Distribution and developmental expression of the AQP7 and AQP8 water channels in rat testis and epididymis.

Abstract

Objective: Fluid homeostasis is a critical process in spermatogenesis and sperm maturation and storage and multiple aquaporin transmembrane water channels (AQPs) have been recognized in the male reproductive tract. Nevertheless, the functional involvement of aquaporins in male reproductive physiology is poorly known. Recent recognition of AQP7 and AQP8, two aquaporins found in rat testis, generated clues about their possible functions in spermatogenesis and sperm maturation and in the protocols of semen freezing. To investigate potential roles for AQP7 and AQP8 we (1) established the distribution and (2) the developmental expression of these aquaporins in rat testis and epididymis. Design: The AQP7 and AQP8 distribution in rat male reproductive tract was assessed by immunohistochemistry. For the ontogeny studies, the specific expression of the AQP7 and AQP8 mRNA and protein in testes and epididymes from rats with ages ranging from embryonic day 10 (E10) through postnatal day 120 (+120) was measured by semi-quantitative RT-PCR and immunoblotting analysis, respectively. Materials/Methods: For immunohistochemistry, 10 μm cryosections of rat testis or epididymis were incubated with affinity purified anti-AQP7 or AQP8 antibodies and staining was visualized with HRP-conjugated secondary antibodies. Rat AQP7 and AQP8 primers were used for semi-quantitative RT-PCR. For immunoblotting, 17000 × g crude membrane fractions from rat testis or epididymis were immunoblotted with a 1:5000 dilution of an anti-AQP7 or AQP8 immune serum. Results: Immunohistochemical studies showed that AQP7 is localized both intracellularly and over the plasma membrane of spermatids throughout spermiogenesis by confirming previous observations (Suzuki-Toyota et al., 1999). AQP7 immunostaining was also seen on the plasma membrane of the cytoplasmic droplet and anterior tail domain of testicular and epididymal spermatozoa. AQP8 labeling was found in the basal portion of Sertoli cell and in the elongating spermatids where it appeared to increase with time until the release of sperm in the lumen of the seminiferous tubule. By RT-PCR, the AQP7 and AQP8 mRNAs were detected in testis starting on days +45 and +15, respectively. By immunoblotting of testis membranes, the AQP7 protein appeared at day +60 and reached its maximal expression from day +75 when the adulthood is attained. AQP8 was detected starting on day +15 coincident with the beginning of spermatogenesis while the highest expression was reached at day +30 by persisting into adulthood. Conclusions: (1) AQP7 and/or AQP8 may mediate the elimination of cytoplasmic water by which spermatids differentiate into spermatozoa during spermiogenesis. (2) AQP7 may be responsible for the high water permeability characterizing the spermatozoon, a feature thought to be critical in sperm-egg fusion, and in the protocols of semen cryopreservation. (3) AQP8 may mediate the secretion of water to form a fluid-filled tubular lumen, a morphological event announcing the beginning of spermatogenesis, and be involved in the generation of seminiferous fluid which serves as a vehicle for sperm transportation and contributes to sperm maturation. Supported by: This project was supported by a EU-TMR network grant and by the Italian ‘Ministero della Ricerca Scientifica e Tecnologica’ (MURST).