Prenatal methamphetamine hydrochloride exposure downregulates miRNA-151-3p and CACNA1C in testis rats' offspring.

Due to the widespread use of methamphetamine (METH) among reproductive-aged women, the effects of intrauterine exposure to METH need to be investigated, as previous studies on this topic have been limited. The goal of this study is to examine the influence of two regulatory genes (miRNA-151-3p and CACNA1C) on the intrauterine life of mice exposed to METH. Pregnant mice received doses of 2 and 5 mg/kg of METH and saline from day 10 of pregnancy until the end. Their offspring were then evaluated for miRNA-151-3p and CACNA1C gene expression levels using real-time PCR. The findings indicated that exposure to METH reduced the expression levels of both miRNA-151-3p and CACNA1C genes in offspring compared to the control group (p≤0.001). In conclusion, intrauterine exposure to METH leads to a decrease in expression levels of both miRNA-151-3p and CACNA1C genes, potentially disrupting regulatory pathways involving these genes and having an impact on male reproductive health.

RNA-DNA differences in variant calls from cattle tissues result in erroneous eQTLs.

BACKGROUND: Association testing between molecular phenotypes and genomic variants can help to understand how genotype affects phenotype. RNA sequencing provides access to molecular phenotypes such as gene expression and alternative splicing while DNA sequencing or microarray genotyping are the prevailing options to obtain genomic variants. RESULTS: We genotype variants for 74 male Braunvieh cattle from both DNA (~ 13-fold coverage) and deep total RNA sequencing from testis, vas deferens, and epididymis tissue (~ 250 million reads per tissue). We show that RNA sequencing can be used to identify approximately 40% of variants (7-10 million) called from DNA sequencing, with over 80% precision. Within highly expressed coding regions, over 92% of expected variants were called with nearly 98% precision. Allele-specific expression and putative post-transcriptional modifications negatively impact variant genotyping accuracy from RNA sequencing and contribute to RNA-DNA differences. Variants called from RNA sequencing detect roughly 75% of eGenes identified using variants called from DNA sequencing, demonstrating a nearly 2-fold enrichment of eQTL variants. We observe a moderate-to-strong correlation in nominal association p-values (Spearman ρ2 ~ 0.6), although only 9% of eGenes have the same top associated variant. CONCLUSIONS: We find hundreds of thousands of RNA-DNA differences in variants called from RNA and DNA sequencing on the same individuals. We identify several highly significant eQTL when using RNA sequencing variant genotypes which are not found with DNA sequencing variant genotypes, suggesting that using RNA sequencing variant genotypes for association testing results in an increased number of false positives. Our findings demonstrate that caution must be exercised beyond filtering for variant quality or imputation accuracy when analysing or imputing variants called from RNA sequencing.

Evidence for NR2F2/COUP-TFII involvement in human testis development.

NR2F2 encodes COUP-TFII, an orphan nuclear receptor required for the development of the steroidogenic lineages of the murine fetal testes and ovaries. Pathogenic variants in human NR2F2 are associated with testis formation in 46,XX individuals, however, the function of COUP-TFII in the human testis is unknown. We report a de novo heterozygous variant in NR2F2 (c.737G > A, p.Arg246His) in a 46,XY under-masculinized boy with primary hypogonadism. The variant, located within the ligand-binding domain, is predicted to be highly damaging. In vitro studies indicated that the mutation does not impact the stability or subcellular localization of the protein. NR5A1, a related nuclear receptor that is a key factor in gonad formation and function, is known to physically interact with COUP-TFII to regulate gene expression. The mutant protein did not affect the physical interaction with NR5A1. However, in-vitro assays demonstrated that the mutant protein significantly loses the inhibitory effect on NR5A1-mediated activation of both the LHB and INSL3 promoters. The data support a role for COUP-TFII in human testis formation. Although mutually antagonistic sets of genes are known to regulate testis and ovarian pathways, we extend the list of genes, that together with NR5A1 and WT1, are associated with both 46,XX and 46,XY DSD.

Tebuconazole Induces Mouse Fetal Testes Damage via ROS Generation in an Organ Culture Method.

The fungicide tebuconazole (TEB) poses risks to human and animal health via various exposure routes. It induces toxicity in multiple organs and disrupts reproductive health by affecting steroid hormone synthesis and fetal development. In this study, we investigated the impact of TEB on fetal testes using in vitro models, focusing on germ, Sertoli, and Leydig cells, and explored the mechanisms underlying cellular damage. The results revealed significant damage to germ cells and disruption of Leydig cell development. TEB exposure led to a decrease in germ cell numbers, as indicated by histological and immunostaining analyses. TEB induced the up- and down-regulation of the expression of fetal and adult Leydig cell markers, respectively. Additionally, TEB-treated fetal testes exhibited increased expression of oxidative-stress-related genes and proteins. However, co-treatment with the antioxidant N-acetylcysteine mitigated TEB-induced germ cell damage and prevented abnormal Leydig cell development. These findings suggest that administration of antioxidants can prevent the intratesticular damage typically caused by TEB exposure.

Understanding testicular single cell transcriptional atlas: from developmental complications to male infertility.

Spermatogenesis is a multi-step biological process where mitotically active diploid (2n) spermatogonia differentiate into haploid (n) spermatozoa via regulated meiotic programming. The alarming rise in male infertility has become a global concern during the past decade thereby demanding an extensive profiling of testicular gene expression. Advancements in Next-Generation Sequencing (NGS) technologies have revolutionized our empathy towards complex biological events including spermatogenesis. However, despite multiple attempts made in the past to reveal the testicular transcriptional signature(s) either with bulk tissues or at the single-cell, level, comprehensive reviews on testicular transcriptomics and associated disorders are limited. Notably, technologies explicating the genome-wide gene expression patterns during various stages of spermatogenic progression provide the dynamic molecular landscape of testicular transcription. Our review discusses the advantages of single-cell RNA-sequencing (Sc-RNA-seq) over bulk RNA-seq concerning testicular tissues. Additionally, we highlight the cellular heterogeneity, spatial transcriptomics, dynamic gene expression and cell-to-cell interactions with distinct cell populations within the testes including germ cells (Gc), Sertoli cells (Sc), Peritubular cells (PTc), Leydig cells (Lc), etc. Furthermore, we provide a summary of key finding of single-cell transcriptomic studies that have shed light on developmental mechanisms implicated in testicular disorders and male infertility. These insights emphasize the pivotal roles of Sc-RNA-seq in advancing our knowledge regarding testicular transcriptional landscape and may serve as a potential resource to formulate future clinical interventions for male reproductive health.

Loss of Function of Vasoactive-intestinal Peptide Alters Sex Ratio and Reduces Male Reproductive Fitness in Zebrafish.

Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess 2 isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa-/- females display normal reproduction. In contrast, we have found that vipa-/- males are severely subfertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with wild-type (WT) males, we show that the testes of vipa-/- are underdeveloped and contain ∼70% less spermatids compared to WT counterparts. The sperm of vipa-/- males displayed reduced potency in terms of fertilization (by ∼80%) and motility span and duration (by ∼50%). In addition, vipa-/- male attraction to WT females was largely nonexistent, indicating decreased sexual motivation. We show that vipa mRNA and protein is present in Leydig cells and in developing germ cells in the testis of WT, raising the possibility that endogenous Vipa contributes to testicular function. Absence of Vipa in vipa-/- males resulted in downregulation of 3 key genes in the androgen synthesis chain in the testis, 3ß-hsd, 17ß-hsd1, and cyp11c1 (11ß-hydrogenase), associated with a pronounced decrease in 11-ketotestosterone production and, in turn, compromised reproductive fitness. Altogether, this study establishes a crucial role for Vipa in the regulation of male reproduction in zebrafish, like in mammals, with the exception that Vipa is also expressed in zebrafish testis.

[Functions and mechanisms of testicular descent in Apodemus agrarius based on transcriptomics and metabolomics].

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.

Heat stress induced piRNA alterations in pachytene spermatocytes and round spermatids.

BACKGROUND: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells. METHODS: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs. RESULTS: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts. CONCLUSIONS: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.

Ursolic acid attenuates oligospermia in busulfan-induced mice by promoting motor proteins.

Background: Oligospermia is one of the most common reasons for male infertility which is troubling numerous couples of child-bearing age. This investigation scrutinizes the implications and mechanistic underpinnings of ursolic acid's effect on busulfan-induced oligospermia in mouse models. Methods: A singular intraperitoneal injection of busulfan at a dosage of 30 mg/kg induced oligospermia. Two weeks subsequent to this induction, mice were subjected to various dosages of ursolic acid (10, 30, and 50 mg/kg body weight, respectively) on a daily basis for four consecutive weeks. Following this treatment period, a meticulous analysis of epididymal sperm parameters, encompassing concentration and motility, was conducted using a computer-assisted sperm analysis system. The histopathology of the mice testes was performed utilizing hematoxylin and eosin staining, and the cytoskeleton regeneration of the testicular tissues was analyzed via immunofluorescent staining. Serum hormone levels, including testosterone, luteinizing hormone, and follicle-stimulating hormone, as well as reactive oxygen species levels (inclusive of reactive oxygen species and malondialdehyde), were gauged employing specific enzyme-linked immunosorbent assay kits. Differentially expressed genes of testicular mRNA between the oligospermia-induced group and the various ursolic acid treatment groups were identified through RNA sequencing analysis. Results: The results revealed that a dosage of 50 mg/kg ursolic acid treatment could increase the concentration of epididymal sperm in oligospermia mice, promote the recovery of testicular morphology, regulate hormone levels and ameliorate oxidative damage. The mechanism research results indicated that ursolic acid increased the expression level of genes related to motor proteins in oligospermia mice.

Transcriptomic changes in the posterior pallium of male zebra finches associated with social niche conformance.

Animals plastically adjust their physiological and behavioural phenotypes to conform to their social environment-social niche conformance. The degree of sexual competition is a critical part of the social environment to which animals adjust their phenotypes, but the underlying genetic mechanisms are poorly understood. We conducted a study to investigate how differences in sperm competition risk affect the gene expression profiles of the testes and two brain areas (posterior pallium and optic tectum) in breeding male zebra finches (Taeniopygia castanotis). In this pre-registered study, we investigated a large sample of 59 individual transcriptomes. We compared two experimental groups: males held in single breeding pairs (low sexual competition) versus those held in two pairs (elevated sexual competition) per breeding cage. Using weighted gene co-expression network analysis (WGCNA), we observed significant effects of the social treatment in all three tissues. However, only the treatment effects found in the pallium were confirmed by an additional randomisation test for statistical robustness. Likewise, the differential gene expression analysis revealed treatment effects only in the posterior pallium (ten genes) and optic tectum (six genes). No treatment effects were found in the testis at the single gene level. Thus, our experiments do not provide strong evidence for transcriptomic adjustment specific to manipulated sperm competition risk. However, we did observe transcriptomic adjustments to the manipulated social environment in the posterior pallium. These effects were polygenic rather than based on few individual genes with strong effects. Our findings are discussed in relation to an accompanying paper using the same animals, which reports behavioural results consistent with the results presented here.

Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development.

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.

ERK/CREB and p38 MAPK/MMP14 Signaling Pathway Influences Spermatogenesis through Regulating the Expression of Junctional Proteins in Eriocheir sinensis Testis.

The hemolymph-testis barrier (HTB) is a reproduction barrier in Crustacea, guaranteeing the safe and smooth process of spermatogenesis, which is similar to the blood-testis barrier (BTB) in mammals. The MAPK signaling pathway plays an essential role in spermatogenesis and maintenance of the BTB. However, only a few studies have focused on the influence of MAPK on crustacean reproduction. In the present study, we knocked down and inhibited MAPK in Eriocheir sinensis. Increased defects in spermatogenesis were observed, concurrently with a damaged HTB. Further research revealed that es-MMP14 functions downstream of ERK and p38 MAPK and degrades junctional proteins (Pinin and ZO-1); es-CREB functions in the ERK cascade as a transcription factor of ZO-1. In addition, when es-MMP14 and es-CREB were deleted, the defects in HTB and spermatogenesis aligned with abnormalities in the MAPK. However, JNK impacts the integrity of the HTB by changing the distribution of intercellular junctions. In summary, the MAPK signaling pathway maintains HTB integrity and spermatogenesis through es-MMP14 and es-CREB, which provides insights into the evolution of gene function during barrier evolution.

Deficiency of ValRS-m Causes Male Infertility in Drosophila melanogaster.

Drosophila spermatogenesis involves the renewal of germline stem cells, meiosis of spermatocytes, and morphological transformation of spermatids into mature sperm. We previously demonstrated that Ocnus (ocn) plays an essential role in spermatogenesis. The ValRS-m (Valyl-tRNA synthetase, mitochondrial) gene was down-regulated in ocn RNAi testes. Here, we found that ValRS-m-knockdown induced complete sterility in male flies. The depletion of ValRS-m blocked mitochondrial behavior and ATP synthesis, thus inhibiting the transition from spermatogonia to spermatocytes, and eventually, inducing the accumulation of spermatogonia during spermatogenesis. To understand the intrinsic reason for this, we further conducted transcriptome-sequencing analysis for control and ValRS-m-knockdown testes. The differentially expressed genes (DEGs) between these two groups were selected with a fold change of ≥2 or ≤1/2. Compared with the control group, 4725 genes were down-regulated (dDEGs) and 2985 genes were up-regulated (uDEGs) in the ValRS-m RNAi group. The dDEGs were mainly concentrated in the glycolytic pathway and pyruvate metabolic pathway, and the uDEGs were primarily related to ribosomal biogenesis. A total of 28 DEGs associated with mitochondria and 6 meiosis-related genes were verified to be suppressed when ValRS-m was deficient. Overall, these results suggest that ValRS-m plays a wide and vital role in mitochondrial behavior and spermatogonia differentiation in Drosophila.

The Functions and Application Prospects of Hepatocyte Growth Factor in Reproduction.

Hepatocyte growth factor (HGF) is expressed in multiple systems and mediates a variety of biological activities, such as mitosis, motility, and morphogenesis. A growing number of studies have revealed the expression patterns and functions of HGF in ovarian and testicular physiology from the prenatal to the adult stage. HGF regulates folliculogenesis and steroidogenesis by modulating the functions of theca cells and granulosa cells in the ovary. It also mediates somatic cell proliferation and steroidogenesis, thereby affecting spermatogenesis in males. In addition to its physiological effects on the reproductive system, HGF has shown advantages in preclinical studies over recent years for the treatment of male and female infertility, particularly in women with premature ovarian insufficiency. This review aims to summarize the pleiotropic functions of HGF in the reproductive system and to provide prospects for its clinical application.

Adeno-associated-virus-mediated delivery of CRISPR-CasRx induces efficient RNA knockdown in the mouse testis.

Rationale: In male mammals, many developmental-stage-specific RNA transcripts (both coding and noncoding) are preferentially or exclusively expressed in the testis, where they play important roles in spermatogenesis and male fertility. However, a reliable platform for efficiently depleting various types of RNA transcripts to study their biological functions during spermatogenesis in vivo has not been developed. Methods: We used an adeno-associated virus serotype nine (AAV9)-mediated CRISPR-CasRx system to knock down the expression of exogenous and endogenous RNA transcripts in the testis. Virus particles were injected into the seminiferous tubules via the efferent duct. Using an autophagy inhibitor, 3-methyladenine (3-MA), we optimized the AAV9 transduction efficiency in germ cells in vivo. Results: AAV9-mediated delivery of CRISPR-CasRx effectively and specifically induces RNA transcripts (both coding and noncoding) knockdown in the testis in vivo. In addition, we showed that the co-microinjection of AAV9 and 3-MA into the seminiferous tubules enabled long-term transgene expression in the testis. Finally, we found that a promoter of Sycp1 gene induced CRISPR-CasRx-mediated RNA transcript knockdown in a germ-cell-type-specific manner. Conclusion: Our results demonstrate the efficacy and versatility of the AAV9-mediated CRISPR-CasRx system as a flexible knockdown platform for studying gene function during spermatogenesis in vivo. This approach may advance the development of RNA-targeting therapies for conditions affecting reproductive health.

The Role of Retinoic Acid in Spermatogenesis and Its Application in Male Reproduction.

Spermatogenesis in mammalian testes is essential for male fertility, ensuring a continuous supply of mature sperm. The testicular microenvironment finely tunes this process, with retinoic acid, an active metabolite of vitamin A, serving a pivotal role. Retinoic acid is critical for various stages, including the differentiation of spermatogonia, meiosis in spermatogenic cells, and the production of mature spermatozoa. Vitamin A deficiency halts spermatogenesis, leading to the degeneration of numerous germ cells, a condition reversible with retinoic acid supplementation. Although retinoic acid can restore fertility in some males with reproductive disorders, it does not work universally. Furthermore, high doses may adversely affect reproduction. The inconsistent outcomes of retinoid treatments in addressing infertility are linked to the incomplete understanding of the molecular mechanisms through which retinoid signaling governs spermatogenesis. In addition to the treatment of male reproductive disorders, the role of retinoic acid in spermatogenesis also provides new ideas for the development of male non-hormone contraceptives. This paper will explore three facets: the synthesis and breakdown of retinoic acid in the testes, its role in spermatogenesis, and its application in male reproduction. Our discussion aims to provide a comprehensive reference for studying the regulatory effects of retinoic acid signaling on spermatogenesis and offer insights into its use in treating male reproductive issues.

Mapping the Development of Human Spermatogenesis Using Transcriptomics-Based Data: A Scoping Review.

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.

Expression of Tshb and Tshr in the ricefield eel Monopterus albus: Potential paracrine/autocrine roles in gonads.

Thyroid stimulating hormone (TSH), a glycoprotein synthesized and secreted from thyrotrophs of the pituitary gland, is composed of a glycoprotein hormone common alpha subunit (CGA) and a specific beta subunit (TSHB). The major biological function of TSH is to stimulate thyroidal follicles to synthesize and secrete thyroid hormones through activating its cognate receptor, the thyroid stimulating hormone receptor (TSHR). In the present study, polyclonal antisera against ricefield eel Tshb and Tshr were generated respectively, and the expression of Tshb and Tshr was examined at mRNA and protein levels. RT-PCR analysis showed that tshb mRNA was expressed mainly in the pituitary as well as in some extrapituitary tissues including the ovary and testis. Tshr mRNA was also expressed in a tissue-specific manner, with transcripts detected in tissues including the kidney, ovary, and testis. The immunoreactive Tshb signals in the pituitary were shown to be localized to the inner areas of adenohypophysis which are close to the neurohypophysis of adult ricefield eels. Tshb-immunoreatvie cells in the pituitary of ricefield eel larvae were firstly observed at hatching. The expression of immunoreactive Tshb and Cga was also detected in ricefield eel ovary and testis together with Tshr. In the ovary, immunoreactive Tshb, Cga, and Tshr were observed in oocytes and granulosa cells. In the testis, immunoreactive Tshb was mainly observed in Sertoli cells while immunoreactive Cga and Tshr were detected in germ cells as well as somatic cells. Results of the present study suggest that Tsh may be synthesized both in the ovary and testis locally, which may play paracrine and/or autocrine roles in gonadal development in ricefield eels.

Intracerebroventricular injection of spexin stimulates the hypothalamic-pituitary-testicular axis and increases the secretion of male reproductive hormones in rats.

BACKGROUND: Male reproductive functions are regulated in the hypothalamic-pituitary-gonadal (HPG) axis. Any problem in this axis would lead to the deterioration of reproductive functions. The present study aimed to investigate the effects of intracerebroventricular (icv) Spexin (SPX) infusion on the HPG axis in detail. METHODS: 40 Wistar albino rats were divided into four groups: control, sham, SPX 30 nmol and SPX 100 nmol (n=10). 30 nmol/1 µl/hour SPX was administered icv to the rats in the SPX 30 nmol group for 7 days, while rats in the SPX 100 nmol group were administered 100 nmol/1 µl/hour SPX. On the 7th day, the rats were decapitated, blood and tissue samples were collected. Serum LH, FSH and testosterone levels were determined with the ELISA method, GnRH mRNA expression level was determined in hypothalamus with the RT-PCR method. Seminiferous tubule diameter and epithelial thickness were determined with the hematoxylin-eosin staining method. RESULTS: SPX infusion was increased GnRH mRNA expression in the hypothalamus tissue independent of the dose (p<0.05). Serum LH, FSH and testosterone levels in the SPX groups were increased when compared to the control and sham groups independent of the dose (p <0.05). Histological analysis revealed that SPX infusion did not lead to any changes in seminiferous epithelial thickness, while the tubule diameter increased in the SPX groups (p<0.05). CONCLUSION: The study findings demonstrated that icv SPX infusion stimulated the HPG axis and increased the secretion of male reproductive hormones.

AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans.

Dynein complexes are large, multi-unit assemblies involved in many biological processes via their critical roles in protein transport and axoneme motility. Using next-generation sequencing of infertile men presenting with low or no sperm in their ejaculates, we identified damaging variants in the dynein-related gene AXDND1. We thus hypothesised that AXDND1 is a critical regulator of male fertility. To test this hypothesis, we produced a knockout mouse model. Axdnd1-/- males were sterile at all ages but presented with an evolving testis phenotype wherein they could undergo one round of histologically replete spermatogenesis followed by a rapid depletion of the seminiferous epithelium. Marker experiments identified a role for AXDND1 in maintaining the balance between differentiation-committed and self-renewing spermatogonial populations, resulting in disproportionate production of differentiating cells in the absence of AXDND1 and increased sperm production during initial spermatogenic waves. Moreover, long-term spermatogonial maintenance in the Axdnd1 knockout was compromised, ultimately leading to catastrophic germ cell loss, destruction of blood-testis barrier integrity and immune cell infiltration. In addition, sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively these data identify AXDND1 as an atypical dynein complex-related protein with a role in protein/vesicle transport of relevance to spermatogonial function and sperm tail formation in mice and humans. This study underscores the importance of studying the consequences of gene loss-of-function on both the establishment and maintenance of male fertility.