Investigation of the Sensitive Target Cell of Spermatocytes Injury Induced by Triptolide in Mice Testes
XIU Xiao-yu1,2, LIU Yong-zhen1,2, LU Heng-lei1,2, QIAO Jun-wen2, TAN Rong-rong2, LAN Xiu-hua2, ZHU Huai-sen2, REN Jin2, QI Xin-ming2*, ZHANG Ze-an1*
1. Shanghai University of Traditional Chinese Medicine,Shanghai 201203, China; 2. Shanghai Institute of Materia Medica, Chinese Academy of Sciences,Shanghai 201203, China
Abstract：OBJECTIVE To investigate the sensitive target cell population of spermatocyte injury induced by TL in mice. METHODS Seven to eight-week-old healthy male C57BL/6 mice were orally administered triptolid(TL) of 0.125, 0.25 and 0.5 mg·kg-1 per day, and dissected on days 3, 7, 11 and 15 respectively. The extracted testes were fixed with formaldehyde. Paraffin sections and hematoxylin-eosin (HE) staining were performed to determine the optimal time point and dose level to be applied for sensitive target cell population analysis of spermatocyte injury induced by TL. The primary spermatocytes in different stages were clearly distinguished and counted based on the characteristic distribution profile of γ-H2AX in spermatocytes under immunohistochemical staining. The sensitive target cell populations of spermatocyte injury were determined according to the decreased percentage of spermatocytes in different stages. RESULTS HE staining showed that the best dose-effect relationship of spermatogenic cell injury in the testes was present on day 11 after TL administration (The severity of the lesion ranged from a minimal degree in the 0.125 mg·kg-1 group to a mild to moderate injury in the 0.25 mg·kg-1 group, and finally to a marked injury in the 0.5 mg·kg-1 group). The degree of injury in the 0.125 and 0.25 mg·kg-1 groups was appropriate and suitable for determination of sensitive target cell populations. γ-H2AX immunohistochemical staining indicated that the γ-H2AX showed different distribution characteristics in nucleus in different stages of spermatocyte differentiation: scattered throughout the nucleus in a few discrete foci to fill the whole nuclear in leptotene; assembled in the chromatin regions in zygotene; located on the edge of the nucleus in a single foci in the pachytene; located in the nucleus in a single foci in the diplotene. The counting results showed that the absolute number of primary spermatocytes in all differentiating stages decreased slightly without statistical significance (P>0.05) in the 0.125 mg·kg-1 dose group; the absolute number of primary spermatocytes decreased significantly with statistical significance (P<0.01 or 0.001) in the 0.25 mg·kg-1 dose group. There was higher decreased percentage of the leptotene primary spermatocyte among the differentiating stages before pachytene stage and with statistical significance (P<0.05) at 0.25 mg·kg-1 when compared with the pachytene primary spermatocyte. CONCLUSION γ-H2AX immunohistochemical staining can clearly distinguish primary spermatocytes at different stages. The leptotene primary spermatocytes are the most likely sensitive target cells in the testicular spermatocyte-injury induced by TL administration.
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2020, Vol. 55 
