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Background: The primary function of testicular Leydig cells (LCs) is to produce testosterone (T). In vitro culture of the cells represents a very important approach to study androgen production and its regulations. Various methods have been developed for the enrichment of the cells from the testes. However, getting cells in large numbers with high purity and viability is still challenging. Here, we describe a new way to isolate LCs from rat testes in large quantity with high purity and viability. Methods: Enzymatic digested testicular cells from adult rats were labelled with prolactin receptor (PRLR) antibody. The positive cells were isolated by magnetic‐activated cell sorting (MACS) protocol. Purified LCs were tested in vitro for their steroidogenic (T production) and non‐steroidogenic (25‐OH‐vitamin D production and Insl3 and Cyp2r1expressions) functions in the presence of Luteinizing Hormone (LH) for up to 24 h. Results: Reanalysis of scRNA‐seq data indicates that Prlr expression is highly specific in LCs of adult rat testis. MACS procedure based on PRLR expression was able to isolate LCs with very high yield (about 106 cells/testis), high purity (about 95%) and viability (> 93%). Purified LCs retained high steroidogenic and non‐steroidogenic functions in responding to maximal LH stimulations, with more than 10‐fold increases in T production in 3 h and 42% and 103% increases in Insl3 and Cyp2r1 expressions in 24 h. Discussion and conclusion: We have established an excellent way to purify high quality LCs from adult rat testis that can serve as a useful tool to study the physiology, pharmacology and toxicology of the cells in vitro. [ABSTRACT FROM AUTHOR]

Background: Midazolam is a neurological drug with diverse functions, including sedation, hypnosis, decreased anxiety, anterograde amnesia, brain-mediated muscle relaxation, and anticonvulsant activity. Since it is frequently used in children and adolescents for extended periods of time, there is a risk that it may affect their pubertal development. Here, we report a potential effect of the drug on the development of Leydig cells (LCs), the testosterone (T)-producing cells in the testis. Methods: Stem LCs (SLCs), isolated from adult rat testes by a magnetic-activated cell sorting technique, were induced to differentiate into LCs in vitro for 3 weeks. Midazolam (0.1–30 μM) was added to the culture medium, and the effects on LC development were assayed. Results: Midazolam has dose-dependent effects on SLC differentiation. At low concentrations (0.1–5 μM), the drug can mildly increase SLC differentiation (increased T production), while at higher concentrations (15–30 μM), it inhibits LC development (decreased T production). T increases at lower levels may be due to upregulations of scavenger receptor class b Member 1 (SCARB1) and cytochrome P450 17A1 (CYP17A1), while T reductions at higher levels of midazolam could be due to changes in multiple steroidogenic proteins. The uneven changes in steroidogenic pathway proteins, especially reductions in CYP17A1 at high midazolam levels, also result in an accumulation of progesterone. In addition to changes in T, increases in progesterone could have additional impacts on male reproduction. The loss in steroidogenic proteins at high midazolam levels may be mediated in part by the inactivation of protein kinase B/cAMP response element-binding protein (AKT/CREB) signaling pathway. Conclusion: Midazolam has the potential to affect adult Leydig cell (ALC) development at concentrations comparable with the blood serum levels in human patients. Further studies are needed to test the effects on human cells. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is widely used by manufacturers and in consumer products. Its release in the environment may affect male reproductive function. In this study, we examined the effect of low dose (0.1 mg/kg BW), short term exposure during puberty (PD21-35) on adult rat male reproduction. The results indicated that such exposure reset growth hormone (GH) and follicular stimulating hormone (FSH) homeostasis and resulted in a significantly higher level of serum testosterone without affecting serum luteinizing hormone level. QPCR and Western blot results showed that BPA significantly up-regulated selective genes/proteins in the Leydig cell steroidogenic pathway, including steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A, and low-density lipoprotein receptor. RNA-Seq analysis of testicular RNAs showed that BPA significantly affected the gene profiles of multiple testicular interstitial populations without affecting germ cells. Also, GO- and KEGG-analysis suggested that IGF1-related PI3K/AKT signaling was activated, which was confirmed by the increased phosphorylation of IRS1, AKT1 and CREB. The results indicated that a low-dose, short-term BPA exposure during puberty affected the adult male rat pituitary (GH and FSH) and testis (testosterone) homeostasis. [Display omitted] • Pubertal low dose BPA exposure resets adult rat GH and FSH homoeostasis. • Pubertal BPA exposure increases adult serum testosterone. • BPA affected the gene profiles of testis interstitial somatic populations. • IRS1/PI3K/AKT signaling pathway may be responsible for testosterone increase. [ABSTRACT FROM AUTHOR]

• CD90 + cells of seminiferous tubules do not express common testicular cell markers. • CD90 + cells of seminiferous tubules do express common stem Leydig cell markers. • CD90 + cells of seminiferous tubules can form Leydig cells in vitro. Adult testicular Leydig cells arise from stem cells in the neonatal and adult testis. The nature of these stem Leydig cells (SLCs) have not been well characterized. We have found previously that a group cells expressing CD90, a cell surface glycoprotein that may play roles in cell-cell and cell-matrix interactions and associated with the seminiferous tubule surface, have the ability to form Leydig cells. As yet, the relationship between this CD90+ cell population and SLCs reported previously by other groups is still unknown. In the present study, we systematically characterized these CD90+ cells by their ability to express multiple potential SLC markers and to proliferate and differentiate into Leydig cells in vitro. First, we have found by qPCR and immunohistochemical staining that the CD90+ cells do not express any of the markers of the common seminiferous tubular cells, including myoid, Sertoli, germ and Leydig cells, as well as macrophages. Moreover, when the CD90+ cells were isolated by fluorescent-sorting, the cells expressed high levels of all the potential SLC marker genes, including Nestin, Cd51, Coup-tf2, Arx, Pdgfra and Tcf21. Also, CD90-positive, but not -negative, cells were able to form Leydig cells in vitro with the proper inducing medium. Overall, the results indicated that the tubule-associated CD90+ cells represent a population of SLC in adult testis. [ABSTRACT FROM AUTHOR]

Studies have shown that phthalates are capable of affecting the development and functions of male reproductive system. The effect of phthalates on Leydig cell functions is well documented. However, little is known about their potential effects on the functions of stem Leydig cells (SLC). In the present study, we have examined the effects of mono-(2-ethylhexyl) phthalate (MEHP) on SLC functions in vitro by culturing seminiferous tubules and isolated SLCs. The results indicate that MEHP can significantly inhibit the proliferation and differentiation of SLCs in both the organ and cell culture systems. Interestingly, the minimal effective concentration that is able to affect SLC function was lower in the tubule culture system (1 μM) than in the isolated cells (10 μM), suggesting a possible involvement of the niche cells. Also, MEHP appeared to affect both the efficiency of SLCs to form Leydig cells and a selected group of Leydig cell-specific genes, including Lhcgr, Scarb1, Hsd3b1, Cyp17a1, Star, Srd5a1, Akr1c14, Insl3, Hao2 and Pah. Since SLCs are multipotent, we also tested the effect of MEHP on the differentiation of SLCs to adipocytes. Though MEHP by itself can not specify SLCs into adipocyte lineage, it indeed significantly increased the adipogenic activity of SLCs if used with an adipocyte inducing medium by up-regulation of multiple adipogenic-related genes, including Pparg and Cebpa. Overall, the results indicate that MEHP inhibits SLCs differentiating into Leydig lineage while stimulates the differentiating potential of SLCs to adipocytes. Image 1 • MEHP suppresses Leydig cell generation from stem cells in cultured tubules. • MEHP inhibits stem Leydig cell proliferation and differentiation into Leydig lineage. • MEHP stimulates stem Leydig cell differentiation into adipocyte lineage. [ABSTRACT FROM AUTHOR]