Five main cell types are present in the Leydig cell lineage, namely the mesenchymal precursor cells, progenitor cells, newly formed adult Leydig cells, immature Leydig cells, and mature Leydig cells. Peritubular mesenchymal cells are the precursors to Leydig cells at the onset of Leydig cell differentiation in the prepubertal rat as well as in the adult rat during repopulation of the testis interstitium after ethane dimethane sulfonate (EDS) treatment. Leydig cell differentiation cannot be viewed as a simple process with two distinct phases as previously reported, simply because precursor cell differentiation and Leydig cell mitosis occur concurrently. During development, mesenchymal and Leydig cell numbers increase linearly with an approximate ratio of 1:2, respectively. The onset of precursor cell differentiation into progenitor cells is independent of LH; however, LH is essential for the later stages in the Leydig cell lineage to induce cell proliferation, hypertrophy, and establish the full organelle complement required for the steroidogenic function. Testosterone and estrogen are inhibitory to the onset of precursor cell differentiation, and these hormones produced by the mature Leydig cells may be of importance to inhibit further differentiation of precursor cells to Leydig cells in the adult testis to maintain a constant number of Leydig cells. Once the progenitor cells are formed, androgens are essential for the progenitor cells to differentiate into mature adult Leydig cells. Although early studies have suggested that FSH is required for the differentiation of Leydig cells, more recent studies have shown that FSH is not required in this process. Anti-Müllerian hormone has been suggested as a negative regulator in Leydig cell differentiation, and this concept needs to be further explored to confirm its validity. Insulin-like growth factor I (IGF-I) induces proliferation of immature Leydig cells and is associated with the promotion of the maturation of the immature Leydig cells into mature adult Leydig cells. Transforming growth factor alpha (TGFalpha) is a mitogen for mesenchymal precursor cells. Moreover, both TGFalpha and TGFbeta (to a lesser extent than TGFalpha) stimulate mitosis in Leydig cells in the presence of LH (or hCG). Platelet-derived growth factor-A is an essential factor for the differentiation of adult Leydig cells; however, details of its participation are still not known. Some cytokines secreted by the testicular macrophages are mitogenic to Leydig cells. Moreover, retarded or absence of Leydig cell development has been observed in experimental models with impaired macrophage function. Thyroid hormone is critical to trigger the onset of mesenchymal precursor cell differentiation into Leydig progenitor cells, proliferation of mesenchymal precursors, acceleration of the differentiation of mesenchymal cells into Leydig cell progenitors, and enhance the proliferation of newly formed Leydig cells in the neonatal and EDS-treated adult rat testes.

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).

The effects of propyl thiouracil (PTU)-induced hypothyroidism on testicular interstitial cells and androgen secretion in vitro in the neonatal rat were investigated using Sprague Dawley rats of 1, 7, 14, and 21 days. The results revealed that the fetal Leydig cell (FLC) number per testis was unchanged between and within treatment groups at all ages tested. FLC size was 50% smaller in 21-day controls than in all other groups. Adult Leydig cells (ALCs) were present at Days 14 and 21 in controls but were absent in PTU rats. ALCs approximated FLCs of 21-day controls in size. ALC number per testis showed a sharp increase at Day 21. 11ss-HSD1-positive cells were absent in 21-day PTU testes, but a few were present in 21-day control testes. Testosterone secretion per testis was unchanged in 1- to 21-day controls and 7- to 21-day PTU rats. However, at Day 21, a significantly lower value was seen in controls compared to PTU rats. Testicular androstenedione secretion was not significantly different between control and PTU rats up to 14 days, but a sharp rise was observed in controls at Day 21. At this age, androstenedione levels in PTU rats were similar to those at younger ages. In summary, histological studies showed that hypothyroidism prevented the hypotrophy of FLC and the emergence of ALC in the neonatal rat testis, and agreed favorably with results concerning testicular androgen secretion in vitro. These findings suggest that thyroid hormones have a regulatory role in precursor cell differentiation into Leydig cells in the neonatal rat testis to establish the ALC population.

Leydig cells in the foetal rat testis are still present at birth and it has been hypothesized that they commence to degenerate immediately after birth, based on the decrease in their volume density (v/v%) with age. In this study the interstitium of the rat testis was studied quantitatively at 1, 5, 10, 15, 20 and 90 days after birth: the latter are considered to be adults. The absolute volumes of connective tissue cells and blood vessels increased with age. The absolute volumes of macrophages and lymphatic spaces were greater at 90 days than at any other age. The absolute volume of foetal Leydig cells per testis was unchanged from 1 to 15 days, despite a decrease in the % volume occupied per testis. The number of foetal Leydig cells per testis did not decline from days 1-20 although on day 20 an average foetal Leydig cell was smaller in volume than at earlier ages (days 1-15). Adult Leydig cells were recognized at day 10 and their absolute volume and number per testis increased from 15 to 90 days. Adult Leydig cells were similar in morphology to foetal Leydig cells at 20 days except for a reduced volume of cytoplasmic lipid.

In this paper we present the state of knowledge on cell-cell interactions in the testis of two groups of anamniote vertebrates--teleosts and elasmobranchs--which include most fish. In these fish, the structural organization of the testis differs fundamentally from that which characterizes amniotes in which the germinal tissue is located in tubules open at both ends and consists of a permanent population of Sertoli cells associated with successive stages of germ cell development. In fish, the spermatogenic unit of testis is the spermatocyst, which corresponds to one germ cell or to a clone of isogenetic germ cells, enclosed by one or several Sertoli cells, which form the wall of the cyst. In fish testis, the Sertoli cells do not represent a permanent population of cells. Although both are of the cystic type, the teleost and elasmobranch testes are differently organized. In elasmobranchs, primary spermatogonia and Sertoli cells lie initially free within the interstitial tissue, before becoming sequestered by a basement membrane; the testis is then composed of a mass of spermatocysts which contain many Sertoli cells, each being associated with a clone of germ cells. In contrast, in teleosts, the cysts are confined to large elongated structures limited by a basement membrane. These structures are either lobules originating under the albuginea or tubules which, in contrast to those of mammals, are anastomosed. In the lobules, the spermatocysts start to develop at the blind end of the lobules and migrate towards the efferent system, whereas in the tubules, the spermatocysts are located against the basement membrane, all along the tubules and do not migrate. In elasmobranchs, unlike teleosts, Leydig cells are either absent from the interstitial tissue or rare and undifferentiated and their role in steroid production is at best marginal. While many studies have focused on topographical and functional interactions between the diverse cell types present in mammalian testis, only a few studies have brought particular attention to these aspects in fish. In fish, like in mammals, testicular cell-cell interactions are based on structural elements and chemical factors. Occasionally, various adhering junctions have been observed, essentially in teleosts, between Sertoli cells, between Sertoli cells and germ cells, between germ cells themselves, and interstitial cells. Furthermore, in some teleost species, using horseradish peroxidase or lanthanum salts, the presence of tight junctions between Sertoli cells has been correlated to the occurrence of a Sertoli barrier. In these species, the barrier develops after meiosis so that only haploid germ cells are shielded from the vascular system. In fish, recent development of techniques which enable the preparation and in vitro culture of enriched populations of testicular cells and of spermatocysts, has allowed investigations on functional aspects of cell-cell interactions. In particular, data have been obtained, in the trout, on the control of spermatogonia proliferation by Sertoli cel...