Dynamic Expression of the Homeobox Factor PBX1 during Mouse Testis Development

Members of the pre-B-cell leukemia transcription factor (PBX) family of homeoproteins are mainly known for their involvement in hematopoietic cell differentiation and in the development of leukemia. The four PBX proteins, PBX1, PBX2, PBX3 and PBX4, belong to the three amino acid loop extension (TALE) superfamily of homeoproteins which are important transcriptional cofactors in several developmental processes involving homeobox (HOX) factors. Mutations in the human PBX1 gene are responsible for cases of gonadal dysgenesis with absence of male sex differentiation while Pbx1 inactivation in the mouse causes a failure in Leydig cell differentiation and function. However, no data is available regarding the expression profile of this transcription factor in the testis. To fill this knowledge gap, we have characterized PBX1 expression during mouse testicular development. Real time PCRs and Western blots confirmed the presence Pbx1 mRNA and PBX1 protein in different Leydig and Sertoli cell lines. The cellular localization of the PBX1 protein was determined by immunohistochemistry and immunofluorescence on mouse testis sections at different embryonic and postnatal developmental stages. PBX1 was detected in interstitial cells and in peritubular myoid cells from embryonic life until puberty. Most interstitial cells expressing PBX1 do not express the Leydig cell marker CYP17A1, indicating that they are not differentiated and steroidogenically active Leydig cells. In adults, PBX1 was mainly detected in Sertoli cells. The presence of PBX1 in different somatic cell populations during testicular development further supports a direct role for this transcription factor in testis cell differentiation and in male reproductive function.

ZDHHC19 localizes to the cell membrane of spermatids and is involved in spermatogenesis

Sperm is the ultimate executor of male reproductive function. Normal morphology, quantity, and motility of sperm ensure the normal reproductive process. Palmitoylation is a posttranslational modification mediated by palmitoyltransferases whereby palmitoyl is added to proteins. Seven palmitoyltransferases have been identified in Saccharomyces cerevisiae and 23 in humans (including ZDHHC1–9 and ZDHHC11–24), with corresponding homologs in mice. We identified two testis-specific palmitoyltransferases ZDHHC11 and ZDHHC19 in mice. The Zdhhc11 and Zdhhc19-knockout mouse models were constructed, and it was found that the Zdhhc11 knockout males were fertile, while Zdhhc19 knockout males were sterile. ZDHHC19 is located in the cell membrane of step 4–9 spermatids in the mouse testis, and phenotypic analysis showed that the testicular weight ratio in the Zdhhc19−/− mice decreased along with the number and motility of the sperm decreased, while sperm abnormalities increased, mainly due to the “folded” abnormal sperm caused by sperm membrane fusion, suggesting the involvement of ZDHHC19 in maintaining membrane stability in the male reproductive system. In addition, Zdhhc19−/− mice showed abnormal sperm morphologies and apoptosis during spermatogenesis, suggesting that spermatogenesis in the Zdhhc19−/− mice was abnormal. These results indicate that ZDHHC19 promotes membrane stability in male germ cells. Summary sentence: ZDHHC19 is located in the cell membrane of Step4–9 spermatids in mouse testis; Zdhhc19 knockout mice showed male infertility, abnormal spermatogenesis, sperm morphology and motility.

Microinjection of antisense oligonucleotides into living mouse testis enables lncRNA function study

Background Long non-coding RNAs (lncRNAs) have been the focus of ongoing research in a diversity of cellular processes. LncRNAs are abundant in mammalian testis, but their biological function remains poorly known. Results Here, we established an antisense oligonucleotides (ASOs)-based targeting approach that can efficiently knock down lncRNA in living mouse testis. We cloned the full-length transcript of lncRNA Tsx (testis-specific X-linked) and defined its testicular localization pattern. Microinjection of ASOs through seminiferous tubules in vivo significantly lowered the Tsx levels in both nucleus and cytoplasm. This effect lasted no less than 10 days, conducive to the generation and maintenance of phenotype. Importantly, ASOs performed better in depleting the nuclear Tsx and sustained longer effect than small interfering RNAs (siRNAs). In addition to the observation of an elevated number of apoptotic germ cells upon ASOs injection, which recapitulates the documented description of Tsx knockout, we also found a specific loss of meiotic spermatocytes despite overall no impact on meiosis and male fertility. Conclusions Our study detailed the characterization of Tsx and illustrates ASOs as an advantageous tool to functionally interrogate lncRNAs in spermatogenesis.

Depletion of HMGB2 causes seminiferous tubule atrophy via aberrant expression of androgen and estrogen receptors in mouse testis

High-mobility group box 2 (HMGB2), a chromatin-associated protein that interacts with DNA, is implicated in multiple biological processes, including gene transcription, replication, and repair. HMGB2 is expressed in several tissues, including the testis; however, its functional role is largely unknown. Here, we elucidated the role of HMGB2 in spermatogenesis using HMGB2 knock-out (KO) mice. Paraffin-embedded testicular tissues were obtained from 8-week-old and 1-year-old wild-type and KO mice. Testis weight and number of seminiferous tubules were decreased, whereas atrophic tubules were increased in HMGB2-depleted mice. Immunohistochemistry revealed that atrophic tubules contained Sertoli cells, but not germ cells. Moreover, decreased cell proliferation and increased apoptosis were demonstrated in HMGB2-depleted mouse testis. To elucidate the cause of tubule atrophy, we examined the expression of androgen and estrogen receptors (AR, ERs, respectively), and the results indicated aberrant expression of AR and ERα in Sertoli and Leydig cells. Southwestern histochemistry detected decreased estrogen response element–binding sites in HMGB2-depleted mouse testis. Expression of HMGB1, which has highly similar structure and function as HMGB2, was examined by immunohistochemistry and western blotting, which indicated increased expression in aged HMGB2 KO mouse testis, especially in spermatocytes. These findings indicate a compensatory increase in HMGB1 expression in HMGB2 KO mouse testis. In summary, depletion of HMGB2 induced aberrant expression of AR and ERα, leading to decreased germ cell proliferation and increased apoptosis that resulted in focal seminiferous tubule atrophy.

DAPL1 is a novel regulator of testosterone production in Leydig cells of mouse testis

Leydig cells in the testes produce testosterone in the presence of gonadotropins. Therefore, male testosterone levels must oscillate within a healthy spectrum, given that elevated testosterone levels augment the risk of cardiovascular disorders. We observed that the expression of death-associated protein-like 1 (DAPL1), which is involved in the early stages of epithelial differentiation and apoptosis, is considerably higher in the testes of sexually mature mice than in other tissues. Accordingly, Dapl1-null mice were constructed to evaluate this variation. Notably, in these mice, the testicular levels of steroidogenic acute regulatory protein (StAR) and serum testosterone levels were significantly elevated on postnatal day 49. The findings were confirmed in vitro using I-10 mouse testis-derived tumor cells. The in vivo and in vitro data revealed the DAPL1-regulated the expression of StAR involving altered transcription of critical proteins in the protein kinase A and CREB/CREM pathways in Leydig cells. The collective findings implicate DAPL1 as an important factor for steroidogenesis regulation, and DAPL1 deregulation may be related to high endogenous levels of testosterone.

Y chromosomal noncoding RNAs regulate autosomal gene expression via piRNAs in mouse testis

Background Deciphering the functions of Y chromosome in mammals has been slow owing to the presence of repeats. Some of these repeats transcribe coding RNAs, the roles of which have been studied. Functions of the noncoding transcripts from Y chromosomal repeats however, remain unclear. While a majority of the genes expressed during spermatogenesis are autosomal, mice with different deletions of the long arm of the Y chromosome (Yq) were previously also shown to be characterized by subfertility, sterility and sperm abnormalities, suggesting the presence of effectors of spermatogenesis at this location. Here we report a set of novel noncoding RNAs from mouse Yq and explore their connection to some of the autosomal genes expressed in testis. Results We describe a set of novel mouse male-specific Y long arm (MSYq)-derived long noncoding (lnc) transcripts, named Pirmy and Pirmy-like RNAs. Pirmy shows a large number of splice variants in testis. We also identified Pirmy-like RNAs present in multiple copies at different loci on mouse Y chromosome. Further, we identified eight differentially expressed autosome-encoded sperm proteins in a mutant mouse strain, XYRIIIqdel (2/3 Yq-deleted). Pirmy and Pirmy-like RNAs have homology to 5′/3′UTRs of these deregulated autosomal genes. Several lines of experiments show that these short homologous stretches correspond to piRNAs. Thus, Pirmy and Pirmy-like RNAs act as templates for several piRNAs. In vitro functional assays reveal putative roles for these piRNAs in regulating autosomal genes. Conclusions Our study elucidates a set of autosomal genes that are potentially regulated by MSYq-derived piRNAs in mouse testis. Sperm phenotypes from the Yq-deleted mice seem to be similar to that reported in inter-specific male-sterile hybrids. Taken together, this study provides novel insights into possible role of MSYq-derived ncRNAs in male sterility and speciation.

Zinc Transporter ZIP12 Maintains Zinc Homeostasis and Protects Spermatogonia From Oxidative Stress During Spermatogenesis

Background: Overwhelming evidences now suggest oxidative stress is a major cause of sperm dysfunction and male infertility. Zinc is an important non-enzyme antioxidant with a wide range of biological functions and plays a significant role in preserving male fertility. Notably, zinc trafficking through the cellular and intracellular membrane is endorsed by precise families of zinc transporters, i.e. SLC39s/ZIPs and SLC30s/ZnTs. However, the expression and function of zinc transporters in the male germ cells were rarely reported. The aim of this study is to determine the crucial zinc transporter responsible for the maintenance of spermatogenesis.Methods: In the present study, we investigated the expression of all fourteen ZIP members in mouse testis and further analyzed the characteristic of ZIP12 expression in testis and spermatozoa by qRT-PCR, immunoblot and immunohistochemistry analyses. To explore the antioxidant role of ZIP12 in spermatogenesis, an obese mouse model fed with high-fat-diet was employed to confirm the correlation between ZIP12 expression level and sperm quality. Furthermore, ZIP12 expression in response to oxidative stress in a spermatogonia cell line, C18-4 cells, was determined and its function involved in regulating cell viability and apoptosis was investigated by RNAi experiment. Results: We initially found that ZIP12 expression in mouse testis was significantly high compared to other members of ZIPs and its mRNA and protein were intensively expressed in testis rather than the other tissues. Importantly, ZIP12 was intensively abundant in spermatogonia and spermatozoa, both in mice and humans. Moreover, ZIP12 expression in testis significantly decreased in obese mice, which associated with reduced sperm zinc content, excessive sperm ROS, poor sperm quality and male subfertility. Similarly, its expression in C18-4 cells significantly declined in response to oxidative stress. Additionally, reduced ZIP12 expression by RNAi associated with a decline in zinc level subsequently caused low cell viability and high cell apoptosis in C18-4 cells. Conclusions: The zinc transporter ZIP12 is intensively expressed in testis, especially in spermatogonia and spermatozoa. ZIP12 may play a key role in maintaining intracellular zinc level in spermatogonia and spermatozoa, by which it resists oxidative stress during spermatogenesis and therefore preserves male fertility.