to be essential for GATA4- and COUP-TFII-dependent activation as well as for the COUP-TFII/GATA4 cooperation. COUP-TFII and GATA4 directly interacted in MA-10 Leydig cell extracts. Chromatin immunoprecipitation revealed that GATA4 and COUP-TFII are recruited to the proximal Amhr2 promoter, which contains binding sites for both factors in addition to the GC-rich element. Cooperation between COUP-TFII and GATA6, but not GATA1 and GATA3, was also observed. Our results establish the importance of physical and functional cooperation between COUP-TFII/GATA4 in the regulation of gene expression in MA-10 Leydig cells, and more specifically the Amhr2 gene.

Recent progress of male sexual differentiation and development in the oriental river prawn (Macrobrachium nipponense): A review

AbstractMany researchers have focused on male sexual differentiation and development of the Macrobrachium nipponense in recent years. In this review, we highlight the status of knowledge about male sexual differentiation and development in this species by summarizing the chromosome analyses, major regulatory factors, identification of male reproduction‐related genes and gene functional analyses. Fifty‐two chromosomes have been identified in the testis of M. nipponense, which led to the identification of sex‐related chromosomes in this prawn. Studies have shown that hormones secreted by the eyestalk, androgenic gland, and testis as well as 17‐estradiol and environmental factors regulate the process of male sexual development in M. nipponense. The sex differentiation and development sensitive period was identified as post‐larva 10 (PL10) to PL22; therefore, this time span is critical in the identification of male reproduction‐related genes from M. nipponense. In this study, 80 male sex‐related homologous genes were identified based on functional analysis in other species, and another 45 genes were considered to be strong male reproduction‐related candidates based on their expression changes through performing multi‐omics profiling analyses. Quantitative polymerase chain reaction analysis revealed 25 genes that were involved in the process of male sexual development in M. nipponense, and RNA interference showed that knockdown of the expression levels of SDHB, PDHE1, NFkBa, HSDL1 and ferritin delayed testis development. Use of Crispr‐Cas9 technology and analysis of noncoding RNA should be further investigated, which will be effective in establishing an artificial technique to regulate the process of male sexual development in M. nipponense.

CeRNA crosstalk mediated by ncRNAs is a novel regulatory mechanism in fish sex determination and differentiation.

Competing endogenous RNAs (ceRNAs) are vital regulators of gene networks in mammals. The involvement of noncoding RNAs (ncRNAs) as ceRNA in genotypic sex determination (GSD) and environmental sex determination (ESD) in fish is unknown. The Chinese tongue sole, which has both GSD and ESD mechanisms, was used to map the dynamic expression pattern of ncRNAs and mRNA in gonads during sex determination and differentiation. Transcript expression patterns shift during the sex differentiation phase, and ceRNA modulation occurs through crosstalk of differentially expressed long ncRNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and sex-related genes in fish. Of note was the significant up-regulation of a circRNA from the sex-determining gene dmrt1 (circular RNA dmrt1) and a lncRNA, called AMSDT (which stands for associated with male sex differentiation of tongue sole) in Chinese tongue sole testis. These two ncRNAs both share the same miRNA response elements with gsdf, which has an up-regulated expression when they bind to miRNA cse-miR-196 and concurrent down-regulated female sex-related genes to facilitate testis differentiation. This is the first demonstration in fish that ceRNA crosstalk mediated by ncRNAs modulates sexual development and unveils a novel regulatory mechanism for sex determination and differentiation.

The Urokinase-Type Plasminogen Activator Contributes to cAMP-Induced Steroidogenesis in MA-10 Leydig Cells

Leydig cells produce androgens which are essential for male sex differentiation and reproductive functions. Steroidogenesis, as well as expression of several genes in Leydig cells, are stimulated by LH/cAMP and repressed by AMP/AMPK. One of those genes is Plau, which codes for the urokinase-type plasminogen activator (uPA), a secreted serine protease. The role of uPA and the regulation of Plau expression in Leydig cells remain unknown. Using siRNA-mediated knockdown, uPA was required for maximal cAMP-induced STAR and steroid hormone production in MA-10 Leydig cells. Analysis of Plau mRNA levels and promoter activity revealed that its expression is strongly induced by cAMP; this induction is blunted by AMPK. The cAMP-responsive region was located, in part, in the proximal Plau promoter that contains a species-conserved GC box at −56 bp. The transcription factor Krüppel-like factor 6 (KLF6) activated the Plau promoter. Mutation of the GC box at −56 bp abolished KLF6-mediated activation and significantly reduced cAMP-induced Plau promoter activity. These data define a role for uPA in Leydig cell steroidogenesis and provide insights into the regulation of Plau gene expression in these cells.

Insights Into the Roles of GATA Factors in Mammalian Testis Development and the Control of Fetal Testis Gene Expression.

Defining how genes get turned on and off in a correct spatiotemporal manner is integral to our understanding of the development, differentiation, and function of different cell types in both health and disease. Testis development and subsequent male sex differentiation of the XY fetus are well-orchestrated processes that require an intricate network of cell-cell communication and hormonal signals that must be properly interpreted at the genomic level. Transcription factors are at the forefront for translating these signals into a coordinated genomic response. The GATA family of transcriptional regulators were first described as essential regulators of hematopoietic cell differentiation and heart morphogenesis but are now known to impact the development and function of a multitude of tissues and cell types. The mammalian testis is no exception where GATA factors play essential roles in directing the expression of genes crucial not only for testis differentiation but also testis function in the developing male fetus and later in adulthood. This minireview provides an overview of the current state of knowledge of GATA factors in the male gonad with a particular emphasis on their mechanisms of action in the control of testis development, gene expression in the fetal testis, testicular disease, and XY sex differentiation in humans.

Morphological and Molecular Gonadal Sex Differentiation in the Wild Japanese eel Anguilla japonica.

Most cultured Japanese eels (Anguilla japonica) show male sex differentiation; however, natural gonadal sex differentiation has not been evaluated. In this study, this process was characterized in wild eels. Differentiated ovaries and testes were observed after the eels grew to 320 and 300 mm in total length, respectively. The youngest ovary and testis appeared at 3 and 4 years old, respectively; however, undifferentiated gonads were found up to 7 years, suggesting that sex differentiation was triggered by growth rather than aging. gsdf, amh, foxl2b and foxl3b were highly expressed in the testes, whereas figla, sox3, foxn5, zar1, and zp3 were highly expressed in the ovaries. The expression of cyp19a1a and foxl2a did not differ significantly between the testis and ovary. In the ovaries, the cyp19a1a and foxl2a levels were highest in the early stages, suggesting that their function is limited to early ovarian differentiation. The foxn5, zar1 and zp3 levels tended to increase in the later stages, suggesting that they function after the initiation of ovarian differentiation. In undifferentiated gonads, dimorphic gene expression was not observed, suggesting that the molecular sex differentiation phase is short and difficult to detect. These findings provide the first demonstration of the whole course of natural gonadal sex differentiation in eels at molecular and morphological levels.

Tle4z1 Facilitate the Male Sexual Differentiation of Chicken Embryos.

BackgroundSex differentiation is a complex and precisely regulated process by multiple genes in chicken. However, it is still unclear on the key genes of sex differentiation.ObjectiveTo explore the function of Tle4z1 screened by RNA-seq sequencing on sex differentiation during the development of chicken embryos.MethodsTle4z1 was differentially expressed from the RNA-seq of ESCs and PGCs in male and female chickens. Then, we established an effective method to overexpression or knocking down the expression of Tle4z1 in ovo and in vitro, respectively. Histomorphological observation, qRT-PCR and ELISA were applied to detect the function of Tle4z1 in the process of male sex differentiation by injecting vectors into embryos at day 0.ResultsIt showed that Tle4z1 has significant male preference in embryonic day 4.5, such phenomenon persisted during the growth period of chicken embryos. Morphological observation results showed that the gonads on both sides of genetic male (ZZ) embryos with Tle4z1 knocking down developed asymmetrically, the gonadal cortex became thicker showing the typical characteristics of genetic female (ZW) gonads. Furthermore, the expression of Cyp19a1, which dominates female differentiation, was significantly increased, while the expression of male marker genes Dmrt1, Sox9, WT1 and AR was significantly downregulated. In addition, the concentration of testosterone also significantly decreased, which was positively correlated with the expression of Tle4z1 (P < 0.01). Conversely, the ZW embryo showed defeminized development when Tle4z1 was overexpressed.ConclusionWe prove that the Tle4z1 is a novel gene through the male sexual differentiation via gene regulation process and synthesis of testosterone, which construct the basis for understanding the molecular mechanism of sex differentiation in chickens.

Transcriptomic profiling and novel insights into the effect of AG ablation on gonad development in Macrobrachium rosenbergii

Androgenic glands (AGs) regulate male sexual differentiation in M. rosenbergii, and AG ablation may induce sex-reversal and male-to-neofemale transformation. However, information on gonad development following AG ablation is scarce. To investigate the effects of gonad development and sex reversal after AG ablation, we utilized transcriptomic profiling, RT–qPCR, and histological and morphological observation. Transcriptomic profiling generated 42.32–42.92 million clean reads. A total of 94,747 unigenes were assembled with a total length, average length, N50, and GC content of 160,460,655 bp, 1693 bp, 3438 bp, and 39.86%, respectively. Hsp70, IGFBP7, Mar-Mrr, Serpin, TUBA3, CREB, EF1a, and BMP7 were identified as differentially expressed by gonad transcriptome profiling after AG ablation. Interestingly, the Rap1, Hippo, PI3K, oxytocin, thyroid hormone, and apoptosis signalling pathways were all found to be involved in the sex-reversal process by regulating cell proliferation and gonad development and maintaining homeostasis. Sexual morphology features and histological changes following AG ablation validated the sex reversal from male to neofemale. Sexual manipulation permits monosex culture while considerably improving output yield. Taken together, these findings provide insight into the sexual reversal mechanism of M. rosenbergii after AG ablation. The findings in this study may aid in the future investigation of the sex-reversal mechanisms in other decapod species and offer a new breeding and culture strategy for M. rosenbergii aquaculture industry.

Complete Androgen Insensitivity Syndrome: Primary Amenorrhoea of Rare Variety

Androgen insensitivity syndrome is an X linked disorder of male sexual differentiation caused by mutation affecting the androgen receptor gene Xq 11-12 resulting in decreased peripheral responsiveness to circulating androgens, with variable phenotypic expression.

Full Functional Sex Reversal Achieved Through Silencing of MroDmrt11E Gene in Macrobrachium rosenbergii: Production of All-Male Monosex Freshwater Prawn.

The freshwater prawn Macrobrachium rosenbergii is one kind of important economic aquaculture species and displays remarkable sexual dimorphism. The molecular mechanism of sexual differentiation in M. rosenbergii has been primarily unraveled through the research efforts of the androgenic gland and its related genes. However, the understanding of conserved genes involved in the molecular mechanism underpinning sex determination and sexual differentiation of M. rosenbergii is still fragmentary. MroDmrt11E is a member of the doublesex and mab-3-related transcription factor (Dmrt) gene family and is prominently expressed in the testis. In the present study, in vivo knockdown of MroDmrt11E at the postlarva stage in male prawn induced a complete and functional sex reversal and achieved the production of an all-male monosex population. Furthermore, a great deal of new information of upregulated and downregulated transcriptions involved in sexual differentiation of MroDmrt11E knockdown was enriched by comparative transcriptomic analysis. The effects of RNAi-mediated gene knockdown of MroDmrt11E on the differentially expressed and sex-related candidate genes, such as transformer, fruitless, feminization, insulin-like androgenic gland gene, Dmrt gene family, were primarily focused on, and their possible molecular regulatory relationships in sexual differentiation were analyzed. Meanwhile, the response of primary Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways was investigated to expound the potential roles of MroDmrt11E in male sexual differentiation, which provided a deeper understanding of the molecular regulatory network underlying sexual differentiation of M. rosenbergii. The finding provided a novel sexual manipulation technique through silencing of Dmrt gene family for achieving a complete and functional sex reversal and offered a new insight regarding the mechanism of the Dmrt gene family in the sexual differentiation of crustaceans.

CHARGE syndrome-associated proteins FAM172A and CHD7 influence male sex determination and differentiation through transcriptional and alternative splicing mechanisms.

To gain further insight into chromatin‐mediated regulation of mammalian sex determination, we analyzed the role of the CHARGE syndrome‐associated proteins FAM172A and CHD7. This study is based on our prior discoveries that a subset of corresponding mutant mice display complete male‐to‐female sex reversal, and that both of these proteins regulate co‐transcriptional alternative splicing in neural crest cells. Here, we report that FAM172A and CHD7 are present in the developing gonads when sex determination normally occurs in mice. The interactome of FAM172A in pre‐Sertoli cells again suggests a role at the chromatin‐spliceosome interface, like in neural crest cells. Accordingly, analysis of Fam172a‐mutant pre‐Sertoli cells revealed transcriptional and splicing dysregulation of hundreds of genes. Many of these genes are similarly affected in Chd7‐mutant pre‐Sertoli cells, including several known key regulators of sex determination and subsequent formation of testis cords. Among them, we notably identified Sry as a direct transcriptional target and WNT pathway‐associated Lef1 and Tcf7l2 as direct splicing targets. The identified molecular defects are also associated with the abnormal morphology of seminiferous tubules in mutant postnatal testes. Altogether, our results thus identify FAM172A and CHD7 as new players in the regulation of male sex determination and differentiation in mice, and further highlight the importance of chromatin‐mediated regulatory mechanisms in these processes.

Leydig and Sertoli cell function in individuals with genital ambiguity, 46,XY karyotype, palpable gonads and normal testosterone secretion: a case-control study.

Because normal male sexual differentiation is more complex than normal female sexual differentiation, there are more cases of disorders of sex development (DSDs) with 46,XY karyotype that have unclear etiology. However, Leydig and Sertoli cell markers are rarely used in distinguishing such individuals. To evaluate the function of Leydig and Sertoli cells in individuals with genital ambiguity, 46,XY karyotype, palpable gonads and normal testosterone secretion. Case-control study with 77 patients, including eight with partial androgen insensitivity syndrome, eight with 5α-reductase deficiency type 2 (5ARD2) and 19 with idiopathic 46,XY DSD, and 42 healthy controls, from the Interdisciplinary Study Group for Sex Determination and Differentiation (GIEDDS), at the State University of Campinas (UNICAMP), Campinas, Brazil. Baseline levels of gonadotropins, anti-Müllerian hormone (AMH), inhibin B, insulin-like 3 (INSL3), testosterone and dihydrotestosterone in cases, and AMH, inhibin B, and INSL3 levels in controls, were assessed. There was no significant difference in age between cases and controls (P = 0.595). AMH and inhibin B levels were significantly lower in cases than in controls (P = 0.031 and P < 0.001, respectively). INSL3 levels were significantly higher in cases than in controls (P = 0.003). Inhibin B levels were lower in 5ARD2 patients (P = 0.045) and idiopathic patients (P = 0.001), in separate comparisons with the controls. According to our findings, we can speculate that inhibin B levels may be used to differentiate among DSD cases.

High Throughput scRNA-Seq Provides Insights Into Leydig Cell Senescence Induced by Experimental Autoimmune Orchitis: A Prominent Role of Interstitial Fibrosis and Complement Activation.

Leydig cells (Lc), located in the interstitial space of the testis between seminiferous tubules, produce 95% of testosterone in male individuals, which is pivotal for male sexual differentiation, spermatogenesis, and maintenance of the male secondary sex characteristics. Lc are prone to senescence in aging testes, resulting in compromised androgen synthesis capability upon aging. However, little is known about whether Lc undergo senescence in a chronic inflammatory environment. To investigate this question, mouse models of experimental autoimmune orchitis (EAO) were used, and Lc were analyzed by high throughput scRNA-Seq. Data were screened and analyzed by correlating signaling pathways with senescence, apoptosis, androgen synthesis, and cytokine/chemokine signaling pathways. EAO did induce Lc senescence, and Lc senescence in turn antagonized androgen synthesis. Based on the correlation screening of pathways inducing Lc senescence, a plethora of pathways were found to play potential roles in triggering Lc senescence during EAO, among which the Arf6 and angiopoietin receptor pathways were highly correlated with senescence signature. Notably, complement and interstitial fibrosis activated by EAO worsened Lc senescence and strongly antagonized androgen synthesis. Furthermore, most proinflammatory cytokines enhanced both senescence and apoptosis in Lc and spermatogonia (Sg) during EAO, and proinflammatory cytokine antagonism of the glutathione metabolism pathway may be key in inducing cellular senescence during EAO.

Dynamic Expression of the Homeobox Factor PBX1 during Mouse Testis Development

Members of the pre-B-cell leukemia transcription factor (PBX) family of homeoproteins are mainly known for their involvement in hematopoietic cell differentiation and in the development of leukemia. The four PBX proteins, PBX1, PBX2, PBX3 and PBX4, belong to the three amino acid loop extension (TALE) superfamily of homeoproteins which are important transcriptional cofactors in several developmental processes involving homeobox (HOX) factors. Mutations in the human PBX1 gene are responsible for cases of gonadal dysgenesis with absence of male sex differentiation while Pbx1 inactivation in the mouse causes a failure in Leydig cell differentiation and function. However, no data is available regarding the expression profile of this transcription factor in the testis. To fill this knowledge gap, we have characterized PBX1 expression during mouse testicular development. Real time PCRs and Western blots confirmed the presence Pbx1 mRNA and PBX1 protein in different Leydig and Sertoli cell lines. The cellular localization of the PBX1 protein was determined by immunohistochemistry and immunofluorescence on mouse testis sections at different embryonic and postnatal developmental stages. PBX1 was detected in interstitial cells and in peritubular myoid cells from embryonic life until puberty. Most interstitial cells expressing PBX1 do not express the Leydig cell marker CYP17A1, indicating that they are not differentiated and steroidogenically active Leydig cells. In adults, PBX1 was mainly detected in Sertoli cells. The presence of PBX1 in different somatic cell populations during testicular development further supports a direct role for this transcription factor in testis cell differentiation and in male reproductive function.

Possible testosterone redundancy for 5α-dihydrotestosterone in the masculinization of mouse external genitalia.

The development of embryonic external genitalia (eExG) into characteristic male structures, such as urethra and penile erectile tissues, depends on 5α-dihydrotestosterone (DHT). Although the corpus cavernosum (CC) is well known as essential for erectile function in adults, its developmental process and its dependency on DHT have been unknown. To reveal the dimorphic formation of the murine CC from the embryonic stage, we first analyzed the production of the protein vascular endothelial growth factor receptor-2 (FLK1) via its expression (hereinafter referred as "expression of FLK1") and the expression of alpha-smooth muscle actin (ACTA2) and collagen type 1 (COL1A1) in developing external genitalia. The 5-α reductase type 2 encoded by the SRD5A2 gene has been suggested to be a crucial enzyme for male sexual differentiation, as it converts testosterone (T) into DHT in the local urogenital organs. In fact, SRD5A2 mutation results in decreased synthesis of DHT, which leads to various degrees of masculinized human external genitalia (ExG). We further investigated the expression profile of SRD5A2 during the formation of the murine CC. We observed that SRD5A2 was expressed in smooth muscle of the CC. To determine the role of SRD5A2 in CC formation, we analyzed the formation of erectile tissue in the male Srd5a2 KO mice and measured the levels of androgens in the ExG by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Intriguingly, there were no obvious defects in the CCs of male Srd5a2 KO mice, possibly due to increased T levels. The current study suggests possible redundant functions of androgens in CC development.

A young infant with complete androgen insensitivity syndrome

Complete androgen insensitivity syndrome (CAIS) is a rare X-linked recessive disorder resulting from maternally inherited or de novo mutations involving the androgen receptor (AR) gene. The AR is a vital steroid hormone receptor that has a critical role in male sexual differentiation and development and preservation of the male phenotype. The diagnosis of CAIS is based on the presence of female external genitalia in an individual with 46, XY karyotype having normally developed but undescended testes and target tissue unresponsiveness to androgen. Our case presented at the age of 2 months with asymmetric labia majora with bilateral labial mass. Ultrasonography revealed absence of female internal genital organs and presence of testes at labial folds. The child was found to have 46, XY karyotype. BIRDEM Med J 2022; 12(1): 74-77

Growth Hormone-induced STAT5B Regulates Star Gene Expression Through a Cooperation With cJUN in Mouse MA-10 Leydig Cells.

Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star messenger RNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (electrophoretic mobility shift assay and supershift) and in vivo (chromatin immunoprecipitation) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.

Roles of Crustacean Female Sex Hormone 1a in a Protandric Simultaneous Hermaphrodite Shrimp

Crustacean female sex hormone (CFSH) plays a pivotal role in the development of secondary sex characteristics in dioecious crustaceans. However, until now the knowledge concerning its functions in hermaphroditic species is scanty. Herein, we explored the function of CFSH (Lvit-CFSH1a) in the peppermint shrimp Lysmata vittata, a species characterized by a rare reproductive system of protandric simultaneous hermaphroditism (PSH). Lvit-CFSH1a cDNA was 1,220-bp in length with a 720-bp ORF encoded a polypeptide of 239-aa. RT-PCR showed that Lvit-CFSH1a was exclusively expressed in the eyestalk ganglion. For female physiology, it was found that Lvit-CFSH1a was indispensable for the development of female gonopores, but it might not involve vitellogenesis of the species. For male physiology, Lvit-CFSH1a suppressed Lvit-IAG2 expression in short-term silencing experiment and recombinant protein injection experiment, but did not affect male sexual differentiation in long-term silencing experiment. In addition, silencing the Lvit-CFSH1a gene impeded individual growth in L. vittata.

P–534 A novel heterozygous mutation in the luteinizing hormone/choriogonadotropin receptor (LHCGR) gene in a patient with ‘genuine’ empty follicle syndrome

Abstract Study question Whether empty follicle syndrome (EFS) in a patient has a genetic basis. Summary answer Our findings would expand the mutational spectrum of LHCGR, in patients with GEFS What is known already The LHCGR gene (OMIM #52790) is located on chromosome 2p21 has 11 exons The LHCGRs present in gonadal cells; granulosa, theca and luteal cells in women and Leydig cells in menandplays a critical role in male sexual differentiation, female ovarian development and fertility (folliculogenesis, ovulation, corpus luteum formation and progesterone secretion) Inactivating mutations in males can lead to Leydig cell hypoplasia, which causes disorders in sexual development (MIM #238 320). Phenotype of women is less severe and variable and has no effect on the secondary sex characteristics, but it could cause amenorrhoea and infertility Study design, size, duration: In the context of clinical genetics, Next Generation Sequencing libraries were prepared in line with the manufacturer’s orders using QIASeq™ Targeted DNA Custom Panel (Qiagen) targeting exons and 20 bp exon-intron boundaries of selected genes (AR, BMP15, CATSPER1, CFTR, CYP21A2, FSHB, FSHR, HESX1, LHB, LHCGR, NR5A1, POU1F1, SRY, ZP1). The variant detected in NGS analysis was further confirmed using Sanger sequencing (MiSeq, Illumina, San Diego, CA). Participants/materials, setting, methods A 27 years old Turkish women with 6 year history of primary unexplained infertility underwent controlled ovarian hyperstimulation and IVF with an antagonist protocole in Ankara City Hospital. Although normal follicular development, E2 levels, and bioavailable β-hCG plasma levels, no oocytes or cumulus-corona complexes were retrieved by follicular aspiration. Main results and the role of chance A novel heterozygous mutation on Exon 5 of LHCGR (NM_000233.4):c.453C&amp;gt;G (p.Phe151Leu). This variant has not been reported in GnomAD database and is a novel variant as per controlled from ClinVar and HGMD mutation databases Also coagulation tests were done Patient was heterozygote for the prothrombin mutation (FXIII) and homozygote for MTHFR C677T mutation. The patient has normal pubertal development and female karyotype (46,XX). Gonadotropin and E2 levels were normal, nor any history of anosmia, primary amenorrhoea, polycystic ovaries, hyperandrogenism, systemic disorder, or neurologic defect. According to ACMG 2015 and HGMD detected variant was classified as unknown clinical significance (VUS) variant In 22nd World Congress COGI in 2015 we have presented another recurrent GEFS case with compound heterozygous frameshift mutations in exon 5 and 11 of LHCGRgene(c.1764_1765insT) and (c.430G&amp;gt;T, p.V144F) who developed premature ovarian failure at the end The different types of LHCGR mutations would lead to the different functional effects and clinical phenotypes in different ages; primary amenorrhea with high levels of LH, poor ovarian response (Poesidon group1), GEFS and secondary amenorrhea (premature ovarian failure) GEFS may be a gradual biological occurrence related to ovarian ageing. Long term follow up is important, since some clinical manifestations appear later in life. Limitations, reasons for caution LH resistance in females were always found due to their effected brothers and only 10 GEFS cases of 46, XX females with LHCGR gene defect reported in literature. Patient was heterozygous for indicated mutation; therefore segregation study should be done in family members and in vitro studies should be performed. Wider implications of the findings: Screening for mutations in the LHCGR gene in Poseidon group 1 and in patients GEFS especially the recurrent ones will provide valuable information and time for clinical management, corresponding treatment and give the opportunity of wise and cost effective counseling of patients about their future reproductive choices. Trial registration number it is a case report