Search

During androgen biosynthesis, the human testes normally produce only small quantities of Δ 4 -C 21 steroids as these are products of the Δ 4 -pathway and healthy human testes preferentially use the Δ 5 -pathway. However, the Δ 4 -C 21 steroid progesterone accumulates in the thickened lamina propria of the seminiferous tubules in testes with deteriorated spermatogenesis. The objectives of this study were to analyse the pregnenolone metabolites in testes with deteriorated spermatogenesis and to establish whether the androgen biosynthesis pathway changes in this condition. Biopsied or orchiectomised testicular samples were obtained from patients with varicocele, non-obstructive azoospermia, obstructive azoospermia, testicular cancer, and cryptorchidism. The samples were segregated into spermatogenesis related Johnsen's score groups: Low-JS (< 5.0) and High-JS (> 7.8). Higher levels of progesterone and 17α-hydroxyprogesterone were metabolised under in vitro conversion in the Low-JS testes than the High-JS testes when cell-free homogenates from each group were separately incubated with 14 C-labelled pregnenolone. Nevertheless, the serum hormone levels did not differ between groups. Two novel pregnenolone metabolites 5β-pregnan-3β-ol-20-one and 5α-pregnan-3α, 21diol-20-one were identified from in vitro conversion in Low-JS testes and by recrystallisation. Immunohistochemistry revealed the higher βHSD expression in the Low-JS than the High-JS testes. However, the CYP17A1 expression levels did not differ between groups. Infertile testes increase the relative βHSD levels in their Leydig cells and synthesised testosterone from pregnenolone via the Δ 4 - rather than the Δ 5 -pathway. A new insight into a change of metabolites in Low-JS testes will be relevant to understand the mechanism of the deteriorated spermatogenesis under the normal range of testosterone level., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflict interests., (Copyright © 2020 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Background: The aim of this study was to evaluate the histochemical characteristics of the thickened inner acellular layer (IL) of the lamina propria specifically present in the human seminiferous tubules of testes showing impaired spermatogenesis., Methods: Eighteen biopsies for the investigation of infertility and 10 orchiectomies for testicular cancer and cryptorchidism were used. Lectin staining [peanut agglutinin (PNA), Maackia amurensis (MAA), Sambuccus nigra (SNA)], PNA lectin staining with sialidase digestion, immunohistochemistry and binding assay of progesterone were performed and analysed quantitatively., Results: The IL of the thickened lamina propria of the seminiferous tubules in the testes showed PNA lectin affinity and binding affinity for progesterone. Both affinities of MAA and SNA were in the IL of only fairly thickened lamina propria. Furthermore, a positive correlation was present between the thickness of the lamina propria and the accumulation of glycoconjugates showing PNA lectin affinity (r = 0.829, P < 0.001) or progesterone (r = 0.629, P < 0.001) in the IL. However, ILs show no immunoreactivities of progesterone receptor, androgen receptor or human serum albumin. Progesterone inhibited the binding affinity of PNA lectin to the IL (P < 0.001), but not the affinity to the spermatogenic cells. In addition, sialidase digestion increased the PNA affinity not in the IL but in the spermatogenic cells (P < 0.001)., Conclusions: These results indicate that the IL of the thickened lamina propria always consists of glycoconjugates with PNA lectin affinity and possible binding affinity to progesterone. In addition, the glycoconjugates in the IL may be predictors of abnormal spermatogenesis in the testes of infertile patients.

For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. Although previous studies described methylation of isolated DNA extracted from cells and tissues using a combination of appropriate restriction endonucleases, no application to tissue cell level has been reported. Here, we report a new method, named histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequences in a tissue section. In this study, we examined changes in the methylation level of CCGG sites during spermatogenesis in paraffin-embedded sections of mouse testis. In principle, the 3'-OH ends of DNA strand breaks in a section were firstly labeled with a mixture of dideoxynucleotides by terminal deoxynucleotidyl transferase (TdT), not to be further elongated by TdT. Then the section was digested with Hpa II, resulting in cutting the center portion of non-methylated CCGG. The cutting sites were labeled with biotin-16-dUTP by TdT. Next, the section was treated with Msp I, which can cut the CCGG sequence irrespective of the presence or absence of methylation of the second cytosine, and the cutting sites were labeled with digoxigenin-11-dUTP by TdT. Finally, both biotin and digoxigenin were visualized by enzyme- or fluorescence-immunohistochemistry. Using this method, we found hypermethylation of CCGG sites in most of the germ cells although non-methylated CCGG were colocalized in elongated spermatids. Interestingly, some TUNEL-positive germ cells, which are frequent in mammalian spermatogenesis, became markedly Hpa II-reactive, indicating that the CCGG sites may be demethylated during apoptosis.

The negative correlation between the thickness of the lamina propria and Johnsen's score count in individual seminiferous tubules was observed and the tubules with thin lamina propria indicated almost complete spermatogenesis. It is suggested that the thin lamina propria may be one of the good indicators of complete spermatogenesis.

Objective: To evaluate the expression patterns of heat shock transcription factor, Y chromosome (HSFY), in the testes showing deteriorated spermatogenesis., Design: Prospective study., Setting: University hospital, its branch hospital, and academic laboratory., Patient(s): Men undergoing testicular biopsy for the investigation of infertility and men undergoing orchiectomy for testicular cancer., Intervention(s): After pathologic evaluation, specimens were subdivided into three groups: normal spermatogenesis (n = 8), maturation arrest (n = 5), and Sertoli cell-only syndrome (n = 4). Immunostaining and Western blotting techniques determined the expression of HSFY., Main Outcome Measure(s): Expression of HSFY in testes., Result(s): Western blotting data revealed HSFY in the testicular tissues with normal spermatogenesis, maturation arrest, and Sertoli cell-only syndrome, but the amount of the protein in the maturation arrest and Sertoli cell-only syndrome samples was altered. The immunohistochemical data demonstrated that HSFY was expressed in spermatogenic cells and Sertoli cells in all specimens. However, the expression of HSFY was low or absent in spermatogenic cells of maturation arrest specimens, and the ratio of HSFY expressed in Sertoli cells was different in the specimens with maturation arrest and with Sertoli cell-only syndrome., Conclusion(s): Altered expression of the HSFY in the testis showing deteriorated spermatogenesis may be associated with alteration of spermatogenic cell differentiation.

Simple Summary: In the abdominal testes of most mammals, with the exception of elephants and a few animal species, spermatogenesis is characterised by an arrest of spermatogenesis due to heat stress. However, the mechanisms underlying normal spermatogenesis in the elephant abdominal testis remain unknown. We hypothesised that comparing the expression of heat stress tolerance molecules of elephant testes with that of other animals and investigating the mechanism of spermatogenesis in elephants might provide clues to overcome the causes of heat stress-induced spermatogenesis dysfunction. The results showed that the elephant's body cells, other than the testes, have a similar mechanism to those of other mammals but that the spermatogenic cells have a different mechanism, with different molecular immunoexpression under heat stress in the abdomen. These findings suggest that these molecules and their relationships may be important in future investigations of the mechanisms that overcome heat stress to maintain spermatogenesis in elephants. The abdominal testes of Asian elephants show normal spermatogenesis. Heat shock in cryptorchid testes elevates heat shock factor (HSF) expression, leading to germ cell apoptosis, while increased heat shock proteins (HSPs) levels provide protection. To investigate how heat shock affects elephant spermatogenic cells, focusing on heat shock-related molecules and the cell death mechanism, immunohistochemistry and TUNEL staining were employed to assess the immunoexpression of several heat shock-related molecules and the status of apoptosis in elephant fibroblasts (EF) induced by heat shock stimulus. Additionally, the immunoexpression of heat shock-related molecules and cell proliferation status in the elephant spermatogenic cells. Our finding indicated that heat shock-induced HSF1 immunoexpression in EF leads to apoptosis mediated by T-cell death-associated gene 51 (TDAG51) while also upregulating HSP70 to protect damaged cells. In elephant spermatogenic cells, immunostaining revealed a predominance of proliferating cell nuclear antigen (PCNA)-positive cells with minimal TDAG51- and TUNEL-positive cells, suggesting active proliferation and apoptosis suppression during normal spermatogenesis in the abdominal testis. Interestingly, spermatogonia co-immunoexpressed HSF1 and HSP90, potentially reducing apoptosis through protective mechanisms different from those observed in other mammals. Spermatogenic cells did not show immunolocalisation of HSP70, and hence, it may not contribute to protecting the spermatogonia from heat shock because the transcriptional activity of HSF1 is suppressed by HSP90A binding. This study provides insight into the specific heat shock response and defence mechanisms in elephant spermatogenic cells and may contribute to our understanding of species-specific adaptation to environmental stresses of the testis. [ABSTRACT FROM AUTHOR]

The balance between proliferation, differentiation and apoptosis is well‐coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridization of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle‐yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling assays. Testicular tissues from yaks (immature: 1 year old, mature: 2–3 years old) and backcrossed hybrids (2 year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells and Leydig cells. Furthermore, hybrid cattle‐yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle‐yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle‐yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle‐yak males. [ABSTRACT FROM AUTHOR]

Although the interspecies hybridization of bovids, such as cattle–yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression‐related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage‐specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5‐methylcytosine (5MC) and acetyl‐histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle–yak spermatogenesis. Testicular tissues from yak (1–3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle–yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same‐aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle–yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage‐specific gene expression modulations in each cell type in yak and cattle–yak to potentially solve the infertility issue in crossbreeding. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate whether the long‐term usage of mirabegron, which was reported to have potential side effects on male reproductive organs in animal studies, was harmful to spermatogenesis in human testis. Methods: Thirty consecutive patients with spinal cord injury (20‐48 years old) who performed clean intermittent catheterization were involved in this study. Ten patients were treated with mirabegron (50 mg/d) for more than 2 years and refrained from using an antimuscarinic agent due to the side effects of constipation and dry mouth. Twenty patients were treated with neither anticholinergic agents nor mirabegron. All underwent conventional testicular sperm extraction. The sperm recovery rate and histopathologic findings of the retrieved testicular tissue were compared between both groups. Results: We found no difference in the sperm recovery rate (P =.083) between both groups. Spinal cord injury patients treated with mirabegron had better spermatogenesis than those not treated with mirabegron (P <.05). Conclusions: From these data, we conclude that the therapeutic dose of mirabegron had no harmful effect on spermatogenesis in spinal cord injury patients of reproductive age. [ABSTRACT FROM AUTHOR]

This study aimed to compare the quality and the penetration ability of frozen–thawed spermatozoa from three microminipigs and Large White boars and to evaluate the effects of caffeine and heparin as well as the sperm–oocyte co-incubation length on the fertilization and embryonic development in vitro. Results showed that the fertilization rates of spermatozoa from three microminipig boars were significantly lower than those of a Large White boar. In the post-thaw spermatozoa from one of three microminipig boars, the sperm quality, penetration ability, and the oocyte development after in vitro fertilization were significantly lower than those of the spermatozoa from other boars. The caffeine supplementation in the fertilization media increased the rates of fertilization and blastocyst formation for the microminipig spermatozoa with low sperm quality. In addition to caffeine supplementation, the rates of fertilization and blastocyst formation after using microminipig spermatozoa were significantly higher with a 10 h sperm–oocyte co-incubation than with 3 h of co-incubation length. Our results indicate that the differences between the males and the breed influence the quality and fertility of frozen–thawed boar spermatozoa. In conclusion, the presence of caffeine in the in vitro fertilization (IVF) medium and adequate length of sperm–oocyte co-incubation may have beneficial effects for improving IVF results when using microminipig spermatozoa with low quality. [ABSTRACT FROM AUTHOR]

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle–yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β‐hydroxysteroid dehydrogenase (3βHSD) and 5α‐reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle–yak spermatogenesis. Testicular tissues from yaks (1–3 years old) and hybrids (F1–F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle–yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle–yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding. [ABSTRACT FROM AUTHOR]