The blood-testis barrier disruption is a prerequisite for toxicant-induced peritubular macrophage increases in the testis of peripubertal rats.

Peritubular macrophages (PTMφ) are predominantly localized near spermatogonial stem cells in the testis. We previously revealed that exposure of peripubertal male Fischer rats to mono-(2-ethylhexyl) phthalate (MEHP) leads to increased PTMφs in the testis. The mechanisms that trigger increases in PTMφs in the testis are poorly understood. However, MEHP exposure is known to both induce spermatocyte apoptosis and to perturb the blood-testis barrier (BTB). This study aims to elucidate the association between the disruption of BTB and the increases of PTMφs in the testis by comparing the effects observed with MEHP to 2 other testicular toxicants with variable effects on the BTB and subtype of germ cell undergoing apoptosis. Methoxyacetic acid (MAA) acts directly on spermatocytes and does not affect BTB function, whereas cadmium chloride (CdCl2) induces profound injury to BTB. The results indicated that MAA exposure significantly increased spermatocyte apoptosis, whereas no significant changes in the numbers of PTMφs in the testis occurred. In contrast, CdCl2 exposure disrupted BTB function and increased the abundance of PTMφs in the testis. To further investigate whether MEHP-induced changes in BTB integrity accounted for the increase in PTMφs, a plasmid for LG3/4/5, the functional component of laminin-alpha 2, was overexpressed in the testis to stabilize BTB integrity before MEHP exposure. The results showed that LG3/4/5 overexpression substantially reduced the ability of MEHP to compromise BTB integrity and prevented the increase in PTMφ numbers after MEHP exposure. These results indicate that BTB disruption is necessary to increase PTMφs in the testis induced by toxicants.

A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies.

OBJECTIVE: To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS). DESIGN: Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA-sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes. SETTING: A laboratory study. PATIENTS: Three men with a diagnosis of obstructive azoospermia (age range, 30-40 years). INTERVENTION: None. MAIN OUTCOME MEASURES: Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue. RESULTS: Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%-8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1-/doublesex and Mab-3 related transcription factor 1-/STRA8+ spermatogonia as well as SYCP3+/protamine 2- spermatocytes. CONCLUSION: This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and in vitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

Loss of SUN1 function in spermatocytes disrupts the attachment of telomeres to the nuclear envelope and contributes to non-obstructive azoospermia in humans.

One of the most severe forms of infertility in humans, caused by gametogenic failure, is non-obstructive azoospermia (NOA). Approximately, 20-30% of men with NOA may have single-gene mutations or other genetic variables that cause this disease. While a range of single-gene mutations associated with infertility has been identified in prior whole-exome sequencing (WES) studies, current insight into the precise genetic etiology of impaired human gametogenesis remains limited. In this paper, we described a proband with NOA who experienced hereditary infertility. WES analyses identified a homozygous variant in the SUN1 (Sad1 and UNC84 domain containing 1) gene [c. 663C > A: p.Tyr221X] that segregated with infertility. SUN1 encodes a LINC complex component essential for telomeric attachment and chromosomal movement. Spermatocytes with the observed mutations were incapable of repairing double-strand DNA breaks or undergoing meiosis. This loss of SUN1 functionality contributes to significant reductions in KASH5 levels within impaired chromosomal telomere attachment to the inner nuclear membrane. Overall, our results identify a potential genetic driver of NOA pathogenesis and provide fresh insight into the role of the SUN1 protein as a regulator of prophase I progression in the context of human meiosis.

Widespread metastasis of a spermatocytic seminoma with concomitant hepatic peliosis in a Southern African hedgehog (Atelerix frontalis).

ABSTRACT: A six-year-old intact male Southern African hedgehog (Atelerix frontalis) presented with a history of chronic mild to moderate weight loss, and sub-acute hind limb ataxia that progressed to complete paralysis, at which point the hedgehog was euthanised. At autopsy, a large multinodular pale mass had completely replaced the left testicle and transcoelomically metastasised to the diaphragm and the peri-renal area, from where it then invaded the vertebral column and spinal cord. Multifocal, irregular to round, well-demarcated, blood-filled, proliferative lesions were also present in the hepatic parenchyma. Histological analysis of both the testis and metastatic lesions revealed diffuse sheets of neoplastic cells with moderate pale cytoplasm, large irregular to round nuclei and mostly one prominent magenta nucleolus, consistent with metastatic seminoma. The neoplastic cells were negative for periodic acid-Schiff (PAS) stain and positive for CD117 by immunohistochemistry (IHC). Taken together with the morphology of the neoplastic cells and the advanced age of the animal, this is suggestive of a spermatocytic seminoma. Histological analysis of the liver revealed multifocal lesions consisting of large anastomosing blood-filled spaces bordered by compressed hepatocytes, consistent with hepatic peliosis. This is the first report of a neoplasm in the Southern African hedgehog (Atelerix frontalis), the first report of a metastatic seminoma in a hedgehog, together with diagnosis of spermatocytic subtype, and the first report of a hedgehog with concomitant hepatic peliosis.

BDE-209 induce spermatocytes arrest at early-pachytene stage during meiotic prophase I in mice.

Deca-brominated diphenyl ether (BDE-209) is a common flame retardant utilized in electronic products, textiles, furniture, and upholstery materials. Environmental BDE-209 exposure results in spermatogenesis disorder, because of the characteristics of bioaccumulation, persistence, and probably toxicity. Meiotic prophase I is a crucial phase during spermatogenesis which is a key influential factor of normal sperm production. However, the effects of BDE-209 on meiotic prophase I during spermatogenesis are poorly understood. The present study aimed to evaluate whether BDE-209 exposure impairs meiotic prophase I during spermatogenesis of spermatocytes. We validated the effects of BDE-209 on alternations of meiotic prophase I in Balb/c male mice. Firstly, we analyzed sperm quality in cauda epididymis with decreasing sperm count, increasing abnormal sperm, and male reproductive dysfunction after exposure to BDE-209. Then, reactive oxygen species (ROS) and malondialdehyde (MDA) levels in testis and GC-2spd cells were significant increased after treated with BDE-209. Furthermore, we found that meiotic prophase I arrest at early-pachytene stage during spermatogenesis with increasing of DSBs damage and trimethylated histone H3 at lysine-4 (H3K4me3) in spermatocytes exposed to BDE-209. Finally, we conducted homologous recombination (HR) analyses to identify the progression of meiosis. The recombination markers, including DMC1 and RAD51, and crossover marker MLH1 were decreased during spermatogenesis after exposure to BDE-209. Collectively, our data indicated that BDE-209 has detrimental impacts on meiotic prophase I of spermatocytes in mice.

Long Noncoding RNAs: Recent Insights into Their Role in Male Infertility and Their Potential as Biomarkers and Therapeutic Targets.

Long noncoding RNAs (lncRNAs) are composed of nucleotides located in the nucleus and cytoplasm; these are transcribed by RNA polymerase II and are greater than 200 nt in length. LncRNAs fulfill important functions in a variety of biological processes, including genome imprinting, cell differentiation, apoptosis, stem cell pluripotency, X chromosome inactivation and nuclear transport. As high throughput sequencing technology develops, a substantial number of lncRNAs have been found to be related to a variety of biological processes, such as development of the testes, maintaining the self-renewal and differentiation of spermatogonial stem cells, and regulating spermatocyte meiosis. These indicate that lncRNAs can be used as biomarkers and potential therapeutic targets for male infertility. However, only a few comprehensive reviews have described the role of lncRNAs in male reproduction. In this paper, we summarize recent findings relating to the role of lncRNAs in spermatogenesis, their potential as biomarkers for male infertility and the relationship between reproductive arrest and transgenerational effects. Finally, we suggest specific targets for the treatment of male infertility from the perspective of lncRNAs.

Responses and coping methods of different testicular cell types to heat stress: overview and perspectives.

To facilitate temperature adjustments, the testicles are located outside the body cavity. In most mammals, the temperature of the testes is lower than the body temperature to ensure the normal progression of spermatogenesis. Rising temperatures affect spermatogenesis and eventually lead to a decline in male fertility or even infertility. However, the testes are composed of different cell types, including spermatogonial stem cells (SSCs), spermatocytes, spermatozoa, Leydig cells, and Sertoli cells, which have different cellular responses to heat stress. Recent studies have shown that using different drugs can relieve heat stress-induced reproductive damage by regulating different signaling pathways. Here, we review the mechanisms by which heat stress damages different cells in testes and possible treatments.

A method for utilizing automated machine learning for histopathological classification of testis based on Johnsen scores.

We examined whether a tool for determining Johnsen scores automatically using artificial intelligence (AI) could be used in place of traditional Johnsen scoring to support pathologists' evaluations. Average precision, precision, and recall were assessed by the Google Cloud AutoML Vision platform. We obtained testicular tissues for 275 patients and were able to use haematoxylin and eosin (H&E)-stained glass microscope slides from 264 patients. In addition, we cut out of parts of the histopathology images (5.0 × 5.0 cm) for expansion of Johnsen's characteristic areas with seminiferous tubules. We defined four labels: Johnsen score 1-3, 4-5, 6-7, and 8-10 to distinguish Johnsen scores in clinical practice. All images were uploaded to the Google Cloud AutoML Vision platform. We obtained a dataset of 7155 images at magnification 400× and a dataset of 9822 expansion images for the 5.0 × 5.0 cm cutouts. For the 400× magnification image dataset, the average precision (positive predictive value) of the algorithm was 82.6%, precision was 80.31%, and recall was 60.96%. For the expansion image dataset (5.0 × 5.0 cm), the average precision was 99.5%, precision was 96.29%, and recall was 96.23%. This is the first report of an AI-based algorithm for predicting Johnsen scores.

The male germline-specific protein MAPS is indispensable for pachynema progression and fertility.

Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a number of mouse genes that were dramatically elevated in spermatocytes, relative to their very low expression in spermatogonia and somatic organs. Here, we investigated in detail 1700102P08Rik, one of these genes, and independently conclude that it encodes a male germline-specific protein, in agreement with a recent report. We demonstrated that it is essential for pachynema progression in spermatocytes and named it male pachynema-specific (MAPS) protein. Mice lacking Maps (Maps-/- ) suffered from pachytene arrest and spermatocyte death, leading to male infertility, whereas female fertility was not affected. Interestingly, pubertal Maps-/- spermatocytes were arrested at early pachytene stage, accompanied by defects in DNA double-strand break (DSB) repair, crossover formation, and XY body formation. In contrast, adult Maps-/- spermatocytes only exhibited partially defective crossover but nonetheless were delayed or failed in progression from early to mid- and late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult Maps-/- pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in Maps-/- pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general.

Food additive-induced oxidative stress in rat male reproductive organs and hippocampus.

As currently defined, the exposome represents the lifetime exposure measure of an individual to all potential external genetic influences and their impact on health. Although intentionally added chemicals (e.g., food additives) and food contact materials (e.g., packaging, pesticides) have been assessed for safety to some degree, the full extent to which they can affect health and reproduction has not been reported. The aim of this study was to determine the in vitro and in vivo effects of food additives on the male rat brain and sperm/testes, particularly through oxidative stress. Results from our in vitro study demonstrated that the administration of the common food additive, stevioside, a major component of the common sweetener stevia, as well as the preservatives, diphenyl and orthophenyl phenol (OPP), induced reactive oxygen species (ROS) production in sperm, and led to sperm dysfunction. These effects were inhibited by the addition of the antioxidant α-tocopherol. Moreover, OPP treatment (1/10,000 of no observed adverse effect) induced ROS production in sperm and lipid peroxidation in the epididymis and hippocampus after two weeks in vivo. Furthermore, 4-hydroxynonenal-positive cells, indicating ROS-generated protein modifications, were detected in spermatocytes in the testes and granular cell layer of the dentate gyrus in the brain. Treatment with α-tocopherol significantly improved oxidative stress. Our study suggests that certain food additives may affect sperm function and induce oxidative stress in the testes and brain, resulting in infertility and short-term memory loss, and some antioxidants may improve these dysfunctions.

Absence of SCAPER causes male infertility in humans and Drosophila by modulating microtubule dynamics during meiosis.

BACKGROUND: Mutation in S-phase cyclin A-associated protein rin the endoplasmic reticulum (SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies. METHODS: Human clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis of short spindle 3 (ssp3) (SCAPER orthologue) Drosophila CAS9-knockout lines. In vitro microtubule (MT) binding assayed by total internal reflection fluorescence microscopy. RESULTS: We show that patients homozygous for a SCAPER mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells. Interestingly, Drosophila null mutants for the ubiquitously expressed ssp3 gene are viable and female fertile but male sterile. We further show that male sterility in ssp3 null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and Ssp3 directly bind MTs. CONCLUSIONS: Our results show that SCAPER null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in ssp3 specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and Drosophila meiosis.

Cold-induced RNA-binding protein (CIRBP) regulates the expression of Src-associated during mitosis of 68 kDa (Sam68) and extracellular signal-regulated kinases (ERK) during heat stress-induced testicular injury.

In this study, the ability of cold-induced RNA-binding protein (CIRBP) to regulate the expression of Src-associated during mitosis of 68 kDa (Sam68) and extracellular signal-regulated kinases (ERK) in the mouse testis and mouse primary spermatocytes (GC-2spd cell line) before and after heat stress was examined to explore the molecular mechanism by which CIRBP decreases testicular injury. A mouse testicular hyperthermia model, a mouse primary spermatocyte hyperthermia model and a low CIRBP gene-expression cell model were constructed and their relevant parameters were analysed. The mRNA and protein levels of CIRBP and Sam68 were significantly decreased in the 3-h and 12-h testicular heat-stress groups, extracellular signal-regulated kinase 1/2 (ERK1/2) protein expression was not significantly affected but phospho-ERK1/2 protein levels were significantly decreased. GC-2spd cellular heat-stress results showed that the mRNA and protein concentrations of CIRBP and Sam68 were reduced 48h after heat stress. In the low CIRBP gene-expression cell model, CIRBP protein expression was significantly decreased. Sam68 mRNA expression was significantly decreased only at the maximum transfection concentration of 50nM and Sam68 protein expression was not significantly affected. These findings suggest that CIRBP may regulate the expression of Sam68 at the transcriptional level and the expression of phospho-ERK1/2 protein, both of which protect against heat-stress-induced testicular injury in mice.

Dual histone methyl reader ZCWPW1 facilitates repair of meiotic double strand breaks in male mice.

Meiotic crossovers result from homology-directed repair of DNA double-strand breaks (DSBs). Unlike yeast and plants, where DSBs are generated near gene promoters, in many vertebrates DSBs are enriched at hotspots determined by the DNA binding activity of the rapidly evolving zinc finger array of PRDM9 (PR domain zinc finger protein 9). PRDM9 subsequently catalyzes tri-methylation of lysine 4 and lysine 36 of Histone H3 in nearby nucleosomes. Here, we identify the dual histone methylation reader ZCWPW1, which is tightly co-expressed during spermatogenesis with Prdm9, as an essential meiotic recombination factor required for efficient repair of PRDM9-dependent DSBs and for pairing of homologous chromosomes in male mice. In sum, our results indicate that the evolution of a dual histone methylation writer/reader (PRDM9/ZCWPW1) system in vertebrates remodeled genetic recombination hotspot selection from an ancestral static pattern near genes towards a flexible pattern controlled by the rapidly evolving DNA binding activity of PRDM9.

Genotoxicity and sperm defects induced by 5-FU in male mice and the possible protective role of Pentas lanceolata-iridoids.

5-Fluorouracil (5-FU) is a widely used antineoplastic drug. In this work, a comprehensive study was performed to detect the extent of chromosomal damage and morphological sperm defects induced by 5-FU in male mice and the possible protective role of the iridoids-rich fraction of Pentas lanceolata leaves (IFPL). Six main groups were examined in micronucleus and chromosomal assays: I- control negative, II- control positive (i.p. treated with single dose of 75 mg/kg 5-FU), III- control plant (orally administrated IFPL, 300 mg/kg, 5 consecutive days), and IV-VI- treated with IFPL (100, 200 and 300 mg/kg, 5 consecutive days) plus 5-FU (i.p. treated at the last day). Samples were taken 24 h post treatment. The study of morphological sperm anomalies, single and repeated treatments were examined and samples were taken after 35 days from the 1st treatment. In bone marrow, 5-FU induced a significant increase in the micro-nucleated polychromatic erythrocytes, chromosome anomalies (CAs) and also cytotoxic effects. A significant percentage of CAs was recorded in spermatocytes after 5-FU treatment reached 22.80 ± 1.32 vs 4.20 ± 0.37 for control (mainly X-Y univalent, 90%). IFPL was recorded to be non-mutagenic in all tests examined. In addition, it alleviated the previous defects in a dose-dependent manner. A significant and dramatic increase in the percentage of morphological sperm defects was recorded after single and repeated treatments with 5-FU reached 13.24 ± 0.24, 30.42 ± 0.32 respectively vs 2.56 ± 0.14 for control. Amorphous head-sperm and sperm with coiled tail were the most pronounced types of abnormalities. Significant protection was detected with the highest tested dose of IFPL. In conclusion: 5-FU demonstrated to be a genotoxic agent. Its genotoxicity in germ cells is serious and may lead to reproductive toxicity, infertility or heritable defects. The results also demonstrated the biosafety of IFPL and its possible protective role in combined treatment with 5-FU.

UHRF1-repressed 5'-hydroxymethylcytosine is essential for the male meiotic prophase I.

5'-hydroxymethylcytosine (5hmC), an important 5'-cytosine modification, is altered highly in order in male meiotic prophase. However, the regulatory mechanism of this dynamic change and the function of 5hmC in meiosis remain largely unknown. Using a knockout mouse model, we showed that UHRF1 regulated male meiosis. UHRF1 deficiency led to failure of meiosis and male infertility. Mechanistically, the deficiency of UHRF1 altered significantly the meiotic gene profile of spermatocytes. Uhrf1 knockout induced an increase of the global 5hmC level. The enrichment of hyper-5hmC at transcriptional start sites (TSSs) was highly associated with gene downregulation. In addition, the elevated level of the TET1 enzyme might have contributed to the higher 5hmC level in the Uhrf1 knockout spermatocytes. Finally, we reported Uhrf1, a key gene in male meiosis, repressed hyper-5hmC by downregulating TET1. Furthermore, UHRF1 facilitated RNA polymerase II (RNA-pol2) loading to promote gene transcription. Thus our study demonstrated a potential regulatory mechanism of 5hmC dynamic change and its involvement in epigenetic regulation in male meiosis.

Reactive oxygen species, not Ca2+ , mediates methotrexate-induced autophagy and apoptosis in spermatocyte cell line.

Methotrexate (MTX) is widely used to treat cancers and systemic autoimmune diseases. However, it is severely toxic to healthy cells, especially those of the reproductive system, and therefore poses a great risk to patient fertility. In addition, the underlying mechanism of MTX-induced reproductive toxicity has not yet been fully elucidated. Here, a spermatocyte cell line (GC2) was used as an in vitro model system to determine whether MTX induces autophagy and apoptosis, and to elucidate the role of reactive oxygen species (ROS) and Ca2+ in these two processes. Treatment with MTX resulted in a dramatic decrease in cell viability, inhibition of cell proliferation, collapse of the mitochondrial membrane potential and activation of caspase 3, suggesting that MTX induced apoptosis. Moreover, MTX activated autophagy, as indicated by conversion of LC3-I to LC3-II (microtubule-associated protein 1 light chain 3) and an increase in the number of LC3 puncta. Furthermore, MTX triggered ROS overproduction, rather than a Ca2+ burst. Intriguingly, eliminating excess ROS significantly alleviated MTX-induced apoptosis and autophagy. In addition, inhibiting autophagy significantly reversed apoptosis and promoted cell survival, indicating that autophagy aggravated MTX-induced apoptosis in GC2 cells. Taken together, these results suggest that ROS signalling, not Ca2+ , is critical in mediating MTX-induced autophagy and apoptosis and autophagy serves as a promoted partner of apoptosis to deteriorate MTX-induced cytotoxicity in GC2 cells. The findings from this study provide new perspectives for evaluating the reproductive toxicity of MTX.

Biochemical, immunohistochemical and morphometrical investigation of the effect of thymoquinone on the rat testis following exposure to a 900-MHz electromagnetic field.

Long-term use of cell phones emitting electromagnetic fields (EMFs) have raised concerns regarding public health in recent year. We aimed to investigate the possible effects of 900 MHz EMF exposure (60 min/day for 28 days) on the rat testis. Another objective was to determine whether the deleterious effect of EMF radiation would be reduced by the administration of thymoquinone (TQ) (10 mg/kg/day). Twenty-four male adult Wistar albino rats were randomly selected, then assigned into four groups as followControl, EMF, TQ and EMF + TQ. Testicular samples were analyzed using histological, stereological, biochemical and immunohistochemical techniques. Total numbers of primary spermatocytes and spermatids as well as Leydig cells were significantly decreased in the EMF group compared to the Control group (p < 0.05). In the EMF + TQ group, the total number of primary spermatocytes was significantly increased compared to the EMF group (p < 0.05). Superoxide dismutase (SOD) activity was significantly increased in the EMF group compared to the Control group (p < 0.05). Also, serum testosterone levels and wet weight of testes were significantly decreased in the EMF group compared to the Control group (p < 0.05). Our findings suggested that exposure to a 900 MHz EMF had adverse effects on rat testicular tissue and that the administration of TQ partially mitigated testicular oxidative damages caused by EMF radiation.

Increasing ERK phosphorylation by inhibition of p38 activity protects against cadmium-induced apoptotic cell death through ERK/Drp1/p38 signaling axis in spermatocyte-derived GC-2spd cells.

Many studies report that cadmium chloride (CdCl2)-induces oxidative stress is associated with male reproductive damage in the testes. CdCl2 also induces mitochondrial fission by increasing dynamin-related protein 1 (Drp1) expression as well as the mitochondria-dependent apoptosis pathway by extracellular signal-regulated kinase (ERK) activation. However, it remains unclear whether mechanisms linked to the mitochondrial damage signal via CdCl2-induced mitogen-activated protein kinases (MAPK) cause damage to spermatocytes. In this study, increased intracellular and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (∆Ψm) depolarization, and mitochondrial fragmentation and swelling were observed at 5 µM of CdCl2 exposure, resulting in increased apoptotic cell death. Moreover, CdCl2-induced cell death is closely associated with the ERK/Drp1/p38 signaling axis. Interestingly, SB203580, a p38 inhibitor, effectively prevented CdCl2-induced apoptotic cell death by reducing ∆Ψm depolarization and intracellular and mitochondrial ROS levels. Knockdown of Drp1 expression diminished CdCl2-induced mitochondrial deformation and ROS generation and protected GC-2spd cells from apoptotic cell death. In addition, electron microscopy showed that p38 inhibition reduced CdCl2-induced mitochondrial interior damage more effectively than N-acetyl-L-cysteine (NAC), an ROS scavenger; ERK inhibition; or Drp1 knockdown. Therefore, these results demonstrate that inhibition of p38 activity prevents CdCl2-induced apoptotic GC-2spd cell death by reducing depolarization of mitochondrial membrane potential and mitochondrial ROS levels via ERK phosphorylation in a signal pathway different from the CdCl2-induced ERK/Drp1/p38 axis and suggest a therapeutic strategy for CdCl2-induced male infertility.

Usp26 mutation in mice leads to defective spermatogenesis depending on genetic background.

Spermatogenesis is a reproductive system process that produces sperm. Ubiquitin specific peptidase 26 (USP26) is an X chromosome-linked deubiquitinase that is specifically expressed in the testes. It has long been controversial whether USP26 variants are associated with human male infertility. Thus, in the present study, we introduced a mutation into the Usp26 gene in mice and found that Usp26 mutant males backcrossed to a DBA/2 background, but not a C57BL/6 background, were sterile or subfertile and had atrophic testes. These findings indicate that the effects of the Usp26 mutation on male reproductive capacity were influenced by genetic background. Sperm in the cauda epididymis of Usp26 mutant mice backcrossed to a DBA/2 background were decreased in number and showed a malformed head morphology compared to those of wild-type mice. Additionally, histological examinations of the testes revealed that the number of round and elongated spermatids were dramatically reduced in Usp26 mutant mice. The mutant mice exhibited unsynapsed chromosomes in pachynema and defective chiasma formation in diplonema, which presumably resulted in apoptosis of metaphase spermatocytes and subsequent decrease of spermatids. Taken together, these results indicate that the deficiencies in fertility and spermatogenesis caused by mutation of Usp26 were dependent on genetic background.

Testis single-cell RNA-seq reveals the dynamics of de novo gene transcription and germline mutational bias in Drosophila.

The testis is a peculiar tissue in many respects. It shows patterns of rapid gene evolution and provides a hotspot for the origination of genetic novelties such as de novo genes, duplications and mutations. To investigate the expression patterns of genetic novelties across cell types, we performed single-cell RNA-sequencing of adult Drosophila testis. We found that new genes were expressed in various cell types, the patterns of which may be influenced by their mode of origination. In particular, lineage-specific de novo genes are commonly expressed in early spermatocytes, while young duplicated genes are often bimodally expressed. Analysis of germline substitutions suggests that spermatogenesis is a highly reparative process, with the mutational load of germ cells decreasing as spermatogenesis progresses. By elucidating the distribution of genetic novelties across spermatogenesis, this study provides a deeper understanding of how the testis maintains its core reproductive function while being a hotbed of evolutionary innovation.