DECREASED CYCLIN A(2) AND INCREASED CYCLIN G(1) LEVELS COINCIDE WITH LOSS OF PROLIFERATIVE CAPACITY IN RAT LEYDIG-CELLS DURING PUBERTAL DEVELOPMENT

Postnatal development of Leydig cells can be divided into three distin ct stages of differentiation: initially they exist as mesenchymal-like progenitors (PLC) by day 21; subsequently, as immature Leydig cells ( ILC) by day 35, they acquire steroidogenic organelle structure and enz yme activities but metabolize most of the testosterone they produce; f inally, as adult Leydig cells (ALC) by day 90 they actively produce te stosterone. The aims of the present study were to determine whether ch anges in proliferative capacity are associated with progressive differ entiation of Leydig cells, and if the proliferative capacity of Leydig cells is controlled by known hormonal regulators of testosterone bios ynthesis: LH, insulin-like growth factor I (IGF-I), androgen, and estr adiol (E-2). Isolated PLC, ILC, and ALC were cultured in DMEM/F-12 for 24 h followed by an additional 24 h in the presence of LH (1 ng/ml), IGF-I (70 ng/ml), 7 alpha-methyl-19-nortestosterone (MENT, 50 nM), a s ynthetic androgen that is not metabolized by 5 alpha-reductase, or E-2 (50 nM). Proliferative capacity was measured by assaying [H-3]thymidi ne incorporation and labeling index (LI). Messenger RNA (mRNA) and pro tein levels for cyclin A(2) and G(1), which are putative intracellular regulators of Leydig cell proliferation and differentiation, were mea sured by RT-PCR and immunoblotting, respectively. Thymidine incorporat ion was highest in PLC (9.24 +/- 0.21 cpm/10(3) cell, mean +/- SE), in termediate in ILC (1.74 +/- 0.07) and lowest in ALC (0.24 +/- 0.03). S imilarly, LI was highest in PLC (13.42 +/- 0.30%, mean +/- SE), interm ediate in ILC (1.95 +/- 0.08%), and undetectable in ALC. Cyclin A(2) m RNA levels, normalized to ribosomal protein S16 (RPS16), were highest in PLC (2.76 +/- 0.21, mean +/- SE), intermediate in ILC (1.79 +/- 0.1 4), and lowest in ALC (0.40 +/- 0.06). In contrast, cyclin G(1) mRNA l evels were highest in ALC (1.32 +/- 0.16), intermediate in ILC (0.47 /- 0.07), and lowest in PLC (0.12 +/- 0.02). The relative protein leve ls of cyclin A(2) and G(1) paralleled their mRNA levels. Increased pro liferative capacity was observed in PLC and ILC, but not ALC, after tr eatment with either LH or IGF-I. Treatment with MENT increased prolife rative capacity only in ILC and had no effect in any other group. Trea tment with E-2 decreased proliferative capacity in PLC but not in ILC or ALC. The changes in proliferative capacity after hormonal treatment paralleled cyclin A(2) mRNA and were the inverse of cyclin G(1) mRNA levels. We conclude that: 1) decreased cyclin A(2) and increased cycli n G(1) are associated with the withdrawal of the Leydig cell from the cell cycle; 2) the proliferative capacity of Leydig cells is regulated differentially by hormones and is progressively lost during postnatal differentiation.