Adult Mouse Testis Research Articles

Stage-specific expression of Toll-like receptors in the seminiferous epithelium of mouse testis

Genes encoding Toll-like receptors (TLRs) are expressed by germ cells in the mouse testis. Nevertheless, the expression of TLRs by germ cells has only been demonstrated for TLR-3, TLR-9, and TLR-11. Furthermore, the expression of each TLR in relation to the stage of spermatogenesis remains uncertain. We aimed in the present study to examine the expression pattern of all TLRs in germ cells throughout the cycle of seminiferous epithelium in the adult mouse testis. Immunohistochemistry was used to evaluate the expression of TLRs. Results of the present study reveal the expression of TLRs by specific populations of germ cells. Expression of TLRs, except for TLR-7, at endosomal compartments, acrosomes, and/or residual bodies was another interesting and novel finding of the present study. We further demonstrate that the expression of TLR-1, -2, -3, -4, -5, -7, -11, -12, and -13 follows a distinct spatiotemporal pattern throughout the cycle of seminiferous epithelium. While TLR-1, -3, -5, -11, and -12 are expressed in all stages, TLR-4 is expressed only in early and middle stages of spermatogenic cycle. On the other hand, TLR-2, -7, and -13 are expressed only in early stage of spermatogenic cycle. Evidence demonstrating the expression of TLRs in a stage specific manner throughout spermatogenesis strengthen the hypothesis that the expression of various TLRs by germ cells is a developmentally regulated process. However, if TLRs play a role in the regulation of proliferation, growth, maturation, and differentiation of germ cells throughout the cycle of the seminiferous epithelium warrants further investigations.

Retinoic Acid Regulates Spermiogenesis Via Hoxb1 and Shh Signaling in Testicular Germ Cells.

Retinoic acid (RA) regulates all four major events in spermatogenesis; spermatogonial differentiation, meiotic entry, spermiogenesis, and spermiation. For the pre-meiotic phase, Sertoli cells are the source of RA and for the post-meiotic phase, pachytene spermatocytes are the source of RA. While the entire spermatogenic process is regulated by RA, how each of these phases is regulated by RA remains completely unknown. Homeobox B1 (Hoxb1) has two retinoic acid response elements (RARE) upstream and downstream of the gene. In this study, we investigated if RA facilitates spermatogenesis by its action on Hoxb1. The expressions of the Hoxb1 and Sonic hedgehog (Shh) genes were analyzed in the post-natal mouse testes and the testicular localizations of Hoxb1, Shh and Gli1 were analyzed by immunohistochemistry in the adult rat testis. To delineate the signaling mechanisms, Hoxb1 expression was altered in vitro and in vivo using retinoic acid and miR-361-3p. Finally, the levels of miR-361-3p and HOXB1 were analyzed in infertile human sperm samples. Hoxb1 and Shh gene expressions were found to be low in the testis of post-natal Swiss mice of 7, 14, 28, 35, and 60days, after which the expressions of both spiked. Immunohistochemistry in the adult mouse testis showed the expressions of Hoxb1, Shh, and Gli1 in the elongating spermatids. Exposure of GC2 cells to RA and in vivoIP RA injection upregulated Hoxb1 andShh signaling in the testiswith increased expressions of Shh, Gli1, and Hdac1. Retinoic acid administration in Swiss mice compromised sperm production and reduced epididymal sperm count. The analysis of infertile human semen samples revealed an increased level of HOXB1 and a decreased level of miR-361-3p as compared to fertile controls. We conclude that retinoic acid regulates late stage of spermatogenesis (spermiogenesis) by affecting Hoxb1 and Shh signaling.

Analysis of platelet-derived growth factor receptor alpha expression in adult mouse testis

Analysis of platelet-derived growth factor receptor alpha expression in adult mouse testis

Adropin promotes testicular functions by modulating redox homeostasis in adult mouse.

Adropin is an emerging metabolic hormone that has a role in regulating energy homeostasis. The present study aimed to explore the impact of adropin on redox homeostasis and its possible role in testicular functions in adult mouse testis. Western blot, flow-cytometry, and TUNEL assay were performed to explore the impact of intra-testicular treatment of adropin (0.5 μg/testis) on testicular functions of adult mice. Hormonal assay was done by ELISA. Further, antioxidant enzyme activities were measured. Adropin treatment significantly increased the sperm count and testicular testosterone by increasing the expression of GPR19 and steroidogenic proteins. Also, adropin treatment reduced the oxidative/nitrosative stress by facilitating the translocation of NRF2 and inhibiting NF-κB into the nucleus of germ cells. Enhanced nuclear translocation of NRF2 leads to elevated biosynthesis of antioxidant enzymes, evident by increased HO-1, SOD, and catalase activity that ultimately resulted into declined LPO levels in adropin-treated mice testes. Furthermore, adropin decreased nuclear translocation of NF-κB in germ cells, that resulted into decreased NO production leading to decreased nitrosative stress. Adropin/GPR19 signaling significantly increased its differentiation, proliferation, and survival of germ cells by elevating the expression of PCNA and declining caspase 3, cleaved caspase 3 expression, Bax/Bcl2 ratio, and TUNEL-positive cells. FACS analysis revealed that adropin treatment enhances overall turnover of testicular cells leading to rise in production of advanced germ cells, notably spermatids. The present study indicated that adropin improves testicular steroidogenesis, spermatogenesis via modulating redox potential and could be a promising target for treating testicular dysfunctions.

Knockdown of type 2 orexin receptor in adult mouse testis potentiates testosterone production and germ cell proliferation

Orexins (OXs) are neuropeptides which regulate various physiological processes. OXs exist in two different forms, mainly orexin A (OXA) and orexin B (OXB) and their effects are mediated via OX1R and OX2R. Presence of OXB and OX2R in mouse testis is also reported. However, the role of OXB/OX2R in the male gonad remains unexplored. Herein we investigated the role of OXB/OX2R system in testicular physiology under in vivo and ex vivo conditions. Adult mice were given a single dose of bilateral intratesticular injection of siRNA targeting OX2R and were sacrificed 96 h post-injection. OX2R-knockdown potentiated serum and intratesticular testosterone levels with up-regulation in the expressions of major steroidogenic proteins. Germ cell proliferation also increased in siRNA-treated mice. Results of the ex vivo experiment also supported the findings of the in vivo study. In conclusion, OX2R may regulate testosterone production and thereby control the fine-tuning between steroidogenesis and germ cell dynamics.

HSD17B1 Compensates for HSD17B3 Deficiency in Fetal Mouse Testis but Not in Adults.

Hydroxysteroid (17β) dehydrogenase (HSD17B) enzymes convert 17-ketosteroids to 17beta-hydroxysteroids, an essential step in testosterone biosynthesis. Human XY individuals with inactivating HSD17B3 mutations are born with female-appearing external genitalia due to testosterone deficiency. However, at puberty their testosterone production reactivates, indicating HSD17B3-independent testosterone synthesis. We have recently shown that Hsd17b3 knockout (3-KO) male mice display a similar endocrine imbalance, with high serum androstenedione and testosterone in adulthood, but milder undermasculinization than humans. Here, we studied whether HSD17B1 is responsible for the remaining HSD17B activity in the 3-KO male mice by generating a Ser134Ala point mutation that disrupted the enzymatic activity of HSD17B1 (1-KO) followed by breeding Hsd17b1/Hsd17b3 double-KO (DKO) mice. In contrast to 3-KO, inactivation of both HSD17B3 and HSD17B1 in mice results in a dramatic drop in testosterone synthesis during the fetal period. This resulted in a female-like anogenital distance at birth, and adult DKO males displayed more severe undermasculinization than 3-KO, including more strongly reduced weight of seminal vesicles, levator ani, epididymis, and testis. However, qualitatively normal spermatogenesis was detected in adult DKO males. Furthermore, similar to 3-KO mice, high serum testosterone was still detected in adult DKO mice, accompanied by upregulation of various steroidogenic enzymes. The data show that HSD17B1 compensates for HSD17B3 deficiency in fetal mouse testis but is not the enzyme responsible for testosterone synthesis in adult mice with inactivated HSD17B3. Therefore, other enzymes are able to convert androstenedione to testosterone in the adult mouse testis and presumably also in the human testis.

Expression profiling of stemness markers in testicular germline stem cells from neonatal and adult Swiss albino mice during their transdifferentiation in vitro

BackgroundSpermatogonial stem cells (SSCs) were considered to be stem cells with limited potencies due to their existence in adult organisms. However, the production of spermatogonial stem cell colonies with broader differentiation capabilities in primary germ cell cultures from mice of select genetic backgrounds (C57BL6/Tg14, ddY, FVB and 129/Ola) indicated that SSCs from these strains were pluripotent.MethodsWe established primary cultures of SSCs from neonatal and adult Swiss 3T3 Albino mice. Stemness of SSC colonies were evaluated by performing real-time PCR and immunofluorescence analysis for a panel of chosen stemness markers. Differentiation potentials of SSCs were examined by attempting the generation of embryoid bodies and evaluating the expression of ectodermal, mesodermal and endodermal markers using immunofluorescence and real-time PCR analysis.ResultsSpermatogonial stem cells from neonatal and mature mice testes colonised in vitro and formed compact spermatogonial stem cell colonies in culture. The presence of stem cell markers ALPL, ITGA6 and CD9 indicated stemness in these colonies. The differentiation potential of these SSC colonies was demonstrated by their transformation into embryoid bodies upon withdrawal of growth factors from the culture medium. SSC colonies and embryoid bodies formed were evaluated using immunofluorescence and real-time PCR analysis. Embryoid body like structures derived from both neonatal and adult mouse testis were quite similar in terms of the expression of germ layer markers.ConclusionThese results strongly suggest that SSC-derived EB-like structures could be used for further differentiation into cells of interest in cell-based therapeutics.

Immunolocalization of apelin receptor (APJ) in mouse seminiferous epithelium.

The apelin receptor (APJ) belongs to the member of the G protein-coupled receptor family, and expression of APJ has been reported in the different cell types of testis. The seminiferous tubules in the testis can be identified as different stages (I-XII). It has been also suggested that different factors could be expressed in stage and cell-specific manner in the seminiferous tubules. Recently, we also shown that expression of APJ is developmentally regulated in the testis from PND1 to PND42. Therefore, we analyzed the expression of APJ in the testis of adult mice by immunohistochemistry. Immunohistochemistry showed that the APJ was highly specific for the round and elongated spermatids with stage-dependent changes. The seminiferous tubules at stages I-VII showed APJ immunostaining in the spermatid steps 1-8, not steps of 13-16. The seminiferous tubules at stages IX-XII showed APJ immunostaining in the spermatid steps 9-12. These results suggested the possible role of APJ in the spermiogenesis process. The intratesticular administration of APJ antagonist, ML221 showed a few round spermatids in the seminiferous tubules and some of the tubules with complete absence of round spermatid. Overall, we present evidence that APJ expression in spermatid is dependent on the stages of the seminiferous epithelium cycle and APJ could be involved in the differentiation of round spermatid to elongated spermatid.

Splicing factor SRSF1 is essential for homing of precursor spermatogonial stem cells in mice.

Spermatogonial stem cells (SSCs) are essential for continuous spermatogenesis and male fertility. The underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Crosslinking immunoprecipitation and sequencing data revealed that spermatogonia-related genes (e.g. Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Specific deletion of Srsf1 in mouse germ cells impairs homing of precursor SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult conditional knockout (cKO) mice, which indicates Sertoli cell-only syndrome in cKO mice. The expression of spermatogonia-related genes (e.g. Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of cKO mice. Moreover, multiomics analysis suggests that SRSF1 may affect survival of spermatogonia by directly binding and regulating Tial1/Tiar expression through AS. In addition, immunoprecipitation mass spectrometry and co-immunoprecipitation data showed that SRSF1 interacts with RNA splicing-related proteins (e.g. SART1, RBM15, and SRSF10). Collectively, our data reveal the critical role of SRSF1 in spermatogonia survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying homing of precursor SSCs.

Evaluation of the Potential Protective Role of Galangin Associated with Gold Nanoparticles in the Histological and Functional Structure of Testes of Adult Male Albino Mice Administrated with Carbon Tetrachloride

Galangin (Gal) is a natural flavonoid sourced from the roots of the plant Albina Kalanga. It possesses a variety of pharmacological activities, such as antioxidant, anticancer, and anti-inflammatory. The current study aims to enhance the efficacy of galangin through the use of gold nanoparticles as a drug delivery system against CCl4-induced toxicity in the testicular tissue of male albino mice. Forty-two albino mice were divided into seven groups (6 mice/group), treatment with carbon tetrachloride solution for two weeks by intraperitoneal injection, (1 ml/kg) once a week for all groups except the control group, after which a group was injected with gold nanoparticles and two groups were injected with galangin in two concentrations, and two groups were injected with AuNPs + Gal conjugation solution with two concentrations. The animals' bodies were weighed and blood samples were obtained for testosterone hormone analysis and testicles for the purpose of weighing and histological study at the end of the experiments. The study showed that the testicular tissue of mice treated with CCl4 had a different pathology compared to the control group, the testosterone hormone levels in the CCl4 group were significantly higher than those of the control group. Results from the AuNPs+Gal group showed a significant reduction in the effects of CCl4 toxicity on mice testicles, with testosterone levels are returning to normal and histological testicle structures improving, the ratio of testicle weights to animal body weight in the group injected with AuNPs was significantly higher than that of the control and CCl4 groups. In conclusion, the results revealed that galangin combined with gold nanoparticles could effectively reduced the histological and functional tissue damages caused by CCl4, presenting a promising natural solution that needs to be further developed

Isolation of Mouse Germ Cells by FACS Using Hoechst 33342 and SYTO16 Double Staining.

In the adult mouse testis, germ cells of various developmental cell states co-exist. FACS isolation of cells stained with the DNA dye Hoechst 33342 has been used for many years to sub-divide these cells based on their DNA content. This approach provides an efficient way to obtain broad categories of male germ cells: pre-meiotic spermatogonia, meiotic spermatocytes and post-meiotic spermatids. The addition of a red filter for Hoechst staining enables further sub-division of spermatocytes depending on sub-stages of meiotic prophase. However, separation of different stage spermatids using Hoechst staining alone is not possible. We recently reported a methodology, combining Hoechst staining with a second DNA dye (SYTO16) that enables the further separation of these cells into three sub-populations: round, early elongating, and late elongating spermatids (Gill et al., Cytometry A 101:529-536, 2022). This method makes it possible to obtain rapidly and simply pure fractions of male germ cells from multiple developental stages from the same animal.

Photobiomodulation Improves Histological Parameters of Testis and Spermatogenesis in Adult Mice Exposed to Scrotal Hyperthermia in the Prepubertal Phase.

Introduction: Heat stress is one of the environmental causes of damage to the testis, whose effects are less known before puberty. The aim of the present study was to investigate the impact of photobiomodulation (PBM) on the testis of prepubertal mice subjected to hyperthermia. Methods: Twenty-four three-week-old prepubertal male mice were allocated to the following groups: I) control, II) scrotal hyperthermia (Hyp), and III) Hyp+PBM (n=8/each group). In order to induce hyperthermia, the scrotum was placed in water at 43 °C for 20 minutes every other day for a total duration of 10 days. In the Hyp+PBM group, after hyperthermia induction, the testis of the mice was subjected to laser irradiation at a wavelength of 890 nm (0.03 J/cm2 for 30 seconds) for 35 days. After the mice were sacrificed, the testis and epididymis were removed for testing. Results: Compared with those of the Hyp group, the sperm parameters of the laser irradiation group improved notably. In addition, histological examinations revealed that the final number of testis cells and the volume of tissue in the Hyp+PBM group were dramatically greater than those in the Hyp group. The analysis of molecular data revealed an increase in the expression of mitotic genes and testosterone levels and a decrease in the formation of reactive oxygen species (ROS) and the expression of the apoptotic gene in the testis subjected to PBM. Conclusion: Based on the present findings, laser therapy can reduce complications caused by scrotal hyperthermia during prepuberty and ameliorate spermatogenesis during puberty.

De novo transcriptome assembly of mouse male germ cells reveals novel genes, stage-specific bidirectional promoter activity, and noncoding RNA expression.

In mammals, the adult testis is the tissue with the highest diversity in gene expression. Much of that diversity is attributed to germ cells, primarily meiotic spermatocytes and postmeiotic haploid spermatids. Exploiting a newly developed cell purification method, we profiled the transcriptomes of such postmitotic germ cells of mice. We used a de novo transcriptome assembly approach and identified thousands of novel expressed transcripts characterized by features distinct from those of known genes. Novel loci tend to be short in length, monoexonic, and lowly expressed. Most novel genes have arisen recently in evolutionary time and possess low coding potential. Nonetheless, we identify several novel protein-coding genes harboring open reading frames that encode proteins containing matches to conserved protein domains. Analysis of mass-spectrometry data from adult mouse testes confirms protein production from several of these novel genes. We also examine overlap between transcripts and repetitive elements. We find that although distinct families of repeats are expressed with differing temporal dynamics during spermatogenesis, we do not observe a general mode of regulation wherein repeats drive expression of nonrepetitive sequences in a cell type-specific manner. Finally, we observe many fairly long antisense transcripts originating from canonical gene promoters, pointing to pervasive bidirectional promoter activity during spermatogenesis that is distinct and more frequent compared with somatic cells.

PFOS-elicited metabolic perturbation in liver and fatty acid metabolites in testis of adult mice.

Multiple factors can contribute to sub-fecundity, including genetics, lifestyle, and environmental contaminants. PFASs are characterized as "forever chemicals" due to their ubiquitous contamination and their persistence in the environment, wildlife, and humans. Numerous studies have demonstrated that PFAS exposure adversely affects multiple bodily functions, including liver metabolism and gonadal function. It is unclear, however, how the disruption of hepatic fatty acid metabolism affects testicular function. In this study, male mice were administered 0.3 and 3 μg/g body weight of PFOS for 21 days. Our data showed that PFOS exposure caused hepatic steatosis, as evidenced by significant increases in triglyceride levels, expression of ATP-citrate lyase, and fatty acid synthase, as well as fasting insulin levels. PFOS perturbed the expression levels of hepatokines, of which fibroblast growth factor-21 (Fgf-21), leukocyte cell-derived chemotaxin-2 (Lect-2), and retinol-binding protein-4 (Rbp-4) were significantly reduced, whereas angiopoietin-like 4 (Angptl4) was noticeably increased. While Rbp-4 and Fgf-21 are known to contribute to spermatogenesis and testosterone synthesis. In PFOS-exposed groups, testicular ATP, and testosterone decreased significantly with a significant increase in the expression of peroxisome proliferator-activated receptor-coactivator 1α. Mass spectrophotometry imaging revealed the localization of PFOS in testes, along with significant increases in fatty acid metabolites. These included arachidonic acid, dihomo-α-linolenic acid, dihomo-γ-linolenic acid, oxidized ceramide, diacylglycerol, phosphatidylcholine, and phosphatidylethanolamine, which are associated with inflammation and post-testicular causes of infertility. This study revealed potential links between PFOS-elicited changes in hepatic metabolism and their impacts on testicular biology. This study provides insights into alternative targets elicited by PFOS that can be used to develop diagnostic and therapeutic strategies for improving testicular dysfunction.

MiR-124-3p regulates the involvement of Ptpn1 in testicular development and spermatogenesis in mouse

Testicular development and spermatogenesis in mouse are a complex process in which phosphorylation modifications and regulation of genes by non-coding RNAs play an important role. However, protein tyrosine phosphatase, non-receptor type 1 (Ptpn1) is widely expressed in mammalian tissues. In this study, we analyzed the expression of Ptpn1 mRNA and its encoded proteins in testicular tissues of juvenile and adult mice by using experimental techniques such as biological information, real-time fluorescence quantitative PCR (RT-qPCR), western blot (WB), immunofluorescence (IF) and transfection, and further analyzed the possible target-regulatory relationship and regulatory mechanisms of miR-124-3p and Ptpn1. We found that Ptpn1 mRNA and its encoded protein were up-regulated in adult mouse testis compared to juvenile mouse testis. The expression trend of miR-124-3p was opposite to that of Ptpn1. In other cell types, Ptpn1 protein is localized in cell membrane, cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. Immunofluorescence showed that Ptpn1 protein was mainly localized in the cytoplasm of male germ cells and was expressed at a high level in early-stage cells (spermatogonia) and at a low level in late-stage cells (sperm). Transfection results showed that the expression levels of Ptpn1 mRNA and its protein were significantly down-regulated after miR-124-3p overexpression in mouse spermatogonia. Bioinformatics analysis showed that Ptpn1 can involved in biological processes such as protein kinase inactivation through peptidyl tyrosine dephosphorylation. The reduction of miR-124-3p may be a key factor in promoting the high expression of Ptpn1 in testicular tissues of adult mice. Increased miR-124-3p may be a key factor in suppressing Ptpn1 expression in the mouse spermatogonia mimics group. The differential expression results from the negative regulation of miR-124-3p.

Efficient isolation and purification of spermatogonia, spermatocytes, and spermatids from mice, piglets, and adult boars using an optimized STA-PUT method

Spermatogenesis is a delicate and complex biological process in which spermatogonial stem cells continue to proliferate and differentiate into mature spermatozoa, maintaining sperm production in male mammals throughout the lifetime. To study the molecular mechanism of spermatogenesis, researchers had to isolate different germ cell subpopulations for in vitro culture and characterization. However, due to the existence of several stages of germ cells and a variety of populations of somatic cells in the testis of male mammals, it is a challenge for us to obtain high-purity germ cell subpopulations for further research. Here, we optimized the STA-PUT device and successfully applied it to isolate and purify spermatogonia populations in piglets, and multiple germ cell populations at different developmental stages in testes of adult mice and boars. This work provides a simple platform for germ cell fractionation to facilitate the molecular mechanistic study of animal spermatogenesis in vitro.

The Effects of Pifithrin-µ on Spermatogonial Stem Cell Viability and Pluripotency

Introduction: Spermatogonial stem cells (SSCs) offer remarkable competencies for animal reproduction and overcoming human disease as a result of their differentiation capability. We evaluated the effect of small molecule pifithrin-mu (PFT-µ), a well-known inhibitor of P53 on SSC biological processes such as viability, apoptosis, and gene expression pattern. Methods: The SSCs were isolated from the testes of adult NMRI mice and then cultured in DMEM/F12 medium containing 10% FBS. Then, they were characterized by the immunocytochemistry technique by high PLZF and low c-Kit expressions. SSC colony formation assay was carried out and their viability was estimated by methylthiazolyldiphenyl-tetrazolium bromide (MTT, or 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay upon exposure to PFT-µ (0, 0.6, 1.2, 2.5, and 5 µm). The apoptosis percentages were also measured using FACS analysis, and finally, Oct4 and Stra8 expression at mRNA levels was assessed using real-time quantitative PCR. Results: The 0.6 and 1.2 µm PFT-µ improved the viability of SSC based on MTT assay results; however, 2.5 and 5 µm PFT-µ reduced SSC viability compared with the control group. Moreover, PFT-µ at lower concentrations enhanced the colony size of SSCs and diminished their apoptosis. As well, exposure to PFT-µ upregulated Oct4 expression while downregulating the meiotic entry marker, Stra8. Conclusion: Based on findings, optimized concentrations of PFT-µ can decrease SSC apoptosis, and conversely potentiate their pluripotency and self-renewal capacities in vitro.

Mouse Pxt1 expression is regulated by Mir6996 miRNA

Mouse Pxt1 gene is expressed exclusively in male germ cells and encodes for a small, cell death inducing protein. However, upon PXT1 interaction with BAG6, cell death is prevented. In transiently transfected cell lines the PXT1 expression triggered massive cell death, thus we ask the question whether the interaction of PXT1 and BAG6 is the only mechanism preventing normal, developing male germ cells from being killed by PXT1. The Pxt1 gene contains a long 3′UTR thus we have hypothesized that Pxt1 can be regulated by miRNA. We have applied Pxt1 knockout and used Pxt1 transgenic mice that overexpressed this gene to shed more light on Pxt1 regulation. Using the ELISA assay we have demonstrated that PXT1 protein is expressed in adult mouse testis, though at low abundance. The application of dual-Glo luciferase assay and the 3′UTR cloned into p-MIR-Glo plasmid showed that Pxt1 is regulated by miRNA. Combining the use of mirDB and the site-directed mutagenesis further demonstrated that Pxt1 translation is suppressed by Mir6996-3p. Considering previous reports and our current results we propose a model for Pxt1 regulation in the mouse male germ cells.

Cycas pectinata stimulates germ cell proliferation in mouse testes

Cycas pectinata has been used traditionally by indigenous tribes in the North Eastern region of India. The seed of Cycas pectinata are eaten by local communities as a source of starch and microsporophylls to improve male sexual potency. However, there is no scientific evidence on its testicular physiology. Thus, present study examined the effect of Cycas pectinata seed extract (CPE) on testicular activity in the testis of adult mice. The methanolic extract of Cycas pectinata seed (CPE) was prepared and its composition has already been reported earlier. CPE was orally given in two doses 100 and 400 mg/kg for 35 days and male reproductive endpoints were monitored. CPE significantly increased sperm parameters, antioxidant status and germ cell proliferation compared to the control. CPE decreased the expression of NF-κB and BCl2 and increased the abundance of active caspase-3. Expression of ERα and β were down-regulated and expression of AR was elevated in the CPE-treated mice testis. Therefore, CPE improves sperm concentration and germ cell proliferation by enhancing the antioxidant status of the testis. CPE might also be involved in the removal of damaged or unhealthy germ cells and the overall effect of CPE could be mediated via AR signalling.

Regulation of Cdh2 by the AP-1 family transcription factor Junb in TM4 Sertoli cells

Cadherins are transmembrane proteins that mediate cell-to-cell adhesion and various cellular processes. In Sertoli cells of the testis, Cdh2 contributes to the development of the testis and the formation of the blood-testis barrier, being essential for germ cells' protection. Analyses of chromatin accessibility and epigenetic marks in adult mouse testis have shown that the region from −800 to +900 bp respective to Cdh2 transcription start site (TSS) is likely the active regulatory region of this gene. In addition, the JASPAR 2022 matrix has predicted an AP-1 binding element at about −600 bp. Transcription factors of the activator protein 1 (AP-1) family have been implicated in the regulation of the expression of genes encoding cell-to-cell interaction proteins such as Gja1, Nectin2 and Cdh3. To test the potential regulation of Cdh2 by members of the AP-1 family, siRNAs were transfected into TM4 Sertoli cells. The knockdown of Junb led to a decrease in Cdh2 expression. ChIP-qPCR and luciferase reporter assays with site-directed mutagenesis confirmed the recruitment of Junb to several AP-1 regulatory elements in the proximal region of the Cdh2 promoter in TM4 cells. Further investigation with luciferase reporter assays showed that other AP-1 members can also activate the Cdh2 promoter albeit to a lesser extent than Junb. Taken together, these data suggest that in TM4 Sertoli cells, Junb is responsible for the regulation of Cdh2 expression which requires its recruitment to the proximal region of the Cdh2 promoter.