Role of p57KIP2 in Stem and Progenitor Leydig Cells of Mouse Testes.

PURPOSE: Precise control of proliferation and differentiation of Leydig cells is important for gonadal androgenesis and spermatogenesis. Though cyclin-dependent kinase inhibitors are crucial for cell proliferation and differentiation, their role in the development of early adult Leydig cells (ALCs) remained unanswered. To understand mechanism for ALC development, functional expression of p57KIP2 (cdkn1c) was investigated in the stem Leydig cells (SLCs) and progenitor Leydig cells (PLCs) in mice. MATERIALS AND METHODS: The roles of p57KIP2 in the proliferation, differentiation, apoptosis, and steroidogenesis in SLCs and PLCs were investigated by antibodies and bromodeoxyuridine (BrdU) labeling in the early neonatal testes and p57kip2 siRNA in the isolated SLCs and PLCs. Steroidogenic differentiation of PLCs was examined by progesterone and testosterone production in cell culture. RESULTS: From postnatal day (PND) 1 to 14, p57KIP2(+) spindle-shaped cells in the testis interstitium were α-smooth muscle actin (αSMA)(-), a peritubular myoid cells marker, suggesting that they are SLCs and PLCs. Besides, p57KIP2 was also expressed in HSD3ß(+) fetal Leydig cells. From PND1 to 14, BrdU(+)/αSMA(-), Ki67(+)/p57KIP2(+), and BrdU(+)/p57KIP2(+) spindle-shaped cells were gradually decreased. From PND1 to 14, p57KIP in the αSMA(-)/p57KIP2(+) cells was peaked at PND7 and decreased thereafter. In THY1(+) isolated SLCs, p57kip2 siRNA significantly increased ki67 and pcna mRNA and pdgfrα mRNA, a differentiation marker and decreased nestin mRNA, a SLC marker. No significant difference in apoptosis related genes mRNA was found after p57kip2 siRNA treatment. In HSD3ß(+) PLCs, p57kip2 siRNA increased proapoptotic genes mRNA, annexin V(+) early-apoptotic cells. Importantly, p57kip2 siRNA significantly decreased hsd3ß6 and cyp17a1 mRNA and progesterone production. CONCLUSIONS: p57KIP2 may suppress proliferation and support stemness of SLCs. In PLCs, p57KIP2 may suppress apoptosis and potentiate the steroidogenic differentiation.

Effects of citrate ester plasticizers and bis (2-ethylhexyl) phthalate in the OECD 28-day repeated-dose toxicity test (OECD TG 407).

Citrate esters are considered functional alternatives to phthalate plasticizers, but their toxicity remains poorly understood. The toxicity of citrate esters, including triethyl 2-acetylcitrate (ATEC) and trihexyl O-acetylcitrate (ATHC), were examined together with that of bis (2-ethylhexyl) phthalate (DEHP) using the Organization for Economic Co-operation and Development Test Guideline 407 (OECD TG407). Following 28-day oral administration, no significant differences in body weight or the weight of the brain, pituitary, heart, epididymis, seminal vesicles, or coagulating gland were found between the vehicle control and DEHP, ATEC or ATHC groups. In the 400 mg/kg day DEHP group, liver, adrenal, thymus, spleen, kidney, testis, and prostate weights were significantly increased. In the 400 mg/kg day ATHC group, kidney, adrenal, thymus, testis and prostate weights were significantly increased. In the 400 mg/kg day ATEC group, kidney, adrenal and testis weights were significantly increased. Hepatocyte size was significantly increased in the 400 mg/kg day DEHP group, suggestive of hepatotoxicity, but was not increased in the ATEC or ATHC groups. There were no significant differences in white blood cell, red blood cell or platelet counts, hemoglobin concentrations, hematocrit, mean corpuscular volume, fasting glucose, insulin, or testosterone concentrations between the vehicle control and DEHP, ATEC and ATHC groups. In the ATHC 400 mg/kg day group, T3 was decreased while T4 was increased, suggestive of disruption of thyroid function. The results of the OECD TG407 subacute repeated dosing toxicity test indicate ATEC is less toxic compared to ATHC or DEHP and could be recommended as an alternative to phthalate plasticizers.