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Stage- and cell type-specific biomarkers are important for understanding spermatogenesis in mammalian testis. The present study identified several testicular cell marker proteins in 6- and 24-month old bovine testes. In 6-month old bovine testes, spermatogonia and spermatocytes were detected but complete spermatogenesis occurred in 24-month old testes. The diameters of the seminiferous tubules increased significantly in the 24-month old testes compared with those in the 6-month old testes. Protein Gene Product 9.5 (PGP9.5), also known as the undifferentiated spermatogonium marker, and GATA4 (GATA binding protein 4), vimentin, and SOX9 (SRY-Box Transcription Factor 9) were detected in the basement membrane region. Interestingly, ID4 (inhibitor of DNA binding protein 4; previously known as the undifferentiated cell marker) proteins were located in the basement membrane region but their expression patterns were different from those of PGP9.5. Co-immunohistochemistry results showed that ID4 was detected in the Sertoli cells expressing vimentin and SOX9 in 6- and 24-month old bovine testes. This result indicated that ID4 is a putative biomarker of Sertoli cell in the bovine system, which is different from the rodent models. Thus, these results will contribute in understanding the process of spermatogenesis that is different in bovines compared to other species., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The reproductive toxicity of 4-octylphenol (4-OP) has been studied in animals such as mouse and fish. In humans, the exposure of sperm to 4-OP has been shown to decrease motility and viability. In this study, we performed an in vitro assessment of the toxic effects of 4-OP on mouse TM4 Sertoli cells and investigated the underlying molecular mechanisms. TM4 cells were treated with four concentrations (0, 10, 30, and 50 µM) of 4-OP at the following time points: 24, 48, and 72 h. Cell viability and apoptosis assays were conducted following 4-OP exposure. We found that 4-OP significantly decreased cell viability in a concentration- and time-dependent manner, and increased apoptosis. Quantitative PCR analysis showed that the mRNA expression levels of BCL2 Associated X, Apoptosis Regulator (Bax) and BCL2 Antagonist/Killer (Bak) increased while that of BCL2 Apoptosis Regulator (Bcl-2) decreased in 4-OP-exposed cells compared with that in the controls. Western blotting revealed that 4-OP induced caspase-3 activity and Bad phosphorylation in a concentration- and time-dependent manner. Additionally, cytochrome C protein did not colocalize with mitochondrial marker dye by 24 h. Cytochrome c protein expression increased in a time-dependent manner upon exposure to 50 µM 4-OP. These results suggest that 4-OP induces mitochondria-mediated apoptosis through regulation of Bcl-2 family proteins and caspase-3 activation in male Sertoli cells.

Chemotherapy is used for childhood cancer but may lead to infertility in patients. Spermatogonia stem cells are present in the testes of prepubertal boys, although they do not produce sperm at this age. Herein, we evaluated the toxicity of cisplatin, a known medicine for cancer treatment, in neonatal mouse testes using in vitro organ culture. Mouse testicular fragments (MTFs) derived from 5.5-d postpartum mouse testes were exposed to 1–10 μg/mL cisplatin. The results showed that cisplatin significantly downregulated the expression of germ cell marker genes, including differentiated and undifferentiated, in a dose-dependent manner. In particular, a high dose of cisplatin (10 μg/mL) led to germ cell depletion. In addition, the expression levels of the Sertoli cell marker gene, the number of SOX9+ Sertoli cells, and the levels of SOX9 protein were markedly decreased in cisplatin-treated MTFs compared to controls. The mRNA expression of steroidogenic enzyme-related genes significantly increased in cisplatin-treated MTFs, except for estrogen receptor 1 (Esr1). Consistently, 3β-hydroxysteroid dehydrogenase protein was also observed in the interstitial regions of cisplatin-treated MTFs. Altogether, our findings showed a significant impairment in germ cell development, Sertoli cell survival, and steroidogenesis in the MTFs of cisplatin-treated mice. [ABSTRACT FROM AUTHOR]

Cryptorchidism, a condition in which testes fail to descend from the abdomen into the scrotum, is a risk factor for infertility and germ cell cancer. Normally, tight junctions between adjacent Sertoli cells in the testes form a blood–testes barrier that regulates spermatogenesis; however, the effect of cryptorchidism on tight junctions is not well-understood. We established a model of heat-induced testicular damage in dogs using surgical cryptorchidism. We sequenced RNA to investigate whether certain transcripts are expressed at higher rates in heat-damaged versus normally descended testes. Claudins, cell adhesion molecules, were relatively highly expressed in cryptorchid testes: claudins 2, 3, 5, 11, and 18 were significantly increased in cryptorchid testes and reduced by orchiopexy. SOX9-positive Sertoli cells were present in the seminiferous tubules in both cryptorchid and control testes. Using real-time PCR and Western blot analysis to compare Sertoli cells cultured at 34 °C and 37 °C, we found that Sertoli cell claudins 2, 3, 5, 11, and 18 were significantly increased at 37 °C; however, accumulation was higher in the G0/G1 phase in Sertoli cells cultured at 34 °C. These results indicate that testicular hyperthermia caused by cryptorchidism affects claudin expression, regulated germ cell death, and the proliferation of Sertoli cells. [ABSTRACT FROM AUTHOR]

Cerium oxide nanoparticles (CeO 2 NPs) are lanthanide element oxides widely used in industrial processes, and environmental exposure to these nanomaterials highlights the potential risks to human health. In the present study, we evaluated the toxicity of CeO 2 NPs in neonatal mouse testes using in vitro organ cultures. Mouse testicular fragments (MTFs) derived from 5-day postpartum mouse testes were exposed to 0–50 μg/mL CeO 2 NPs for 5 days. We observed that CeO 2 NPs significantly reduced the number of undifferentiated and differentiated germ cells in MTFs in a dose-dependent manner. Compared with the control, CeO 2 NPs downregulated expression levels of both undifferentiated and differentiated germ cell marker genes. Although treatment with 10–30 μg/mL CeO 2 NPs did not alter Sertoli cell numbers, 50 μg/mL CeO 2 NP reduced the cell number and mRNA expression of Sox9 and AMH in MTFs. Accordingly, protein levels of Sox9 were also reduced in MTFs exposed to 50 μg/mL CeO2NPs when compared with those exposed to other treatments. Exposure of MTFs to 50 μg/mL CeO2NPs decreased mRNA expression levels of steroidogenic enzymes, such as Cyp17α1 and HSD3b1. Furthermore, the expression of Insl3, a Leydig cell-specific marker gene, decreased with increasing CeO 2 NP concentrations, although interstitial cells were still observed in MTFs exposed to 50 μg/mL CeO2NPs. Collectively, our findings revealed that CeO 2 NPs could negatively impact prepubertal spermatogenesis and germ cell maintenance via germ cell depletion, and high doses could damage Sertoli cells and disturb steroidogenesis in MTFs. • In vitro cerium oxide nanoparticle (CeO2NP) toxicity was assessed in neonatal testes. • CeO2NPs dose-dependently reduced undifferentiated and differentiated germ cells. • CeO2NPs dose-dependently downregulated the expression of germ cell markers. • High-dose CeO2NPs (50 μM) downregulated Sertoli and Leydig cell-specific markers. • Low-dose CeO2NPs (10–30 µM) did not impact Sertoli and Leydig cell-specific markers. [ABSTRACT FROM AUTHOR]

Resmethrin is a widely used pyrethroid insecticide, which causes low toxicity in mammals. However, its toxicity in testes has not been fully investigated. Therefore, we evaluated the toxicity of resmethrin in mouse testes using an in vitro organ culture. Mouse testicular fragments (MTFs) derived from neonates were cultured in medium containing resmethrin for 30 days. Effects on spermatogenesis in the cultured testes were investigated as functions of both time and dose. Resmethrin significantly downregulated the transcription levels of marker genes for spermatogonia and the number of spermatogenic germ cells relative to those of the controls, according to quantitative PCR and immunostaining. In addition, spermatocyte was observed in the control, but not in 50 μM resmethrin-exposed cultures. Levels of the SYCP3 meiotic marker and phosphorylated H2AX decreased by resmethrin treatment, as observed by Western blotting. Toxic or apoptotic effects of resmethrin in Sertoli and Leydig cells from MTFs were not observed by immunostaining and Tunnel assay. No changes in the expression of steroidogenic enzymes were noted. Apoptosis was only detected in the germ cells of resmethrin-treated MTFs. Thus, the highest dose of resmethrin tested (50 μM) completely inhibited spermatogenesis, because of apoptosis of germ cells and spermatocytes. Although the in vivo toxicity of resmethrin has not yet been studied in detail, significant evidence for cytotoxicity was observed in our organ cultures. This methodological approach is useful for the study of reproductive toxicity before proceeding to animal models, as it greatly reduces the use of laboratory animals. [ABSTRACT FROM AUTHOR]

• Spermatozoa were not detected in cryptorchid pig testes. • Undifferentiated spermatogonia were significantly reduced in cryptorchid pig testes. • Spermatocyte and spermatid were significantly reduced in cryptorchid pig testes. • Vimentin expressed Sertoli cells not changed in cryptorchid pig testes. Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility. [ABSTRACT FROM AUTHOR]

T-2 mycotoxin, a type A trichothecene toxin that, specifically, causes male and female reproductive toxicity. We evaluated T-2 toxin toxicity in testes from neonatal testes after in vitro tissue cultured. Additionally, current study focuses on the molecular mechanism of toxicity and germ cell damage in GC-1 spermatogonial cells. Mouse testicular fragments were subjected to T-2 toxin (0–20 nM) during days 5 of in vitro culture. Testicular germ cell number were reduced and downregulated the expression of corresponding markers depending on the exposure concentration of T-2 toxin; however, Sertoli cell markers and steroidogenic enzyme expression increased when treated with 20 nM T-2 toxin. The cell viability decreased, apoptosis increased, and pro-apoptotic protein expression increased in 5–20 nM T-2 toxin–exposed spermatogonia. Moreover, T-2 toxin generated reactive oxygen species (ROS) and induced mitochondrial dysfunction, indicating that activation of p38 MAPK signaling triggered by ROS is involved in the apoptotic molecular mechanism of T-2 toxin. T-2 toxin induced the phosphorylation of ERK1/2, c-Jun, JNK/SAPK, p38, and p53, and the subsequent inhibition of AKT phosphorylation. The upregulation of genes related to apoptosis and MAPK/JNK signaling was consistently observed in cells exposed to T-2 toxin. These results indicate that T-2 toxin triggers apoptotic cell death in germ cells through the triggering of ROS-mediated JNK/p38-MAPK signaling pathways. [Display omitted] • T-2 toxin induced Germ damage in neonatal testes using in vitro organ culture. • T-2 toxin exposed spermatogonia, cell viability and pro-apoptotic protein expression increased. • Mitochondrial ROS contribute to T-2 toxin induced spermatogonia apoptosis. • Activation of p38/MAPK in response to T-2 toxin triggers cell death by apoptosis. [ABSTRACT FROM AUTHOR]

Humans are exposed to a multitude of endocrine disruptor chemicals (EDCs) that can interfere with the action of endogenous hormones and the normal development of reproductive organs. Bisphenol A (BPA) is one of the most common EDCs found in the environment. Here, we evaluated BPA toxicity on fetal testes using an in vitro organ culture system. Mouse fetal testes sampled at 15.5 days post coitus were cultured in a medium containing BPA for 5 days. The number of germ cells was reduced by BPA treatment, whereas the number of Sertoli cells was slightly increased by BPA at the highest dose (100 μM). Consistently, BPA treatment reduced the protein and gene expression levels of germ cell markers, but it increased the expression levels of Sertoli cell markers. The expression levels of fetal Leydig cell markers such as Cyp11a1, Thbs2, Cyp17a1, and Pdgf-α were significantly increased, whereas those of adult Leydig cell markers such as Hsd17b3, Ptgds, Sult1e1, Vcam1, and Hsd11b1 were decreased in the testes exposed to BPA. Generally, Notch signaling restricts Leydig cell differentiation from progenitor cells during fetal testis development. The expression levels of Notch1, Notch2, Notch3, Hes1, Ptch1, Jag1, Jag2, c-Myc, Hey1, and Hey2, which are involved in Notch signaling, were markedly higher in BPA-treated fetal testes than in the controls, indicating that BPA interrupts fetal Leydig cell development. BPA also disrupted steroidogenesis in the fetal testis organ culture system. In conclusion, our study showed that BPA inhibits fetal germ cell growth, Leydig cell development, and steroidogenesis. Image 1 • BPA toxicity in fetal testes was assessed in an in vitro organ culture system. • Culture for 5 days in BPA reduced germ cells and slightly increased Sertoli cells. • BPA downregulated the expression of germ cell markers. • BPA upregulated the expression of Sertoli cell markers. • BPA increased Notch signaling gene levels to suppress fetal Leydig cell development. [ABSTRACT FROM AUTHOR]

Diuron is a phenylurea herbicide widely used in the agricultural industry. In recent years, the risk of infertility and developmental defects has increased due to exposure to environmental pollutants. In this study, we investigated the toxicity of diuron in fetal mouse testes using three-dimensional organ cultures. Fetal testes derived from embryonic day (E) 14.5 were cultured with 200 µM diuron for 5 days. The results revealed that diuron did not impair fetal germ cell proliferation or the expression levels of germ cell markers such as Ddx4, Dazl, Oct 4, Nanog, Plzf, and TRA 98. Similarly, the gene or protein expression of the Sertoli cell markers Sox9 and Wt1 in diuron-exposed fetal testes did not change after 5 days of culture. In contrast, diuron increased fetal Leydig cell markers (FLC), Cyp11a1, Cyp17a1, Thbs2, and Pdgf α, and decreased adult Leydig cell (ALC) markers, Sult1e1, Hsd173, Ptgds, and Vcam1. However, 3-βHSD, an FLC and ALC marker, was consistently maintained upon exposure to diuron in fetal testes compared to non-treated groups. In conclusion, our study demonstrates that diuron negatively impacts Fetal Leydig cell development, although it does not affect germ and Sertoli cells. • Germ cell and Sertoli cell proliferation were not affected by Diuron in in vitro cultured testes. • No significant difference in gene and protein expression of Germ cell and Sertoli cell marker by Diuron. • Diuron increased the expression of Fetal Leydig cells marker in in vitro cultured testes. • Diuron suppressed the expression of Adult Leydig cell marker in in vitro cultured testes. [ABSTRACT FROM AUTHOR]