Kinetic Studies on the Development of the Adult Population of Leydig Cells in Testes of the Pubertal Rat*

Abstract

The objective of this study was to determine whether postnatal increases in rat Leydig cell number result from differentiation of precursor cells, division of existing Leydig cells, or both. Our approach was 1) to examine changes in the absolute number of Leydig cells and potential precursor cells (macrophages, pericytes, and mesenchymal, endothelial, and myoid cells) per testis on day 19 of gestation (day -2) and days 7, 14, 21, 28, and 56 postpartum; 2) to examine the frequency with which mesenchymal and Leydig cells divide during prenatal and postnatal development; and 3) to identify and examine the fate of the progeny of Leydig and mesenchymal cell divisions during prenatal and postnatal development. Stereological methods were used to show that mesenchymal cells comprised 44% of the total interstitial cell population and Leydig cells 16% on day -2, whereas by day 56 postpartum the relationship had reversed; mesenchymal cells comprised 3% and Leydig cells 49%. These results suggested a precursor-product relationship between mesenchymal and Leydig cells because no such reciprocal relationship was observed between Leydig cells and macrophages, pericytes, endothelial, or myoid cells. Autoradiographic analysis of [3H]thymidine incorporation into mesenchymal and Leydig cells was consistent with this interpretation. In a series of pulse-chase experiments, the percentage of labeled mesenchymal and Leydig cells was measured after a single injection of [3H]thymidine on days 2, 14, 28, and 56 postpartum, each followed by sampling at timed intervals (between 1 h and 14 days) thereafter. Starting on day 14, the percentage of labeled Leydig cells was approximately 1% immediately after injection of [3H]thymidine and increased significantly to approximately 6% by 6 days after injection. No such increase was observed when rats were similarly injected starting on days 2, 28, and 56 postpartum. The rise in Leydig cell labeling between days 14 and 28 postpartum did not result in a decline in the number of silver grains over labeled Leydig cell nuclei, indicating that the increase in the percentage of labeled cells was not caused by Leydig cell division. These observations led us to conclude that the increase in Leydig cell labeling from days 14 to 28 was the result of recruitment from a compartment of labeled mesenchymal cells. In contrast, our analysis indicated that from day 28 postpartum and thereafter until the mature number of Leydig cells is attained, Leydig cells are generated by division of morphologically recognizable Leydig cells.

Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring

“…This observation is also in full agreement with the estimate of total Leydig cell numbers per testis, as well as with the secretion profiles for the peptide hormone INSL3. Leydig cell numbers were close to those previously reported by Zirkin and Ewing 31 and Hardy et al 17 Close inspection of the testicular histology (data not shown) indicated that the increase in Leydig cell numbers did not appear to be associated with any kind of focal hyperplasia as has been reported for the fetal testis under DBP treatment. 32 Nor was there any evidence for focal atrophy in any of the testes examined, which, however, did exclude any of the grossly dysgenic testes resulting from maternal DBP treatment.…”

“…Microscope images were captured on Spot analysis software (Diagnostic Instruments Inc., Sterling Heights, MI, USA) using a 3100 oil-free Figure 1 Diagram to illustrate the treatment and sampling regimen applied (small dots above the bar indicate approximate blood sampling times). The morphological changes during pubertal Leydig cell differentiation (after [16][17][18] ) are illustrated at their corresponding times in the upper section of the figure. GD, gestational day; PND, postnatal day; DBP, dibutyl phthalate; DES, diethylstilbestrol.…”

“…Leydig cells were recognized by their oval to spherical nucleus with a thin rim of heterochromatin and prominent nucleolus. [16][17][18] Finally, the entire testis cross-section was captured with 31.25 objective for determination of the surface area of each testis section. The total number of Leydig cells within the whole testis was estimated by the formula 19 : Nˆ5Q31/f 1 31/f 2 31/f 3 , where Nˆis the estimated total number, Q is the number of Leydig cells counted in the disector, f 1 is the fraction of tissue (i.e., number of randomly selected slices/number of total slices), f 2 is the fraction of sections (i.e., total number of randomly selected sections/number of total sections) and f 3 is the sampling fraction (i.e., S volume of dissectors/S volume of the entire testis section).…”

In search of rat stem Leydig cells: Identification, isolation, and lineage-specific development

“…The mitotic activity of PLCs gradually is reduced, and the cells enlarge in transition to another intermediate, the immature LC (ILC), during days 28-56 postpartum. ILCs undergo a final division and transform into testosterone-secreting adult LCs (ALCs) by day 56 (10).…”

Expression of Connexin43 in mouse Leydig, Sertoli, and germinal cells at different stages of postnatal development

“…In the prepubertal animal, mesenchymal cells represent the largest interstitial cell population. With the onset of puberty, their relative number decreases whereas LC number increases to become the most abundant interstitial cell type in the mature animal (Hardy et al, 1989;Vergoween et al, 1993).…”