Single-cell and spatial transcriptomic investigation reveals the spatiotemporal specificity of the beta-defensin gene family during mouse sperm maturation.

Low sperm motility is a significant contributor to male infertility. beta-defensins have been implicated in host defence and the acquisition of sperm motility; however, the regulatory mechanisms governing their gene expression patterns and functions remain poorly understood. In this study, we performed single-cell RNA and spatial transcriptome sequencing to investigate the cellular composition of testicular and epididymal tissues and examined their gene expression characteristics. In the epididymis, we found that epididymal epithelial cells display a region specificity of gene expression in different epididymal segments, including the beta-defensin family genes. In particular, Defb15, Defb18, Defb20, Defb25 and Defb48 are specific to the caput; Defb22, Defb23 and Defb26 to the corpus; Defb2 and Defb9 to the cauda of the epididymis. To confirm this, we performed mRNA fluorescence in situ hybridisation (FISH) targeting certain exon region of beta-defensin genes, and found some of their expression matched the sequencing results and displayed a close connection with epididimosome marker gene Cd63. In addition, we paid attention to the Sertoli cells and Leydig cells in the testis, along with fibroblasts and smooth muscle cells in the epididymis, by demonstrating their gene expression profile and spatial information. Our study provides a single-cell and spatial landscape for analysing the gene expression characteristics of testicular and epididymal environments and has important implications for the study of spermatogenesis and sperm maturation.

Retinoic acid mitigates the NSC319726-induced spermatogenesis dysfunction through cuproptosis-independent mechanisms.

Copper ionophore NSC319726 has attracted researchers' attention in treating diseases, particularly cancers. However, its potential effects on male reproduction during medication are unclear. This study aimed to determine whether NSC319726 exposure affected the male reproductive system. The reproductive toxicity of NSC319726 was evaluated in male mice following a continuous exposure period of 5 weeks. The result showed that NSC319726 exposure caused testis index reduction, spermatogenesis dysfunction, and architectural damage in the testis and epididymis. The exposure interfered with spermatogonia proliferation, meiosis initiation, sperm count, and sperm morphology. The exposure also disturbed androgen synthesis and blood testis barrier integrity. NSC319726 treatment could elevate the copper ions in the testis to induce cuproptosis in the testis. Copper chelator rescued the elevated copper ions in the testis and partly restored the spermatogenesis dysfunction caused by NSC319726. NSC319726 treatment also decreased the level of retinol dehydrogenase 10 (RDH10), thereby inhibiting the conversion of retinol to retinoic acid, causing the inability to initiate meiosis. Retinoic acid treatment could rescue the meiotic initiation and spermatogenesis while not affecting the intracellular copper ion levels. The study provided an insight into the bio-safety of NSC319726. Retinoic acid could be a potential therapy for spermatogenesis impairment in patients undergoing treatment with NSC319726.

MiRNA-seq and mRNA-seq revealed the mechanism of fluoride-induced cauda epididymal injury.

The widespread presence of fluoride in water, food, and the environment continues to exacerbate the impact of fluoride on the male reproductive health. However, as a critical component of the male reproductive system, the intrinsic mechanism of fluoride-induced cauda epididymis damage and the role of miRNAs in this process are still unclear. This study established a mouse fluorosis model and employed miRNA and mRNA sequencing; Evans blue staining, Oil Red O staining, TEM, immunofluorescence, western blotting, and other technologies to investigate the mechanism of miRNA in fluoride-induced cauda epididymal damage. The results showed that fluoride exposure increased the fluoride concentration in the hard tissue and cauda epididymis, altered the morphology and ultrastructure of the cauda epididymis, and reduced the motility rate, normal morphology rate, and hypo-osmotic swelling index of the sperm in the cauda epididymis. Furthermore, sequencing results revealed that fluoride exposure resulted in differential expression of 17 miRNAs and 4725 mRNAs, which were primarily enriched in the biological processes of tight junctions, inflammatory response, and lipid metabolism, with miR-742-3p, miR-141-5p, miR-878-3p, and miR-143-5p serving as key regulators. Further verification found that fluoride damaged tight junctions, raised oxidative stress, induced an inflammatory response, increased lipid synthesis, and reduced lipid decomposition and transport in the cauda epididymis. This study provided a theoretical basis for developing miRNA as potential diagnostic markers and therapeutic target drugs for this injury.

RUNX transcription factors are essential in maintaining epididymal epithelial differentiation.

Apart from the androgen receptor, transcription factors (TFs) that are required for the development and formation of the different segments of the epididymis have remained unknown. We identified TF families expressed in the developing epididymides, of which many showed segment specificity. From these TFs, down-regulation of runt related transcription factors (RUNXs) 1 and 2 expression coincides with epithelial regression in Dicer1 cKO mice. Concomitant deletion of both Runx1 and Runx2 in a mouse epididymal epithelial cell line affected cell morphology, adhesion and mobility in vitro. Furthermore, lack of functional RUNXs severely disturbed the formation of 3D epididymal organoid-like structures. Transcriptomic analysis of the epididymal cell organoid-like structures indicated that RUNX1 and RUNX2 are involved in the regulation of MAPK signaling, NOTCH pathway activity, and EMT-related gene expression. This suggests that RUNXs are master regulators of several essential signaling pathways, and necessary for the maintenance of proper differentiation of the epididymal epithelium.

A Single-Cell Landscape of Spermioteleosis in Mice and Pigs.

(1) Background: Spermatozoa acquired motility and matured in epididymis after production in the testis. However, there is still limited understanding of the specific characteristics of sperm development across different species. In this study, we employed a comprehensive approach to analyze cell compositions in both testicular and epididymal tissues, providing valuable insights into the changes occurring during meiosis and spermiogenesis in mouse and pig models. Additionally, we identified distinct gene expression signatures associated with various spermatogenic cell types. (2) Methods: To investigate the differences in spermatogenesis between mice and pigs, we constructed a single-cell RNA dataset. (3) Results: Our findings revealed notable differences in testicular cell clusters between these two species. Furthermore, distinct gene expression patterns were observed among epithelial cells from different regions of the epididymis. Interestingly, regional gene expression patterns were also identified within principal cell clusters of the mouse epididymis. Moreover, through analysing differentially expressed genes related to the epididymis in both mouse and pig models, we successfully identified potential marker genes associated with sperm development and maturation for each species studied. (4) Conclusions: This research presented a comprehensive single-cell landscape analysis of both testicular and epididymal tissues, shedding light on the intricate processes involved in spermatogenesis and sperm maturation, specifically within mouse and pig models.

Distinct actions of testicular endocrine and lumicrine signaling on the proximal epididymal transcriptome.

The epididymal function and gene expression in mammals are under the control of the testis. Sex steroids are secreted from the testis and act on the epididymis in an endocrine manner. There is another, non-sex steroidal secreted signaling, named lumicrine signaling, in which testis-derived secreted proteins go through the male reproductive tract and act on the epididymis. The effects of such multiple regulations on the epididymis by the testis have been investigated for many genes. The recent development of high-throughput next-generation sequencing now enables us a further comparative survey of endocrine and lumicrine action-dependent gene expression. In the present study, testis-derived endocrine and lumicrine actions on epididymal gene expression were comparatively investigated by RNA-seq transcriptomic analyses. This investigation utilized experimental animal models in which testis-derived endocrine and/or lumicrine actions were interfered with, such as unilateral or bilateral orchidectomy. By bilateral orchidectomy, which interferes with both endocrine and lumicrine actions, 431 genes were downregulated. By unilateral orchidectomy, which also interferes with endocrine and lumicrine actions by the unilateral testis, but the endocrine action was compensated by the contralateral testis, 283 genes were downregulated. The content of such genes downregulated by unilateral orchidectomy was like those of lumicrine action-interfered efferent duct-ligation, W/Wv, and Nell2-/- mice. When genes affected by unilateral and bilateral orchidectomy were compared, 154 genes were commonly downregulated, whereas 217 genes were specifically downregulated only by bilateral orchidectomy, indicating the distinction between endocrine and lumicrine actions on the proximal epididymal transcriptome. Comparative transcriptome analyses also showed that the expressions of genes emerging since Amniota were notably impacted by bilateral orchidectomy, unilateral orchidectomy, and lumicrine action-interfering treatments; the degree of influence from these treatments varied based on the evolutionary stage beyond Amniota. These findings unveil an evolutional transition of regulated gene expression in the proximal epididymis by two different testis-derived signaling mechanisms.

Vas deferens to rete testis anastomosis for obstructive azoospermia.

PURPOSE: This video aims to present an in-depth, step-by-step tutorial on microsurgical reconstruction for obstructive azoospermia, featuring a distinctive case involving anastomosis from vas deferens to rete testis. The primary aim of this endeavor is to offer thorough and practical insights for healthcare professionals and researchers within the realm of reproductive medicine. The video endeavors to disseminate expertise, methodologies, and perspectives that can be advantageous to individuals grappling with obstructive azoospermia, providing a significant contribution to the progress of reproductive medicine and the augmentation of existing treatment alternatives. MATERIALS AND METHODS: Surgical footage was recorded using the ORBEYE 4K 3D Orbital Camera System by Olympus America, with patient consent acquired for research purposes. Additionally, a retrospective examination of patient records was undertaken to compile relevant medical histories. RESULTS: This video furnishes an exhaustive guide to microsurgical reconstruction for obstructive azoospermia, encompassing a distinctive instance of anastomosis from vas deferens to rete testis. State-of-the-art technology, such as the ORBEYE 4K 3D Orbital Camera, heightens procedural transparency, accentuating the significance of advanced instrumentation. The ethical underpinning is emphasized by obtaining patient consent for footage utilization, and a retrospective chart review augments the repository of valuable patient data. This comprehensive approach serves as an invaluable reservoir of knowledge for medical professionals and underscores excellence in clinical and ethical healthcare research. CONCLUSIONS: Anastomosis from vas deferens to rete testis emerges as a viable surgical reconstruction alternative for obstructive azoospermia, particularly when confronted with non-dilated tubules within the epididymis.

The challenges in diagnosing isolated epididymal tuberculosis (TB) in an adolescent male: a case report.

BACKGROUND: Genitourinary tuberculosis (GUTB) is a common form of extrapulmonary TB (EPTB) in children. An example of GUTB is epididymal TB, which usually presents unspecific chronic clinical manifestations. Definitive diagnosis can be conducted based on bacteriologic confirmation and histopathologic results, but this is challenging due to the paucibacillary nature of EPTB. Therefore, we reported the challenges in diagnosing isolated epididymal TB in an adolescent male. CASE PRESENTATION: A 16-year-old male presented to respirology clinic with painful swelling of the left scrotum for 3 months before visiting to the hospital. The symptoms were associated with persistent coughing for 2 months, and physical examination of the left scrotum showed swelling accompanied by cardinal signs. A palpable hard mass was found on the left scrotum, with firm borders, measuring 7 × 4 cm. Laboratory examination and tumor markers were within normal limits, although leukocyturia was found, and the urine culture was negative. Genital ultrasound (US) showed epididymitis sinistra with septal hydrocele, while magnetic resonance imaging (MRI) indicated inhomogeneous left epididymitis with bilateral inguinal lymph node enlargement. Although TB evaluation presented a negative purified protein derivative (PPD) test and bacteriologic examination, chest X-ray (CXR) showed perihilar lymphadenopathy. Based on the clinical and radiologic results suggesting TB, the patient was diagnosed with isolated epididymal TB and received quadruple antituberculosis therapy (ATT) for 6 months. After treatment, the left testicle size started to shrink and was equal to the right testicle, also, there were no signs of inflammation, the body weight increased by 5 kg, and cough disappeared. Sperm analysis at the end of treatment indicated teratozoospermia, which was subsequently treated by the urologic surgery department. CONCLUSIONS: Biopsy and bacteriologic confirmation for TB epididymitis were challenging to perform in the clinical setting. Epididymal TB should be considered in adolescent males with complaints of chronic scrotal swelling and pain. Clinical judgment based on history taking, physical examination, and radiologic features supporting TB features could be helpful in accurate and fast diagnosis for favorable outcome.

Antioxidant effects of α-lipoic acid against epididymal oxidative damage in adult offspring rats exposed to maternal hypothyroidism stress.

It is well known that the epididymis promotes post-testicular sperm maturation events. However, its malfunction during congenital hypothyroidism is relatively less understood as compared to the testis. The present study evaluated the probable effect of α-lipoic acid on epididymal oxidative stress parameters in rats exposed to antithyroid drug, carbimazole during fetal period. Time-mated pregnant rats in unexposed and carbimazole (1.35 mg/Kg body weight exposed were allowed to deliver pups and weaned. At postnatal day 100, the F1 male pups were assessed for epididymal endpoints. Among the epididymal regions, significant elevation of lipid peroxidation levels, superoxide anion, and hydrogen peroxide contents with a concomitant reduction in the activity levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and reduced glutathione levels were observed in cauda epididymis of carbimazole exposed rats over controls. Significant elevation in sperm DNA fragmentation (comet assay), accelerated cauda epididymal sperm transit time and reduction in epididymal sialic acid content was observed in carbimazole exposed rats. RT-qPCR studies revealed that embryonic exposure to carbimazole resulted in down regulation of androgen receptor, nuclear factor eryrthoid 2 like 2, 5α-reducatse 1 mRNA levels, while up regulation of caspase 3 mRNA was observed in epididymal regions of rats. In addition, fetal exposure to carbimazole resulted in disorganization of cauda epididymal architecture in rats. Conversely, supplementation of α-lipoic acid (70 mg/Kg bodyweight) during PND 3 to 14 restored epididymal functions in carbimazole exposed rats and the ameliorative effects of lipoic acid could be attributed to its antioxidant and steroidogenic effects.

Busulfan administration replicated the characteristics of the epididymal initial segment observed in mice lacking testis-epididymis lumicrine signaling.

The physiological functions of the mammalian epididymis are typically regulated by the testes. In addition to sex steroids secreted by testicular Leydig cells, which act on the epididymis in an endocrine manner, there is a non-sex-steroidal signaling pathway known as the lumicrine pathway. This lumicrine signaling pathway involves ligand proteins secreted from germ cells within the testicular seminiferous tubules traversing the male reproductive tract, which induce epithelial differentiation in the epididymis. These findings prompted an inquiry into whether treatments influencing testis physiology can disrupt epididymal function by interfering with testis-epididymis communication. Busulfan, an alkylating agent commonly used to deplete testicular germ cells in reproductive biology, has not been sufficiently explored because of its effects on the epididymis. This study investigated the effects of busulfan administration on the proximal epididymis using histological and transcriptomic analyses. Notably, busulfan, as opposed to the vehicle dimethyl sulfoxide (DMSO), altered the morphology of the initial segment of the epididymis, leading to a reduction in the cell height of the luminal epithelium. RNA sequencing identified 185 significantly downregulated genes in the proximal epididymis of busulfan-administered mice compared to DMSO-administered mice. Comparative transcriptome analyses revealed similarities between the epididymal transcriptome of busulfan-administered mice and lumicrine-deficient mice, such as efferent-duct-ligated W/Wv and Nell2-/- mice. However, this differed from that of bilaterally orchidectomized mice, in which both the endocrine and lumicrine signaling pathways were simultaneously ablated. Collectively, these results suggested that the harmful effects of busulfan on the proximal epididymis are secondary consequences of the ablation of testis-epididymis lumicrine signaling.

Morphometric study applied to testicular and epididymis hydatids torsion.

Twisted testicular appendages had difficult differential diagnosis with testicular torsion. The objective of this paper is to evaluate the number, shape, size and determine the laterality pattern of the testicular and epididymal hydatids and evaluate the correlations between the length and width of the testicular and epididymal hydatids with testicular measurements. We analyzed 60 fixed cadavers and 16 patients with prostate cancer without previous hormonal treatment undergoing bilateral orchiectomy, totalizing 76 units and 152 testicles. In relation to the testicular appendices, we analyzed the following situations: absence of testicular and epididymis appendages, presence of a testicular appendix, presence of epididymis appendix, and presence of testicular and epididymis appendix. We measured the length, width and thickness of the testis and classified the appendages as sessile or pedicled. Chi-square test was used to verify associations between categorical variables. McNemar Test was used to verify differences between the percentages of right and left appendages. Correlations between quantitative measures were evaluated using the Pearson Correlation Coefficient (p < 0.05). In 50 cases (65.78%) we observed the presence of some type of appendices, in 34 cases (44.72%) we observed the presence of testicular appendices and in 19 cases (25%) the presence of epididymal appendices. We observed the presence of pedicled appendices in 39 cases (51.32%), with 25 of the cases (32.89%) of pedicled testicular appendices and 14 of the cases (18.42%) of pedicled epididymal appendages, with a significant association between the occurrence of appendices on the right and left sides (p < 0.001). Testicular hydatids were present in around two thirds of our sample being pedunculated in almost half of the cases with bilateral similarity. There is a significant chance in cases of twisted appendices that the same anatomical characteristics are present on the opposite side, which is a factor that tends to indicate the need for contralateral surgical exploration in cases of torsion, however studies with larger samples are needed to confirm these findings.

Kaempferol mitigates reproductive dysfunctions induced by Naja nigricollis venom through antioxidant system and anti-inflammatory response in male rats.

Naja nigricollis Venom (NnV) contains complex toxins that affects various vital systems functions after envenoming. The venom toxins have been reported to induce male reproductive disorders in envenomed rats. This present study explored the ameliorative potential of kaempferol on NnV-induced male reproductive toxicity. Fifty male wistar rats were sorted randomly into five groups (n = 10) for this study. Group 1 were noted as the control, while rats in groups 2 to 5 were injected with LD50 of NnV (1.0 mg/kg bw; i.p.). Group 2 was left untreated post envenomation while group 3 was treated with 0.2 ml of polyvalent antivenom. Groups 4 and 5 were treated with 4 and 8 mg/kg of kaempferol, respectively. NnV caused substantial reduction in concentrations of follicle stimulating hormone, testosterone and luteinizing hormone, while sperm motility, volume and counts significantly (p < 0.05) decreased in envenomed untreated rats. The venom enhanced malondialdehyde levels and substantially decreased glutathione levels, superoxide dismutase and glutathione peroxidase activities in the testes and epididymis of envenomed untreated rats. Additionally, epididymal and testicular myeloperoxidase activity and nitric oxide levels were elevated which substantiated severe morphological defects noticed in the reproductive organs. However, treatment of envenomed rats with kaempferol normalized the reproductive hormones with significant improvement on sperm functional parameters. Elevated inflammatory and oxidative stress biomarkers in testis and epididymis were suppressed post kaempferol treatment. Severe histopathological lesions in the epididymal and testicular tissues were ameliorated in the envenomed treated groups. Results highlights the significance of kaempferol in mitigating reproductive toxicity induced after snakebite envenoming.

Morphology of the epididymal duct of the domestic pig (Sus scrofa domesticus).

The study provides a general overview of the morphology of the epididymal duct in pigs. Four epididymides from two sexually mature boars were dissected into 32 segments and examined histologically. Duct lumen and wall thickness were measured and relative surface area of different components was assessed by Chalkley's random hit method. The epithelial lining was characterized at X1000. Lumen diameter and wall thickness of efferent ductules averaged 177 and 30 µm, respectively. Of the epididymal duct from caput to distal corpus the luminal diameter was 332 µm, with a narrower section in the proximal corpus. Wall thickness averaged 70 µm. In the cauda, luminal diameter and wall thickness increased to 717 and 751 µm, respectively. The epithelial lining of the efferent ductules consists of a single layer of columnar cells with average height 21 µm. The lining of the epididymal duct consists of ciliated, pseudo-stratified columnar epithelium composed of "basal cells" and "principal cells." Particularly tall principal cells (96 µm) were found in the proximal caput. Height decreased to 40 µm at the distal cauda. Microvilli from principal cells were 14-17 µm long in the distal caput but decreased to 5 µm in the distal cauda. The epithelial lining was folded in the proximal caput and more so in the distal cauda. Secretory granules (epididymosomes) were present in small amounts in efferent ductules and epididymal duct; the largest quantities occurred in the distal cauda. Leukocytes were present throughout the duct, albeit in insignificant numbers. Chalkley's random hit method showed rapid spermatozoan transport through efferent ductules and proximal caput in large amounts of fluid. Sperm concentration increased due to fluid resorption in the proximal caput, was highest from caput flexure to proximal cauda and decreased at the caudal flexure, indicating secretory activity.

Comparative evaluation of double- and single-armed two-suture longitudinal intussusception techniques in microsurgical vasoepididymostomy: An updated systematic review and meta-analysis.

BACKGROUND: This study aimed to compare the outcomes of double-armed two-suture longitudinal intussusception microsurgical vasoepididymostomy (LIVE) and single-armed two-suture LIVE techniques in patients with epididymal obstructive azoospermia (EOA). The main outcomes assessed were patency rates, patency time, semen quality and natural pregnancy rates. METHODS: Data from patients with EOA who underwent two-suture LIVE were obtained from databases including PubMed, EMBASE, and Web of Science. Weighted data were analyzed using a random-effects model, and weighted mean differences were reported. RESULTS: A total of 1574 patients with EOA from 24 studies were included. The overall patency rate was approximately 68% (95% confidence interval [CI]: 63-72%), with a patency time of approximately 4.63 months (95% CI: 4.15-5.12). The sperm concentration reached 26.90 million/ml and the sperm motility was 23.74%. The natural pregnancy rate was 38% (95% CI: 31-46%). The different definitions of patency do not seem to have any meaningful impact when comparing patency rates. There was no significant difference in patency rates, patency time, semen quality and natural pregnancy rates between the double-armed and single-armed LIVE techniques. CONCLUSION: The single-armed LIVE is a potential alternative surgical option when high quality double-needle sutures are not easily accessible.

Cellular Distribution of Aquaporin 3, 7 and 9 in the Male Reproductive System: A Lesson from Bovine Study (Bos taurus).

The increasing incidence of male infertility in humans and animals creates the need to search for new factors that significantly affect the course of reproductive processes. Therefore, the aim of this study was to determine the temporospatial expression of aquaglyceroporins (AQP3, AQP7 and AQP9) in the bovine (Bos taurus) reproductive system using immunohistochemistry and Western blotting. The study also included morphological analysis and identification of GATA-4. In brief, in immature individuals, AQP3 and AQP7 were found in gonocytes. In reproductive bulls, AQP3 was observed in spermatocytes and spermatogonia, while AQP7 was visible in all germ cells and the Sertoli cells. AQP7 and AQP9 were detected in the Leydig cells. Along the entire epididymis of reproductive bulls, aquaglyceroporins were visible, among others, in basal cells (AQP3 and AQP7), in epididymal sperm (AQP7) and in the stereocilia of the principal cells (AQP9). In males of all ages, aquaglyceroporins were identified in the principal and basal cells of the vas deferens. An increase in the expression of AQP3 in the testis and cauda epididymis and a decrease in the abundance of AQP7 in the vas deferens with age were found. In conclusion, age-related changes in the expression and/or distribution patterns of AQP3, AQP7 and AQP9 indicate the involvement of these proteins in the normal development and course of male reproductive processes in cattle.

Epididymectomy Is an Effective Treatment for Chronic Epididymal Pain.

INTRODUCTION: Chronic scrotal pain is difficult to manage, and epididymectomy is a treatment option for a subset of men with pain localized to the epididymis. We sought to evaluate the efficacy of epididymectomy at our institution. METHODS: Between 2000 and 2020, 225 men underwent epididymectomy at our institution for pain localized to the epididymis and not part of a greater constellation of pelvic pain or urinary symptoms. Our primary outcome measure was change in pain after epididymectomy, categorized as cured/improved or no change/worsened. Multivariable logistic regression compared the impact of pain duration, and surgical and psychiatric histories on postoperative pain. RESULTS: Patients in both outcome categories-cured/improved and no change/worsened-were similar in age and BMI. Overall, 162 patients (72%) reported cured/improved pain at the last documented follow-up visit. Median follow-up time was 12 (IQR 1-364) weeks. About half of the cohort (n = 117, 52%) had a prior vasectomy, and there was no difference in outcome based on vasectomy history on multivariate analysis (OR 0.625, P = .3). Men with pain duration > 1 year (OR 0.46, P = .03), diagnosed psychiatric conditions (OR 0.44, P = .04), or prior scrotal/inguinal/abdominal surgeries other than vasectomy (OR 0.47, P = .03) had decreased odds of pain relief after epididymectomy. CONCLUSIONS: This 20-year analysis is the largest review of postepididymectomy outcomes reported. Among carefully screened men, 72% had resolution or improvement of scrotal pain. Epididymectomy is most effective for men with < 1 year of focal epididymal pain, with no history of psychiatric conditions or scrotal/inguinal/abdominal surgery other than vasectomy.