Primary Culture of Purified Leydig Cells Isolated from Adult Rat Testes*

Abstract

Methods for isolating highly purified Leydig cells permit the study of acute responses and biochemical properties of Leydig cells independent of other testicular cell types. The present study describes the development of a primary culture system for purified Leydig cells from adult rats in which the cells retain their ability to secrete testosterone for at least 72 h in culture. When Leydig cells were cultured in tissue culture medium 199--0.1% BSA (M199-BSA), basal testosterone secretion declined by 72 h, whereas hCGB-stimulated testosterone secretion was reduced by 48 h. Changing the culture medium twice daily or adding 0.5% fetal calf serum (fcs) enhanced basal and gonadotropin-stimulated testosterone secretion at 72 h in culture, although responsiveness to hCG was reduced to 57% of that in freshly isolated cells. Incubation of Leydig cells in the defined culture medium Dulbecco's Modified Eagles-Ham's F-12 (1:1, vol/vol) supplemented with 15 mM Hepes buffer, transferrin, insulin, and epidermal growth factor (DHG:F12 + Hepes + TIE) in either the presence or absence of 0.5% fcs yielded functional Leydig cells for longer intervals in culture. Furthermore, testosterone secretion was greater in DHG:F12 + Hepes + TIE than in M199-BSA at all time intervals tested. In DHG:F12 + Hepes + TIE, basal and gonadotropin-stimulated testosterone production by Leydig cells were maintained for 72 h in culture. Degenerative changes in morphology were apparent in some cells at 72 h, but not at earlier times in culture. This primary culture system for isolated Leydig cells provides a valuable tool to examine the temporally regulated events in Leydig cell function.

Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin‐C and insulin in cultured pig Leydig cells

“…This contention is supported by the demonstration of high affinity specific insulin binding sites on testicular membrane preparation (Posner et al, 1974;Saucier et al, 1981;Abele et al, 1986). Indeed, previous studies have shown that addition of insulin to cultured rat interstitial cells (Adashi et al, 1982) or to purified rat Leydig cells (Browning et al, 1983;Abele et al, 1985a) enhanced the hCG-induced testosterone production. However, since the conentrations used in these studies were high (0.1 to 10 pg/ml), the possibility exists that the effect of insulin may reflect the consequence of its action through the Sm-C receptor.…”

“…The original rationale for this investigation was the previous in vitro demonstrations of effects of insulin at high concentrations in several cell models (Froesch et al, 1985) including rat Leydig cells (Adash et al, 1982;Browning et al, 1983;Abele et al, 1985). Our results indicated that Sm-C at nanomolar concentrations stimulated Leydig cell DNA synthesis and cell mutiplication while micromolar concentrations of insulin were required to obtain the same effects.…”

“…Moreover testicular hances the steroidogenic responsiveness to hCG of culinsulin receptors seem to be regulated mainly LWhCG tured rat interstitial cells (Adashi et al, 1982), purified (Abelb andTremblay, 1985, 1986). In contrast, experi-rat Leydig cells (Browning et al, 1983; Abel6 and Tremmentally induced diabetes in the male rat produce a blay, 19861, and pig Leydig cells @lather et al, 1982b). marked decrease in testicular LH receptor number However, as the insulin concentrations used in these in and in Leydig cell steroidogenesis, both under basal conditions and following acute human chorionic gonadotropin (hCG) sitmulation (Calvo Received February 11,1986;accepted June 10,1986accepted June 10, .…”

Construction of a Leydig cell line synthesizing testosterone under gonadotropin stimulation: a complex endocrine function immortalized by cell hybridization.

“…Leydig cells in primary culture have been used to study the mechanism of action of gonadotropin, but testosterone production is not sustained more than a few days (2)(3)(4)(5)(6). Clonal cell lines derived from mouse (7)(8)(9) and rat (10) Leydig cell tumors, although they may retain LH/hCG receptors, are unable to pursue steroidogenesis beyond progesterone synthesis.…”

Proliferated Leydig cells for engineered testis-like tissue regeneration with testosterone-secreting ability

“…Density gradient centrifugation has been reported as the classical method for harvesting LCs. [18][19][20][21][22][23] However, the cell number of harvested LCs in this method was very limited with complicated procedures and the obtained cells showed very poor proliferative potential in vitro. 24 Therefore, it is urgent and necessary to establish a modified harvesting and expanding system for obtaining adequate LCs.…”