17 beta-estradiol activates rapid signaling pathways involved in rat pachytene spermatocytes apoptosis through GPR30 and ER alpha.

Aim of the present study was to investigate whether estrogens were able to directly activate rapid signaling pathways controlling spermatogenesis in rat pachytene spermatocytes (PS). Classically, estrogens act by binding to estrogen receptors (ERs) alpha and beta. Recently, it has been demonstrated that rapid estrogen action can also be activated through the G-protein-coupled receptor (GPR)-30. Herein, we demonstrated that rat PS express ER alpha, ER beta and GPR30. Treatment of PS with estradiol (E2), the selective GPR30 agonist G1 and the selective ER alpha agonist PPT determined activation of ERK1/2 which are part of GPR30 signaling cascade. ERK1/2 activation in response to E2 and G1 was correlated to an increased phosphorylation of c-Jun. All treatments failed to induce these responses in the presence of EGFR inhibitor AG1478, ERK inhibitor PD98059 and ER inhibitor ICI182780. mRNA expression of cell cycle regulators cyclin A1 and B1 was downregulated by E2 and G1 while an up-regulation of proapoptotic factor Bax was observed in the same conditions. These data demonstrate that E2, working through both ER alpha and/or GPR30, activates in PS the rapid EGFR/ERK/c-Jun pathway, modulating the expression of genes involved in the balance between cellular proliferation and apoptosis.

Protective effects of estrogens and caloric restriction during aging on various rat testis parameters.

AIM: To investigate the effects of 17beta-estradiol (E2), Peganum harmala extract (PHE) and caloric restriction (CR) on various testis parameters during aging. METHODS: Twelve month-old male rats were treated for 6 months with either E2 or PHE, or submitted to CR (40%). RESULTS: Our results show that estrogens and CR are able to protect the male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase activities and decreased the activity of testicular enzymes: gamma-glutamyl transferase, alkaline phosphatase, lactate deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at 18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats. CONCLUSION: Besides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the reproductive functions from the adverse effects exerted by reactive oxygen species (ROS) produced in large quantities in the aged testis.

Age-related decrease in aromatase and estrogen receptor (ERalpha and ERbeta) expression in rat testes: protective effect of low caloric diets.

AIM: To examine the effects on rat aging of caloric restriction (CR1) and undernutrition (CR2) on the body and on testicular weights, on two enzymatic antioxidants (superoxide dismutase and catalase), on lipid peroxidation and on the expression of testicular aromatase and estrogen receptors (ER). METHODS: CR was initiated in 1-month-old rats and carried on until the age of 18 months. RESULTS: In control and CR2 rats an age-related decrease of the aromatase and of ER (alpha and beta) gene expression was observed; in parallel a diminution of testicular weights, and of the total number and motility of epididymal spermatozoa was recorded. In addition, aging in control and CR2 rats was accompanied by a significant decrease in testicular superoxide dismutase, catalase activities, and an increase in lipid peroxidation level (thiobarbituric acid reactive substance), associated with alterations of spermatogenesis. Conversely, caloric restriction-treatment exerted a protective effect and all the parameters were less affected by aging. CONCLUSION: These results indicate that during aging, a low caloric diet (not undernutrition) is beneficial for spermatogenesis and likely improves the protection of the cells via an increase of the cellular antioxidant defense system in which aromatase/ER could play a role.

Three promoters PII, PI.f, and PI.tr direct the expression of aromatase (cyp19) gene in male rat germ cells.

Aromatase is the key enzyme responsible for the irreversible transformation of androgens into estrogens. It is encoded by the cyp19 gene and is expressed in the mammalian testis under the control of the proximal promoter PII. Since both somatic and germ cells contain a biologically active aromatase, we looked for the existence of other promoters that may direct the expression of aromatase in adult rat germ cells. Besides the promoter II, we have shown the presence of transcripts derived from the brain promoter PI.f in spermatogonia-preleptotene spermatocytes (G-PL), pachytene spermatocytes (PS), and round spermatids (RS). A new aromatase cDNA has been isolated by 5'-rapid amplification of cDNA ends that we named I.tr (testis rat). The I.tr transcripts are found in all the testicular cell populations studied with a greater expression in PS. Because of the utilization of these three promoters in the adult rat testis, we studied their putative involvement according to the age. At 10 days, aromatase expression was very low and then strongly increased between 10 and 20 days, with a preferential activation of PII and PI.tr. Transcripts coming from PI.f were found starting from 20 days onwards. The new promoter PI.tr, localized between promoters PI.f and PII, is devoid of a TATA box but contains a transcriptional initiator (Inr) and putative regulatory sequences. Therefore, the identification of the specific trans-activating factors should bring some enlightenments to understand the regulation of these three promoters in germ cells especially according to their stage of development.

Effects of maternal undernutrition during lactation on aromatase, estrogen, and androgen receptors expression in rat testis at weaning.

The goal of this study was to evaluate the effects of maternal malnutrition during lactation on serum levels of testosterone and estradiol, testicular testosterone concentration, aromatase, testicular androgen (AR) and estrogen alpha (ERalpha) receptors expression in the pups at weaning. From parturition until weaning, Wistar rats were separated into three groups: (C) control group, with free access to a standard laboratory diet containing 23% protein; protein-energy restricted (PER) group, with free access to an isoenergy and protein-restricted diet containing 8% protein; and energy-restricted (ER) group, receiving standard laboratory diet in restricted quantities, which were calculated according to the mean ingestion of the PER group. All pups were killed at weaning, corresponding to 21 days post partum. Compared with the C group, body weights (C=48 +/- 2.3 g; PER=20 +/- 1.3 g; ER=25.4 +/- 0.9 g; P<0.01) and testicular weights (C=0.15 +/- 0.02 g, PER=0.05 +/- 0.01 g, ER=0.06 +/- 0.02 g, P < 0.001) of both PER and ER groups were lower. However, there was no significant difference in the testicular/body weight ratio in PER and ER groups compared with the C group. The testosterone serum concentration (ng/ml) was significantly higher in the PER group compared with ER and C groups (C=0.09 +/- 0.012; PER=0.45 +/- 0.04; ER=0.15 +/- 0.03, P < 0.01). Testicular testosterone concentration (C=2.1 +/- 0.43; PER=6.5 +/- 0.7; ER=13 +/- 2.3, P < 0.01) was increased in treated groups when compared with controls. The estradiol serum concentration (pg/ml) was lower in both dietary groups (C=74 +/- 4.6; PER=49 +/- 3.2; ER=60 +/- 5.5, P < 0.01). The amounts of aromatase mRNA and ERalpha transcripts were significantly lower (P<0.05) in PER and ER groups; conversely AR (both mRNA and protein) was significantly enhanced (P<0.05) in treated animals. The nutritional state in early phases of development is important since we have demonstrated here that the maternal malnutrition during lactation leads to alterations in estradiol and testosterone serum concentrations, testicular testosterone concentration, AR and ERalpha expression together with a decrease of aromatase expression. All together, these changes of steroid status may be deleterious for future germ cell development and reproductive function of these male pups submitted to early malnutrition.

beta-Nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes.

NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously. betaNGF did not modify the number of Sertoli cells, pachytene spermatocytes, secondary spermatocytes nor the half-life of round spermatids, but increased the number of secondary meiotic metaphases and decreased the number of round spermatids formed in vitro. These effects of betaNGF were reversible and maximal at about 4 x 10(-11) M. Conversely, K252a, a Trk-specific kinase inhibitor, enhanced the number of round spermatids above that of control cultures. The presence of betaNGF and its receptors TrkA and p75(NTR) was investigated in testis sections, in Sertoli cell and germ cell fractions, and in germ cell and Sertoli cell co-cultures. betaNGF was detected only in germ cells from pachytene spermatocytes of stages VII up to spermatids of stages IX-X. TrkA and p75(NTR) were detected in Sertoli cells and in these germ cells. Taken together, these results indicate that betaNGF should participate in an auto/paracrine pathway of regulation of the second meiotic division of rat spermatocytes in vivo.

Estrogens: a new player in spermatogenesis.

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them, estrogens are the end products obtained from the irreversible transformation of androgens by aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rat the aromatase activity is mainly located in Sertoli cells of immature animals and then in Leydig cells of adults. Moreover rat germ cells represent an additional source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes (PS) compared to gonocytes or round spermatids (RS); conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank vole, bear and monkey express also aromatase. In man besides Leydig cells, we have shown the presence of a biologically active aromatase and of estrogen receptors in ejaculated spermatozoa and in immature germ cells. Concerning aromatase, a 30% decrease of the amount of mRNA is observed in immotile compared to motile sperm fraction from the same sample; moreover the aromatase activity is also diminished of 34%. In asthenoteratozoospermic and teratozoospermic patients the aromatase gene expression is decreased by 67 and 52%, respectively when compared to normospermic controls. Statistical analyses between the sperm morphology and the aromatase/GAPDH ratio have revealed a high degree of correlation (r=-0.64) between the ratio and the percentage of abnormal spermatozoa (especially microcephaly and acrosmome malformations). Alterations of sperm number and motility have been described in men genetically deficient in aromatase, which together with our data, suggest a likely role for aromatase/estrogens in the acquisition of sperm motility. Therefore besides gonadotrophins and testosterone, estrogens produced locally should be considered as a physiologically relevant hormone involved in the regulation of spermatogenesis and spermiogenesis.

Aromatase and estrogen receptors in male reproduction.

Aromatase is a terminal enzyme which transforms irreversibly androgens into estrogens and it is present in the endoplasmic reticulum of numerous tissues. We have demonstrated that mature rat germ cells express a functional aromatase with a production of estrogens equivalent to that of Leydig cells. In humans in addition to Leydig cells, we have shown the presence of aromatase in ejaculated spermatozoa and in immature germ cells. In most tissues, high affinity estrogen receptors, ERalpha and/or ERbeta, mediate the role of estrogens. Indeed, in human spermatozoa, we have successfully amplified ERbeta mRNA but the protein was not detectable. Using ERalpha antibody we have detected two proteins in human immature germ cells: one at the expected size 66 kDa and another at 46 kDa likely corresponding to the ERalpha isoform lacking exon 1. In spermatozoa only the 46 kDa isoform was present, and we suggest that it may be located on the membrane. In addition, in men genetically deficient in aromatase, it is reported that alterations of spermatogenesis occur both in terms of the number and motility of spermatozoa. All together, these observations suggest that endogenous estrogens are important in male reproduction.

The promoter(s) of the aromatase gene in male testicular cells.

Aromatase is the terminal enzyme responsible for estrogen biosynthesis in mammals; it is present in various testicular cells including germ cells. The aromatase gene (Cyp19) is unique in humans and its expression is regulated in a tissue and more precisely, in a cell-specific manner via the alternative use of various promoters located in the first exon. Nevertheless, there is little information concerning the regulation of the testicular aromatase especially in germ cells. This prompted us to study the control of Cyp19 gene expression and its role in the regulation of the testicular androgen/estrogen ratio. Gonadotrophins and cAMP modulate aromatase expression in somatic cells which confirms that promoter II is controlled via CRE. Moreover, we have demonstrated that in highly purified germ cells from adult rats (pachytene spermatocytes and round spermatids), transforming growth factor beta (TGFbeta) inhibited the expression of Cyp19 in both germ cell types. In contrast, tumor necrosis factor alpha (TNFalpha) stimulated Cyp19 expression in pachytene spermatocytes. The effect of TNFalpha is amplified in presence of dexamethasone. Therefore, we suggest that in germ cells, TNFalpha enhances expression of aromatase through promoter PI.4 in pachytene spermatocytes, possibly via an AP1 site upstream the GAS element, while in round spermatids TNF requires glucocorticoids as a co-stimulator to increase Cyp19 gene expression. In addition, we have shown that androgens and estrogens by themselves modulate Cyp19 gene expression in all testicular cell types studied suggesting the presence of ARE and ERE on the Cyp19 gene promoter(s). Finally, in presence of seminiferous tubules or Sertoli cell-conditioned media, aromatase transcripts are increased in both Leydig cells and germ cells suggesting that other locally produced modulators (e.g. LRH-1) are involved in the regulation of the aromatase gene expression especially in Leydig cells. Using RACE (Rapid Amplification of cDNA Ends)-PCR, we have confirmed that promoter II mainly directs expression of the aromatase gene in all testicular cell types studied in the rat. However, involvement of another promoter such as PI.4 is suggested as well.