Morphology and function of human Leydig cells in vitro. Immunocytochemical and radioimmunological analyses

The aim of our study was to show whether the cells isolated from testes of patients underwent bilateral orchiectomy for prostatic cancer are able to grown in vitro, and if so, are functionally active. Immuncytochemistry was performed to show the functional status of human cultured cells. In detail, immunolocalization of luteinizing hormone receptors (LHR), mitochondria, and cytoskeletal elements was demonstrated. Moreover, radioimmunological assay was used to measure testosterone secretion by cultured Leydig cells. Using Nomarski interference contrast and fine immunofluorescence analysis the positive immunostaining for LHR was observed in almost all Leydig cells, however it was of various intensity in individual cells. Testosterone measurement revealed significant difference between testosterone secretion by hCG-stimulated and unstimulated Leydig cells (p<0.05). Moreover, testosterone levels were significantly higher in 24- and 48-hour-cultures than in those of 72 hrs (p<0.05). Morphological analysis of Leydig cells in culture revealed the presence of mononuclear and multinucleate cells. The latter cells occurred in both hCG-stimulated and unstimulated cultures. In Leydig cells labeled with a molecular marker MitoTtracker, an abundance of mitochondria and typical distribution of microtubules and microfilaments were observed irrespective of the number of nuclei within the cell, suggesting no functional differences between mono- and multinucleate human Leydig cells in vitro. Since the percentage of multinucleate cells was similar in both hCG-stimulated and unstimulated cultures (23.70% and 22.80%), respectively, the appearance of these cell population seems to be independent of hormonal stimulation

Connexin 43 expression in human and mouse testes with impaired spermatogenesis

Connexin 43 (Cx43) belongs to a family of proteins that form gap junction channels. The aim of this study was to examine the expression of Cx43 in the testis of a patient with Klinefelter’s syndrome and of mice with the mosaic mutation and a partial deletion in the long arm of the Y chromosome. These genetic disorders are characterized by the presence of numerous degenerated seminiferous tubules and impaired spermatogenesis. In mouse testes, the expression and presence of Cx43 were detected by means of immunohistochemistry and Western blot analysis, respectively. In testes of Klinefelter’s patient only immunoexpression of Cx43 was detected. Regardless of the species Cx43 protein was ubiquitously distributed in testes of reproductively normal males, whereas in those with testicular disorders either a weak intensity of staining or no staining within the seminiferous tubules was observed. Moderate to strong or very strong staining was confined to the interstitial tissue. In an immunoblot analysis of testicular homogenates Cx43 appeared as one major band of approximately 43 kDa. Our study adds three more examples of pathological gonads in which the absence or apparent decrease of Cx43 expression within the seminiferous tubules was found. A positive correlation between severe spermatogenic impairment and loss of Cx43 immunoreactivity observed in this study supports previous data that gap junctions play a crucial role in spermatogenesis. Strong Cx43 expression detected mostly in the interstitial tissue of the Klinefelter’s patient may presumably be of importance in sustaining Leydig cell metabolic activity. However, the role of gap junction communication in the control of Leydig cell function seems to be more complex than originally thought

Complementary approaches demonstrate that cellular aromatization in the bank vole testis is related to photoperiod

A growing body of evidence indicates that germ cells, at least in several mammalian species, are responsible for estrogen formation since they possess active aromatase. In seasonally breeding rodent, the bank vole, the length of photoperiod seems to be the primary environmental factor regulating annual changes in the reproductive activity. However, in this species gonadal steroidogenesis is still not well understood, neither the site of aromatization in testicular cells. In the bank vole testis, aromatase visualized by immunohistochemistry was found in Leydig cells, Sertoli cells, and germ cells: especially in spermatocytes and spermatids. Moreover, in the immuno-electron microscopic study, gold particles indicating aromatase were observed over the cytoplasm of elongated spermatids. The presence of aromatase and the activity of this enzyme were found in microsomal preparations of the whole testes and those of seminiferous tubules. This was measured by means of Western blot and the biochemical assay with tritiated androstenedione, respectively. Additionally, using radioimmunological assays testosterone and estradiol concentrations in homogenates were detected. All the studied parameters revealed close correlation with the length of photoperiod being evidently higher in animals kept in the long day conditions when compared with those from short light cycles

Teasing out the role of aromatase in the healthy and diseased testis

Scientific discoveries over the past decade have shifted the stereotypical view of androgens as male hormones and estrogens as female hormones. It is now recognized that a delicate balance of both androgens and estrogens, a process controlled by aromatase, is fundamental for normal testicular development and fertility. While the site-specific actions of these two classes of steroids within the testis are becoming better documented, the role and regulation of estrogen biosynthesis by aromatase within the testis remains unclear. The majority of data comes from a wide range of animal species, particularly genetically modified mouse models; aromatase knockout (ArKO) and overexpressing (AROM+), with limited information on humans, however the existence of congenital aromatase mutations has provided some insight into its effects on individual parameters of the testis. This review dissects out the localization and activity of aromatase in the healthy and diseased testis, addresses the cellular insult to the testis that occurs in its absence and over abundance and proposes potential molecular mechanisms of aromatase regulation in the testis