The effects of neonatal treatment with the reversible goitrogen, 6-n-propyl-2-thiouracil (PTU) on the volumes of testicular interstitial components, the number and average volume of Leydig cells, and the steroid secretory capacity of testis and Leydig cells of rats at 135 days of age were investigated. Rat pups were hypothyroid from birth to 25 days of age following the addition of 0.1% (w/v) PTU to the drinking water of the mother. Treatment was stopped at 25 days and the pups subsequently returned to a euthyroid state. Control rat pups were raised without adding PTU to drinking water of the mother. On day 135, one testis from each rat (n = 5 per group) was fixed by whole body perfusion for microscopy and stereology, and the ipsilateral testis was used for steroid secretion analysis using an in vitro testis perfusion system. Average testis volume was 84% greater in PTU-treated rats than in controls. This increase resulted from increases in both the seminiferous tubule (86%) and the interstitial (80%) volumes. Moreover, absolute volumes of all testis components in PTU-treated rats were significantly (P < 0.05) greater than those of controls; the highest volume increase was observed in the lymphatic space (147%). The number of Leydig cells per testis was nearly doubled, but the average volume of a Leydig cell was decreased by 25% in PTU-treated rats compared with controls. Steroid secretion per testis was not significantly different between control and PTU-treated rats; however, steroid secretion per Leydig cell was significantly lower in PTU-treated rats than in controls. These results demonstrate that the neonatal administration of PTU causes Leydig cell hyperplasia. However, the normal androgenic status of these animals is maintained by hypotrophy associated with reduced steroid secretion of individual Leydig cells at maturity.

Fetal (FLC) and adult Leydig cells (ALC) secrete insulin-like peptide 3 (INSL3), which is linked to cryptorchidism in the newborn rat. Its gene regulation appears to be independent of that for most steroidogenic enzymes, and may thus be a marker for other aspects of ALC differentiation. Our study examined the following on INSL3 peptide expression in ALC lineage (i) timing, (ii) which cell stage, and (iii) effects of triiodothyronine (T3). Male Sprague–Dawley (SD) rats of postnatal days (pd) 1, 5, 7–21, 28, 40, 60, and 90 were used for the objectives (i) and (ii). For the objective (iii), control and T3-treated (daily T3 SC, 50 μg/kg bw) SD rats of pd7–16 and 21 were used. INSL3 was immunolocalized in Bouin’s-fixed testes. FLC were positive and mesenchymal and Leydig progenitor cells were negative for INSL3 at tested ages. INSL3 in ALC lineage was first detected in newly formed ALC on pd16, although they were present from pd10. The intensity of INSL3 label was greater in ALC of pd40–90. ALC were present in T3-treated testes at pd9, but INSL3 first detected in them was on pd12. While INSL3 in FLC regulates testicular descent, INSL3 in ALC still has no well-defined function. However, its pattern of expression correlates temporally with the development of steroidogenic function and spermatogenesis. Thus, the delay between ALC differentiation and INSL3 expression in them implies that INSL3 in ALC is associated with maturation. The advancement of INSL3 expression in the ALC of T3-treated rats implies that this function is established earlier with T3-treatment.