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The mouse has been used as a model of human organogenesis with the tacit assumption that morphogenetic and molecular mechanisms in mice are translatable to human organogenesis. While many morphogenetic and molecular mechanisms are shared in mice and humans, many anatomic, morphogenetic, and molecular differences have been noted. Two critical gaps in our knowledge prevent meaningful comparisons of mouse versus human testicular development: (a) human testicular development is profoundly under-represented in the literature, and (b) an absence of a detailed day-by-day ontogeny of mouse testicular development from E11.5 to E16.5 encompassing the ambisexual stage to seminiferous cord formation. To address these deficiencies, histologic and immunohistochemical studies were pursued in comparable stages of mouse and human testicular development with a particular emphasis on Leydig, Sertoli and myoid cells through review of the literature and new observations. For example, an androgen-receptor-positive testicular medulla is present in the developing human testis but not in the developing mouse testis. The human testicular medulla and associated mesonephros were historically described as the source of Sertoli cells in seminiferous cords. Consistent with this idea, the profoundly androgen receptor (AR)-positive human testicular medulla was shown to be a zone of mesenchymal to epithelial transition and a zone from which AR-positive cells appear to migrate into the human testicular cortex. While mouse Sertoli and Leydig cells have been proposed to arise from coelomic epithelium, Sertoli (SOX9) or Leydig (HSD3B1) cell markers are absent from the immediate coelomic zone of the developing human testis, perhaps because Leydig and Sertoli cell precursors are undifferentiated when they egress from the coelomic epithelium. The origin of mouse and human myoid cells remains unclear. This study provides a detailed comparison of the early stages of testicular development in human and mouse emphasizing differences in developmental processes., (Copyright © 2022 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 μM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function., (Copyright © 2021. Published by Elsevier B.V.)

Insulin-like peptide 3 (INSL3) plays a key role in testicular descent in rodents, whereas in domestic animals, many aspects of the roles of INSL3 in reproductive organs after puberty are still unknown. This study was undertaken to (1) determine the quantitative changes of gene expression of testicular INSL3, its receptor (RXFP2), LH receptor, and 3β-hydroxysteroid dehydrogenase during and after puberty in normal male dogs; (2) compare the expressions of these substances in normal and cryptorchid dogs; and (3) localize the cells expressing INSL3 in normal and retained canine testes. Testes were obtained from small-breed normal male dogs (n = 56) and cryptorchid dogs (n = 22). Normal scrotal testes from the normal dogs (normal testes), retained testes from both the unilateral and bilateral cryptorchid dogs (retained testes), and scrotal testes of the unilateral cryptorchid dogs (cryptorchid scrotal testes) were used. We measured the concentrations of these testicular messenger RNAs (mRNAs) by quantitative real-time reverse transcription polymerase chain reaction, and an enzyme immunoassay was used for measuring INSL3 peptide. Immunohistochemistry for INSL3 peptide was done in paraformaldehyde-fixed frozen testicular tissue. In the normal dogs, total amount of INSL3 mRNA per testis tended to decrease (P = 0.05) from pubertal (6-12 months) to postpubertal (1-5 years) and decreased (P < 0.01) to middle age (5-10 years), but total amount of INSL3 peptide per testis did not change among age groups. Concentrations of INSL3 mRNA were higher (P < 0.01) in retained testes than those in the normal testes and cryptorchid scrotal testes, and similar differences were observed for INSL3 peptide. Reversely, total amounts of INSL3 mRNA and peptide per retained testis were lower (P < 0.01) than those per normal testis because of smaller weight of retained testes. Concentrations and total amount of RXFP2 mRNA in the retained testes were almost nil and lower (P < 0.01) than those in the normal testes and in the cryptorchid scrotal testes. Total amount of LH receptor mRNA per retained testis was lower (P < 0.01) than that per normal testis. The immunohistochemical analysis revealed that INSL3 was expressed only in Leydig cells of both the normal and retained canine testes. These results suggest that INSL3 in retained testes of cryptorchid dogs is substantially expressed per unit-weight basis but may be produced with lower amount as a whole testis. Also, this study provides findings that RXFP2 gene is expressed scarcely in the retained testes but normally in cryptorchid scrotal testes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The main objective of this study was to examine the modulatory in vitro effects of gonadotropin-releasing hormone (GnRH) on isolated Leydig cells of adult alpaca (Lama pacos) testis. We first evaluated the presence of GnRH receptor (GnRHR) and cyclooxygenase (COX) 1 and COX2 in alpaca testis. We then studied the in vitro effects of buserelin (GnRH analogue), antide (GnRH antagonist), and buserelin plus antide or inhibitor of phospholipase C (compound 48/80) and COXs (acetylsalicylic acid) on the production of testosterone, PGE(2), and PGF(2α) and on the enzymatic activities of COX1 and COX2. Immunoreactivity for GnRHR was detected in the cytoplasm of Leydig cells and in the acrosomal region of spermatids. COX1 and COX2 immunosignals were noted in the cytoplasm of spermatogonia, spermatocytes, spermatids, Leydig cells, and Sertoli cells. Western blot analysis confirmed the GnRHR and COX1 presence in alpaca testis. The in vitro experiments showed that buserelin alone increased (P < 0.01) and antide and buserelin plus acetylsalicylic acid decreased (P < 0.01) testosterone and PGF(2α) production and COX1 activity, whereas antide and compound 48/80 counteracted buserelin effects. Prostaglandin E(2) production and COX2 activity were not affected by buserelin or antide. These data suggest that GnRH directly up-regulates testosterone production in Leydig cells of adult alpaca testis with a postreceptorial mechanism that involves PLC, COX1, and PGF(2α)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The aims of this study were to determine the cellular and subcellular localization of aquaporin-9 (AQP9) in different rat organs by immunoblotting, immunohistochemistry and immunoelectron microscopy. To analyze this, we used rabbit antibodies to rat AQP9 raised against three different AQP9 peptides (amino acids 267-287, 274-295, and 278-295). In Cos7 cells transfected with rat AQP9, the affinity-purified antibodies exhibited marked labeling, whereas nontransfected cells and cells transfected with aquaporin-8 (AQP8) exhibited no labeling, indicating the specificity of the AQP9 antibodies. Immunoblotting revealed a predominant band of 28 kDa in membranes of total rat liver, epididymis, testes, spleen, and brain. Preabsorption with the immunizing peptides eliminated the labeling. Immunohistochemistry showed strong anti-AQP9 labeling in liver hepatocytes. The labeling was strongest at the sinusoidal surface, and there was little intracellular labeling. Immunoelectron microscopy revealed that the labeling was associated with the plasma membrane of the hepatocytes. In testes Leydig cells exhibited anti-AQP9 labeling, and in epididymis, the stereocilia of the ciliated cells (principal cells) exhibited significant labeling, whereas there was no labeling of the nonciliated cells (basal cells). This was confirmed by immunoelectron microscopy. In spleen strong labeling of cells was observed of leukocytes in the red pulp, whereas there was no labeling of cells in the white pulp. In rat brain, AQP9 immunolabeling was confined to ependymal cells lining the ventricles and to the tanycytes of the mediobasal hypothalamus. Antibody preabsorbed with the immunizing peptide revealed no labeling. In conclusion, AQP9 proteins is strongly expressed in rat liver, testes, epididymis, spleen, and brain., (Copyright 2000 Academic Press.)

A study was conducted to identify the cell types that express retinol-binding protein (RBP) in the bovine testis and to compare relative steady-state levels of RBP mRNA expression at different times of testicular development. At the ages of 10 (n = 3), 20 (n = 8), and 34 (n = 7) wk, Angus bulls were bled three times at 1.5-hr intervals, then surgically castrated. Blood samples were analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T), by radioimmunoassay and the degree of seminiferous tubule development was evaluated histologically in sections of fixed tissue samples stained with hematoxylin and eosin. Immunolocalization of RBP was based on the biotin-strepavidin-horseradish peroxidase method. Testis weight and concentrations of LH and T increased with age (P < 0.05), but those of FSH did not change (P > 0.05) between 10 and 34 wk. Seminiferous tubules at 10 wk contained immature Sertoli cells and gonocytes whereas, at 20 wk, spermatogonia and few spermatocytes were detected. At 34 wk, Sertoli cells appeared differentiated and spermatids were observed. RBP was immunolocalized in Sertoli, Leydig, and peritubular cells at the ages of 10, 20, and 34 wk. Furthermore, no differences in staining between Sertoli cells from tubules with or without germ cells were detected. Northern hybridization of testicular RNA with an RBP cDNA probe revealed the presence of a 1.4-Kb mRNA, which was similar to previous RBP transcripts found in other bovine tissues. Quantitative slot blot analysis revealed that steady-state RBP mRNA levels were 50% higher at 10 wk (P < 0.05) than at 20 and 34 wk of age.

The adipose differentiation-related protein (ADRP) was first characterized as a mRNA induced early during adipocyte differentiation (Jiang, H. P., and G. Serrero. 1992. Proc. Natl. Acad. Sci. USA. 89:7856-7860). The present study demonstrates that ADRP mRNA is expressed in a variety of tissues and cultured cell lines. Immunocytochemical examination revealed that ADRP localizes to neutral lipid storage droplets in cultured murine 3T3-L1 adipocytes, murine MA-10 Leydig cells, Chinese hamster ovary (CHO) fibroblasts, and human HepG2 hepatoma cells; the association of ADRP with lipid droplets was confirmed by subcellular fractionation of MA-10 Leydig cells. In addition to ADRP, steroidogenic cells and adipocytes express the perilipins, a family of lipid droplet-associated proteins that share a highly related sequence domain with ADRP. ADRP and perilipins co-localize on lipid droplets in MA-10 Leydig cells. While ADRP was found on small lipid droplets in 3T3-L1 preadipocytes and early differentiated adipocytes, it was absent in maturing adipocytes. In contrast, perilipins were absent early during differentiation, but were found on small and large lipid droplets at later stages. The transition in surface protein composition of adipocyte lipid droplets from ADRP to perilipins occurred 3 days after the initiation of differentiation when cells displayed co-localizatioin of both proteins on the same lipid droplets. The specific localization of adipose differentiation-related protein to lipid droplets in a wide variety of cells suggests that ADRP plays a role in management of neutral lipid stores.

Heat shock protein 60 (HSP60), a member of the chaperonin family, has an essential role in mediating correct folding of nuclear encoded proteins imported to mitochondria. We have investigated immunocytochemical expression of HSP60 in developing fetal, newborn, postnatal, and pubertal testis and ovary, and in the adult ovary of the rat. In the fetal gonads, HSP60 was expressed in the germ cells organized into sex cords and in the developing Leydig cells of the testis. In the pubertal testis, Leydig cells were strongly, spermatogonia and premeiotic spermatocytes moderately labeled, spermatids unlabeled. In the postnatal ovary, oocytes at all stages of folliculogenesis were positive for HSP60. In the pubertal ovary, glandular theca cells, and in the mature ovary, also the cells of the corpora lutea exhibited intense cytoplasmic labeling. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix, and in the Western blot analysis the antibody detected one single band of 60 kDa. Anti-HSP60 labeling in male and female sex steroid producing cells and their progenitors seems to be coordinated with the functional differentiation of these endocrine cells of the gonad. In the oocytes, a key element required for proper folding of imported mitochondrial proteins seems to be constitutively expressed throughout folliculogenesis. However, the data suggest that in the male germ cells mitochondrial chaperonin HSP60 is either not needed during the haploid phase of spermatogenesis or its level becomes extensively reduced and therefore undetectable by the methods used in the study.

The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. Cd induces predominantly testicular interstitial cell (TIC) tumors. In order to clarify the molecular mechanism underlying tissue differences in Cd sensitivity, we compared Cd-induced metallothionein (MT) gene expression, MT protein accumulation, and Cd retention in freshly isolated TICs and liver. Adult male Fischer rats received a s.c. injection of 4.0 micromol Cd/kg or vehicle and 24 h later tissues were sampled and TICs isolated. MT-I and MT-II mRNA levels were determined by slot-blot analysis followed by densitometry scanning, and MT was estimated by the Cd-heme method. Testicular lesions were not grossly or histologically observed in rats treated with 4 micromol Cd/kg. Both MT mRNA and MT (as determined by Cd-binding capacity) were constitutively present in TICs as well as the liver. TICs isolated from Cd-treated rats accumulated more Cd (4-fold), and had higher levels of MT-I (1.9-fold) and MT-II (1.4-fold) mRNAs over control, but contained less MT (30% decrease) than TICs isolated from control animals. Cd exposure substantially increased hepatic Cd content (6000-fold), MT (58-fold), and MT-I mRNA (5.3-fold), but did not increase MT-II mRNA. Thus, our findings indicate that, although low-dose Cd exposure results in increases of MT mRNA in TICs it does not enhance MT synthesis within these cells. The inability to induce the metal-detoxicating MT-protein, in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver.

RAD6, a member of the expanding family of ubiquitin-conjugating (E2) enzymes, functions in the so-called "N-rule" protein breakdown pathway of Saccharomyces cerevisiae. In vitro, the protein can attach one or multiple ubiquitin (Ub) moieties to histones H2A and B and trigger their E3-dependent degradation. Rad6 mutants display a remarkably pleiotropic phenotype, implicating the protein in DNA damage-induced mutagenesis, postreplication repair, repression of retrotransposition, and sporulation. RAD6 transcription is strongly induced upon UV exposure and in meiosis, suggesting that it is part of a damage-induced response pathway and that it is involved in meiotic recombination. It is postulated that the protein exerts its functions by modulating chromatin structure. Previously, we have cloned two human homologs of this gene (designated HHR6A and HHR6B) and demonstrated that they partially complement the yeast defect. Here we present a detailed characterisation of their expression at the transcript and protein levels. Both HHR6 proteins, resolved by 2-dimensional immunoblot analysis, are expressed in all mammalian tissues and cell types examined, indicating that both genes are functional and constitutively expressed. Although the proteins are highly conserved, the UV induction present in yeast is not preserved, pointing to important differences in damage response between yeast and mammals. Absence of alterations in HHR6 transcripts or protein upon heat shock and during the cell cycle suggests that the proteins are not involved in stress response or cell cycle regulation. Elevated levels of HHR6 transcripts and proteins were found in testis. Enhanced HHR6 expression did not coincide with meiotic recombination but with the replacement of histones by transition proteins. Immunohistochemistry demonstrated that the HHR6 proteins are located in the nucleus, consistent with a functional link with chromatin. Electron microscopy combined with immunogold labeling revealed a preferential localisation of HHR6 in euchromatin areas, suggesting that the protein is associated with transcriptionally active regions. Our findings support the idea that both HHR6 genes have overlapping, constitutive functions related to chromatin conformation and that they have a specific role in spermatogenesis, involving Ub-mediated histone degradation.

Thyrotropin-releasing hormone receptor (TRH-R) mRNA was detected in cryostat sections of the mouse testis using biotinylated oligonucleotides complementary to the cDNA encoding the mouse pituitary TRH-R by in situ hybridization. Hybridization signals were detected exclusively in the Leydig cells. The intensity of the signal was probe-concentration dependent. This result suggests that testicular TRH may serve as an autocrine regulator of reproductive function and development via TRH-R in a fashion that is similar or identical to that in the pituitary.

In situ localization and developmental changes in expression of testis-specific calcineurin B subunit isoform beta 1 was examined in rat testis. Two different sizes of mRNA signal, 4.0 kb and 0.9 kb, were detected by Northern blot hybridization. Both signals were expressed synchronously with the start of meiosis at 3 weeks after birth, and increased depending on the maturation of spermatogenesis. In situ hybridization using non-radioactive riboprobes showed that the beta 1 mRNA was specifically localized to spermatocytes where meiosis occurs but none or very little was observed in spermatogonia, spermatids, Sertoli or Leydig cells.

Histopathological and biochemical effects of gossypol acetate (GA) on pituitary-gonadal axis were investigated. 10 and 25 mg GA/kg were administered orally to sexually mature adult male Wistar rats for 4 and 5 weeks, respectively. STH and LTH/PRL cells showed no significant changes as compared to those of controls while TSH cells showed hypertrophy, hyperplasia and degranulation in both experimental groups. Pituitary FSH, LH/ICSH cells showed progressive regression. Gonosomatic indices of sex accessory glands at 25 mg showed significant reduction in the experimental animals as compared to those of controls. The diameter of seminiferous tubules reduced and azoospermia developed. Sertoli and Leydig cells also regressed. At 10 and 25 mg GA treatment, spermatogenesis ceased at secondary spermatocytes and spermatogonia stages, respectively. Epididymis and prostate regressed. Seminal vesicle showed no significant histological variations as compared to that of control except reduction in secretion. Biochemical observations revealed increased levels of acid phosphatase, fructose and citric acid and significant reduction in glycerylphosphoryl choline in reproductive glands of both experimental groups as compared to those of controls. Possible mechanism of antifertility action of GA is discussed.