Endocrinology, Vol 130, 599-606, Copyright © 1992 by Endocrine Society

ARTICLES

Steroidogenesis-inducing protein promotes deoxyribonucleic acid synthesis in Leydig cells from immature rats

SA Khan, K Teerds and J Dorrington
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.

A protein [steroidogenesis-inducing protein (SIP)] has been isolated from human ovarian follicular fluid and shown previously to stimulate steroidogenesis in Leydig cells, adrenal cells, and early luteal cells. Since proteins and peptides known to regulate steroidogenesis, such as gonadotropins and growth factors, also influence the growth of gonadal cells, the present study was designed to assess the effects of SIP on the synthesis of DNA by Leydig cells in vitro. Leydig cells were isolated from 10- and 20-day-old rats and cultured in serum-free medium for 48 h. The cells were then treated with the test materials for 18 h. Incorporation of [3H]thymidine into DNA was measured during the final 4 h of the culture. SIP significantly stimulated DNA synthesis in Leydig cells in a dose- and time-dependent manner, and the response to SIP was higher than that obtained with maximal concentrations of LH/hCG. The stimulatory effects of SIP were significantly enhanced when the cell cultures were preincubated in the presence of low levels of ovine LH (2 ng/ml). Cultures treated with SIP, followed by incubation with [3H]thymidine, contained 22 times as many labeled cells as control cultures, as assessed by autoradiography. The cells that were labeled were identified morphologically as Leydig cells. Insulin/insulin-like growth factor-I and/or transforming growth factor-alpha alone stimulated DNA synthesis and enhanced the effects of SIP on DNA synthesis. Dramatic changes in the morphology of cultured Leydig cells treated with SIP were observed; cells became flattened and developed extended projections which connected adjacent cells. LH/hCG, insulin, and transforming growth factor-alpha did not induce effects comparable to those of SIP on the morphology of Leydig cells. The effects of SIP on the synthesis of DNA and the morphology of Leydig cells were blocked in the presence of cycloheximide. It is concluded that SIP not only stimulates steroid production in Leydig cells, as shown previously, but also stimulates DNA synthesis and induces morphological changes in these cells. The latter properties of SIP combined with the magnitude of the responses elicited identify SIP as a unique gonadal protein.

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