Relationship Between The Expression Of Aromatase In Human Ejaculated Spermatozoa And Their Function In Fertilization

In recent decades, an increase of incidence of infertility and a decrease of male fertility potential and semen quality have been reported with the development of industrialization and urbanization and the deterioration of environmental pollution. World health organization (WHO) has predicted that infertility will be one of the most common diseases worldwide, rating the third in occurrence following tumourous diseases and angiocardiopathy in 21st century. So, it's significant for us to understand the relevant factors involved in infertility.The biological conversion of androgens into estrogens is catalyzed irreversibly by aromatase, a microsomal enzymatic complex, which is composed of a glycoprotein, cytochrome P450 aromatase (P450arom) and a flavoprotein, NADPH cytochrome P450 reductase. P450arom is the component responsible for its catalytic activity. It has been well established that estrogens are essential not only in sexual differentiation and reproduction of the female, but also in development and maintenance of fertility of themale. In testis, estrogens play stimulatory roles in the proliferation in prepuberty, onset of spermatogenesis in puberty, spermatogenesis and the post-meiotic maturation of germ cells in mature. Recently, Aquila et al. have reported that in addition to Leydig cells and Sertoli cells in testis, ejaculated human spermatozoa also contain active aromatase, which is related to the acquisition of sperm motility. Concerning the relationship between expression of P450aorm in ejaculated human spermatozoa and their functions in fertilization, limited data are available in these days.In the present study, a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and a flow cytometry (FCM) method were carried out respectively to determine the amount of P450arom mRNA transcripts and the proportion of P450arom-positive spermatozoa. The ability of fertilization and acrosome reaction (AR) of ejaculated human spermatozoa were assessed by a sperm penetration of zona-free hamster egg assay (SPA) and a triple-stain technique, respectively. To investigate the relationship between the expression of P450 aromatase and sperm functions, correlation analyses were performed primarily between the amount of P450arom mRNA, the percentage of P450arom-positive spermatozoa, and the penetration rate of SPA, then between the amount of P450arom mRNA, the percentage of P450arom-positive spermatozoa, and the rate of acrosome-reacted spermatozoa. The P450arom mRNA level and the proportion of P450arom-positive spermatozoa of infertile men were respectively compared to those of fertile men to investigate if the aberrant expression of P450arom is involved in unexplained male infertility.Spermatozoa from fertile men and unexplained infertile men with normal semen parameters were obtained. Samples were allowed to completely liquefy, then subjected to centrifugation on discontinuous Percoll gradients.Total RNA extracted from purified spermatozoa underwent RT-PCR to amplify specific mRNA transcripts of P450arom and those of control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The staining intensities of the P450arom and GAPDH signals were determined respectively and the ratio of them was used to indicate the amount of P450 aromatase transcripts in spermatozoa. The results revealed that the P450arom mRNA content was correlated with the penetration rate of SPA(r =0.5907) and the rate of acrosome-reacted spermatozoa (r =0.5924). The P450arom/GAPDH ratio was 0.39±0.16 in infertile men (n =18) and 0.60±0.29 in fertile men (n=16), respectively. There was significant difference between them (P<0.02).Following an indirect immunocytochemistry, flow cytometry was performed to detect the proportion of P450arom-positive spermatozoa. Similarly, the percentage of P450arom-positive spermatozoa was correlated with either the penetration rate of SPA or the rate of acrosome-reacted spermatozoa, with correlation coefficient in 0.5319 and 0.5114, respectively. Compared with fertile men (n =18), the percentage of P450arom-positive spermatozoa of infertile men (n =21) was decreased (27.72±11.69% vs. 41.32±18.99%, p<0.01).Two possible explanations could be proposed for the relationship mentioned above. Firstly, the presence of P450arom in ejaculated spermatozoa may be the remnants during development and maturation ofgerm cells, it suggests that P450 aromatase defection possibly reflect the impairment of estrogens production and spermatogenesis to a certain degree, since estrogens are involved in spermatogenesis in testis. Secondly, functional P450 aromatase in ejaculated spermatozoa is certainly involved in local production of estrogens, which influence functions of themselves or adjacent spermatozoa via an autocrinal or a paracrinal pathway.In summary, our findings demonstrated that a link existed between the P450arom expression in ejaculated human spermatozoa and their functions. It also showed that the amount of P450 aromatase transcripts and proportion of spermatozoa expressing P450arom in spermatozoa of infertile men was significantly lower than those of fertile men. It suggests that P450 aromatase in spermatozoa exert important roles in acquirement of normal sperm function. Disturbance of P450arom expression in spermatozoa may be one of infertile causes.