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BACKGROUND: Blotched snakehead (Channa maculata) displays significant sexual dimorphism, with males exhibiting faster growth rates and larger body sizes compared to females. The cultivation of the all-male population of snakeheads holds substantial economic and ecological value. Nonetheless, the intricate processes governing the development of bipotential gonads into either testis or ovary in C. maculata remain inadequately elucidated. Therefore, it is necessary to determine the critical time window of sex differentiation in C. maculata, providing a theoretical basis for sex control in production practices. METHODS: The body length and weight of male and female C. maculata were measured at different developmental stages to reveal when sexual dimorphism in growth initially appears. Histological observations and spatiotemporal comparative transcriptome analyses were performed on ovaries and testes across various developmental stages to determine the crucial time windows for sex differentiation in each sex and the sex-related genes. Additionally, qPCR and MG2C were utilized to validate and locate sex-related genes, and levels of E2 and T were quantified to understand sex steroid synthesis. RESULTS: Sexual dimorphism in growth became evident starting from 90 dpf. Histological observations revealed that morphological sex differentiation in females and males occurred between 20 and 25 dpf or earlier and 30-35 dpf or earlier, respectively, corresponding to the appearance of the ovarian cavity or efferent duct anlage. Transcriptome analyses revealed divergent gene expression patterns in testes and ovaries after 30 dpf. The periods of 40-60 dpf and 60-90 dpf marked the initiation of molecular sex differentiation in females and males, respectively. Male-biased genes (Sox11a, Dmrt1, Amh, Amhr2, Gsdf, Ar, Cyp17a2) likely play crucial roles in male sex differentiation and spermatogenesis, while female-biased genes (Foxl2, Cyp19a1a, Bmp15, Figla, Er) could be pivotal in ovarian differentiation and development. Numerous biological pathways linked to sex differentiation and gametogenesis were also identified. Additionally, E2 and T exhibited sexual dimorphism during sex differentiation and gonadal development. Based on these results, it is hypothesized that in C. maculata, the potential male sex differentiation pathway, Sox11a-Dmrt1-Sox9b, activates downstream sex-related genes (Amh, Amhr2, Gsdf, Ar, Cyp17a2) for testicular development, while the antagonistic pathway, Foxl2/Cyp19a1a, activates downstream sex-related genes (Bmp15, Figla, Er) for ovarian development. CONCLUSIONS: This study provides a comprehensive overview of gonadal dynamic changes during sex differentiation and gametogenesis in C. maculata, establishing a scientific foundation for sex control in this species.

Blotched snakehead (Channa maculata) exhibits significant sexual dimorphism, as males display faster growth rates and larger body sizes compared to females. The cultivation of the all-male population of snakeheads holds substantial economic and ecological value. However, the mechanisms underlying sex determination and differentiation in C. maculata remain insufficiently elucidated. In this study, sexual dimorphism in growth became evident starting from 90 dpf through the measurement of body length and weight of male and female C. maculata at different developmental stages. Histological observations indicated that morphological sex differentiation in females and males occurred at 20­25 dpf or earlier and 30­35 dpf or earlier, respectively, corresponding to the appearance of the ovarian cavity or efferent duct anlage. Transcriptome analyses revealed divergent gene expression patterns in male and female gonads after 30 dpf, suggesting that the period preceding 30 dpf might be the critical time window for sex control in C. maculata. The periods of 40­60 dpf and 60­90 dpf marked the initiation of molecular sex differentiation in females and males, respectively. Male-biased genes (Sox11a, Dmrt1, Amh, Amhr2, Gsdf, Ar, Cyp17a2) likely play crucial roles in testicular differentiation and spermatogenesis, while female-biased genes (Foxl2, Cyp19a1a, Bmp15, Figla, Er) could be pivotal in ovarian differentiation and oogenesis. Additionally, numerous biological pathways linked to sex differentiation and gametogenesis were identified. Moreover, sexual dimorphism was observed in the levels of E₂ and T during gonadal differentiation and development. Based on these findings, it is hypothesized that in C. maculata, the potential male sex differentiation pathway, Sox11a­Dmrt1­Sox9b, activates downstream sex-related genes (Amh, Amhr2, Gsdf, Ar, Cyp17a2) for testicular development, while the antagonistic pathway, Foxl2/Cyp19a1a, activates downstream sex-related genes (Bmp15, Figla, Er) for ovarian development. This study provides a comprehensive overview of gonadal dynamic changes during sex differentiation and gametogenesis in C. maculata, thereby establishing a scientific foundation for sex control in this species.

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Heat stress due to climate warming can significantly affect the synthesis of sex hormones in male adolescents, which can impair the ability of the hypothalamus to secrete gonadotropin-releasing hormone on the hypothalamic-pituitary-gonadal axis, which leads to a decrease in luteinizing hormone and follicle-stimulating hormone, which ultimately negatively affects spermatogenesis and testosterone synthesis. For optimal spermatogenesis, the testicular temperature should be 2-6 °C lower than body temperature. Heat stress directly affects the testes, damaging them and reducing testosterone synthesis. Additionally, chronic heat stress abnormally increases the level of aromatase in Leydig cells, which increases estradiol synthesis while decreasing testosterone, leading to an imbalance of sex hormones and spermatogenesis failure. Low levels of testosterone in male adolescents lead to delayed puberty and incomplete sexual maturation, negatively affect height growth and bone mineral density, and can lead to a decrease in lean body mass and an increase in fat mass. In order for male adolescents to acquire healthy reproductive capacity, it is recommended to provide sufficient nutrition and energy, avoid exposure to heat stress, and provide foods and supplements to prevent or repair testosterone reduction, germ cell damage, and sperm count reduction caused by heat stress so that they can enter a healthy adulthood.

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BACKGROUND: The Testis is an important reproductive organ in male mammals and the site for spermatogenesis, androgen synthesis, and secretion. Non-coding RNAs (ncRNAs) play an important regulatory role in various biological processes. However, the regulatory role of ncRNAs in the development of yak testes and spermatogenesis remains largely unclear. RESULT: In this study, we compared the expression profiles of circular RNAs (circRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in yak testicular tissue samples collected at 6 months (Y6M), 18 months (Y18M), and 4 years (Y4Y). Using RNA sequencing (RNA-Seq), we observed a significant difference in the expression patterns of ncRNAs in the samples collected at different testicular development stages. Twenty-two differentially expressed (DE) circRNAs, 69 DE miRNAs, and 64 DE mRNAs were detected in Y6M, Y18M, and Y4Y testicular samples, respectively. The results of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the source genes of DE circRNAs, predicted target genes of DE miRNAs, and DE mRNAs were specifically associated with signaling pathways and GO terms that were related to sperm synthesis, sperm vitality, and testicular development, such as cell cycle, Wnt signaling pathway, MAPK signaling pathway, GnRH signaling pathway, and spermatogenesis. The analysis of the circRNA-miRNA-mRNA network revealed that some DE ncRNAs, including miR-574, miR-449a, CDC42, and CYP11A1, among others, may be involved in testicular spermatogenesis. Concurrently, various circRNA-miRNA interaction pairs were observed. CONCLUSION: Our findings provide a database of circRNAs, miRNAs, and mRNAs expression profiles in testicular tissue of yaks at different developmental stages and a detailed understanding of the regulatory network of ncRNAs in yak testicular development and provide data that can help elucidate the molecular mechanisms underlying yak testicular development.

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DNA methylation plays a critical role in regulating gene expression during testicular development. However, few studies report on candidate genes related to the DNA methylation regulation of porcine testicular development. This study examined the differentially expressed genes (DEGs) and their methylation levels in testicular tissues from pigs at 60 days of age (60 d) and 180 days of age (180 d) using RNA-Seq and whole genome bisulfite sequencing (WGBS). It was determined that DNA methylation primarily occurs in the cytosine-guanine (CG) context, and the analysis identified 106,282 differentially methylated regions (DMRs) corresponding to 12,385 differentially methylated genes (DMGs). Further integrated analysis of RNA-Seq and WGBS data revealed 1083 DMGs negatively correlated with the expression of DEGs. GO analysis showed that these genes were significantly enriched in spermatogenesis, germ cell development, and spermatid differentiation. The screening of enriched genes revealed that hyper-methylation repressed ADAM30, ADAM3A, DPY19L2, H2BC1, MAK, RPL10L, SPATA16, and YBX2, while hypo-methylation elevated CACNA1I, CADM1, CTNNB1, JAM2, and PAFAH1B3 expression. Additionally, the methylation status of the key genes ADAM3A, ADAM30, YBX2, JAM2, PAFAH1B3, and CTNNB1 was detected by bisulfite sequencing PCR (BSP). This study offers insights into the epigenetic regulation mechanisms underlying porcine testicular development.

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Male reproductive health is largely determined already in the early development of the testis. Although much work has been carried out to study the mechanisms of testicular development and spermatogenesis, there was previously no information on the differences in the protein composition of yak testicles during early development. In this study, the protein profiles in the testicles of 6- (M6), 18- (M18), and 30-month-old (M30) yaks were comparatively analyzed using TMT proteomics. A total of 5521 proteins were identified, with 13, 1295, and 1397 differentially expressed proteins (DEPs) in 30- vs. 18-, 18- vs. 6-, and 30- vs. 6-month-old testes, respectively. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that DEPs were mainly involved in signaling pathways related to testicular development and spermatogenesis, including the MAPK, PI3K-Akt, Wnt, mTOR, TGF-ß, and AMPK signaling pathways. Furthermore, we also identified eight potential proteins (TEX101, PDCL2, SYCP2, SYCP3, COL1A1, COL1A2, ADAM10, and ATF1) that may be related to the testicular development and spermatogenesis of yaks. This study may provide new insights into the molecular mechanisms of the testicular development and spermatogenesis of yaks.

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Epilepsy is the chronic non-communicable disease of the nervous system most prevalent in the world. Valproic acid (VPA) is one of the most used drugs in the treatment of epilepsy but with various side effects. One of the organs that can be affected is the testis, where it has been seen that men treated with VPA reduce their fertility rates, in addition to causing endocrine disorders by decreasing androgens and gonadotropins. In animal models, it has been shown to reduce the weights of the glands attached to the male reproductive tract, as well as at the testicular level, decreasing sperm concentration and increasing apoptotic cell count. These effects are because VPA increases reactive oxygen species (ROS), causing damage to macromolecules and affecting all cellular processes sensitive to oxide reduction. Throughout testicular development, in utero, it has been seen that the expression of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase, are lower during early embryonic development, as well as vitamin E (VE) is decreased. Therefore, they are not sufficient to reverse the toxic effects of ROS. The objective of this study was to review the use of VPA during pregnancy, its effect on testicular development, and to explore the potential protective role of vitamin E.

La epilepsia es una enfermedad crónica no transmisible que afecta al sistema nervioso más prevalente en el mundo. Dentro de los tratamientos, uno de los fármacos más utilizados es el ácido valproico (AVP), el que ocasiona diversos efectos secundarios. Entre los órganos que se pueden ver afectados se encuentra la gónada masculina, en donde se ha visto que hombres en tratamiento con AVP reducen sus tasas de fecundidad, además de causar trastornos endocrinos disminuyendo andrógenos y gonadotrofinas. En modelos animales, se ha visto que disminuye los pesos de las glándulas anexas al tracto reproductor masculino, como también a nivel testicular, disminuyendo la concentración espermática y aumentando el recuento de células apoptóticas. Estos efectos se deberían a que el AVP aumenta las especies reactivas de oxígeno (ROS), ocasionando daño en macromoléculas, afectando todos los procesos celulares sensibles a óxido reducción. A lo largo del desarrollo testicular, in utero se ha visto que la expresión de enzimas antioxidantes como superóxido dismutasa, catalasa y glutatión peroxidasa, son más bajos durante el desarrollo embrionario temprano, como también la vitamina E (VE) se encuentra disminuida. Por tanto, no resultan suficientes para revertir los efectos tóxicos de las ROS. El objetivo de esta revisión fue asociar el uso de AVP durante la gestación y sus efectos a nivel del desarrollo testicular y describir el potencial rol protector de la VE.

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The estrogen receptor (ER), a ligand-dependent transcription factor, is critical for vertebrate reproduction. However, its role in bivalves is not well understood, with ongoing debates regarding its function in regulating reproduction similarly to vertebrates. To investigate ER's function, we conducted a 21-day RNA interference experiment focusing on its role in gonadal development in bivalves. Histological analyses revealed that ER inhibition significantly suppressed ovarian development in females and, conversely, promoted gonadal development in males. Additionally, levels of 17ß-estrogen (E2) were markedly reduced in the gonads of both sexes following ER suppression. Transcriptomic analysis from RNA-seq of testes and ovaries after ER interference showed changes in the expression of key genes such as Vtg, CYP17, 3ß-HSD, and 17ß-HSD. These genes are involved in the estrogen signaling pathway and steroid hormone biosynthesis. Furthermore, ER suppression significantly affected the expression of genes linked to gametogenesis and the reproductive cycle. Our findings highlight ER's crucial, yet complex and sex-specific roles in gonadal development in bivalves, emphasizing the need for further detailed studies.

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This study aims to explore the functions and mechanisms of testicular descent in Apodemus agrarius, and analyze the changes in genes and metabolite levels in this process. Illumina NovaSeq and liquid chromatography-mass spectrometry were used for the transcriptomic analysis and metabolomic analysis, respectively, of the normal and descending testis of A. agrarius. Gene ontology (GO) enrichment of the transcriptomic results revealed 240 differentially expressed genes (DEGs), such as Spesp1, Izumo1, Hyal5, and Fabp9. Kyoto encyclopedia of genes and genomes (KEGG) enrichment showed 52 DEGs, including Pcyt1, Pla2g4e, Gpd1l, and Lypla3. The qRT-PCR results were consistent with the transcriptomic results in terms of the expression patterns of six randomly selected genes in the normal and descending testis. The metabolomic results revealed 28 differential metabolites associated with the testicular function, including 3-dehydroquinic acid, α-linolenic acid, dihydroxyacetone phosphate, and fructose 1,6-bisphosphate. The conjoint analysis showcased that glycerophospholipid metabolism, α-linolenic acid metabolism, and arachidonic acid metabolism may be the key metabolic pathways regulating testicular descent in A. agrarius. This study will help to understand the mechanism of testicular descent and lay a theoretical foundation for exploring the mechanisms of the population changes of A. agrarius and developing laboratory animal resources.

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BACKGROUND: Exploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases. METHODS: Diaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development. RESULTS: Based on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3ß-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated. CONCLUSIONS: Overall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

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Male infertility is a major public health concern globally with unknown etiology in approximately half of cases. The decline in total sperm count over the past four decades and the parallel increase in childhood obesity may suggest an association between these two conditions. Here, we review the molecular mechanisms through which obesity during childhood and adolescence may impair future testicular function. Several mechanisms occurring in obesity can interfere with the delicate metabolic processes taking place at the testicular level during childhood and adolescence, providing the molecular substrate to hypothesize a causal relationship between childhood obesity and the risk of low sperm counts in adulthood.

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In vitro maturation (IVM) is a promising fertility restoration strategy for patients with nonobstructive azoospermia or for prepubertal boys to obtain fertilizing-competent spermatozoa. However, in vitro spermatogenesis is still not achieved with human immature testicular tissue. Knowledge of various human testicular transcriptional profiles from different developmental periods helps us to better understand the testis development. This scoping review aims to describe the testis development and maturation from the fetal period towards adulthood and to find information to optimize IVM. Research papers related to native and in vitro cultured human testicular cells and single-cell RNA-sequencing (scRNA-seq) were identified and critically reviewed. Special focus was given to gene ontology terms to facilitate the interpretation of the biological function of related genes. The different consecutive maturation states of both the germ and somatic cell lineages were described. ScRNA-seq regularly showed major modifications around 11 years of age to eventually reach the adult state. Different spermatogonial stem cell (SSC) substates were described and scRNA-seq analyses are in favor of a paradigm shift, as the Adark and Apale spermatogonia populations could not distinctly be identified among the different SSC states. Data on the somatic cell lineage are limited, especially for Sertoli cells due technical issues related to cell size. During cell culture, scRNA-seq data showed that undifferentiated SSCs were favored in the presence of an AKT-signaling pathway inhibitor. The involvement of the oxidative phosphorylation pathway depended on the maturational state of the cells. Commonly identified cell signaling pathways during the testis development and maturation highlight factors that can be essential during specific maturation stages in IVM.

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The testes serve as the primary source of androgens and the site of spermatogenesis, with their development and function governed by hormonal actions via endocrine and paracrine pathways. Male fertility hinges on the availability of testosterone, a cornerstone of spermatogenesis, while follicle-stimulating hormone (FSH) signaling is indispensable for the proliferation, differentiation, and proper functioning of Sertoli and germ cells. This review covers the research on how androgens, FSH, and other hormones support processes crucial for male fertility in the testis and reproductive tract. These hormones are regulated by the hypothalamic-pituitary-gonad (HPG) axis, which is either quiescent or activated at different stages of the life course, and the regulation of the axis is crucial for the development and normal function of the male reproductive system. Hormonal imbalances, whether due to genetic predispositions or environmental influences, leading to hypogonadism or hypergonadism, can precipitate reproductive disorders. Investigating the regulatory network and molecular mechanisms involved in testicular development and spermatogenesis is instrumental in developing new therapeutic methods, drugs, and male hormonal contraceptives.

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The testis-specific double sex and mab-3-related transcription factor 1 (DMRT1) has long been recognized as a crucial player in sex determination across vertebrates, and its essential role in gonadal development and the regulation of spermatogenesis is well established. Here, we report the cloning of the key spermatogenesis-related DMRT1 cDNA, named Tc-DMRT1, from the gonads of Tridacna crocea (T. crocea), with a molecular weight of 41.93 kDa and an isoelectric point of 7.83 (pI). Our hypothesis is that DMRT1 machinery governs spermatogenesis and regulates gonadogenesis. RNAi-mediated Tc-DMRT1 knockdown revealed its critical role in hindering spermatogenesis and reducing expression levels in boring giant clams. A histological analysis showed structural changes, with normal sperm cell counts in the control group (ds-EGFP) but significantly lower concentrations of sperm cells in the experimental group (ds-DMRT1). DMRT1 transcripts during embryogenesis exhibited a significantly high expression pattern (p < 0.05) during the early zygote stage, and whole-embryo in-situ hybridization confirmed its expression pattern throughout embryogenesis. A qRT-PCR analysis of various reproductive stages revealed an abundant expression of Tc-DMRT1 in the gonads during the male reproductive stage. In-situ hybridization showed tissue-specific expression of DMRT1, with a positive signal detected in male-stage gonadal tissues comprising sperm cells, while no signal was detected in other stages. Our study findings provide an initial understanding of the DMRT1 molecular machinery controlling spermatogenesis and its specificity in male-stage gonads of the key bivalve species, Tridacna crocea, and suggest that DMRT1 predominantly functions as a key regulator of spermatogenesis in giant clams.

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Normal testicular development ensures the process of spermatogenesis, which is a complex biological process. The sustained high productivity of spermatogenesis throughout life is predominantly attributable to the constant proliferation and differentiation of spermatogonial stem cells (SSCs). The self-renewal and differentiation processes of SSCs are strictly regulated by the SSC niche. Therefore, understanding the developmental pattern of SSCs is crucial for spermatogenesis. The Shaziling pig is a medium-sized indigenous pig breed originating from central China. It is renowned for its superior meat quality and early male sexual maturity. The spermatogenic ability of the boars is of great economic importance to the pig industry. To investigate testicular development, particularly the pattern of SSC development in Shaziling pigs, we used single-cell transcriptomics to identify gene expression patterns in 82,027 individual cells from nine Shaziling pig testes at three key postnatal developmental stages. We generated an unbiased cell developmental atlas of Shaziling pig testicular tissues. We elucidated the complex processes involved in the development of SSCs within their niche in the Shaziling pig. Specifically, we identified potential marker genes and cellular signaling pathways that regulate SSC self-renewal and maintenance. Additionally, we proposed potential novel marker genes for SSCs that could be used for SSC isolation and sorting in Shaziling pigs. Furthermore, by immunofluorescence staining of testicular tissues of different developmental ages using marker proteins (UCHL1 and KIT), the developmental pattern of the spermatogonia of Shaziling pigs was intensively studied. Our research enhances the comprehension of the development of SSCs and provides a valuable reference for breeding Shaziling pigs.

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The East Asian common octopus (Octopus sinensis) is an economically important species among cephalopods. This species exhibits a strict dioecious and allogamous reproductive strategy, along with a phenotypic sexual dimorphism, where the third right arm differentiates into hectocotylus in males. However, our understanding of the molecular mechanisms that underlie sex determination and differentiation in this species remains limited. In the present study, we surveyed gene-expression profiles in the immature male and female gonads of O. sinensis based on the RNA-seq, and a total of 47.83 Gb of high-quality data were generated. Compared with the testis, we identified 8302 differentially expressed genes (DEGs) in the ovary, of which 4459 genes were up-regulated and 3843 genes were down-regulated. Based on the GO enrichment, many GO terms related to sex differentiation were identified, such as sex differentiation (GO: 0007548), sexual reproduction (GO: 0019953) and male sex differentiation (GO: 0046661). A KEGG classification analysis identified three conserved signaling pathways that related to sex differentiation, including the Wnt signaling pathway, TGF-ß signaling pathway and Notch signaling pathway. Additionally, 21 sex-related DEGs were selected, of which 13 DEGs were male-biased, including Dmrt1, Foxn5, Foxj1, Sox30, etc., and 8 DEGs were female-biased, including Sox14, Nanos3, ß-tubulin, Suh, etc. Ten DEGs were used to verify the expression patterns in the testis and ovary using the RT-qPCR method, and the results showed that the expression level shown by RT-qPCR was consistent with that from the RNA-seq, which confirmed the reliability of the transcriptome data. The results presented in this study will not only contribute to our understanding of sex-formation mechanisms in O. sinensis but also provide the foundational information for further investigating the molecular mechanisms that underline its gonadal development and facilitate the sustainable development of octopus artificial breeding.

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Sex determination mechanisms differ widely among vertebrates, particularly in fish species, where diverse sex chromosomes and sex-determining genes have evolved. However, the sex-differentiation pathways activated by these sex-determining genes appear to be conserved. Gonadal soma-derived growth factor (Gsdf) is one of the genes conserved across teleost fish, especially in medaka fishes of the genus Oryzias, and is implicated in testis differentiation and germ cell proliferation. However, its role in sex differentiation remains unclear. In this study, we investigated Gsdf function in Oryzias hubbsi, a species with a ZW sex-determination system. We confirmed its male-dominant expression, as in other species. However, histological analyses revealed no male-to-female sex reversal in Gsdf-knockout fish, contrary to findings in other medaka species. Genetic sex determination remained intact without Gsdf function, indicating a Gsdf-independent sex-differentiation pathway in O. hubbsi. Instead, Gsdf loss led to germ cell overproliferation in both sexes and accelerated onset of meiosis in testes, suggesting a role in germ cell proliferation. Notably, the feminizing effect of germ cells observed in O. latipes was absent, suggesting diverse germ cell-somatic cell relationships in Oryzias gonad development. Our study highlights species-specific variations in the molecular pathways governing sex determination and differentiation, emphasizing the need for further exploration to elucidate the complexities of sexual development.

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PURPOSE: This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice. METHODS: Epididymosomes were extracted and characterized using SEM and western blotting. After cryopreservation, thawed SSCs were cultured in a hydrogel-based three-dimensional (3D) culture containing 10 ng/mL GDNF or 20 µg/mL epididymosomes. SSCs were assessed using the MTT assay, flow cytometry, and qRT-PCR after two weeks of culture. The isolated SSCs were microinjected into the efferent ducts of busulfan-treated mice. DiI-labeled SSCs were followed, and cell homing was assessed after two weeks. After 8 weeks, the testes were evaluated using morphometric studies and immunohistochemistry. RESULTS: The expression of PLZF, TGF-ß, and miR-10b did not increase statistically significantly in the 3D + GDNF and 3D + epididymosome groups compared to the 3D group. Among the groups, the GDNF-treated group exhibited the highest expression of miR-21 (*P < 0.05). Caspase-3 expression was lower in the epididymosome-treated group than in the other groups (***P < 0.001). Compared to the 3D and negative control groups, the 3D + epididymosomes and 3D + GDNF groups showed an increase in spermatogenic cells. Immunohistochemical results confirmed the growth and differentiation of spermatogonial cells into spermatids in the treatment groups. CONCLUSION: The DTM hydrogel containing 20 µg/mL epididymosomes or 10 ng/mL GDNF is a novel and safe culture system that can support SSC proliferation in vitro to obtain adequate SSCs for transplantation success. It could be a novel therapeutic agent that could recover deregulated SSCs in azoospermic patients.

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Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2-/- and Garin4-/- males are normal, Garin5a-/- and Garin5b-/- males are subfertile, and Garin3-/- males are infertile. Further analysis revealed that Garin3-/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b-/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility.

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AIM: To assess testicular volume at puberty for boys who underwent orchidopexy at 9 or at 36 months compared to boys with spontaneous postnatal descent. METHODS: At age 6 months, boys with congenital unilateral cryptorchidism were randomised to surgery at 9 or 39 months of age and followed to 16 years in parallel with boys with spontaneous postnatal descent. Ultrasound was done at 11 and 16 years to determine testicular volume. The ratio of the initially undescended testis to its scrotal counterpart was used to assess testicular growth. RESULTS: At age 16, the ratio was lower (p < 0.00) in the late group compared to the early group. At 16 years, the spontaneously descended testes were significantly smaller than their scrotal counterparts but larger than the operated groups (early p < 0.01 and late p < 0.00). CONCLUSION: Our data at 16 years show that orchidopexy at 9 months results in better testicular growth compared to 3 years but did not reach the corresponding volumes of their scrotal counterparts. This indicates that earlier surgery is beneficial to testicular growth. At age 16, the postnatally descended testes were not only larger than the surgically treated testes but also exhibited impaired testicular growth.

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DNA methyltransferases (DNMTs) are important epigenetic modification during spermatogenesis. To further evaluate the pattern of DNMTs in horse testes during development, we investigated the expression and localization of DNMT1, DNMT3a and DNMT3b at different time points. The qRT-PCR results showed that DNMT1 expression was maintained in testes tissue from 6-month-old (0.5y) to 2-year-old (2y) of age and decreased after 3-year-old (3y) (P < 0.01). The expression levels of DNMT3a and DNMT3b peaked in testes tissue at 3y (P < 0.01). At 4-year-old (4y), the expression of DNMT3a and DNMT3b was decreased and became similar to that at 0.5y. Immunofluorescence of DNMT1, DNMT3a and DNMT3b on testis samples confirmed the differential expression and localization of these three DNA methylation transferases during horse development. Further molecular biological studies are needed to understand the implications of the expression patterns of these DNMTs in horse testes.

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