Bazi Bushen alleviates reproductive aging in aged male mice.

Bazi Bushen (BZBS), a traditional Chinese medicine (TCM), has demonstrated therapeutic efficacy in testicular dysfunction within D-galactose and NaNO2 mouse models. This study aimed to ascertain if BZBS could also mitigate the decline in testicular function associated with natural aging. Therefore, male aged mice were employed to evaluate the preventive effects of BZBS on male reproductive aging. This was achieved by assessing sex hormone production, testicular histomorphology, and spermatogenesis. Relative to the untreated aged control group, BZBS administration elevated the levels of sex hormones and spermatocyte populations and preserved normal testicular structure in aged mice. Notably, spermatogenesis was maintained. Further analyses, including malondialdehyde (MDA) assays and real-time PCR, indicated that BZBS diminished testicular oxidative stress and the inflammatory burden. Corroborating these findings, mice treated with BZBS exhibited reductions in the populations of senescent and apoptotic cells within the seminiferous tubules, suggesting alleviated cellular damage. In contrast, we observed that rapamycin, a drug known for its longevity benefits, induced excessive testicular apoptosis and did not decrease lipid peroxidation. Collectively, our results highlight BZBS's promising clinical potential in counteracting male reproductive aging, underlining its mechanisms of action.

The crosstalk between telomeres and DNA repair mechanisms: an overview to mammalian somatic cells, germ cells, and preimplantation embryos.

Telomeres are located at the ends of linear chromosomes and play a critical role in maintaining genomic stability by preventing premature activation of DNA repair mechanisms. Because of exposure to various genotoxic agents, telomeres can undergo shortening and genetic changes. In mammalian cells, the basic DNA repair mechanisms, including base excision repair, nucleotide excision repair, double-strand break repair, and mismatch repair, function in repairing potential damages in telomeres. If these damages are not repaired correctly in time, the unfavorable results such as apoptosis, cell cycle arrest, and cancerous transition may occur. During lifespan, mammalian somatic cells, male and female germ cells, and preimplantation embryos experience a number of telomeric damages. Herein, we comprehensively reviewed the crosstalk between telomeres and the DNA repair mechanisms in the somatic cells, germ cells, and embryos. Infertility development resulting from possible defects in this crosstalk is also discussed in the light of existing studies.

CDX2 alleviates hypoxia-induced apoptosis and oxidative stress in spermatogenic cells through suppression of reactive oxygen species-mediated Wnt/ß-catenin pathway.

Hypoxia-induced apoptosis and oxidative stress in spermatogenic cells are considered to be important factors leading to male infertility. It was reported that CDX2 expression was downregulated in hypoxia-stimulated spermatogenic cells. However, the effects of CDX2 on hypoxia-induced apoptosis and oxidative stress in spermatogenic cells are still unknown. This study aimed to explore the roles of CDX2 in hypoxia-induced injury of spermatogenic cells, as well as its mechanism of action. Spermatogenic cells were cultured under 1% oxygen for 48 h to established hypoxia damage model. Reactive oxygen species (ROS) generation was determined using 2',7'-dichlorofluorescein diacetate assay. Apoptosis was assessed using flow cytometry. Enzyme-linked immunosorbent assay was used to evaluate oxidative stress markers, including malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidases (GSH-Px). Protein levels were detected using western blotting. Hypoxia exposure induced increase in ROS generation, apoptosis rate, and oxidative stress in spermatogenic cells. ROS scavenger inhibited hypoxia-induced apoptosis, oxidative stress, and Wnt/ß-catenin pathway activation. Hypoxia exposure induced CDX2 downregulation. CDX2 overexpression suppressed hypoxia-induced ROS generation, apoptosis rate, oxidative stress, and Wnt/ß-catenin pathway activation. Moreover, CDX2 knockdown restores the inhibitory effects of si-ß-catenin or NAC on hypoxia-induced activation of the Wnt/ß-catenin pathway, apoptosis, and oxidative stress. In conclusion, our study suggests that CDX2 overexpression alleviates hypoxia-induced apoptosis and oxidative stress by suppression of ROS-mediated Wnt/ß-catenin pathway in spermatogenic cells.

Effects of cadmium exposure during pregnancy on genome-wide DNA methylation and the CREB/CREM pathway in the testes of male offspring rats.

This experimental study explored the multigenerational and transgenerational effects of cadmium (Cd) exposure during pregnancy on the testicular tissue and spermatogenesis of male offspring rats. CdCl2 at different doses (0, 0.5, 1, 2 mg/kg/day) were dispensed to pregnant SD rats, thus producing generation F1. Adult females in F1 (PND 56) were mated with untreated fertile males so as to produce generation F2. Likewise, adult females in F2 were mated to produce generation F3. Damages to testicular tissue were observed in all the three generations, with serum testosterone (T) increased in F2 and F3. Notably, the genome-wide DNA methylation level in the testicular tissue of F1 was altered, as was the expression of F1-F3 methyltransferases. In addition, the expression of Creb/Crem pathway, a pathway critical for the metamorphosis from postmeiotic round spermatocytes to spermatozoa, was also remarkably altered in the three generations. In concludion, prenatal Cd exposure might bring multigenerational and transgenerational toxic effects to testes via genome-wide DNA methylation and the regulation of CREB/CREM pathway.

Tembusu virus induced apoptosis in vacuolate spermatogenic cells is mediated by Cytc-mediated mitochondrial apoptotic signaling pathway.

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that infects mainly poultry and has caused huge economic losses to the poultry farming industry in China. Also known as duck hemorrhagic ovarian disease, DTMUV principally destroys ovarian tissue in ducks, causing a dramatic drop in egg production. and can also invade the male reproductive system causing lesions. Currently, little research has been done to reveal the underlying mechanisms of reproductive dysfunction in ducks caused by DTMUV infection. In this study, histopathological analysis and electron microscopy of testes of ducks infected with DTMUV showed that DTMUV caused testicular atrophy and cytoplasmic vacuolation in ducks. Terminal Deoxynucleotidyl Transferase-Mediated Nick-End Labeling (TUNEL) staining and real-time quantitative PCR(RT-qPCR) results further indicated that DTMUV induced spermatogenic cells apoptosis. After DTMUV infection, a large amount of cytochrome c(Cytc) was released from the mitochondrial matrix into the cytoplasm, activating downstream target proteins and causing apoptosis. To sum up, DTMUV induces spermatogenic cell apoptosis through the Cytc-induced mitochondrial apoptosis pathway, our study provides evidence for DTMUV infection-induced male reproductive disorders.

Fine ultrastructural features of germ cells and spermatozoa during spermatogenesis in the European grayling, Thymallus thymallus (Teleostei, Salmoniformes, Salmonidae).

This study aimed to examine the ultrastructure of spermatogenic stages and mature spermatozoa in the European grayling, Thymallus thymallus. The testes were examined microscopically with a transmission electron microscope to find out details of the structure and morphology of the grayling germ cells, spermatozoa and some somatic cells. The grayling testis has a tubular shape, with cysts or clusters of germ cells within seminiferous lobules. The spermatogenic cells, including spermatogonia, spermatocytes, and spermatids, can be found along seminiferous tubules. There are electron-dense bodies in germ cells from the primary spermatogonia to secondary spermatocyte stages. These undergo mitosis to reach the secondary spermatogonia stage, when they form primary and secondary spermatocytes. Spermatids undergo three different stages of differentiation during spermiogenesis, characterized by the level of chromatin condensation, elimination of cytoplasm, and the occurrence of the flagellum. The midpiece of spermatozoa is short and contains spherical or ovoid mitochondria. The sperm flagellum has an axoneme with nine doublets of peripheral microtubules and two central microtubules. The result of this study is valuable to be used as a standard reference for germ cell development, which is of great importance to get a clear insight into the process of grayling breeding practice.

Time-resolved ICP-MS analysis of mineral element contents and distribution patterns in spermatogenic cells of different types.

Mineral elements play an important role in the spermatogenesis, maturation, and fertilization of sperm. It is of great scientific significance to study the role of mineral elements in spermatogenesis by accurately measuring the content of elements in different spermatogenic cells and analyzing the distribution pattern of elements in spermatogenesis. Here, time-resolved inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze the content and distribution patterns of mineral elements in spermatogenic cells of different types at the single cell level. Firstly, spermatogonia, spermatocytes, round spermatids and elongating spermatids were successfully isolated from testis of mice of different weeks of age by differential adherent method and discontinuous bovine serum albumin (BSA) density gradient method. Then, signal profiles and elemental distributions of 24Mg, 31P, 52Cr, 55Mn, 56Fe and 66Zn in spermatogenic cells were obtained with dwell time at 0.1 ms. Based on the results of acid digestion, we derived a formula to calculate element content in single cell from peak area for each element, and the feasibility and universality of the formula in the quantitative detection of single cell elements were verified by sperm samples to a certain extent. The detection results of element content in single cell showed that the content of 31P in elongating spermatids was significantly higher than that in spermatogonia, spermatocytes and round spermatids (P < 0.01), and the distribution range was wider. However, the 52Cr and 56Fe content of elongating spermatids was lower than that of spermatogonia, spermatocytes and round spermatids (P < 0.05). When spermatogonia developed into round spermatids, the contents of 55Mn and 66Zn in single cell increased significantly (P < 0.05), then decreased to the lowest in elongating spermatids. In addition, the significant decrease of 52Cr, 55Mn, 56Fe and 66Zn content in elongating spermatids also be visually observed from the center of the fitting curve of the element signal intensity distribution moving to the left. This study provides an elemental view of the changes in elemental content at various stages of spermatogenesis at the single-cell level. Time-resolved ICP-MS is used to detect mineral elements content and distribution patterns in spermatogenic cells of testis, which is helpful to better explore the stages and modes of action of various elements in spermatogenesis, and provide direct evidence for revealing the effects of element content changes on spermatogenesis and semen quality regulation.

Effects of imidacloprid-contaminated feed exposure on spermatogenic cells and Leydig cells in testes of adult male rabbits (Oryctolagus cuniculus).

This research was undertaken to assess the results of repeated exposure to the insecticide; imidacloprid (IMI)-contaminated feed on testicular tissue, spermatogenic cell population, Leydig cell number, and sperm morphology in adult male rabbits (n = 24). The treatment groups received IMI (Bildor® 100 mg/L water spray on green grass)-contaminated green grass without wash (n = 8, not-washed-feed rabbit group) and after wash (n = 8, washed-feed rabbit group) once daily for two weeks on an alternate day basis. The rest of the rabbits, as control, received a normal pesticide-free standard feed. During the exposure time, there was no evident toxic symptom found on regular monitoring of IMI-treated rabbits. Histopathologically, the thickness of tunica albuginea of testes reduced significantly with loosely arranged connective tissues in IMI-treated rabbits. Within the testes, the bizarre-shaped seminiferous tubules were seen with increased lumen diameter in IMI-treated rabbits. The spermatogenic cells were disorganized and detached from the basement membrane in seminiferous tubules of IMI-exposed testes of rabbits. The spermatogenic cell population decreased significantly (P < 0.05) in IMI-treated rabbits compared to control rabbits. Leydig cell number decreased significantly (P < 0.05) in IMI-treated rabbits. A high percentage of morphologically abnormal spermatozoa was seen in IMI-treated rabbits. The degree of the histopathological changes was more prominent in the testes of IMI-exposed not-washed-feed rabbits. The results showed that insecticide-IMI has toxicological effects on testicular tissues, mainly spermatogenic and Leydig cell population of adult rabbits which may cause infertility. A short running title: Effect of imidacloprid on testicular tissue of rabbits.

[Expression of Zfx in mouse testicular spermatogenic cells].

OBJECTIVE: To investigate the expression of Zfx gene in spermatogenic cells. METHODS: The testes of d6, d8, d17 and adult mice were collected, single cell suspension was prepared by combinatorial enzyme digestion, spermatogenic cells were isolated by BSA density gradient method, and Zfx expression was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western Blot (WB). RESULTS: Single cell suspension prepared by combination enzyme digestion method and density gradient method laid with BSA can obtain various types of spermatogenic cells with purity>85%; The expression level of the Zfx gene is low in primitive type A spermatogonia, type A spermatogonia, and type B spermatogonia, whereas it is high in preleptotene spermatocytes, pachytene spermatocytes, and round spermatid cells. It is not expressed in elongating spermatids and mature sperm. CONCLUSION: Zfx gene exhibits periodic expression in various levels of spermatogenic cells and may be an important transcription factor involved in regulating meiosis in spermatogenic cells.

Regulation of spermatogenic cell apoptosis by the pro-apoptotic proteins in the testicular tissues of mammalian and avian species.

Apoptosis of germ cells is an important feature of spermatogenesis, as this process allows the removal of excess germ cells from testicular tissue. This is crucial to control the number of germ cells that can be supported and nourished by the Sertoli cells. It has been established that up to 75 % of germ cells are lost during the development of spermatogonia. In this process, germ cells with defective genes are removed. Also, apoptosis regulates homeostasis of testicular tissue by maintaining a balance between germ cell proliferation and cell death. This is necessary as it guarantees normal spermatogenesis. Apoptosis also occurs during maturation divisions of spermatocytes and spermatids but albeit to a lesser extent. Several factors, known pro-apoptotic proteins, play a critical role in the process of apoptosis. The most vital pro-apoptotic proteins are caspase-3, B-cells lymphoma 2 (Bcl2), truncated BH3 interacting death domain (tBID), tumor suppressor protein (p53), and Bcl-2 associated protein (BAX). Execution of apoptosis may be triggered by either an extrinsic or an intrinsic pathway. The extrinsic pathway is initiated by death receptors and death ligands. Death receptors trigger pro-apoptotic proteins such as caspase-3 for the execution of apoptosis. The intrinsic pathway, on the other hand, is triggered by nutrient deprivation, stress, or DNA damage, which in turn activates Bcl2 families of pro-apoptotic proteins that foster apoptosis. The present review focuses on pro-apoptotic proteins and their mechanisms of action, with special emphasis on their involvement in germ cell apoptosis in the testicular tissues of mammalian and avian species.

Exposure to automobile exhaust-derived PM2.5 induces spermatogenesis dysfunction by damaging UPRmt of prepubertal rats.

Automobile exhaust-derived particulate matter 2.5 (PM2.5) can cause spermatogenic cell damage, potentially resulting in male infertility. This study uses male prepubertal Sprague Dawley (SD) rats to explore the molecular mechanisms by which automobile exhaust-derived PM2.5 causes spermatogenic cell damage and induces spermatogenesis dysfunction during sexual maturity by disrupting the mitochondrial unfolded protein response (UPRmt) in spermatogenic cells. Male prepubertal SD rats were randomly divided into four groups: control (intratracheal instillation of normal saline), low-dose PM2.5 (5 mg/kg), high-dose PM2.5 (10 mg/kg), and PM2.5 10 mg/kg +Vit (100 mg/kg of vitamin C and 50 mg/kg of vitamin E). The rats were treated for four weeks, with five consecutive treatment days and two non-treatment days, followed by cohabitation. Testicular and epididymal tissues were harvested for analysis. The mitochondria in spermatogenic cells were observed under an electron microscope. UPRmt-, oxidative stress-, and apoptosis-related markers in spermatogenic cells were examined. Spermatogenic cell numbers and conception rate declined significantly with increasing PM2.5 dose, with their mitochondria becoming vacuolated, swollen, and degenerated to varying degrees. The apoptosis of spermatogenic cells was abnormally enhanced in PM2.5 exposed groups compared to the control group. Spermatogenic cell numbers of conception rate gradually recovered, mitochondrial damage in spermatogenic cells was alleviated, and spermatogenic cell apoptosis was significantly reduced after vitamin intervention. In addition, protein levels of superoxide dismutase 1 (Sod1), nuclear factor erythroid 2-related factor 2 (Nrf2), and B-cell lymphoma 2 (Bcl-2) were significantly lower, while those of Bcl2-associated X apoptosis regulator (Bax), cleaved caspase 3 (Casp3), and cytochrome c (Cyt-c) and malondialdehyde (MDA) levels were significantly higher in the high-dose PM2.5 group than in the control group. The levels of UPRmt-related proteins C/EBP homologous protein (Chop), heat shock protein 60 (Hsp60), and activating transcription factors 4 (Atf4) and 5 (Atf5) were higher in the low-dose PM2.5 group, lower in the high-dose PM2.5 group, and gradually recovered in PM2.5 10 mg/kg +Vit group. Our results show that exposure to automobile exhaust-derived PM2.5 induces oxidative stress responses, leads to post-sexual maturation UPRmt dysfunction and mitochondrial impairment, and abnormally enhances spermatogenic cell apoptosis in prepubertal rats, resulting in male infertility.

Quantitative evaluation of spermatogenesis by fluorescent histochemistry.

Identifying the types of spermatogenic cells that compose seminiferous tubules, as well as qualitative confirmation of the presence or absence of disorders, has been regarded as crucial in spermatogenesis. Sperm count and fertilizing capacity, both of which depend on the quality as well as quantity of spermatogenesis, are factors critical to fertilization. However, the quantitative assessment of spermatogenesis is not commonly practiced. Spermatogenesis has species-specific stages; when the specific stage in the seminiferous tubules is precisely determined, the types of spermatogenic cells in each stage can be spontaneously identified. Thereafter, a unique marker is used to classify the cells observed in each stage. Quantitative assessment of spermatogenesis has the potential to detect inapparent spermatogenesis disorders or numerically indicate the degree of the disorder. To this end, a histochemical approach using unique markers is indispensable for the quantitative assessment of spermatogenesis. Future developments in techniques to measure cell populations using computer software will further facilitate the establishment of quantitative assessment of spermatogenesis as a standard analysis method that can contribute significantly to advance our understanding of spermatogenesis.

[Expressions of FOXO1/3a/4/6 in human testis and their roles in testicular aging].

OBJECTIVE: To investigate the expression rules of FOXO1a, FOXO3a, FOXO4 and FOXO6 proteins in the human testis, and explore their roles in the development and progression of testicular aging. METHODS: We collected the para-carcinoma testis tissue from 4 testis cancer patients aged 28, 31, 32 and 46 years, and the testis tissue from another 2 PCa patients aged 66 and 81 years after castration surgery from January 2018 to December 2020. We detected the expressions of FOXO1a, FOXO3a, FOXO4 and FOXO6 proteins in the testis tissue by Western blot, determined the locations of FOXO1a, FOXO3a, FOXO4 and FOXO6 in the testis cells by immunofluorescence staining, and performed semi-quantitative and statistical analyses using image J and SPSS 23.0 software, respectively. RESULTS: The expression levels of FOXO1a and FOXO3a proteins were significantly decreased in the testis tissue of the elderly patients (P < 0.01), with an age-dependent reduction in the proportion of the positive cells. No statistically significant difference was observed in the expression levels of FOXO4 and FOXO6 between different age groups. FOXO1a was mainly expressed at the base of the seminiferous tubules, FOXO3a and FOXO4 in the Leydig cells, and FOXO6 in the seminiferous tubules. In addition, FOXO4 underwent age-related nuclear translocation in the senescent Leydig cells, suggesting its involvement in the aging of Leydig cells. CONCLUSION: FOXO1a/3a/4 may be closely related to human testicular aging and corresponding pathological changes, but its underlying mechanism remains to be further explored.

Structural observations on spermatogenic cells of Japanese quail (Coturnix coturnix japonica) pre-pubertally exposed to dibutyl phthalate: A light and transmission electron microscopy study.

Exposure to dibutyl phthalate (DBP) induces testicular damage in mammals. However, studies on the effects of DBP on spermatogenic cells in birds are grossly lacking. Therefore, this study was designed to determine the effects of the pre-pubertal exposure to DBP on the histology and ultrastructure of spermatogenic cells in the testis of adult Japanese quail (Coturnix coturnix japonica). The birds were randomly divided into five dosage groups at the age of 4 weeks. The control group received a corn oil vehicle only (a dose of 1 mL/kg body weight), while the other four experimental groups received a daily dosage of 10, 50, 200, 400 mg/kg body weight of DBP (dissolved in corn oil), respectively with the aid of gastric lavage, for 30 days. Testicular samples were processed and examined by light microscopy and transmission electron microscopy. Histopathological evaluation revealed vacuole formation, germ cell degenerations, and the absence of spermatogenic cell series. Ultrastructurally, chromatin clumps in spermatocyte and degenerated spermatogonia with ruptured nuclear membranes resting on the distorted basement membranes were observed. Others were intracytoplasmic vacuoles in round spermatids and fragments of dense apoptotic bodies. In conclusion, the findings of the present study reveal that spermatogenic cells of Japanese quails seem to be more sensitive to DBP-induced degeneration compared to mammalian species studied. The Japanese quail could be used to monitor environmental contamination with low doses of DBP.

[Antagonistic effect of modified Dahuang Zhechong Granule against the apoptosis of epididymal spermatogenic cells in varicocele rats based on Nrf2/HO-1 pathways].

OBJECTIVE: To investigate the effects of modified Dahuang Zhechong Granule (DZG) on the epididymal tissue of varicocele (VC) rats and the expressions of the nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) and heme oxygenase-1 (HO-1) protein. METHODS: Sixty SD rats were randomly divided into six groups of an equal number: sham operation, VC model control, aescuven forte (AF) and low-, medium- and high-dose DZG. The VC model was established by ligation of the left renal vein with the Turner's method, followed by intragastrical administration of normal saline to the rats in the sham operation and VC model control groups, AF Tablets at 54 mg/kg to those in the AF group, and modified DZG at 0.6, 1.2 and 2.4 g/ml to those in the low-, medium- and high-dose DZG groups respectively, all once daily for 8 weeks. Then, all the animals were sacrificed and their left epididymides harvested for examination of semen quality, observation of local ultrastructural changes, measurement of the apoptosis of spermatogenic cells by Annexin V-FITC, and determination of the expressions of Nrf2 and HO-1 in the epididymal tissue by immunohistochemistry. RESULTS: Evident pathological damage was observed in the left epididymal tissue of the VC model controls, with significantly reduced numbers of spermatogenic cells and sperm at all levels, partially destroyed cellular structure, and disappearance of some subcellular structures such as the lysosome, mitochondrion, endoplasmic reticulum, nucleus and cell membrane, which were all improved to some extent in the DZG and AF group. Sperm concentration and motility in the left epididymis were significantly higher in the medium- and high-dose DZG and AF groups than in the VC model controls (P < 0.05), even more significantly in the high-dose DZG than in the AF group (P < 0.05). The apoptosis rate of spermatogenic cells was markedly higher in the VC model control than in the sham operation group (P < 0.05), but lower in the medium- and high-dose DZG and AF groups than in the VC model controls (P < 0.05). Immunohistochemistry showed positive expressions of Nrf2 and HO-1 proteins, brown, scattered and with a low luminance of the cells, in the left epididymis tissue of the VC model control rats, but with a significantly higher cell luminance in the high-dose DZG and AF groups. CONCLUSIONS: Modified Dahuang Zhechong Granule can effectively repair pathological damage to the epididymis of varicocele rats, increase the expressions of Nrf2 and HO-1 proteins, antagonize the apoptosis of spermatogenic cells and provide a favorable condition for sperm maturation.

[Efficacy of Zhibai Dihuang Decoction on energy metabolism of spermatogenic cells in Ureaplasma urealyticum-infected rats: A study based on AMPK and PPARα signaling pathways].

OBJECTIVE: To study the effect of the serum containing Zhibai Dihuang Decoction (ZDD) on the energy metabolism of spermatogenic cells infected with Ureaplasma urealyticum (UU) in rats and its action mechanism. METHODS: Healthy male SD rats were randomly divided into six groups, normal control, UU-infection (UUI) model control, doxycycline, and low-, medium- and high-dose ZDD-containing serum. After successful establishment of the UUI model in vivo in the latter five groups, the rats in the normal control group were treated with simple serum and those in the latter five with respective agents. Then primary spermatogenic cells were harvested from the rats for examination of the apoptosis of spermatogenic cells, contents of lactate dehydrogenase (LDH) and adenosine triphosphate (ATP), glucose disposal rate (GDR) and expressions of AMPK and PARα proteins in the spermatogenic cells, and other related parameters. RESULTS: The apoptosis rate of the spermatogenic cells was dramatically increased in the UUI model controls compared with that in the normal controls (ï¼»49.24 ± 0.86ï¼½% vs ï¼»10.09 ± 0.52ï¼½%, P < 0.01), but significantly decreased in the doxycycline and low-, medium- and high-dose ZDD groups (ï¼»11.21 ± 1.02ï¼½%, ï¼»30.64 ± 0.99ï¼½%, ï¼»35.54 ± 1.17ï¼½% and ï¼»42.95 ± 1.31ï¼½%) in comparison with that in the UUI model control group (P < 0.01).The content of LDH in the spermatogenic cells was also remarkably increased in the UUI model controls compared with that in the normal controls (ï¼»201.12 ± 2.88ï¼½ vs ï¼»60.72 ± 1.83ï¼½) mU/ml, P < 0.01), but significantly decreased in the doxycycline and low-, medium- and high-dose ZDD groups (ï¼»90.66 ± 1.61ï¼½, ï¼»94.74 ± 1.20ï¼½, ï¼»101.24 ± 2.03ï¼½ and ï¼»111.04 ± 3.35ï¼½ mU/ml) in comparison with that in the UUI model control group (P < 0.01). The GDR in the spermatogenic cells was markedly reduced in the UUI model controls compared with that in the normal controls (ï¼»49.42 ± 1.70ï¼½% vs ï¼»99.86 ± 1.26ï¼½%, P < 0.01), but significantly elevated in the doxycycline and low-, medium- and high-dose ZDD groups (ï¼»86.90 ± 2.03ï¼½%, ï¼»84.14 ± 1.21ï¼½%, ï¼»80.30 ± 1.37ï¼½% and ï¼»75.18 ± 1.76ï¼½% in comparison with that in the UUI model control group (P < 0.01). The content of ATP was also dramatically decreased in the UUI model controls compared with that in the normal controls (ï¼»19.76 ± 1.46ï¼½ vs ï¼»58.94 ± 1.95ï¼½ µmol/L, P < 0.01), but significantly increased in the doxycycline and low-, medium- and high-dose ZDD groups (ï¼»48.34 ± 1.34ï¼½, ï¼»42.82 ± 1.30ï¼½, ï¼»38.70 ± 2.03ï¼½ and ï¼»34.78 ± 0.82ï¼½ µmol/L) in comparison with that in the UUI model control group (P < 0.01). The mitochondrial membrane potential was remarkably elevated in the UUI model controls compared with that in the normal controls (ï¼»8.53 ± 0.71ï¼½% vs ï¼»2.43 ± 0.25ï¼½%, P < 0.01), but markedly reduced in the doxycycline and low-, medium- and high-dose ZDD groups (ï¼»3.92 ± 0.36ï¼½%, ï¼»4.43 ± 0.27ï¼½%, ï¼»4.65 ± 0.22ï¼½% and ï¼»4.88 ± 0.10ï¼½% in comparison with that in the UUI model control group (P < 0.01). The phosphorylation levels of AMPK and PPARα proteins were significantly up-regulated in the UUI model controls compared with that in the normal controls (P < 0.01), but down-regulated in a dose-dependent manner in the ZDD groups. CONCLUSIONS: Zhibai Dihuang Decoction can significantly improve the damage to the mitochondrial structure and inhibit UU infection-induced apoptosis of spermatogenic cells and secretion of LDH by increasing the ATP content and GDR and regulating the phosphorylation of AMPK and PARα signaling pathway.

Effects of Environmental and Pathological Hypoxia on Male Fertility.

Male infertility is a widespread health problem affecting approximately 6%-8% of the male population, and hypoxia may be a causative factor. In mammals, two types of hypoxia are known, including environmental and pathological hypoxia. Studies looking at the effects of hypoxia on male infertility have linked both types of hypoxia to poor sperm quality and pregnancy outcomes. Hypoxia damages testicular seminiferous tubule directly, leading to the disorder of seminiferous epithelium and shedding of spermatogenic cells. Hypoxia can also disrupt the balance between oxidative phosphorylation and glycolysis of spermatogenic cells, resulting in impaired self-renewal and differentiation of spermatogonia, and failure of meiosis. In addition, hypoxia disrupts the secretion of reproductive hormones, causing spermatogenic arrest and erectile dysfunction. The possible mechanisms involved in hypoxia on male reproductive toxicity mainly include excessive ROS mediated oxidative stress, HIF-1α mediated germ cell apoptosis and proliferation inhibition, systematic inflammation and epigenetic changes. In this review, we discuss the correlations between hypoxia and male infertility based on epidemiological, clinical and animal studies and enumerate the hypoxic factors causing male infertility in detail. Demonstration of the causal association between hypoxia and male infertility will provide more options for the treatment of male infertility.

Histology and structure of the testicles in three species of Atelopus frogs (Anura: Bufonidae) endemic to the Sierra Nevada de Santa Marta, Colombia / Histología y estructura de los testículos en tres especies de ranas Atelopus (Anura: Bufonidae) endémicas de la Sierra Nevada de Santa Marta, Colombia

Abstract Introduction: Testicular histology constitutes one of the least explored aspects in frogs of the genus Atelopus. This taxonomic group shows an alarming population decline; therefore, its reproductive biology is one of the greatest topics of interest for its conservation. Objective: To describe the testicular morphology and the spermatogenetic lineage cells in adult males of Atelopus laetissimus, Atelopus nahumae, and Atelopus carrikeri in the Sierra Nevada de Santa Marta, Colombia. Methods: During June - July 2017 and 2018, sampling was conducted in the localities of San Lorenzo and Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), to collect 15 adult males, 5 per species. Testes samples were fixed in Bouin to be processed by the standard paraffin-embedding technique. Histological sections (3 μm) were stained with Hematoxylin-eosin and Mallory-Heidenhain-Azan-Gomori's. For the description and photographic register of the germ cells, the photonic microscopy technique was used with the differential interference contrast system. Results: The testes are oval organs, compact, light yellow color, and with little vascularization. Externally, they are surrounded by a thin albuginea tunic constituted by regular dense connective tissue. Inside this layer, they are composed of numerous seminiferous tubules of hexagonal contour, in which germ cell cysts are distinguished at different stages of spermatogenesis (spermatogonia I and II, spermatocyte I and II, and early and late spermatids) and spermiogenesis (spermatozoa in fascicles and free spermatozoa). Separating the seminiferous structures is the interstitial tissue in which Leydig cells and blood vessels stand out. Additionally, in the cranial part of the testis, the Bidder's organ was found, formed by two distinguishable regions, the cortex and the medulla. In the cortex, there are previtellogénic oocytes of different sizes surrounded by a monolayer of flat follicular cells. For its part, the medullary region is the connective tissue that nourishes the oocytes and is constituted by blood capillaries. Conclusions: The gonads of the three species analyzed present a cystic cellular organization similar to other anurans, where all stages of spermatogenesis and spermiogenesis were identified, possibly indicating a continuous reproductive activity. Likewise, the Bidder's organ is reported for the first time in the three Atelopus species, which allows suggesting a possible sexual reversion in case of a population decrease of females as a reproductive strategy.

Resumen Introducción: La histología testicular constituye uno de los aspectos menos explorados en las ranas del género Atelopus. Este grupo taxonómico ostenta un declive poblacional alarmarte, es por ello, que su biología reproductiva resulta uno de los temas de mayor interés para su conservación. Objetivo: Describir la morfología testicular y las células del linaje espermatogénico en machos adultos de Atelopus laetissimus, Atelopus nahumae y Atelopus carrikeri en la Sierra Nevada de Santa Marta, Colombia. Métodos: Durante Junio - Julio de 2017 y 2018 se realizaron muestreos en las localidades de San Lorenzo y Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), para recolectar 15 machos adultos, 5 por especie. Las muestras de testículo se fijaron en Bouin para ser procesadas mediante la técnica estándar de inclusión en parafina. Las secciones histológicas (3 μm) se tiñeron con Hematoxilina-eosina y Mallory-Heidenhain-Azan-Gomori's. Para la descripción y registro fotográfico de las células germinales, se utilizó la técnica de microscopía fotónica con el sistema de contraste diferencial de interferencia. Resultados: Los testículos son órganos ovalados, compactos, de color amarillo claro y con poca vascularización. Externamente, están rodeados por una delgada túnica albugínea constituida por tejido conectivo denso regular. Al interior de esta capa se componen por numerosos túbulos seminíferos de contorno hexagonal, en los que se distinguen quistes de células germinativas en diferentes etapas de la espermatogénesis (espermatogonia I y II, espermatocito I y II y espermátidas tempranas y tardías) y espermiogénesis (espermatozoides en fascículos y espermatozoides libres). Separando las estructuras seminíferas se halla el tejido intersticial en el que se destacan las células de Leydig y los vasos sanguíneos. Adicionalmente, en la parte craneal del testículo se encontró el órgano de bidder formado por dos regiones diferenciables, la corteza y la medula. En la corteza se aprecian ovocitos previtelogénicos en diferente tamaño rodeados por una monocapa de células foliculares planas. Por su parte, la región medular es el tejido conectivo que nutre los ovocitos y está constituido por capilares sanguíneos. Conclusiones: Las gónadas de las tres especies analizadas presentan una organización celular quística de manera similar con otros anuros, donde se identificó todos los estadios de la espermatogénesis y espermiogénesis indicando posiblemente una actividad reproductiva continua. Así mismo, se reporta por primera vez el órgano de bidder en las tres especies de Atelopus, lo cual permite sugerir una posible reversión sexual en caso de una disminución poblacional de las hembras como una estrategia reproductiva.

Sub-fertility in crossbred bulls: Identification of proteomic alterations in spermatogenic cells using high throughput comparative proteomics approach.

The present study was carried out to compare the proteomic profiles of spermatogenic cells of crossbred and zebu cattle in an effort to understand the possible reasons for a higher incidence of sub-fertility in crossbred bulls. The spermatogenic cells collected from the testes of pre-pubertal (6 mo) and adult (24 mo) crossbred and zebu males through fine needle aspiration were proliferated in vitro, and proteomic profiling was done using a shotgun proteomics approach. The age- and species-specific variations in the expression level of proteins were identified in spermatogenic cells. The number of differentially expressed proteins (DEPs) identified in pre-pubertal zebu and crossbred was 546, while 579 DEPs were identified between adult zebu and crossbred bulls. Out of these, 194 DEPS were common to these groups and 40 DEPs displayed a fold change ≥2. However, only 20 proteins exhibited similar expression variation trends (upregulated or downregulated) among pre-pubertal as well as adult zebu and crossbred bulls. Out of these 20 DEPs, 13 proteins were upregulated, and 7 proteins were downregulated in spermatogenic cells of zebu compared to crossbred bulls. Among the upregulated proteins were RPLP2, PAXIP1, calumenin, prosaposin, GTF2F1, TMP2, ubiquitin conjugation factor E4A, COL1A2, vimentin, protein FAM13A, peripherin, GFPT2, and GRP78. Seven proteins that were downregulated in zebu bulls compared to crossbred included APOA1, G patch domain-containing protein 1, NAD P transhydrogenase mitochondrial, glutamyl aminopeptidase, synaptojanin 1 fragment, Arf GAP with SH3 domain ANK repeat and PH domain-containing protein 1, and protein transport protein sec16B. It was inferred that the proteins associated with sperm function and fertilization processes, such as calumenin, prosaposin, vimentin, GRP78, and APOA1 could be studied further to understand the precise cause of subfertility in crossbred bulls.

CAG repeat instability in embryonic stem cells and derivative spermatogenic cells of transgenic Huntington's disease monkey.

PURPOSE: The expansion of CAG (glutamine; Q) trinucleotide repeats (TNRs) predominantly occurs through male lineage in Huntington's disease (HD). As a result, offspring will have larger CAG repeats compared to their fathers, which causes an earlier onset of the disease called genetic anticipation. This study aims to develop a novel in vitro model to replicate CAG repeat instability in early spermatogenesis and demonstrate the biological process of genetic anticipation by using the HD stem cell model for the first time. METHODS: HD rhesus monkey embryonic stem cells (rESCs) were cultured in vitro for an extended period. Male rESCs were used to derive spermatogenic cells in vitro with a 10-day differentiation. The assessment of CAG repeat instability was performed by GeneScan and curve fit analysis. RESULTS: Spermatogenic cells derived from rESCs exhibit progressive expansion of CAG repeats with high daily expansion rates compared to the extended culture of rESCs. The expansion of CAG repeats is cell type-specific and size-dependent. CONCLUSIONS: Here, we report a novel stem cell model that replicates genome instability and CAG repeat expansion in in vitro derived HD monkey spermatogenic cells. The in vitro spermatogenic cell model opens a new opportunity for studying TNR instability and the underlying mechanism of genetic anticipation, not only in HD but also in other TNR diseases.