Crlz-1 Is Prominently Expressed in Spermatogonia and Sertoli Cells during Early Testis Development and in Spermatids during Late Spermatogenesis

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.     

Asynchronous expression of the homeodomain protein CUX1 in Sertoli cells and spermatids during spermatogenesis in mice

The homeodomain CUX1 protein exists as multiple isoforms that arise from proteolytic processing of a 200-kDa protein or an alternate splicing or from the use of an alternate promoter. The 200-kDa CUX1 protein is highly expressed in the developing kidney, where it functions to regulate cell proliferation. Transgenic mice ectopically expressing the 200-kDa CUX1 protein develop renal hyperplasia associated with reduced expression of the cyclin kinase inhibitor p27. A 55-kDa CUX1 isoform is expressed exclusively in the testes. We determined the pattern and timing of CUX1 protein expression in developing testes. CUX1 expression was continuous in Sertoli cells from prepubertal testes but became cyclic when spermatids appeared. In testes from mature mice, CUX1 was highly expressed only in round spermatids at stages IV-V of spermatogenesis, in both spermatids and Sertoli cells at stages VI-X of spermatogenesis, and only in Sertoli cells at stage XI of spermatogenesis. While most of the seminiferous tubules in wild-type mice were between stages VI and X of spermatogenesis, there was a significant reduction in the percentage of seminiferous tubules between stages VI and X in Cux1 transgenic mice and a significant increase in the percentage of seminiferous tubules in stages IV-V and XI. Moreover, CUX1 was not expressed in proliferating cells in testes from either wild-type or transgenic mice. Thus, unlike the somatic form of CUX1, which has a role in cell proliferation, the testis-specific form of CUX1 is not involved in cell division and appears to play a role in signaling between Sertoli cells and spermatids.     

Quantitative Analysis of the Cellular Composition in Seminiferous Tubules in Normal and Genetically Modified Infertile Mice

The aim of this study was to establish a quantitative standard for the cellular composition in seminiferous tubules at each stage of spermatogenesis in the mouse testis, and thereby evaluate abnormalities in the infertile mouse testis. We applied a combination of lectin histochemistry for acrosomes and immunohistochemistry for various specific cell markers, both of which were visualized with fluorescence, on paraffin sections of the testis. We first examined seminiferous tubules from normal mice and counted the number of each cell type at each stage of spermatogenesis. We then examined seminiferous tubules from genetically modified mice deficient (-/-) for one of the cell adhesion molecules, nectin-2 or nectin-3, and compared the number of each cell type at each stage of spermatogenesis with the corresponding value in normal mice. In both nectin-2^-/- and nectin-3^-/- mice, which are infertile despite the apparently normal morphology of the seminiferous epithelia, we measured a progressive loss in the later-step spermatids, with significantly lower numbers of step 11–16 spermatids in nectin-3^-/- mice and step 15–16 spermatids in nectin-2^-/- mice as compared with that in normal control mice. The present study demonstrated that a quantitative analysis of cellular compositions at different stages in seminiferous tubules was useful for evaluating abnormalities in spermatogenesis.     

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

Background

The C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1 ^-/- mice using a Cre/loxP system.

Results

Both male and female Ehd1 ^-/- mice survived at sub-Mendelian ratios. A proportion of Ehd1 ^-/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1 ^-/- adult males were infertile and displayed decreased testis size, whereas Ehd1 ^-/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1 ^-/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1 ^-/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1 ^-/- mice.

Conclusions

Contrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.     

Correlation of appearance of metastasis-associated protein1 (Mta1) with spermatogenesis in developing mouse testis

Mta1, a representative of the MTA gene family, is believed to be involved in the metastasis of malignant tumors. However, a systematic study of its physiological function has not been performed. It has been found in normal mouse organs at relatively low levels, except for in testis, suggesting a potential function in the male reproductive system. In order to explore the role of Mta1 protein during spermatogenesis, its expression in adult mouse testis was compared with that in developing mouse testis and in testis from adult mice treated with methoxyacetic acid, which selectively depletes primary spermatocytes. Quantitative analysis revealed that Mta1 protein gradually increased in the testis from 14 days postnatally. Immunolocalization analysis demonstrated strong signals in the seminiferous tubules, and Mta1 was predominantly present in the nucleus of primary spermatocytes and spermatogonia from 14 days postnatally. The most intensive staining was located in the nucleus of pachytene spermatocytes in mature testes. The expression pattern of Mta1 during spermatogenesis was also shown to be stage-specific by immunohistochemistry analysis. Finally, dramatic loss of Mta1 expression from pachytene spermatocytes was observed in the spermatogenic-arrested adult mouse testis. These results collectively demonstrate that Mta1 appears during postnatal testis development and suggest that this expression may be crucial for spermatogenesis. This study was supported by the Natural Science Foundation of China (2006: 30570982; 2003: 30370750; 2003: 30371584).     

Expression and function of the testis-predominant protein LYAR in mice

Mammalian spermatogenesis is a complex process involving an intrinsic genetic program of germ cell-specific and -predominant genes. In the present study, we analyzed the Ly-1 reactive clone (Lyar) gene in the mouse. Lyar, which is known to be expressed abundantly in the testis, encodes a nucleolar protein that contains a LYAR-type C2HC zinc finger motif and three nuclear localization signals. We herein confirmed that Lyar is expressed predominantly in the testis, and further showed that this expression is specific to germ cells. Protein analyses with an anti-LYAR antibody demonstrated that the LYAR protein is present in spermatocytes and spermatids, but not in sperm. To assess the functional role of LYAR in vivo, we used a genetrap mutagenesis approach to establish a LYAR-null mouse model. Lyar mutant mice were born live and developed normally. Male mutant mice lacking LYAR were fully fertile and showed intact spermatogenesis. Taken together, our results demonstrate that LYAR is strongly preferred in male germ cells, but has a dispensable role in spermatogenesis and fertility.     

Lipocalin-2 (LCN2) regulates PLIN5 expression and intracellular lipid droplet formation in the liver

Lipocalin-2 (LCN2) belongs to the superfamily of lipocalins and plays critical roles in the control of cellular homeostasis during inflammation and in responses to cellular stress or injury. In the liver, LCN2 triggers protective effects following acute or chronic injury, and its expression is a reliable indicator of liver damage. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis. In this study, we fed wild type (WT) and LCN2-deficient (Lcn2^−/−) mice a methionine- and choline-deficient (MCD) diet as a nutritional model of non-alcoholic steatohepatitis, and compared intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins that regulate cellular lipid metabolism. We found that Lcn2^−/− mice fed an MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5, also known as OXPAT) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. Restoration of LCN2 in Lcn2^−/− primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. Our data indicate that LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with elevated fat accumulation and steatosis.     

Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.     

Disruption of Chtf18 Causes Defective Meiotic Recombination in Male Mice

CHTF18 (chromosome transmission fidelity factor 18) is an evolutionarily conserved subunit of the Replication Factor C-like complex, CTF18-RLC. CHTF18 is necessary for the faithful passage of chromosomes from one daughter cell to the next during mitosis in yeast, and it is crucial for germline development in the fruitfly. Previously, we showed that mouse Chtf18 is expressed throughout the germline, suggesting a role for CHTF18 in mammalian gametogenesis. To determine the role of CHTF18 in mammalian germ cell development, we derived mice carrying null and conditional mutations in the Chtf18 gene. Chtf18-null males exhibit 5-fold decreased sperm concentrations compared to wild-type controls, resulting in subfertility. Loss of Chtf18 results in impaired spermatogenesis; spermatogenic cells display abnormal morphology, and the stereotypical arrangement of cells within seminiferous tubules is perturbed. Meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double-strand breaks is delayed and incomplete; both RAD51 and γH2AX persist in prophase I. In addition, MLH1 foci are decreased in pachynema. These findings demonstrate essential roles for CHTF18 in mammalian spermatogenesis and meiosis, and suggest that CHTF18 may function during the double-strand break repair pathway to promote the formation of crossovers.     

Infection of SARS-CoV-2 causes severe pathological changes in mouse testis

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected more than 600 million people worldwide. Several organs including lung, intestine, and brain are infected by SARS-CoV-2. It has been reported that SARS-CoV-2 receptor angiotensin-converting enzyme-2 (ACE2) is expressed in human testis. However, whether testis is also affected by SARS-CoV-2 is still unclear. In this study, we generate a human ACE2 (hACE2) transgenic mouse model in which the expression of hACE2 gene is regulated by hACE2 promoter. Sertoli and Leydig cells from hACE2 transgenic mice can be infected by SARS-CoV-2 pseudovirus in vitro, and severe pathological changes are observed after injecting the SARS-CoV-2 pseudovirus into the seminiferous tubules. Further studies reveal that Sertoli and Leydig cells from hACE2 transgenic mice are also infected by authentic SARS-CoV-2 virus in vitro. After testis interstitium injection, authentic SARS-CoV-2 viruses are first disseminated to the interstitial cells, and then detected inside the seminiferous tubules which in turn cause germ cell loss and disruption of seminiferous tubules. Our study demonstrates that testis is most likely a target of SARS-CoV-2 virus. Attention should be paid to the reproductive function in SARS-CoV-2 patients.     

BNC1 is required for maintaining mouse spermatogenesis

Basonuclin (BNC1) is a zinc finger protein expressed primarily in gametogenic cells and proliferative keratinocytes. Our previous work suggested that BNC1 is present in spermatogonia, spermatocytes, and spermatids, but absent in the Sertoli cells. BNC1's role in spermatogenesis is unknown. Here, we show that BNC1 is required for the maintenance of spermatogenesis. Bnc1-null male mice were sub-fertile, losing germ cells progressively with age. The Bnc1-null seminiferous epithelia began to degenerate before 8 weeks of age and eventually became Sertoli cell-only. Sperm count and motility also declined with age. Furthermore, Bnc1 heterozygotes, although fertile, showed a significant drop in sperm count and in testis weight by 24 weeks of age, suggesting a dosage effect of Bnc1 on testis development. In conclusion, our data demonstrate for the first time BNC1's essential role in maintaining mouse spermatogenesis.