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Decreased Cyclin A₂ and Increased Cyclin G₁ Levels Coincide with Loss of Proliferative Capacity in Rat Leydig Cells During Pubertal Development¹

The Population Council (R.-S.G., M.P.H) and Rockefeller University (M.P.H.), 1230 York Avenue, New York, New York 10021

Address all correspondence and requests for reprints to: Matthew P. Hardy, The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: hardy{at}popcbr.rockefeller.edu

Postnatal development of Leydig cells can be divided into three distinct 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 enzyme activities but metabolize most of the testosterone they produce; finally, as adult Leydig cells (ALC) by day 90 they actively produce testosterone. The aims of the present study were to determine whether changes in proliferative capacity are associated with progressive differentiation of Leydig cells, and if the proliferative capacity of Leydig cells is controlled by known hormonal regulators of testosterone biosynthesis: LH, insulin-like growth factor I (IGF-I), androgen, and estradiol (E₂). 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-methyl-19-nortestosterone (MENT, 50 nM), a synthetic androgen that is not metabolized by 5-reductase, or E₂ (50 nM).

Proliferative capacity was measured by assaying [³H]thymidine incorporation and labeling index (LI). Messenger RNA (mRNA) and protein levels for cyclin A₂ and G₁, which are putative intracellular regulators of Leydig cell proliferation and differentiation, were measured by RT-PCR and immunoblotting, respectively. Thymidine incorporation was highest in PLC (9.24 ± 0.21 cpm/10³ cell, mean ± SE), intermediate in ILC (1.74 ± 0.07) and lowest in ALC (0.24 ± 0.03). Similarly, LI was highest in PLC (13.42 ± 0.30%, mean ± SE), intermediate in ILC (1.95 ± 0.08%), and undetectable in ALC. Cyclin A₂ mRNA levels, normalized to ribosomal protein S16 (RPS16), were highest in PLC (2.76 ± 0.21, mean ± SE), intermediate in ILC (1.79 ± 0.14), and lowest in ALC (0.40 ± 0.06). In contrast, cyclin G₁ mRNA levels 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 levels of cyclin A₂ and G₁ paralleled their mRNA levels. Increased proliferative capacity was observed in PLC and ILC, but not ALC, after treatment with either LH or IGF-I. Treatment with MENT increased proliferative capacity only in ILC and had no effect in any other group. Treatment with E₂ decreased proliferative capacity in PLC but not in ILC or ALC. The changes in proliferative capacity after hormonal treatment paralleled cyclin A₂ mRNA and were the inverse of cyclin G₁ mRNA levels. We conclude that: 1) decreased cyclin A₂ and increased cyclin G₁ 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.

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