Mature Testis Research Articles

Variation in the Oxidative Status of Testicular Tissues During Critical Pubertal Age Under Influence of Bisphenol A

Pubertal development of testis requires stringent regulations and precise expansion of germ cells. Reactive oxygen species (ROS) are involved in regulation and proliferation of spermatogonia. Bisphenol A (BPA) is well known for the induction of oxidative stress in testicular tissues leading to major adversities including reduced fertility in male. In the present study, BPA led response of antioxidants was assessed during critical period of pubertal maturation of testis. Four groups of Wistar albino rats were formed containing 15 animals each; Group I-Control, Group II-administered with 5 mg/kg/d BPA, Group III- administered with 25 mg/kg/d BPA and Group IV- administered with 50 mg/kg/d BPA. These groups were further bifurcated into three distinct periods of exposure i.e. 42-63 PND, 42-91 PND and 42-105 PND, containing 5 animals each. Level of CAT, SOD, GSH, GPx, and LPO was analysed. Linear regression for individual antioxidants and Pearson’s correlation between antioxidants were applied for age-wise analysis of variance. Results showed that during first three weeks (i.e. 42-63 PND) of BPA administration were better tolerated, irrespective of doses. Later intervals indicated significant decline (p<0.05) in the activity and level of antioxidants. Relatedness between variables of antioxidants following BPA exposure and control were extremely low (R2<0.1) indicating differential activity. Nonetheless, between antioxidants strong strength of association (r>0.9) was evident. Although initial toleration against BPA’s oxidative insult was evident, it could not be sustained following 91 or 105 PND. Extremely lower activity of antioxidant at later stage suggested potential delay in testicular maturity.

Cloning and Expression of Sox2 and Sox9 in Embryonic and Gonadal Development of Lutraria sieboldii

The Sox family plays essential roles as transcription factors in vertebrates; however, little is known about the Sox family in Lutraria sieboldii. L. sieboldii are pleasant to eat with a short growth cycle and have become one of the best bottom-seeded enrichment species in Guang Xi. In this study, Sox2 (named LsSox2) and Sox9 (named LsSox9) from L. sieboldii were cloned, and their expression patterns were analyzed. The length of the LsSox2 gene coding sequence was 1011 bp, encoding 336 amino acids, and LsSox9 was 1449 bp, encoding 482 amino acids. LsSox2 had its highest expression levels in the ovary, which were 356 times those in testis, whereas LsSox9 presented higher expression in testis, which was 6 times more highly expressed than in the ovary. LsSox2 exhibited the highest expression during the morula stage, which was 20 times that of the D-shaped larvae or zygote. LsSox9 exhibited two expression peaks, one at the four-cell stage and the other at the trochophore stage, while the lowest expression was in the zygote. LsSox9 was 73 times more highly expressed in the four-cell stage than in the zygote stage. During gonadal development, LsSox2 presented the highest expression in the mature ovary, which was 756 times more highly expressed than in mature testis. LsSox9 presented higher expression in testis at the emission stage which was 6 times more highly expressed than in the ovary. These results indicate that LsSox2 and LsSox9 may play important roles in embryonic and gonadal development.

Involvement of GLCCI1 in mouse spermatogenesis.

Spermatid production is a complex regulatory process in which coordination between hormonal control and apoptosis plays a pivotal role in maintaining a balanced number of sperm cells. Apoptosis in spermatogenesis is controlled by pro-apoptotic and anti-apoptotic molecules. Hormones involved in the apoptotic process during spermatogenesis include gonadotrophins, sex hormones, and glucocorticoid (GC). GC acts broadly as an apoptosis inducer by binding to its receptor (glucocorticoid receptor: GR) during organ development processes, such as spermatogenesis. However, the downstream pathway induced in GC-GR signaling and the apoptotic process during spermatogenesis remains poorly understood. We reported previously that GC induces full-length glucocorticoid-induced transcript 1 (GLCCI1-long), which functions as an anti-apoptotic mediator in thymic T cell development. Here, we demonstrate that mature murine testis expresses a novel isoform of GLCCI1 protein (GLCCI1-short) in addition to GLCCI1-long. We demonstrate that GLCCI1-long is expressed in spermatocytes along with GR. In contrast, GLCCI1-short is primarily expressed in spermatids where GR is absent; instead, the estrogen receptor is expressed. GLCCI1-short also binds to LC8, which is a known mediator of the anti-apoptotic effect of GLCCI1-long. A luciferase reporter assay revealed that β-estradiol treatment synergistically increased Glcci1-short promotor-driven luciferase activity in Erα-overexpressing cells. Together with the evidence that the conversion of testosterone to estrogen is preceded by aromatase expression in spermatids, we hypothesize that estrogen induces GLCCI1-short, which, in turn, may function as a novel anti-apoptotic mediator in mature murine testis.

A Comprehensive Sequencing Analysis of Testis-Born miRNAs in Immature and Mature Indigenous Wandong Cattle (Bos taurus).

Micro RNAs (miRNAs) have been recognized as important regulators that are indispensable for testicular development and spermatogenesis. miRNAs are endogenous transcriptomic elements and mainly regulate the gene expression at post-transcriptional levels; however, the key role of miRNA in bovine testicular growth is not clearly understood. Thus, supposing to unveil the transcriptomics expression changes in the developmental processes of bovine testes, we selected three immature calves and three sexually mature bulls of the local Wandong breed for testicular-tissue sample collection. The cDNA libraries of experimental animals were established for RNA-sequencing analysis. We detected the miRNA expression in testes by using high-throughput sequencing technology, and bioinformatics analysis followed. The differentially expressed (DE) data showed that 151 miRNAs linked genes were significantly DE between immature and mature bull testes. Further, in detail, 64 were significantly up-regulated and 87 were down-regulated in the immature vs. mature testes (p-value < 0.05). Pathway analyses for miRNA-linked genes were performed and identified JAG2, BCL6, CFAP157, PHC2, TYRO3, SEPTIN6, and BSP3; these genes were involved in biological pathways such as TNF signaling, T cell receptor, PI3KAkt signaling, and functions affecting testes development and spermatogenesis. The DE miRNAs including MIR425, MIR98, MIR34C, MIR184, MIR18A, MIR136, MIR15A, MIR1388 and MIR210 were associated with cattle-bull sexual maturation and sperm production. RT-qPCR validation analysis showed a consistent correlation to the sequencing data findings. The current study provides a good framework for understanding the mechanism of miRNAs in the development of testes and spermatogenesis.

Identification of spermatogenesis-related lncRNA in Holstein bull testis after sexual maturity based on transcriptome analysis

Under the large-scale breeding model, the performance of the Holstein bull is directly related to the economic efficiency of the whole dairy farm and is the key factor affecting the genetic quality of the herd. Although the number of reported studies on the association of long noncoding RNAs (lncRNAs) with male reproduction is increasing, there is a lack of research on how lncRNAs regulate Holstein bull testicular development and spermatogenesis. To explore the molecular mechanisms between lncRNAs and spermatogenesis, three 8-week-old Holstein bull (young bull, YB) testes and three 80-week-old Holstein bull (adult bull, AB) testes from the same herd were randomly chosen, and transcriptome analysis was performed to find associations between spermatogenesis and transcriptome profiles. About 16,956 differentially expressed lncRNAs (DELs) and 7970 differentially expressed mRNAs (DEMs) were identified, and 3642 significantly relationship pairs were screened out based on co-expression analysis. Further Hub analysis obtained 6 lncRNAs, and 71 mRNAs regulated by them were enriched subsequently. Functional analysis results of differentially expressed Hub LncRNA-mRNA pairs showed that more positive cell cycle checkpoints, spindle assembly and the sister chromatids separation in AB testes indicated higher production rates of sperm in AB as compared to YB. This study reveals physiological changes and further broaden our understanding of lncRNA regulation of spermatogenesis in the fully mature testis according to the transcriptome profiles.

Cancer-testis antigen lactate dehydrogenase C4 as a novel biomarker of male infertility and cancer.

A unique lactate dehydrogenase (LDH) isoenzyme designated as lactate dehydrogenase C4 (LDH-C4) is found in mammalian mature testis and spermatozoa. Thus far, LDH-C4 has been well studied with regard to its gene and amino acid sequences, structure, biological properties, and peptide synthesis. Accumulating evidence has shown that LDH-C4 is closely related to spermatic energy metabolism and plays a critical role in sperm motility, capacitation, and fertilization. Defects in the catalytic activity of LDH-C4 are key to pathophysiological abnormalities underlying infertility. LDH-C4 was originally thought to be present only in mature testis and spermatozoa; however, recent studies have implicated LDH-C4 as a cancer-testis antigen (CTA), owing to its aberrant transcription in a broad spectrum of human neoplasms. This review highlights the recent findings on LDH-C4 with particular emphasis on its role in male infertility and tumors.

The hsp70 new functions as a regulator of reproduction both female and male in Ophraella communa.

Heat shock proteins (Hsps) function as molecular chaperones that enable organisms to withstand stress and maintain normal life activities. In this study, we identified heat shock protein 70 (encoded by hsp70), which exhibits a higher expression in the mature male testis than in the unmature testis of Ophraella communa. Tissue expression profile revealed that Ochsp70 levels in males were highest in the testis, whereas those in females were highest in the head. Moreover, the expression of Ochsp70 was found to be significantly induced in female bursa copulatrix after mating. Double-stranded RNA dsOchsp70 was injected into males to performance RNA interference, which significantly decreased the male Ochsp70 expression levels within 20 d post-injection, whereas no effect was observed on the Ochsp70 expression level in the females after mating with dsOchsp70-injected males. However, significant downregulation of female fertility was marked simultaneously. Furthermore, knockdown of female Ochsp70 expression also led to a significant reduction in fertility. Finally, comparative transcriptomic analysis identified glucose dehydrogenase and insulin-like growth factor binding protein as putative downstream targets of Ochsp70. Overall, we deduced that Ochsp70 is an indispensable gene and a potential male mating factor in O. communa, which regulates reproduction.

Transcriptome Sequencing and Analysis of Male Fourfinger Threadfin, Eleutheronema tetradactylum (Shaw, 1804)

Fourfinger threadfin, Eleutheronema tetradactylum (Shaw, 1804), is one of the country’s most popular table fish species. This is a priority species for mariculture in India. Reproductive biology, life cycle, culture, and management are some budding research areas, while there is a lacuna in the species' genetic information. Using Illumina next-generation sequencing technique, 82,072 mRNA transcripts in the mature testis of fourfinger threadfin were obtained. The present study attempted to understand the expression of genes that facilitate sex determination, sex differentiation and gonadal maturation. From 82,072 transcripts, 50,943 were predicted by comparing them to proteins in UniProt using BLASTX. A total of 41 genes involved in sex determination and sex differentiation, spermatogenesis, steroid receptors in testis, steroidogenesis and gonadotropin-releasing hormone (GnRH) regulation are reported for the first time in E. tetradactylum. The entire raw sequencing reads of transcripts were made available in NCBI under BioProject PRJNA770837.

BCL2-associated athanogene 6 exon24 contributes to testosterone synthesis and male fertility in mammals.

ObjectivesBCL2‐associated athanogene 6 (BAG6) plays critical roles in spermatogenesis by maintaining testicular cell survival. Our previous data showed porcine BAG6 exon24‐skipped transcript is highly expressed in immature testes compared with mature testes. The objective of this study is to reveal the functional significance of BAG6 exon24 in mammalian spermatogenesis.Materials and MethodsCRISPR/Cas9 system was used to generate Bag6 exon24 knockout mice. Testes and cauda epididymal sperm were collected from mice. TMT proteomics analysis was used to discover the protein differences induced by Bag6 exon24 deletion. Testosterone enanthate was injected into mice to generate a high‐testosterone mice model. H&E staining, qRT‐PCR, western blotting, vector/siRNA transfection, immunofluorescence, immunoprecipitation, transmission electron microscopy, TUNEL and ELISA were performed to investigate the phenotypes and molecular basis.Results Bag6 exon24 knockout mice show sub‐fertility along with partially impaired blood‐testis barrier, increased apoptotic testicular cell rate and abnormal sperm morphology. Endoplasmic reticulum stress occurs in Bag6 exon24‐deficient testes and sterol regulatory element‐binding transcription factor 2 is activated; as a result, cytochrome P450 family 51 subfamily A member 1 expression is up‐regulated, which causes a high serum testosterone level. Additionally, serine/arginine‐rich splicing factor 1 down‐regulates BAG6 exon24‐skipped transcripts in porcine Sertoli cells by binding to 35–51 nt on BAG6 exon24 via its N‐terminal RNA‐recognition domain.ConclusionsOur findings reveal the critical roles of BAG6 exon24 in testosterone biosynthesis and male fertility, which provides new insights into the regulation of spermatogenesis and pathogenesis of subfertility in mammals.

Disruption of cell proliferation and apoptosis balance in the testes of crossbred cattle-yaks affects spermatogenic cell fate and sterility.

The balance between proliferation, differentiation and apoptosis is well-coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridization of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle-yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays. Testicular tissues from yaks (immature: 1year old, mature: 2-3 years old) and backcrossed hybrids (2year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells and Leydig cells. Furthermore, hybrid cattle-yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle-yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle-yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle-yak males.

Changes in lipid and fatty acid contents of gonad during the reproductive cycle of the Mediterranean swordfish Xiphias gladius

Swordfish Xiphias gladius is a large pelagic fish distributed worldwide and exploited for human consumption, however there is limited knowledge about its reproductive biology, especially regarding lipid dynamic in gonads. In teleost fish, reproductive success and offspring survival are associated to lipid availability for gamete synthesis. This study investigated the lipid composition, including lipid classes and fatty acids (FA) of cell membrane and reserve lipids (i.e., polar and neutral lipids, respectively; PL and NL), along female and male gonad development of a swordfish population from waters surrounding Corsica Island in the Mediterranean Sea. Overall, swordfish gonads contained <2% wet weight of total lipids, with testes and ovaries having similar fat content. Lipid classes and FA concentrations remained unchanged during testes maturation. However, concentrations of phosphatidyl choline (PL), triacylglycerol (NL), and some FA (16:0, 18:1n-9, and 22:6n-3) followed an “inverted U-shaped” relationship with the ovarian maturation. In both PL and NL, 22:6n-3 was the main polyunsaturated FA (>20% of total FA), while 20:5n-3 and 20:4n-6 were minor (3–6% of total FA) and varied little with maturation. 22:6n-3 and 18:1n-9 were selectively allocated to the ovarian maturation (increased in concentration and in proportion with maturation) until spawning. Finally, swordfish gonads might represent a good food source for humans given that 150 g of swordfish ovaries can cover the daily requirements in omega-3 for humans, but research on pollutants should also be conducted to evaluate their implications on the reproduction output of this species, and on the safety of swordfish gonads for human consumption.

Proteomic changes induced by ascorbic acid treatment on porcine immature Sertoli cells

Somatic Sertoli cells constitute the microenvironment and produce essential substances, to support male germ cell development and maturation in testis. We previously found that ascorbic acid treatment of porcine immature Sertoli cells enhances its proliferation and secretion of reproductive hormones and metabolites, and reprograms the global transcriptome. Proteomics is a powerful tool to systematically profile the underlying protein changes. Here, by employing the TMT-based quantitative proteomics method, we identified 96 and 64 significantly up- and down-regulated proteins in porcine immature Sertoli cells treated by ascorbic acid, respectively. Gene enrichment (GO and KEGG) and protein-protein interaction analyses revealed important molecular pathways (dioxygenase activity, sterol biosynthetic process, PI3K-Akt, negative regulation of peptide hormone secretion, extracellular matrix etc.). Further validation of three proteins, HMGCS1 (cholesterol synthesis), P4HA1 (glycolysis) and KDM5A (demethylation of histone 3 at lysine 4), confirmed their significant differential abundance, respectively. Taken together, our findings show that ascorbic acid can alter multiple important protein molecules and related signaling pathways, which could explain partially phenotypic changes (proliferation, apoptosis, nucleic acid methylation, lactate and reproductive hormone secretion) of porcine immature Sertoli cells as induced by ascorbic acid.

Open reading frame dominance indicates protein‐coding potential of RNAs

Recent studies have identified numerous RNAs with both coding and noncoding functions. However, the sequence characteristics that determine this bifunctionality remain largely unknown. In the present study, we develop and test the open reading frame (ORF) dominance score, which we define as the fraction of the longest ORF in the sum of all putative ORF lengths. This score correlates with translation efficiency in coding transcripts and with translation of noncoding RNAs. In bacteria and archaea, coding and noncoding transcripts have narrow distributions of high and low ORF dominance, respectively, whereas those of eukaryotes show relatively broader ORF dominance distributions, with considerable overlap between coding and noncoding transcripts. The extent of overlap positively and negatively correlates with the mutation rate of genomes and the effective population size of species, respectively. Tissue‐specific transcripts show higher ORF dominance than ubiquitously expressed transcripts, and the majority of tissue‐specific transcripts are expressed in mature testes. These data suggest that the decrease in population size and the emergence of testes in eukaryotic organisms allowed for the evolution of potentially bifunctional RNAs.

Regeneration of spermatogenesis by mouse germ cell transplantation into allogeneic and xenogeneic testis primordia or organoids.

SummaryGametogenesis requires close interactions between germ cells and somatic cells. Derivation of sperm from spermatogonial stem cells (SSCs) is hampered by the inefficiency of spermatogonial transplantation technique in many animal species because it requires a large number of SSCs and depletion of endogenous spermatogenesis. Here we used mouse testis primordia and organoids to induce spermatogenesis from SSCs. We microinjected mouse SSCs into embryonic gonads or reaggregated neonatal testis organoids, which were transplanted under the tunica albuginea of mature testes. As few as 1 × 104 donor cells colonized both types of transplants and produced sperm. Moreover, rat embryonic gonads supported xenogeneic spermatogenesis from mouse SSCs when transplanted in testes of immunodeficient mice. Offspring with normal genomic imprinting patterns were born after microinsemination. These results demonstrate remarkable flexibility of the germ cell-somatic cell interaction and raise new strategies of SSC manipulation for animal transgenesis and analysis of male infertility.

Liposome-Encapsulated Rec8 and Dmrt1 Plasmids Induce Red-Spotted Grouper (Epinephelus akaara) Testis Maturation.

In fish, the maturity of gonads plays an important role in the development and reproduction of the population, and it also dictates the success of captive breeding. Therefore, finding ways to promote gonadal maturation is an important goal in aquaculture. In this study, we injected recombinant dmrt1 and rec8 overexpression plasmids packaged in liposomes into the immature testis of red-spotted grouper (Epinephelus akaara) and measured the expression of Dmrt1 and Rec8 protein in vivo. Gonadosomatic index (GSI) and gonadal histology analyses showed that the testis developed from the immature to the mature state within 7days after plasmid injection. Additionally, the spermatozoa concentration and motility in plasmid-injected fish was the same as that of naturally mature fish. These results provided evidence that delivery of dmrt1 and rec8 expression plasmids into the testis via injection induced testis maturation in vivo.

Pituitary Gonadotropin Gene Expression During Induced Onset of Postsmolt Maturation in Male Atlantic Salmon: In Vivo and Tissue Culture Studies.

Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17β-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.

Comparative transcriptomics analysis of testicular miRNA from indicine and taurine cattle

Numerous studies have shown that several microRNAs (miRNAs) are specifically expressed in testis, play an essential role in regulating testicular spermatogenesis. Hainan and Mongolian cattle are two representative Chinese native cattle breeds representing Bos indicus (indicine cattle) and Bos taurus (taurine cattle), respectively, which are distributed in hot Hainan and cold Inner Mongolia province. To study the functional differences of miRNA in spermatogenesis between indicine and taurine cattle, six mature testes samples from indicine cattle (n = 3) and taurine cattle (n = 3) were collected, respectively. We detected miRNA expression using small RNA sequencing technology following bioinformatic analysis. A total of 578 known miRNAs and 132 novel miRNAs were detected in the six libraries. Among the 710 miRNAs, 564 miRNAs were expressed in both indicine and taurine cattle, 73 miRNAs were found solely in indicine cattle and 73 miRNAs were found solely in taurine cattle. After further analysis, among the miRNAs were identified in both indicine and taurine cattle, 184 miRNAs were differentially expressed (|log2 fold change| ≥ 1 and corrected p-value <0.05). Among the miRNAs that were only expressed in indicine cattle, 10 miRNAs were differentially expressed, whereas, among the miRNAs that were only expressed in taurine cattle, six miRNAs were differentially expressed. The enrichment analysis result showed that predicted target genes of a total of 200 differentially expressed miRNAs were enriched on some testicular spermatogenesis-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially mitogen-activated protein kinase (MAPK) signaling pathway. These findings identify miRNAs as key factors to regulate spermatogenesis in both indicine and taurine cattle, which may also be helpful for improving cattle reproductive performance in future studies.

Aromatase inhibitor induces sex reversal in the protogynous hermaphroditic rice field eel (Monopterus albus)

The rice field eel (Monopterus albus) is a protogynous hermaphroditic fish reared commercially as an aquaculture species in East Asia. Artificial breeding of rice field eels requires an abundance of adult females and males; however, the eels need to undergo a long period of female and intersexual stages before they develop into males, which results in a shortage of functional males in production practice. This study developed an artificial method for producing functional males through the oral administration of an aromatase inhibitor (AI, letrozole: 300 mg/kg feed) in juvenile fish. Histological analysis of gonadal sections of treated fish revealed the transition of primary ovaries to mature testes. During the initial period of the AI treatment, 2-month-old rice field eels were exclusively female. AI administration resulted in a significant reduction in 17β-estradiol levels and increase in testosterone and 11-ketotestosterone levels in vivo. Cell proliferation occurred in the outer layer of the gonadal epithelium, and oocytes degenerated in the ovaries. After 120 days of AI treatment, the ovaries were completely converted to testes. The sexually reversed fish maintained the male sex and produced functional sperm after cessation of drug treatment. Notably, artificially sex-reversed males' fecundity did not differ significantly from wild-type males. Quantitative PCR analysis revealed that the expression of cyp19a1a decreased, whereas the mRNA levels of estrogen receptors esr1, esr2a, and esr2b increased significantly during AI treatment. Moreover, the expression of foxl2 was downregulated, whereas dmrt1, amh, and sox9 were upregulated in the gonads of fish administered AI. This study revealed that endogenous estrogen synthesis plays a key role in the sex change of rice field eels, with ovaries undergoing a reversal to testes in response to estrogen insufficiency. Such artificially induced sex conversion will be of considerable value in breeding rice field eels.

Sertoli, Leydig, and Spermatogonial Cells' Specific Gene and Protein Expressions as Dog Testes Evolve from Immature into Mature States.

Simple SummarySertoli-, Leydig-, and spermatogonial-cells proliferate and differentiate from birth to puberty and then stay stable in adulthood. We hypothesized that expressions of spermatogenesis-associated genes are not enhanced with the increase of these cells’ numbers. To accomplish this postulation, we investigated the abundances of Sertoli cell-specific FSHR and AMH, Leydig cell-specific LHR and INSL3, and spermatogonia-specific THY1 and CDH1 markers in immature and mature canine testis. Four biological replicates of immature and mature testes were processed, and RT-PCR was performed to elucidate cells’ specific markers. Results showed that the gene expressions of all the studied cells’ specific markers were downregulated in adult testis compared with immature testis. Western blot and immunohistochemistry showed the presence of these proteins in the testis. Protein expressions of these markers were greater in immature compared with mature testis. The results support the postulation that the gene expressions do not directly correlate with the increase of the cell numbers during post-natal development but changes in gene expressions show functional significance.Sertoli, Leydig, and spermatogonial cells proliferate and differentiate from birth to puberty and then stay stable in adulthood. We hypothesized that expressions of spermatogenesis-associated genes are not enhanced with a mere increase of these cells’ numbers. To accept this postulation, we investigated the abundances of Sertoli cell-specific FSHR and AMH, Leydig cell-specific LHR and INSL3, and spermatogonia-specific THY1 and CDH1 markers in immature and mature canine testis. Four biological replicates of immature and mature testes were processed, and RT-PCR was performed to elucidate the cells’ specific markers. The data were analyzed by ANOVA, using the 2−∆∆Ct method to ascertain differences in mRNA expressions. In addition, Western blot and IHC were performed. Gene expressions of all the studied cells’ specific markers were down-regulated (p < 0.05) in adult testis compared with immature testis. Western blot and immunohistochemistry showed the presence of these proteins in the testis. Protein expressions were greater in immature testis compared with mature testis (p < 0.05). Despite the obvious expansion of these cells’ numbers from immature to adult testis, the cells’ specific markers were not enriched in mature testis compared with immature dog testis. The results support the postulation that the gene expressions do not directly correlate with the increase of the cell numbers during post-natal development but changes in gene expressions show functional significance.

Characterization, expression and CpG methylation analysis of Dmrt1 and its response to steroid hormone in blotched snakehead (Channa maculata).

Dmrt1 is an important transcriptional regulator that plays critical role in male gonadogenesis, testicular differentiation and development. In this study, Dmrt1 was cloned from blotched snakehead (Channa maculata), which is designated as CmDmrt1. CmDmrt1 encoded a putative protein with 293 amino acids and presented an extremely conserved DM domain. It was nearly expressed in the gonads, and the expression was more than 15 times higher in the testis than in the ovary. 1851bp promoter sequence of CmDmrt1 was characterized and the methylation levels of the CpG sites were analyzed to detect sex-related differences. A significant negative correlation between CmDmrt1 expression and CpG methylation level of its promoter was found in the testis and ovary. During gonadal development, CmDmrt1 transcription displayed strong male-biased expression patterns, increased with the maturation of testis and reached the peak at 195days after hatching (dah), which indicates a significant role of Dmrt1 in spermatogenesis. Steroid treatment could influence CmDmrt1 expression, and long-term 17β-estradiol (E2) treatment could induce the male-to-female secondary sex reversal (SSR), which resulted in the differentiated testis transformed to ovary or ovotestis. Meanwhile, CmDmrt1 expression was down-regulated to fairly low level in the ovary of the SSR XY fish, which was similar to that in normal XX females ovary. Our research illustrates that Dmrt1 is linked to testis differentiation and spermatogenesis in blotched snakehead, providing information for functional studies on sex differentiation and gonadal development of C. maculata, and scientific basis for the production practice of all-male snakehead breeding.