Differentiation and development of the reproductive system in the iguanid lizard, Sceloporus undulatus.

Embryos of the lizard Sceloporus undulatus were sampled throughout incubation, and the differentiation and development of the reproductive system was documented histologically. The undifferentiated gonads possess both a cortex and medulla, both of which contain germ cells until embryonic stage 34. Beginning at stage 34, the cortex of the presumptive ovary thickens, and cortical germ cells are more abundant. By the time of hatching, the ovarian cortex is 6 to 10 cells thick and filled with oogonia and oocytes; primordial follicles, however, are not yet present. In males at embryonic stage 34, seminiferous tubules appear in the medulla of the testis, and Sertoli cells begin to differentiate. Seminiferous tubule formation is complete by hatching, and both Sertoli and Leydig cells are apparent. The mullerian ducts develop in both sexes but begin regressing in the male at embryonic stage 37. The wolffian ducts also develop in both sexes and are present in males and females at hatching.

Expression of SPARC/osteonectin transcript in murine embryos and gonads.

A cDNA clone, p2-4, was isolated from mouse teratocarcinoma-derived parietal endoderm-like cells and used to analyze expression of the corresponding transcript during mouse embryogenesis. Nucleotide-sequence analysis revealed extensive homology between this clone and SPARC/osteonectin cDNA cloned from mouse parietal endoderm and bovine bone cells. The SPARC/osteonectin transcript became more abundant when embryonal carcinoma (EC) cells differentiated into parietal endoderm-like cells. In embryos, the transcript began to appear in the embryo proper on day 11 and continued to be expressed throughout the gestation period. The transcript was also present in extraembryonic membranes and placenta from days 9 and 11 onward, respectively. Thus, expression of the transcript was regulated during differentiation of EC cells and during embryogenesis. In adult mice, several non-bone tissues, including testis, also expressed the transcript. Analysis of germ-cell-deficient mice indicated that non-germ-cell components of the testis expressed the transcript. Analysis of mouse testicular cell lines further suggested that the transcript was abundant in Sertoli cells and Leydig cells. Cumulus oophorus cells that envelope the ovulated egg also expressed high levels of the transcript.

Hormonal regulation of differentiation of human fetal prostate and Leydig cells in vitro.

Trowell type of organ culture was used for correlative study of the human fetal prostate and Leydig cell differentiation at the ultrastructural level. Androgens accelerated the differentiation of human urethral epithelial cells into secretory prostatic cells and the ultrastructure resembled that in vivo. Also Leydig cells retained in organ culture the same ultrastructural features as in vivo and human chorionic gonadotropin (hCG) accelerated their differentiation. It is concluded that this type of culture technic can be used in the study of early differentiation of human genital organ and androgenes and hCG take part of human prostatic and Leydig cell differentiation.

Acquisition of hormone-mediated mechanisms regulating testicular steroidogenesis during development.

Our studies have demonstrated that in the fetal rat Leydig cell, estradiol causes an up-regulation of its receptor and an induction of the regulatory mechanism (late steroidogenic lesion) that is similar to that observed in the adult rat Leydig cell. The absence of this regulation in fetal life is due to a very low level of aromatization capacity, with lack of up-regulation and/or induction of estrogen receptor by estradiol. Higher doses or frequent administration of LH is able to elevate aromatase activity and consequent E2-receptor-mediated action for the induction of gonadotropin-mediated desensitization in fetal cells. Our studies have revealed a small population of adult-like Leydig cells in the fetal testis and the emergence of a functional adult-like population from the fetal Leydig cell induced by gonadotropin treatment. The in vitro fetal Leydig cell culture system has permitted the analysis of cellular actions of gonadotropin with particular reference to the role of tropic hormone and estrogen in the development of late steroidogenic lesions during Leydig cell maturation. Future research with this system will help to clarify further the modulatory mechanisms responsible for emergence of the adult cell population.

Pro-opiomelanocortin-derived peptides in the testis: evidence for a possible role in Leydig and Sertoli cell function.

Pro-opiomelanocortin (POMC)-derived peptides such as beta-endorphin, ACTH, and MSHs were identified in the testis where they were exclusively localized in Leydig cells. Examination of testicular extracts by a variety of physicochemical and immunological techniques indicates that the processing of the POMC in the testis is very similar to that in the brain. By using a cDNA probe, the POMC-like mRNA present in total testis and cultured Leydig cells was 150-200 bases shorter than that in the hypothalamus and pituitary. In addition, POMC mRNA was localized to Leydig cells using in situ hybridization. The expression of the POMC-like gene and the accumulation of POMC-derived peptides in Leydig cell were shown to be under the control of gonadotropin. As the testis contains low concentrations of POMC-derived peptides, we suggested that they may be implicated in local regulatory events within this organ. This postulate was supported by results from in vivo and in vitro experiments suggesting that different portions of the POMC-molecule may have opposite effects on Sertoli cell functions. For example, MSHs increased cAMP accumulation and aromatase activity in these cells, while opioids inhibited Sertoli cell proliferation and androgen binding protein (ABP) secretion. Furthermore, following intratesticular administration of opiate antagonists, testosterone production was reduced, suggesting that Leydig cell function may be also modulated by beta-endorphin and/or other related peptides. Taken together, these studies support the hypothesis of a possible role of POMC-derived peptides in testicular function.

Percoll-gradient separation of Leydig cells from postnatal rat testes.

The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.

The onset of testicular differentiation in the rat: An ultrastructural study.

The structure of developing rat testes was studied by light and electron microscopy. Ultrastructural differentiation of Sertoli and Leydig cells was followed from 14 days of gestation through birth. Specialized morphology in Sertoli cells was first seen at 16 days of gestation. In these cells the rough endoplasmic reticulum increased and became organized as numerous short cisternae loaded with a homogenous material. Typical Leydig cells were found among the stromal cells, around day 17 of gestation. There is good correlation between the time of appearance of ultrastructural specialization and published data on secretory capacity of the fetal testes, in respect to the inhibition and differentiation of the Müllerian and Wolffian ducts.

Hypothalamic control of the pituitary-testicular relation in fetal rats: measurement of collective volume of Leydig cells.

On the 20th day of gestation in pregnant rats, the male fetuses were subjected to surgical hypophysectomy by decapitation or to intracranial paraffin injection which compressed the fetal brain. Autopsy was done on the 22nd day of gestation. The collective volume of Leydig cells in the left fetal testis was estimated by Chalkley's method. Decapitation of a fetus caused a significant retardation in increase of the collective volume of Leydig cells 2 days later. In fetuses given an intracranial paraffin injection, the Leydig cell volume was increased significantly compared with normal fetuses on the 20th day of gestation but was far smaller than that in their intact littermates. This effect of paraffin compression was completely prevented by injections of LRH. The Leydig cell volume remained extremely small in decapitated fetuses given LRH. The observations suggest that in the male rat the hypothalamus begins to govern a pituitary gonadotropic function before birth.