The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function-in vivo and in vitro evaluation.

In this study, G-coupled estrogen receptor (GPER) was inactivated, by treatment with antagonist (G-15), in testes of C57BL/6 mice: immature (3 weeks old), mature (3 months old) and aged (1.5 years old) (50 µg/kg bw), as well as MA-10 mouse Leydig cells (10 nM/24 h) alone or in combination with 17ß-estradiol or antiestrogen (ICI 182,780). In G-15-treated mice, overgrowth of interstitial tissue was found in both mature and aged testes. Depending on age, differences in structure and distribution of various Leydig cell organelles were observed. Concomitantly, modulation of activity of the mitochondria and tubulin microfibers was revealed. Diverse and complex GPER regulation at the mRNA level and protein of estrogen signaling molecules (estrogen receptor α and ß; ERα, ERß and cytochrome P450 aromatase; P450arom) in G-15 Leydig cells was found in relation to age and the experimental system utilized (in vivo and in vitro). Changes in expression patterns of ERs and P450arom, as well as steroid secretion, reflected Leydig cell heterogeneity to estrogen regulation throughout male life including cell physiological status.We show, for the first time, GPER with ERs and P450arom work in tandem to maintain Leydig cell architecture and supervise its steroidogenic function by estrogen during male life. Full set of estrogen signaling molecules, with involvement of GPER, is crucial for proper Leydig cell function where each molecule acts in a specific and/or complementary manner. Further understanding of the mechanisms by which GPER controls Leydig cells with special regard to male age, cell of origin and experimental system used is critical for predicting and preventing testis steroidogenic disorders based on perturbations in estrogen signaling.

Differences in aromatase expression and steroid hormone concentrations in the reproductive tissues of male domestic turkeys (Meleagris gallopavo) with white and yellow semen.

1. To show hormonal differences between male turkeys with yellow semen syndrome (YSS) and white, normal semen (WNS), the expression of aromatase, oestrogen receptor α (ERα), and oestrogen receptor ß (ERß) as well as testosterone and oestradiol concentrations in YSS and WNS testes, epididymis, and ductus deferens were examined. 2. To measure gene expression levels of aromatase and oestrogen receptors (ERs), three complementary techniques (real-time PCR, Western blot, and immunohistochemistry) were used, whereas steroid hormone levels were determined radio-immunologically. 3. Upregulation of aromatase and ERα mRNAs in YSS testes (P < 0.05; P < 0.01), epididymis (P < 0.001; P < 0.001), and ductus deferens (P < 0.05; P < 0.01) compared to those of WNS tissues was detected. Significant increases in the levels of aromatase and ERα proteins were detected in YSS testes (P < 0.001; P < 0.05), epididymis (P < 0.001; P < 0.001), and ductus deferens (P < 0.001; P < 0.05). The expression of ERß mRNA and protein level was upregulated in the testes (P < 0.05; P < 0.01) and epididymis (P < 0.001; P < 0.01) but not in ductus deferens where it was downregulated (P < 0.01; P < 0.01). Increased intensity of immunoreactive proteins in YSS versus WNS reproductive tissues corroborated gene expression results. 4. Testosterone concentration diminished in YSS epididymis (P < 0.05) and ductus deferens (P < 0.05), but not in the testes, remaining at high level (P < 0.05) compared to WNS values. Concomitantly, increased oestradiol concentration was found in YSS testes (P < 0.05) and epididymis (P < 0.05) but decreased in the ductus deferens (P < 0.05). 5. From the published literature, this study is the first to demonstrate the ability for androgen aromatisation in the turkey reproductive tissues and to show the cellular targets for locally produced oestrogens. The data suggested that the androgen/oestrogen ratio is a mechanistic basis for amplification of differences between turkeys with white and yellow semen and that these results can have a relevance in applied sciences to widen the knowledge on domestic bird reproduction.

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.

Sources of oestrogen in the testis and reproductive tract of the male.

The cytochrome P450 aromatase (P450arom) is the terminal enzyme responsible for the irreversible transformation of androgens into oestrogens and is present in the endoplasmic reticulum of various tissues throughout at least the phylum of vertebrates. The CYP 19 gene is unique and its expression is regulated in a tissue and more precisely in a cell-specific fashion via the alternative use of several promoters located in the first exons. The P450arom has been immunolocalized in germ cells of the mouse, brown bear and rooster. According to age, aromatase activity has been measured in immature and mature rat Leydig cells as well as in Sertoli cells, whereas in the pig, ram and human aromatase is mainly present in Leydig cells. In the adult rat testis, four complementary approaches (RTPCR, in situ hybridization, immunocytochemistry and the tritiated water assay) demonstrate that not only somatic cells but also mature germ cells represent a source of oestrogen synthesis. Taking into account the widespread distribution of oestrogen receptors (ER alpha & ER beta) in testicular cells and the genital tract of the male on the one hand, and the cross-talk between sex steroids and growth factors, and between membrane receptors and nuclear receptors for steroids on the other hand, it is anticipated that understanding of the pathophysiological roles of these 'female' hormones in the male will advance understanding of the hormonal regulation of male reproductive function. One of the future goals is to define oestrogen-targeted genes in the male gonad and indeed, a lot of work is now focused on this specific area in order to clarify the role of oestrogens in the reproductive tract of the male as well as to elucidate the regulation of aromatase gene expression.

[Rat testicular germ cells as a new source of estrogens]. / Plemniki szczura, potencjalnym zródlem estrogenów.

The presence of estrogens, their function and targets in male reproductive tract are still not fully understood. Recent studies, basic and clinical indicate an important role of estrogens during germ cell development. In this study the efforts have been made to immunolocalize aromatase P450 as well as estrogen receptors within rat testis. Spermatids and spermatozoa have been found as aromatase immunopositive cells. Colocalization of estrogen receptors have also been observed. To prove the site of functional aromatase the authors used molecular complementary techniques, as described previously. In the light of literature, this article discusses also a novel, nontraditional site of estrogen synthesis and potential estrogen action within male reproductive tract.

Effect of estradiol and progesterone on growth of porcine myometrial smooth muscle cells and phospholipase C and adenylate cyclase signalling systems in vitro.

The objective of presented studies was to investigate whether estradiol and progesterone administered in vivo and/or added in vitro can influence the primary myometrial cell culture and how these steroid hormones can affect hCG stimulated cAMP and inositol phosphate production in the porcine uterine myocytes. Myometrial smooth muscle cells were obtained from six ovariectomized gilts pretreated (n = 4) or not (n = 2) with oestradiol benzoate and progesterone for 5 consecutive days. Immunocytochemical staining proved that the pattern of filamentous actin in the cytoplasm of the myometrial fibroblasts (basketlike network) was different from that of myometrial smooth muscle cells (long parallel fibres). The myocytes derived from steroid treated pigs and supplemented with estradiol and progesterone in vitro formed a hillock pattern on days 4-5 day of culture whereas cells obtained from not steroid pretreated gilts were smaller and did not create confluent form. Myocytes were treated in vitro with two doses of estradiol/progesterone (low - 0.2 nM/50 nM and high - 2 nM/500 nM, respectively) and two doses of hCG - 0.1 mU and 1000 mU/ml to study hCG action on the second messenger system in myocytes. The myometrial smooth muscle cells treated with low dose of estradiol and progesterone in vivo responded with much higher accumulation of inositol phosphates to strong (1000 mU/ml) hCG stimulation when compared with those receiving high dose of both steroids. The different doses of estradiol and progesterone caused a similar increase in basal cAMP accumulation as compared to control cells cultured without steroid hormones. hCG (0.1 mU/ml) had usually the additive effect on cAMP production in porcine myometrial cells. The presented paper shows that estradiol and progesterone administration in vivo followed by steroid hormone treatment in vitro affects the primary myometrial cells culture and that both steroid hormones modify the basal accumulation of the second messengers: cAMP and IP3 and their answer to hCG stimuli in pigs.