Testis Development Research Articles

Neonatal vitamin A supplementation improves sheep fertility potential

This study aimed to explore the effects of neonatal vitamin A (VA) supplementation on testis development and spermatogenesis. A total of 32 newborn lambs were intramuscularly injected with corn oil (control group) or corn oil + 2500 IU/kg BW VA (VA group). They were slaughtered and sampled at 3 weeks and 8 months of age to analyze spermatogenesis, cell proliferation, hormone secretion, antioxidant status of the testis, and adult sheep sperm parameters. Compared with the control group, the expression of spermatogonial differentiation-related genes in VA group was up-regulated (P < 0.05). Testis weight, seminiferous tubule diameter, number of spermatogonium and spermatocyte, and sperm density increased significantly in VA group at 8 months of age (P < 0.05). Neonatal VA injection upregulated the expression of the cell proliferation marker PCNA and cell cycle-related genes in the testis (P < 0.05). VA increased the concentrations of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum and upregulated steroidogenesis-related genes in the testis (P < 0.05). The antioxidant levels in the VA group were maintained at high levels. The total antioxidant capacity (T-AOC), antioxidant enzyme content and antioxidant-related genes were increased in the testis (P < 0.05). Furthermore, neonatal VA injection activated retinoic acid (RA) signaling to maintain the blood-testosterone barrier (BTB) in the testis of 3-week-old sheep. AMP-activated protein kinase (AMPK) and protein kinase B (AKT) signaling were also modulated in the sheep testis (P < 0.05). Taken together, VA supplementation in newborn rams promotes testis development and spermatogenesis to improve fertility.

Testicular differentiation in 46,XX DSD: an overview of genetic causes

In mammals, the development of male or female gonads from fetal bipotential gonads depends on intricate genetic networks. Changes in dosage or temporal expression of sex-determining genes can lead to differences of gonadal development. Two rare conditions are associated with disruptions in ovarian determination, including 46,XX testicular differences in sex development (DSD), in which the 46,XX gonads differentiate into testes, and 46,XX ovotesticular DSD, characterized by the coexistence of ovarian and testicular tissue in the same individual. Several mechanisms have been identified that may contribute to the development of testicular tissue in XX gonads. This includes translocation of SRY to the X chromosome or an autosome. In the absence of SRY, other genes associated with testis development may be overexpressed or there may be a reduction in the activity of pro-ovarian/antitesticular factors. However, it is important to note that a significant number of patients with these DSD conditions have not yet recognized a genetic diagnosis. This finding suggests that there are additional genetic pathways or epigenetic mechanisms that have yet to be identified. The text will provide an overview of the current understanding of the genetic factors contributing to 46,XX DSD, specifically focusing on testicular and ovotesticular DSD conditions. It will summarize the existing knowledge regarding the genetic causes of these differences. Furthermore, it will explore the potential involvement of other factors, such as epigenetic mechanisms, in developing these conditions.

A Novel Investigation for Early Sex Determination in Alive Adult European Seabass (Dicentrarchus labrax) Using cyp19a1a, dmrt1a, and dmrt1b Genes Expression in Tail Fin tissues.

This study is the first investigation for using sex-related gene expression in tail fin tissues of seabass as early sex determination without killing the fish. The European seabass (Dicentrarchus labrax) is gonochoristic and lacks distinguishable sex chromosomes, so, sex determination is referred to molecular actions for some sex-related genes on autosomal chromosomes which are well known such as cyp19a1a, dmrt1a, and dmrt1b genes which play crucial role in gonads development and sex differentiation. cyp19a1a is expressed highly in females for ovarian development and dmrt1a and dmrt1b are for testis development in males. In this study, we evaluated the difference in the gene expression levels of studied genes by qPCR in tail fins and gonads. We then performed discriminant analysis (DA) using morphometric traits and studied gene expression parameters as predictor tools for fish sex. The results revealed that cyp19a1a gene expression was significantly higher in future females' gonads and tail fins (p ≥ 0.05). Statistically, cyp19a1a gene expression was the best parameter to discriminate sex even the hit rate of any other variable by itself could not correctly classify 100% of the fish sex except when it was used in combination with cyp19a1a. In contrast, Dmrt1a gene expression was higher in males than females but there were difficulties in analyzing dmrt1a and dmrt1b expressions in the tail because levels were low. So, it could be used in future research to differentiate and determine the sex of adult fish using the cyp19a1a gene expression marker without killing or sacrificing fish.

Multiple transcriptome analyses reveal mouse testis developmental dynamics

The testes are the organs of gamete production and testosterone synthesis. Up to date, no model system is available for mammalian testicular development, and only few studies have characterized the mouse testis transcriptome from no more than three postnatal ages. To describe the transcriptome landscape of the developing mouse testis and identify the potential molecular mechanisms underlying testis maturation, we examined multiple RNA-seq data of mouse testes from 3-week-old (puberty) to 11-week-old (adult). Sperm cells appeared as expected in 5-week-old mouse testis, suggesting the proper sample collection. The principal components analysis revealed the genes from 3w to 4w clustered away from other timepoints, indicating they may be the important nodes for testicular development. The pairwise comparisons at two adjacent timepoints identified 7,612 differentially expressed genes (DEGs), resulting in 58 unique mRNA expression patterns. Enrichment analysis identified functions in tissue morphogenesis (3-4w), regulation of peptidase activity (4-5w), spermatogenesis (7-8w), and antigen processing (10-11w), suggesting distinct functions in different developmental periods. 50 hub genes and 10 gene cluster modules were identified in the testis maturation process by protein-protein interaction (PPI) network analysis, and the miRNA-lncRNA-mRNA, miRNA-circRNA-mRNA and miRNA-circRNA-lncRNA-mRNA competing endogenous RNA (ceRNA) networks were constructed. The results suggest that testis maturation is a complex developmental process modulated by various molecules, and that some potential RNA-RNA interactions may be involved in specific developmental stages. In summary, this study provides an update on the molecular basis of testis development, which may help to understand the molecular mechanisms of mouse testis development and provide guidance for mouse reproduction.

Identification and characterization of the Dmrt1B gene in the oriental river prawn, Macrobrachium nipponense.

Dmrt (doublesex and mab-3 related transcription factor) is a protein family of transcription factors implicated in sexual regulation. Dmrt proteins are widely conserved and known for their involvement in sex determination and differentiation across species, from invertebrates to humans. In this study, we identified a novel gene with a DM (doublesex/Mab-3)-domain gene in the river prawn, Macrobrachium nipponense, which we named MniDmrt1B due to its similarities and close phylogenetic relationship with Dmrt1B in Macrobrachium rosenbergii. Through amino acid alignments and structural predictions, we observed conservation and identified putative active sites within the DM domain. qRT-PCR analysis revealed that MniDmrt1B exhibited high expression levels in the testis, with consistently higher expression in males compared to females during development. Additionally, similar to other sex-regulated genes, the MniDmrt1B gene exhibited high expression levels during the sex differentiation-sensitive periods in M. nipponense. These results strongly indicated that MniDmrt1B probably plays an important role in testis development and sex differentiation in M. nipponense.

Expression pattern of germ cell markers in cryptorchid stallion testes.

Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression patterns through reverse transcription quantitative PCR and immunofluorescence assays. We also compared the cross-sectional ST area between groups. Six post-pubertal Thoroughbred unilateral cryptorchid stallions were used. The relative abundance of the mRNA transcripts of PGP9.5 and VASA was significantly upregulated in the NST group than in the CST group. Additionally, the cross-sectional ST area and localization of PGP9.5 and VASA in germ cells were significantly higher in the NST group than in the CST group. Regarding Leydig cells, PGP9.5 staining was observed in both groups. Spermatogonia, primary spermatocytes and secondary spermatocytes were immunostained with VASA in the NST group, while immunostaining was only observed in spermatogonia in the CST group. These results indicate long-term exposure to heat stress conditions, such as cryptorchidism, directly impacts germ cell proliferation and differentiation, leading to impaired spermatogenesis and compromised fertility in stallions.

Development of gonad and brood pouch and expression profiles of cgα, fshβ and lhβ transcripts and plasma 17β-E2 and 11-KT during the first reproductive cycle of farmed big-belly seahorse (Hippocampus abdominalis)

Limited data are available regarding the gonadal development of farmed seahorse along with the physiological role of gonadotropins and sex steroids during gonadal maturation. In the present study, we conducted an examination of the developmental stages of gonad and brood pouch during the initial reproductive cycle of the farmed big-belly seahorse (Hippocampus abdominalis), and our results showed that males sexually matured half of a month later than females, and the brood pouch emerged at 3.5-month old and then reached the late-stage at 5.5-month old. Meanwhile, the open reading frame (ORF) of gonadotropin subunits (cgα, fshβ and lhβ) of the big-belly seahorse were cloned and characterized, and tissue distribution profiles of their transcripts were analyzed, revealing their detectability across various tissues, with a notable presence observed in the brain tissues. Furthermore, expression profiles of cgα, fshβ and lhβ transcripts and the plasma levels of 17β-estradiol (E2) and 11-ketotestosterone (KT) in farmed big-belly seahorse during the first reproductive cycle were also investigated. The findings indicated that cgα, fshβ and lhβ transcripts exhibited low expressions at the initial development of ovary or testis, raised continuously and reached their peak levels at the period of spawning or spermation, and they kept gradually increasing accompany with brood pouch growth. It is noteworthy that variation of mRNA levels of fshβ were slightly higher than those of lhβ. Besides, the results showed that plasma 11-KT levels presented an increasing trend during testicular development, peaking in fish with III and IV-stage testis and early-stage brood pouch, and plasma 17β-E2 levels were higher in vitellogenic stage of fish with II-stage ovary and early III-stage ovary. Taken together, these data reveal that gonadotropins and sex steroids likely play a role in the regulation of gonad and brood pouch growth in seahorse. These observations not only contribute to clarifying function of reproductive hormones on growth of gonad and brood pouch of seahorse, but also providing an important theoretical foundation for the artificial breeding of seahorses.

A conserved NR5A1-responsive enhancer regulates SRY in testis-determination

The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5’ regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process.

Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Two redundant transcription factor binding sites in a single enhancer are essential for mammalian sex determination.

Male development in mammals depends on the activity of the two SOX gene: Sry and Sox9, in the embryonic testis. As deletion of Enhancer 13 (Enh13) of the Sox9 gene results in XY male-to-female sex reversal, we explored the critical elements necessary for its function and hence, for testis and male development. Here, we demonstrate that while microdeletions of individual transcription factor binding sites (TFBS) in Enh13 lead to normal testicular development, combined microdeletions of just two SRY/SOX binding motifs can alone fully abolish Enh13 activity leading to XY male-to-female sex reversal. This suggests that for proper male development to occur, these few nucleotides of non-coding DNA must be intact. Interestingly, we show that depending on the nature of these TFBS mutations, dramatically different phenotypic outcomes can occur, providing a molecular explanation for the distinct clinical outcomes observed in patients harboring different variants in the same enhancer.

Identification of testis development-related genes by combining Iso-Seq and RNA-Seq in Zeugodacus tau.

Introduction: Zeugodacus tau (Walker) is an invasive pest. An effective method to control this pest is the sterile insect technique (SIT). To better apply this technique, it is necessary to understand testis development progression. Methods: Differentially expressed genes (DEGs) during testis development were analyzed by PacBio Iso-Seq and RNA-seq. Results: RNA-Seq library of Z. tau testes on day 1, 6, and 11 post eclosion were constructed. We identified 755 and 865 differentially expressed genes in the comparisons of T6 (testes on day 6) vs. T1 and T11 vs. T1, respectively. The KEGG pathway analysis showed that the DEGs were significantly enriched in retinol metabolism, vitamin B6 metabolism, and ascorbate and aldarate metabolism pathways. Knockdown of retinol dehydrogenase 12-like (rdh12-like), pyridoxal kinase (pdxk) and regucalcin (rgn), the representative gene in each of the above 3 pathways, reduced the hatching rate of Z. tau offspring. In addition, we identified 107 Drosophila spermatogenesis-related orthologous genes in Z. tau, of which innexin 2 (inx2) exhibited significantly up-regulated expression throughout testis development, and the knockdown of this gene reduced offspring hatching rate. Discussion: Our data indicated that rdh12-like, pdxk, rgn, and inx2 genes were related to testis development, and they were conserved in tephritid species. These results suggested that this gene might have the same function in tephritid. The findings provide an insight into testis development and spermatogenesis in tephritid species.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

A temperature‐controlled, circular maintenance system for studying growth and development of pelagic tunicates (salps)

AbstractSalps have attracted attention as zooplankton organisms that may be able to expand their habitat range and increase their ecological importance in the face of ongoing global warming. Due to their gelatinous nature, unique feeding strategy, and reproductive ecology such changes could have profound impacts on regional marine ecosystems. While their role in the regional carbon cycle is receiving attention, our knowledge of their physiology and life cycle is still limited. This knowledge gap is mainly due to their fragile gelatinous nature, which makes it difficult to capture and maintain intact specimen in the laboratory. We present here a modified kreisel tank system that has been tested onboard a research vessel with the Southern Ocean salp Salpa thompsoni and at a research station with Salpa fusiformis and Thalia democratica from the Mediterranean Sea. Successful maintenance over days to weeks allowed us to obtain relative growth and developmental rates comparable to in situ field samples of S. thompsoni and S. fusiformis, and provided insights into previously unknown features of their life cycle (e.g., testes development). Our results show that traditional methods of estimating growth, such as cohort analysis, may lead to a general overestimation of growth rates and neglect individual strategies (e.g., shrinkage), which can affect the results and conclusions drawn from population dynamic models. By providing a starting point for the successful maintenance of different species, comparable experiments on the physiology of salps is made possible. This will contribute to refining model parameters and improving the reliability of the predictions.

Bioaccumulation and Male Reproductive Toxicity of the New Brominated Flame Retardant Tetrabromobisphenol A-Bis(2,3-dibromo-2-methylpropyl ether) in Comparison with Hexabromocyclododecane.

Tetrabromobisphenol A-bis(2,3-dibromo-2-methylpropyl ether) (TBBPA-DBMPE) has come into use as an alternative to hexabromocyclododecane (HBCD), but it is unclear whether TBBPA-DBMPE has less hazard than HBCD. Here, we compared the bioaccumulation and male reproductive toxicity between TBBPA-DBMPE and HBCD in mice following long-term oral exposure after birth. We found that the concentrations of TBBPA-DBMPE in livers significantly increased with time, exhibiting a bioaccumulation potency not substantially different from HBCD. Lactational exposure to 1000 μg/kg/d TBBPA-DBMPE as well as 50 μg/kg/d HBCD inhibited testis development in suckling pups, and extended exposure up to adulthood resulted in significant molecular and cellular alterations in testes, with slighter effects of 50 μg/kg/d TBBPA-DBMPE. When exposure was extended to 8 month age, severe reproductive impairments including reduced sperm count, increased abnormal sperm, and subfertility occurred in all treated animals, although 50 μg/kg/d TBBPA-DBMPE exerted lower effects than 50 μg/kg/d HBCD. Altogether, all data led us to conclude that TBBPA-DBMPE exerted weaker male reproductive toxicity than HBCD at the same doses but exhibited bioaccumulation potential roughly equivalent to HBCD. Our study fills the data gap regarding the bioaccumulation and toxicity of TBBPA-DBMPE and raises concerns about its use as an alternative to HBCD.

A role for TRPC3 in mammalian testis development.

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

MAPKs signaling is obligatory for male reproductive function in a development-specific manner.

Mitogen-activated protein kinases (MAPKs) represent widely expressed and evolutionarily conserved proteins crucial for governing signaling pathways and playing essential roles in mammalian male reproductive processes. These proteins facilitate the transmission of signals through phosphorylation cascades, regulating diverse intracellular functions encompassing germ cell development in testis, physiological maturation of spermatozoa within the epididymis, and motility regulation at ejaculation in the female reproductive tract. The conservation of these mechanisms appears prevalent across species, including humans, mice, and, to a limited extent, livestock species such as bovines. In Sertoli cells (SCs), MAPK signaling not only regulates the proliferation of immature SCs but also determines the appropriate number of SCs in the testes at puberty, thereby maintaining male fertility by ensuring the capacity for sperm cell production. In germ cells, MAPKs play a crucial role in dynamically regulating testicular cell-cell junctions, supporting germ cell proliferation and differentiation. Throughout spermatogenesis, MAPK signaling ensures the appropriate Sertoli-to-germ cell ratio by regulating apoptosis, controlling the metabolism of developing germ cells, and facilitating the maturation of spermatozoa within the cauda epididymis. During ejaculation in the female reproductive tract, MAPKs regulate two pivotal events-capacitation and the acrosome reaction essential for maintaining the fertility potential of sperm cells. Any disruptions in MAPK pathway signaling possibly may disturb the testicular microenvironment homeostasis, sperm physiology in the male body before ejaculation and in the female reproductive tract during fertilization, ultimately compromising male fertility. Despite decades of research, the physiological function of MAPK pathways in male reproductive health remains inadequately understood. The current review attempts to combine recent findings to elucidate the impact of MAPK signaling on male fertility and proposes future directions to enhance our understanding of male reproductive functions.

Transcriptome sequencing reveals the characteristics of spermatogenesis and testis development in Amphioctopus fangsiao

Spermatogenesis is the production of spermatozoa by the testes, a process that is associated with reproduction in males. Amphioctopus fangsiao is an economically important species from the northern coast of China, and little research has been done on spermatogenesis in this species. In this study, we performed transcriptome analysis of testis and other somatic tissues. The maturing stage of testis development was selected, in which the emergence of spermatozoa was observed. A total of 15,773 differentially expressed genes were identified, including 8,922 upregulated genes and 6,851 downregulated genes. GO and KEGG enrichment analyses were performed to explore the functions of genes related to spermatogenesis, and the interactions between genes were examined by constructing protein interaction networks. Pathways and genes related to spermatogenesis were identified, including the oxytocin signaling pathway, the calcium signaling pathway, the FOXO signaling pathway, the DNAH family, and other key genes. The expression levels of 25 key genes were verified by qRT-PCR. The results for spermatogenesis-related genes will provide insights into the processes of spermatogenesis and testis development in A. fangsiao.

Novel molecular aberrations involved in testicular germ cell tumor (TGCT) development and cisplatin resistance.

520 Background: Although highly curable, a proportion of testicular germ cell tumors (TGCT) develop resistance to cisplatin-based chemotherapy with subsequent disease relapses and possible patient death. The causes of disease progression and cisplatin (CDDP) resistance have not yet been well elucidated. Methods: We analyzed 56 samples of 15 patients with advanced and relapsing TGCT including primary tumors, metastases persisting after CDDP-containing chemotherapy, and circulating free tumor DNA (cfDNA) from the disease progression, with gDNA from blood leucocytes used as a background control. The quantity and quality of cfDNA was checked by capillary electrophoresis. All samples were sequenced by whole exome sequencing with SureSelect libraries on Illumina platform. Only mutations that were found in malignant samples or cfDNA with variant allele frequency (VAF) ≥ 5% and at least in 3 reads, not present in gDNA, with a minimum total read depth of 5 quality reads, excluding synonymous variants, were further analyzed. Results: Mutations of 6 genes with a significant role in carcinogenesis and/or testis development were identified in more than one patient: RBMX (in 4 pts), TPTE2 and ANKRD30A (in 3 pts), CDC27, PRAMEF8, and PRDM9 (in 2 pts). All the mutations were missense, with VAF 5 - 86%. They were all detectable in patients’ cfDNA, with increasing VAF during the tumor progression; the VAFs in cfDNA collected at advanced disease stages were even higher than those in primary tumors. PRDM9 mutations were found only in cfDNA from disease progression, but not in the primary testicular tumors. Of the identified genes, only CDC27 mutations have been previously described in TGCT. The genes newly associated with TGCT in this study are involved in genomic stability maintenance ( RBMX), spermiogenesis ( TPTE2), cell proliferation and apoptosis ( PRAMEF8), and several of them show testis-specific expression ( TPTE2, PRAMEF8, ANKRD30A, PRDM9). PRDM9 encodes a zinc-finger protein with histone methyltransferase activity, responsible for H3 methylation during meiosis. As epigenetic changes are supposed to play a crucial role in TGCT pathogenesis and resistance, and aberrant H3 methylation has been previously related to abnormal expression of OCT3/4 transcription factor promoting TGCT development, this gene is a strong candidate for TGCT progression and evolving CDDP resistance. Conclusions: Mutations of 5 novel genes have been identified in association with TGCT progression and cisplatin resistance. Their impact is strongly suggested by their functions in normal and malignant regulatory pathways and testis-specific expression patterns but has to be confirmed in experimental studies. The National Institute for Cancer Research project (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Supported by grants MH CZ - DRO (00064190, 00064203).

Transcriptome analysis of reproductive tract tissues of male river prawn Macrobrachium americanum.

The river prawn, Macrobrachium americanum (M. americanum), is one of the largest prawns of the genus in Latin America and is an amphidromous species distributed along the Pacific coast of America. This prawn has commercial value due to its size and taste, making it a good option for aquaculture production. Its culture has been attempted in ponds and concrete tanks, but no successful technique can still support commercial production. Understanding the mechanisms that regulate reproduction at the molecular level is very important. This knowledge can provide tools for manipulating transcripts, which could increase the number or size of animals in the culture. Our understanding of the mechanism that regulates the reproduction of M. americanum at the molecular level is limited. Perform and analyze the transcriptome assembly of the testes, vas deferens, and terminal ampulla of M. americanum. to provide new molecular information about its reproduction. The cDNA library was constructed and sequenced for each tissue to identify novel transcripts. A combined transcriptome with the three tissues was assembled using Trinity software. Unigenes were annotated using BLASTx and BLAST2GO. The transcriptome assembly generated 1,059,447 unigenes, of which 7222 genes had significant hits (e-value < 1 × 10-5) when compared against the Swiss-Prot database. Around 75 genes were related to sex determination, testis development, spermatogenesis, spermiogenesis, fertilization, maturation of testicular cells, neuropeptides, hormones, hormone receptors, and/or embryogenesis. These results provide new molecular information about M. americanum reproduction, representing a reference point for further genetic studies of this species.

Loss of Fshr Prevents Testicular Maturation in Atlantic Salmon (Salmo salar L.).

Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr-/-) or in-frame mutations (fshrif/if). Nearly all wt males matured while all fshr-/- males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr-/- males compared with maturing males. More than half of the fshrif/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.