An acute exposure to glyphosate-based herbicide alters aromatase levels in testis and sperm nuclear quality.

Roundup is the major pesticide used in agriculture worldwide; it is a glyphosate-based herbicide. Its molecular effects are studied following an acute exposure (0.5%) of fifteen 60-day-old male rats during an 8-day period. Endocrine (aromatase, estrogen and androgen receptors, Gper1 in testicular and sperm mRNAs) and testicular functions (organ weights, sperm parameters and expression of the blood-testis barrier markers) were monitored at days 68, 87, and 122 after treatment, spermiogenesis and spermatogenesis. The major disruption is an increase of aromatase mRNA levels at least by 50% in treated rats at all times, as well as the aromatase protein. We have also shown a similar increase of Gper1 expression at day 122 and a light modification of BTB markers. A rise of abnormal sperm morphology and a decrease of the expression of protamine 1 and histone 1 testicular in epididymal sperm are observed despite a normal sperm concentration and motility.

Aromatase, oestrogens and human male reproduction.

In most mammalian species aromatase is encoded by a single gene (Cyp19), which contains 18 exons, nine of them being translated. In man, the presence of a biologically active aromatase and oestrogen receptors (ERalpha and ERbeta) has been reported in Leydig cells, and also in immature germ cells and ejaculated spermatozoa. Concerning aromatase, the amount of transcript and enzymatic activity are decreased in immotile compared with motile sperm. We have amplified aromatase mRNA by real-time polymerase chain reaction in spermatozoa from asthenospermic, teratospermic and asthenoteratospermic men and recorded, respectively, 44, 52 and 67 per cent decreases of the amount of transcripts compared with fertile donors. A high degree of correlation (r = -0.64) between the abnormal spermatozoa (especially microcephaly and acrosome malformations) and aromatase/GAPDH transcript ratio has been observed. Idiopathic infertility is a wide health problem and no treatment is currently available. In humans, even if the role of oestrogens in spermatogenesis is still a matter of debate, the observations of decreased sperm number and motility in men genetically deficient in aromatase, together with our data and those reported in the literature, may suggest a role for aromatase/oestrogens not only during the development and maintenance of spermatogenesis but also in the final maturation of spermatozoa.

Mammalian sperm quality and aromatase expression.

In most mammalian species the aromatase is encoded by a single gene (cyp19), which contains 18 exons, 9 of them being translated. In adult rats, together with Leydig cells germ cells represent an additional source of estrogens. The amount of P450arom transcript is threefold higher in pachytene spermatocytes compared to younger cells (spermatogonia-preleptotene spermatocyte) or round spermatids; conversely, aromatase activity is more intense in haploid cells. In man besides Leydig cells, we have shown the presence of a biologically active aromatase and of estrogen receptors (ERalpha and ERss) in immature germ cells and ejaculated spermatozoa. 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 diminished. We have amplified aromatase mRNA by RT-real time PCR in spermatozoa from asthenospermic, teratospermic, and asthenoteratospermic men and recorded respectively 44, 52, and 67% decreases of the amount of transcripts as compared to controls. Statistical analyses between the sperm morphology and the aromatase/GAPDH ratio have revealed a high degree of correlation (r = -0.64) with the percentage of abnormal spermatozoa (especially microcephaly and acrosome 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 mammalian spermatogenesis.

Relationship between chromatin organization, mRNAs profile and human male gamete quality.

Spermiogenesis is a complex process leading to the formation of motile spermatozoa characterized by a highly stable chromatin compaction that transfers the paternal genome into the oocyte. It is commonly held that these haploid cells are devoid of transcriptional and translational activities and that the transcripts represent remnants of stored mRNAs. Recently, the chromatin organization of mature spermatozoa has been revisited as a double nucleoprotamine-nucleohistone structure possessing less-condensed regions sensitive to nuclease activity, which could be implicated in the expression of genes involved in the early embryo development. The existence of a complex population of mRNAs in human sperm is well-documented, but their role is not yet elucidated. Evidence for a latent transcriptional capacity and/or a potential de novo translation in mature spermatozoa from fertile men are essential for understanding the last steps of sperm maturation, such as capacitation and acrosome reaction. As such, we have documented the relationship between sperm quality and the distribution of sperm RNAs by showing divergent levels of transcripts encoding for proteins involved in either nuclear condensation (protamines 1 and 2) or in capacitation (eNOS and nNOS, c-myc) or in motility and sperm survival (aromatase) between low and high motile sperm issued from the same sample. Therefore, analyzing the profile of mRNAs could be helpful either as a diagnostic tool for evaluating male fertility after spermatogenesis or for prognosis use for fertilization.

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.

Expression of aromatase in human ejaculated spermatozoa: a putative marker of motility.

Cytochrome p450 aromatase (p450arom) is a key enzyme responsible for the irreversible transformation of androgens into estrogens. In the present study, we have analysed the ability of human ejaculated spermatozoa to produce estrogens and for that purpose we have looked for the expression of specific aromatase transcript and protein. We have confirmed the presence of p450arom transcript in all normospermic purified samples by nested PCR. The sequence of PCR products from purified spermatozoa shares 98% identity with published human p450arom sequence. Using a semi-quantitative approach, we have observed in immotile sperm a significant decrease (28%) of the aromatase/glyceraldehyde-3-phosphate dehydrogenase ratio compared with the motile sperm fraction. On Western blot with a monoclonal antibody directed against aromatase, we have detected two bands (53 and 49 kDa) in microsome preparations from purified spermatozoa. In total protein extracts of purified spermatozoa (with and without cytoplasmic droplets), we have only found the aromatase as a 49 kDa band with a stronger intensity when cytoplasmic droplets are present. Moreover, the band seems to be weaker in immotile spermatozoa (with and without cytoplasmic droplets). Our data demonstrate the expression of aromatase both in terms of mRNA and protein in each sample of human purified spermatozoa and in addition, our results suggest that aromatase could be concerned with the acquisition of sperm motility.

[Testicular aromatase]. / L'aromatase testiculaire.

The cytochrome P450 aromatase (P450arom) is the terminal enzyme involved in the irreversible transformation of androgens into estrogens. The P450arom plays a role in development, reproduction, sexual differentiation and behaviour, but also in bone and lipid metabolisms, brain functions and diseases such as breast and testicular tumors. Besides testicular somatic cells, where the aromatase gene is expressed via promoter II and I.4, this gene is transduced in a fully active protein in rat germ cells providing evidences for an additional site of estrogen production within the male gonad of rodents (our results and these in the literature). In addition we provided evidence for the expression of P450arom in ejaculated human spermatozoa. Together with the widespread distribution of estrogen receptors (ER alpha and ER beta) in various testicular cells as well as in the other parts of the genital tract, these data suggest a physiological role for these female hormones in the regulation of spermatogenesis especially in the postmeiotic steps.

Reproductive system: aromatase and estrogens.

The cytochrome P450 aromatase (P450arom) is the terminal enzyme responsible for the formation of estrogens from androgens. In the rat testis we have immunolocalized the P450arom not only in Leydig cells but also in germ cells and especially in elongated spermatids. Related to the stage of germ cell maturation, we have shown that the level of P450arom transcripts decreases, it is much more abundant in pachytene spermatocytes (PS) than in mature germ cells whereas the aromatase activity is two- to fourfold greater in spermatozoa when compared to younger germ cell preparations. In rat germ cells, the aromatase gene expression is not only under androgen and cyclic AMP control but also subjected to cytokine (TNFalpha) and growth factor (TGFbeta) regulation. In the bank-vole testis we have evidenced a positive correlation between a fully developed spermatogenesis and a strong immunoreactivity for both P450arom and estrogen receptor (ERbeta) not only in Sertoli cells but also in PS and round spermatids (RS). Therefore, the aromatase gene expression and its translation in a fully active protein in rodent germ cells evidence an additional site for estrogen production within the testis. Our recent data showing that human ejaculated spermatozoa expressed specific transcripts for P450arom reinforced the observations reported in germ cells of other mammalian species. Together with the widespread distribution of ERs in testicular cells these data bring enlightenment on the hormonal regulation of spermatogenesis.