Testis Cells Research Articles

Dexamethasone is a regulator of clock genes in testicular peritubular cells.

We recently found that peritubular cells of the human testis are a dominant site of expression of the glucocorticoid receptor (GR; encoded by NR3C1). Activation of GR by dexamethasone (Dex) strongly influences the phenotype of cultured human testicular peritubular cells (HTPCs), causing massive changes of their proteome and secretome. As glucocorticoids (GC) are also known to set the internal clock of peripheral organs by regulating clock genes, we tested such an influence of Dex in HTPCs. We performed cellular studies with HTPCs and immortalized nonhuman primate (Callithrix jacchus; Cj)-derived peritubular cells, organotypic incubations of testicular fragments of Cj, qPCR and proteomic, as well as immunohistochemical studies. Basal clock gene expression levels, when monitored by qPCR under standard culture conditions, showed alterations over 24 h, suggesting an endogenous circadian rhythm, especially for BMAL1. Dex (1 µM) when added to cells, caused a strong and significant increase of PER1, followed by elevations of BMAL1, and other clock genes. This action was observed as early as 4 h after the addition of Dex. Immunohistochemistry and data mining revealed GR in testicular peritubular cells and other somatic cells of Cj, in situ. We therefore performed organotypic incubations of testicular fragments of Cj (n = 3) and found that upon addition of Dex (1 µM), mRNA levels of BMAL1 and PER1 also increased in samples of two out of three animals after 6 h. Mass spectrometry did, however, not reveal significant alterations of the testicular proteome, possibly due to the short time point and/or the fact that the somatic GR-expressing cells represent only a small portion of the testis. In support for this assumption, Dex (1 µM; 6 h) significantly increased mRNA levels of BMAL1 and PER1 in Cj-derived immortalized testicular peritubular cells. The results indicate that an internal clock system likely exists in peritubular cells of the testis and that Dex, via testicular GR expressed by peritubular cells and other somatic cells, is a strong regulator of this system. In a physiological situation, GC thus may be important regulators of the testicular clock, while in a situation of prolonged stress or GC-medication, derangements in clock gene expression may result.

Ferritinophagy is involved in hexavalent chromium-induced ferroptosis in Sertoli cells

Hexavalent chromium [Cr(VI)] has significant adverse effects on the environment and human health, particularly on the male reproductive system. Previously, we observed ferroptosis and autophagy in rat testicular injury induced by Cr(VI). In the present study, we focused on the association between ferroptosis and autophagy in mouse Sertoli cells (TM4) exposed to concentrations of 2.5 μМ, 5 μМ, and 10 μМ Cr(VI). Cr(VI) exposure altered mitochondrial ultrastructure; increased intracellular iron, malondialdehyde, and reactive oxygen species (ROS) levels; decreased glutathione content; increased TfR1 protein expression; and decreased GPX4, FPN1, and SLC7A11 protein expression, ultimately resulting in ferroptosis. Additionally, we observed ferritinophagy, increased expression of BECLIN1, LC3B, and NCOA4, and decreased expression of FTH1 and P62. Inhibition of autophagy and ferritinophagy via 3-MA and small interfering RNA (siRNA)-mediated silencing of NCOA4 ameliorated changes in ferritinophagy- and ferroptosis-associated protein expression, and reduced ROS levels. Rats exposed to Cr(VI) exhibited atrophy of testicular seminiferous tubules, a reduction in germ and Sertoli cells, and the occurrence of ferritinophagy and ferroptosis in cells of the rat testes. These results indicate that ferroptosis, triggered by NCOA4-mediated ferritinophagy, is one of the mechanisms that contribute to Cr(VI)-induced damage in Sertoli cells.

Pathogenicity and phylogenetic analysis of ovine contagious ecthyma virus isolated during a sheeppox outbreak in Morocco

Contagious ecthyma (CE), also known as ORF is a highly contagious zoonotic viral skin disease that affects humans, sheep, goats and other domesticated and wild animals. As reported here-in, the objective of this study was to investigate a suspected outbreak of both sheeppox and ORF diseases in a sheep herd during the winter of 2020 in Northwest Morocco. The affected sheep showed nodules and proliferative scabby skin lesions around the mouth and hairless area of the body. Samples of skin crust were collected for virus identification and isolation. A virus was isolated in Vero cells, lamb testis and heart cells and the cytopathic effect was characterized by cells aggregation, ballooning, and detachment. Initially, the suspensions of skin crust were positive for sheeppox virus (SPPV) by PCR. Subsequent testing of the isolated virus from skin crust of affected animals indicated that the virus was SPPV-negative and ORFV-positive by PCR. Furthermore, nucleotide sequences of the B2L aligned with reference ORFV isolates for genetic analysis. Phylogenetic analyses results confirmed that the isolated virus was ORFV and that the virus was closely related to ORFV strains isolated in Sudan and Malaysia.In conclusion, this study is the first reported detection of ORFV in Morocco, and therefore, poses as an imminent threat to the health of humans, domestic and wild animals.

Ultrastructural Characterization of Testis in Non-descript Goat (Capra hircus)

Background: The proposed experiment was aimed at locating the spermatogonial stem cell niches inside the testis and to characterise the development of germ cells of testis in the non- descript goats at the different stages of pre and post natal life, which could be useful in selecting the goats for breeding at appropriate time and for other interventions related to reproductive developments in the goats. Methods: The present study was conducted on the testes of non-descript goats divided into three groups, viz. neonatal (within 1 month), prepubertal (from 1-18 months) and pubertal (above 18 months) with 10 healthy animals in each group. The collected samples of testis were processed for scanning electron microscopic study and subsequently, the samples were viewed and the photographs were taken in the facility available at Central Instrumentation Facility (CIF), OUAT, Bhubaneswar. Result: The present study revealed that the testis was surrounded by the tunica albuginea, a thin capsule of connective tissue, from which thin septum originated and extended into the inside of the organ. Among these septa, the seminiferous tubules were found. The seminiferous epithelium consisted of spermatogonial cells at different stages of development separated from each other by interstitial connective tissue. The interstitium was made up of randomly arranged collagen bundles. The intertubular vascular connective tissue contained Leydig cells mainly at wider locations where they were bounded by more than two seminiferous tubules. The mediastinal rete testis area contained many cavities of many intercommunicating channels supported by connective tissue pillars. The present study helped in developing a baseline data on the ultrastructural characteristics of seminiferous epithelium, rete restis, peritubular cells, Lyedig cells and other components of testis in non-descript goat at different post natal stages under study. The appearance of seprmatogonial cell lineage at the various post natal stages in non-descript goat would help in accessing the reproductive status of the animal.

Microanatomy of the Testes and Male Genital Ducts of Banded Krait Bungarus fasciatus (Schneider, 1801)

The Banded krait is a species of venomous snakes that ranks among the top 5 in Thailand, with a significance role as a predator for controlling other animals. Despite numerous studies on the ecology, the microanatomy of body systems has still not reported clearly. The present study was to investigate the microanatomy of the testes and male genital ducts of Banded krait that collected from Phatthalung Province, Thailand. The snakes were collected after anesthetized, and their testes and genital ducts were fixed in Bouin’s fixative. The reproductive organs were collected during July to October 2022, and were sectioned of 5-µm-thick pieces for staining with Harris’s hematoxylin and eosin (H&E), periodic acid Schiff’s (PAS), alcian blue (AB) pH 2.5 and pH 1.0, bromophenol blue (BB) and Masson’s trichrome (MT). The testes contained the seminiferous tubules with 7 stages of germ cells: (I) type A spermatogonium, (II) type B spermatogonium, (III) primary spermatocyte, (IV) secondary spermatocyte, (V) round spermatid, (VI) elongated spermatid and (VII) spermatozoa. Besides, Sertoli and Leydig’s cells were appeared in the seminiferous tubules and the interstitial area, respectively. During the sampling period, the spermatozoa were observed in the seminiferous tubules and genital ducts. The extra-testicular rete testis, ductuli efferentes and ductus epididymis were embedded in the epididymal sheath. The epithelial cells of rete testis, ductus epididymis, as well as ductus deferens possessed microvilli, whereas those of the ductuli efferentes composed of cilia. The epithelial cells of all portions showed the positive staining with PAS and BB due to the presence of neutral glycoproteins and proteins, respectively, and the negative staining for AB pH 2.5 and pH 1.0 because of the absence of acidic glycoproteins. HIGHLIGHTS During the sampling period from July to October, the Banded Krait exhibited active spermatogenesis The rete testis could be divided into 2 portions: intra-rete testis and extra-rete testis The ductus epididymis was divided into 3 portions: proximal, middle and distal ductus epididymis All portions of the male genital ducts, the epithelium showed the secretion of neutral glycoproteins and proteins without sulfated and carboxylated glycoproteins GRAPHICAL ABSTRACT

The interactome of cystic fibrosis transmembrane conductance regulator and its role in male fertility: A critical review.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic adenosine monophosphate (cAMP)-regulated chloride and bicarbonate ion channel found in many human cells. Its unique biochemical characteristics and role as a member of the adenosine triphosphate (ATP)-binding cassette transporters superfamily are pivotal for the transport of several substrates across cellular membranes. CFTR is known to interact, physically and functionally, with several other cellular proteins. Hence, its properties are essential for moving various substances across cell membranes and ensuring correct cell functioning. Genetic mutations or environmental factors may disrupt CFTR's function resulting in different possible phenotypes due to gene variations that affect not only CFTR's function, localization, and processing within cells, but also those of its interactors. This has been reported as an underlying cause of various diseases, including cystic fibrosis. The severe clinical implications of cystic fibrosis have driven intense research into the role of CFTR in lung function but its significance to fertility, particularly in men, has been comparatively understudied. However, ongoing and more recent research into CFTR and its interacting proteins in the testis or specific testicular cells is beginning to shed light on this field. Herein, we provide a comprehensive and up-to-date overview of the CFTR, its interactome, and its crucial role in male reproduction, highlighting recent discoveries and advancements in understanding the molecular mechanisms involved. The comprehension of these complex interactions may pave the way for potential therapeutic approaches to improve fertility of men suffering from alterations in the function of CFTR.

Structural and network analysis of ADAM9 and ADAM10 and its transcript expression in the male reproductive tract of Bos Taurus

A disintegrin and metalloproteinases (ADAMs) are a family of proteins expressed as transmembrane and secreted proteins in tissues. This study investigated the expression of glycoproteins and transcripts of ADAM9 and ADAM10 in different regions of the bovine male reproductive tract (MRT). Fresh bovine samples were collected from the testis, epididymis, and vas deferens of MRT for this study. Special stains were utilized for identifying glycoproteins depending on the type of mucopolysaccharides in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. Quantitative real-time PCR (qPCR) was used to assess the mRNA concentrations of ADAMs9 and 10. Additionally, structural and network analysis was performed to investigate the similarity between human and bovine ADAM9 and ADAM10. Variable glycoprotein (acidic and neutral mucopolysaccharides) expression was observed in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. ADAM9 and 10 mRNA were detected in the testis epididymis and vas deferens, with ADAM9 expression being significantly higher than ADAM10 mRNA expression in the testis and tail of epididymis. Significant similarity between the protein sequence of human and bovine ADAM9 (85.75%) and ADAM10 (96.66%) was observed, while the similarity between protein sequence of ADAM 9 and ADAM10 in humans and bovine was <26%. Bovine ADAM9 showed 4 binding sites, while bovine ADAM10, human ADAM9, and human ADAM10 showed 7 binding pockets, each with considerable variations in the affinity of specific antibody sequences to these binding pockets. Network analysis confirmed that ADAM9 and ADAM10 have critical roles in physiological processes involving cell migration, protease activity, and secretase-related functions. In addition, they have large numbers of hydrogen bonds to most other members of the ADAM family. Our study reports similarities in the expression of glycoproteins and ADAM9 and ADAM10 mRNA in the MRT of Bos Taurus. Structural and network analysis of ADAM9 and ADAM10 suggested functional similarities in these proteins, which are likely to be in facilitating spermatogenesis, transcervical sperm migration, egg-sperm fusion, and fertilization. Further, the similarity between human and bovine ADAM9 and ADAM10 proteins suggests that bovine sperms can be considered as a model system for comparative medicine studies.

Effects of anti-rainbow trout germ-cell monoclonal antibody on germ cells and gonadal tissues in rainbow trout (Oncorhynchus mykiss)

The development of sterilization technology is important to improve the production efficacy of aquaculture and prevent genetically modified fish from escaping. Immunosterilization is a novel sterilization method that has already been used in mammals. In this study, anti-rainbow trout germ-cell monoclonal antibodies (anti-GC mAbs) were injected into immature rainbow trout Oncorhynchus mykiss, and their effects were analyzed. First, anti-GC mAbs labeled with Alexa Fluor 488 were injected into the body cavity of larvae 20 days (group 1) and 102 days (group 2) after fertilization. Second, anti-GC mAbs were injected into the body cavity of yearling trout (group 3), and their impacts on gonadal tissue were analyzed in terms of histology and gene expression levels. As a result, anti-GC mAbs were observed to bind to some primordial GCs in group 1, whereas there were no obvious positive cells in male testes of group 2. In contrast, green fluorescent signals were observed in an arrangement parallel to the ovarian lamina in female ovaries of group 2, which was similar to the arrangement of positive signals on ovarian tissue sections immunostained with anti-GC mAbs. GCs in group 1 were observed again after 76 days, and double-positive cells remained. In group 3, there were changes in the expression levels of GC-specific genes, but the changes were not seen as biologically meaningful. Moreover, there was no histological abnormality in gonadal tissues after immunization. In conclusion, these results suggested that anti-GC mAbs could only reach GCs in early developmental stages or in the ovary, and injection of anti-GCs mAbs did not have harmful effects on GCs after binding.

Single-Cell RNA Sequencing Reveals an Atlas of Hezuo Pig Testis Cells.

Spermatogenesis is a complex biological process crucial for male reproduction and is characterized by intricate interactions between testicular somatic cells and germ cells. Due to the cellular heterogeneity of the testes, investigating different cell types across developmental stages has been challenging. Single-cell RNA sequencing (scRNA-seq) has emerged as a valuable approach for addressing this limitation. Here, we conducted an unbiased transcriptomic study of spermatogenesis in sexually mature 4-month-old Hezuo pigs using 10× Genomics-based scRNA-seq. A total of 16,082 cells were collected from Hezuo pig testes, including germ cells (spermatogonia (SPG), spermatocytes (SPCs), spermatids (SPTs), and sperm (SP)) and somatic cells (Sertoli cells (SCs), Leydig cells (LCs), myoid cells (MCs), endothelial cells (ECs), and natural killer (NK) cells/macrophages). Pseudo-time analysis revealed that LCs and MCs originated from common progenitors in the Hezuo pig. Functional enrichment analysis indicated that the differentially expressed genes (DEGs) in the different types of testicular germ cells were enriched in the PI3K-AKT, Wnt, HIF-1, and adherens junction signaling pathways, while the DEGs in testicular somatic cells were enriched in ECM-receptor interaction and antigen processing and presentation. Moreover, genes related to spermatogenesis, male gamete generation, sperm part, sperm flagellum, and peptide biosynthesis were expressed throughout spermatogenesis. Using immunohistochemistry, we verified several stage-specific marker genes (such as UCHL1, WT1, SOX9, and ACTA2) for SPG, SCs, and MCs. By exploring the changes in the transcription patterns of various cell types during spermatogenesis, our study provided novel insights into spermatogenesis and testicular cells in the Hezuo pig, thereby laying the foundation for the breeding and preservation of this breed.

Lumpy skin disease virus isolation, experimental infection, and evaluation of disease development in a calf

Lumpy skin disease (LSD) is one of the most economically significant viral diseases of cattle caused by the Lumpy Skin Disease Virus (LSDV), classified as a member of the genus Capripoxvirus and belongs to the family Poxviridae. Nodular skin samples were collected from clinically sick cattle in the districts of Amuru and Wara Jarso Ethiopia to isolate LSD virus. The virus was isolated using primary lamb testis and kidney cells. The isolated LSDV was infected into a healthy calf while maintaining the necessary biosecurity measures to generate skin lesions and to assess disease progression using postmortem examinations. On the fourth day after virus inoculation, the calf developed typical LSD skin nodules with increased rectal temperature, which lasted until the 12th day, when they began to decrease. Viral shedding was detected in nasal, oral, and conjunctival swabs from 6 to 14 days after infection using real-time PCR. Post-mortem tissue specimens tested positive for LSD virus using real-time PCR and virus isolation. This study showed that LSDV were responsible for the LSD outbreaks, and the appearance of typical skin nodules accompanied by fever (> 39.5 °C) defined the virus’s virulent status. The experimental infection with the isolated infectious LSDV could serve as a platform for future vaccine evaluation study using an LSDV challenge model.

Trehalose attenuates testicular aging by activating autophagy and improving mitochondrial quality.

Reproductive aging can adversely affect male fertility and the health of offspring. The aging process is accompanied by impaired autophagy. Recent studies have shown that Trehalose plays an important role in the prevention of various diseases by regulating autophagy. However, the roles of Trehalose in testicular aging and reproductive decline remain to be clarified. The present study aimed to evaluate the protective effects of Trehalose on testes in an aging mouse model. In this study, an in vivo aging model in mice by administering D-galactose was established to explore the protective effect of Trehalose on testicular aging. We examined histological changes and related indicators of apoptosis, autophagy, mitochondrial biogenesis, and sperm quality. D-galactose treatment induced oxidative stress, apoptosis, and impairment of autophagy of testicular cells in mouse testes. Trehalose administration significantly reduced germ cell apoptosis and DNA damage caused by D-galactose-induced oxidative stress. Notably, Trehalose activated autophagy activity and improved mitochondrial function in testicular cells. Furthermore, Trehalose treatment increased the expression level of the tight junction protein ZO-1, and accelerated clearance of damaged mitochondria in Sertoli cells, indicating that Trehalose ameliorated Sertoli cell function in D-galactose-induced aging testes. These findings suggest that Trehalose administration activated the autophagy activity in testicular cells and improved mitochondrial function, thereby effectively preventing testicular aging. Trehalose and its activated autophagy are crucial for preventing testicular aging, thus restoring autophagy activity by administering Trehalose could be a promising means to delay aging.

Single-cell transcriptomic analysis reveals that the APP-CD74 axis promotes immunosuppression and progression of testicular tumors.

Testicular tumors represent the most common malignancy among young men. Nevertheless, the pathogenesis and molecular underpinning of testicular tumors remain largely elusive. We aimed to delineate the intricate intra-tumoral heterogeneity and the network of intercellular communication within the tumor microenvironment. A total of 40,760 single-cell transcriptomes were analyzed, encompassing samples from six individuals with seminomas, two patients with mixed germ cell tumors, one patient with a Leydig cell tumor, and three healthy donors. Five distinct malignant subclusters were identified in the constructed landscape. Among them, malignant 1 and 3 subclusters were associated with a more immunosuppressive state and displayed worse disease-free survival. Further analysis identified that APP-CD74 interactions were significantly strengthened between malignant 1 and 3 subclusters and 14 types of immune subpopulations. In addition, we established an aberrant spermatogenesis trajectory and delineated the global gene alterations of somatic cells in seminoma testes. Sertoli cells were identified as the somatic cell type that differed the most from healthy donors to seminoma testes. Cellular communication between spermatogonial stem cells and Sertoli cells is disturbed in seminoma testes. Our study delineates the intra-tumoral heterogeneity and the tumor immune microenvironment in testicular tumors, offering novel insights for targeted therapy. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Chronic dietary exposure to glyphosate-induced connexin 43 autophagic degradation contributes to blood-testis barrier disruption in roosters

Glyphosate (GLY) is the most universally used herbicide worldwide and its application has caused extensive pollution to the ecological environment. Increasing evidence has revealed the multi-organ toxicity of GLY in different species, but its male reproductive toxicity in avian species remains unknown. Thus, in vivo and in vitro studies were conducted to clarify this issue. Data firstly showed that chronic GLY exposure caused testicular pathological damage. Intriguingly, we identified and verified a marked down-regulation gap junction gene Connexin 43 (Cx43) in GLY-exposed rooster testis by transcriptome analysis. Cx43 generated by Sertoli cells acts as a key component of blood-testis barrier (BTB). To further investigate the cause of GLY-induced downregulation of Cx43 to disrupt BTB, we found that autophagy activation is revealed in GLY-exposed rooster testis and primary avian Sertoli cells. Moreover, GLY-induced Cx43 downregulation was significantly alleviated by ATG5 knockdown or CQ administration, respectively, demonstrating that GLY-induced autophagy activation contributed to Cx43 degradation. Mechanistically, GLY-induced autophagy activation and resultant Cx43 degradation was due to its direct interaction with ER-α. In summary, these findings demonstrate that chronic GLY exposure activates autophagy to induce Cx43 degradation, which causes BTB damage and resultant reproductive toxicity in roosters.

Fluorene-9-bisphenol impaired male fertility through disrupting the testicular function and local microenvironment

Past studies have observed that BHPF induces multi-organ toxicity, however, whether it induces damage to male reproductive system and the specific mechanism remains unclear. In the present study, male mice were given 0, 2, 10 or 50 mg/kg/day of BHPF by gavage for 35 days to observe its effect on reproductive organ and sperm quality. The results indicated that BHPF decreased sperm count and sperm motility in a dose-dependent manner. Besides, our results demonstrated that BHPF triggered the proliferation inhibition and cell death of germ cells in vivo and in vitro. Also, BHPF reduced the expression of function markers for germ cells, Sertoli cells, and Leydig cells, indicating its damage to function of testis cells. Simultaneously, testicular microenvironment was found to be altered by BHPF, as presented with declined testosterone level and decreased expression of local microenvironment regulators. Overall, our findings indicated the detrimental effects of BHPF on male reproductive function in mice, suggesting testicular function and local microenvironment disturbance as mechanism underlying testicular damage.

A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells

Germ cells preferentially induce apoptosis in response to DNA damage to avoid genomic mutations. Apoptosis of germ cells is closely related to cancer development and chemotherapy resistance; however, its regulatory mechanism is unclear. Here, we suggest that testis-specific lncRNA LINC03074 is involved in male germ cell apoptosis by regulating the expression of the proto-oncogene MDM2. LINC03074 is highly expressed in the sperm of healthy adult testes and cancer cells of testes with testicular germ cell tumors (TGCTs). LINC03074 binds to MDM2 mRNA via an Alu element, thereby reducing MDM2 protein levels. LINC03074 stimulates STAU1-mediated nuclear export of MDM2 mRNA by increasing STAU1 binding to MDM2 mRNA in the cell nucleus, thereby promoting PKR-mediated translational repression in the cytoplasm. The induction of apoptosis in TGCT cells and their responsiveness to the anticancer drug cisplatin is enhanced by LINC03074. Notably, LINC03074 increased E2F1 expression without increasing p53, the primary target of MDM2, and upregulated the apoptotic gene p73, the target gene of E2F1. LINC03074-mediated regulation of apoptosis contributes to the responsiveness of TGCTs to anticancer drug-induced DNA damage.

Porcine deltacoronavirus nucleocapsid protein interacts with the Grb2 through its proline-rich motifs to induce activation of the Raf-MEK-ERK signal pathway and promote virus replication.

Porcine deltacoronavirus (PDCoV), an enteropathogenic coronavirus, causes severe watery diarrhoea, dehydration and high mortality in piglets, which has the potential for cross-species transmission in recent years. Growth factor receptor-bound protein 2 (Grb2) is a bridging protein that can couple cell surface receptors with intracellular signal transduction events. Here, we investigated the reciprocal regulation between Grb2 and PDCoV. It is found that Grb2 regulates PDCoV infection and promotes IFN-β production through activating Raf/MEK/ERK/STAT3 pathway signalling in PDCoV-infected swine testis cells to suppress viral replication. PDCoV N is capable of interacting with Grb2. The proline-rich motifs in the N- or C-terminal region of PDCoV N were critical for the interaction between PDCoV-N and Grb2. Except for Deltacoronavirus PDCoV N, the Alphacoronavirus PEDV N protein could interact with Grb2 and affect the regulation of PEDV replication, while the N protein of Betacoronavirus PHEV and Gammacoronavirus AIBV could not interact with Grb2. PDCoV N promotes Grb2 degradation by K48- and K63-linked ubiquitin-proteasome pathways. Overexpression of PDCoV N impaired the Grb2-mediated activated effect on the Raf/MEK/ERK/STAT3 signal pathway. Thus, our study reveals a novel mechanism of how host protein Grb2 protein regulates viral replication and how PDCoV N escaped natural immunity by interacting with Grb2.

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.

Identification of Catecholamine and Drug Target α2A-Adrenoceptor in Human Testis and Human Testicular Peritubular Cells.

Background: Clonidine has been used in clinical medicine, e.g., to treat high blood pressure and other conditions. Animal studies have linked its use to impairments of male reproductive functions, and although only a few reports exist for the human species, such actions may exist in man as well. The underlying reasons and, specifically, possible actions of clonidine at the level of the testis are not known. Introduction: Clonidine is an agonist at the α2A-adrenoceptor (ADRA2A), which, as data bank mining indicated, is expressed by several cells of the human testis. The human testis and most of its cells are, however, not readily accessible to experimental testing. Cells from the peritubular wall compartment (human testicular peritubular cells; HTPCs) are the exception. Methods and Results: As shown by immunohistochemical/immunocytochemical and PCR techniques these cells express ADRA2A and retain expression upon isolation and culture. When tested over a concentration range (1-1000 µM) and 24 h, clonidine did not visibly affect HTPC morphology but significantly stimulated IL6 mRNA levels in a concentration-dependent manner. ELISA measurements of cell culture supernatants confirmed a stimulatory action of clonidine (10 µM) on secreted IL6. When examined in collagen gel contraction assays of HTPCs, clonidine (10 µM) exerted a slight relaxing action, while a proteomic study revealed that clonidine (10 µM) did not significantly change cellular protein abundance of HTPCs after 24 h (data available via ProteomeXchange with identifier PXD052220). Conclusion: Thus, ADRA2A-bearing cells in the human testis are targets for catecholamines and drugs such as clonidine. The results of this HTPCs-focused study only show the tip of the iceberg. It is likely that catecholamines/catecholaminergic drugs have the potential to interfere with human testicular functions.

CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells.

DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.

P-005 Could early life stress be related to the hippo signaling pathway in rat testis?

Abstract Study question Is there a change in the Hippo signaling pathway in the rat testis exposed to early life stress for 14 days in the postnatal period? Summary answer It was observed that the YAP and p-YAP signaling levels decreased in rats exposed to early-life stress when compared to the control groups. What is known already Hippo signaling pathway has been reported that it participates in many events such as cell division, proliferation, differentiation, and programmed cell death. YAP (Yes-Associated Protein) and p-YAP (phospho-YAP) are the main components of this pathway. Deterioration in sperm production and decreasing the serum testosterone levels were observed in rats exposed to stress stimulation in literature. However, the molecular mechanisms underlying these problems in the male reproductive system caused by stress have not yet been elucidated. Study design, size, duration 180-minute maternal deprivation and isolation model was applied to the early-life stress group (ELS) every day between the 1st and 14th postnatal day. Tail blood was collected before and after stress conditions on 14th postnatal day. Serum corticosterone levels were detected. On the 14th day, the control and ELS groups were sacrificed and the testicles were placed in appropriate fixatives. Participants/materials, setting, methods In total of 10 newborn Wistar albino male rats were used. Two groups were formed as Control (n = 5) and ELS (n = 5) groups. On postnatal 14th day, the groups were sacrificed. For immunohistochemical analyses, testis were taken into buffered formalin, paraffin blocks were made and the sections were taken. ELISA kit was used for serum corticosterone level. Main results and the role of chance The post-stress serum corticosterone levels of the ELS group were found to be significantly higher compared to the control group (p < 0.05). At least 60 seminiferous tubules were evaluated for immunohistochemical analyzes in the testis. H-score evaluation was applied. It was determined that YAP and p-YAP expressions decreased in the ELS group when compared to control group (p < 0.05) on the 14th postnatal day when mitosis and proliferation are high in testis cell groups. In addition the seminiferous tubule diameter was smaller in the experimental group. As we conducted in the literature research, stress conditions were generally based on adult subjects. There are insufficient data on the effects of stress on the testis in the early postnatal periods. This suggests that stress created in the early period may cause various defects as the cells enter the adult period. Limitations, reasons for caution This study was performed in vivo rat model in the postnatal 14th days. In further studies, groups including the adulthood period will be added. Wider implications of the findings After the 14th day of the postnatal period day, the blood-testis barrier is formed and spermatogenesis processes begin. Any defect that occurs in this day can cause problems in sperm development. This study can bring a new perspective to researching the effects of stress on testis in prepubertal period. Trial registration number not applicable