- Email: [email protected]
Possible Involvement of Aquaporin-7 and -8 in Rat Testis Development and Spermatogenesis
Biochemical and Biophysical Research Communications 288, 619 – 625 (2001) doi:10.1006/bbrc.2001.5810, available online at http://www.idealibrary.com on
Possible Involvement of Aquaporin-7 and -8 in Rat Testis Development and Spermatogenesis Giuseppe Calamita,* ,1 Amelia Mazzone,* Antonella Bizzoca,† and Maria Svelto* *Department of General and Environmental Physiology and †Department of Pharmacology and Human Physiology, University of Bari, Bari, Italy
Received September 17, 2001
Fluid secretion and reabsorption are of central importance in male reproductive (MR) physiology. However, the related molecular mechanisms are poorly known. Here, potential roles for AQP7 and AQP8, two aquaporin water channels abundantly expressed in the MR tract, were investigated by studying their expression and distribution in the developing testis of the Wistar rat. By semiquantitative RT-PCR and immunoblotting, first expression of AQP7 was noted at postnatal day 45 (P45), with levels increasing substantially at P90 and remaining at high levels thereafter. AQP8 began to be expressed at P15, rapidly increased until P20, and remained fairly stable thereafter. Immunohistochemical analyses demonstrated AQP7 in elongated spermatids, testicular spermatozoa, and residual bodies at P45 with increased signal intensity thereafter. AQP8 was observed in primary spermatocytes from P20 to P30 and, in elongated spermatids, residual bodies and Sertoli cells at P30 and thereafter. The ontogeny and distribution of AQP7 and AQP8 in rat testis suggest involvement in major physiologic changes in testis development and spermatogenesis. © 2001 Academic Press
Key Words: AQP7; AQP8; aquaporin water channel; fluid; spermatid; Sertoli cell; spermatogenesis; testis.
Fluid homeostasis is of major importance in a number of processes in the male reproductive physiology, including testis development, spermatogenesis, sperm maturation and storage, secretion of seminal liquid and egg fertilization. During postnatal testis development, Sertoli cells secrete fluid to form a fluid-filled tubular lumen (1) which is one of the first morphological events characterizing the formation of the bloodtestis barrier (Sertoli cell barrier) and the beginning of spermatogenesis (2, 3). In the adult animal, the semi1 To whom correspondence and reprint requests should be addressed at Dipartimento di Fisiologia Generale ed Ambientale, Universita` degli Studi di Bari, via Amendola 165/A, 70126 Bari, Italy. Fax: 39 0805443388. E-mail: [email protected].
niferous tubule fluid serves as a vehicle for sperm transportation and possible further maturation of sperm (4). A remarkable efflux of water from the cell has been evoked to explain the striking cytoplasm condensation needed for differentiation of round spermatids into elongating spermatids during spermiogenesis (5, 6). The seminiferous fluid is mostly reabsorbed in the efferent ducts (7) while fluids rich in nutrients are secreted by seminal vesicles and prostate and are needed for sperm to survive and fertilize eggs. Alterations in fluid balance in the male reproductive tract have been already reported to result in long-term atrophy of testes (8) and it is very likely that other forms of male infertility will be discovered. However, while the transporters accounting for the salt movements in the male reproductive physiology begin to be characterized (9), the molecular mechanisms by which water is transported in the MR tract remain poorly defined. Identification of multiple aquaporin water channels (see Ref. 10 for review on aquaporins), variously expressed in the secretory and absorptive portions of the MR tract, suggests roles for these proteins in MR physiology. The cDNAs of two aquaporins, AQP7 and AQP8, were recently reported in the MR and gastrointestinal tracts (11 and 12, 13, and 14, respectively). AQP7 has been found to be permeable to small neutral solutes, such as glycerol and urea in addition to water (11). AQP8 has been shown to transport water (12–15) and (in mouse, but not in rat or human) also urea (14). In the MR tract of adult rat, AQP7 was observed in spermatids and testicular (16) and epididymal (17) spermatozoa while abundant AQP8 was noted to be variously distributed in testis (17, 18). The AQP7 and 8 genes have been cloned and characterized structurally in human (19) and mouse (20) and human (21), respectively. In spite of their strong expression in MR tract, no information is available regarding the regulation and function of AQP7 and AQP8 in reproductive biology. This study was undertaken to determine the developmental expression and distribution of AQP7 and
619
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AQP8 in Wistar rat testis, in order to evaluate possible roles in developing testis, spermatogenesis and sperm maturation. As this work was nearing completion, the developmental expression of AQP7 and AQP8 in Sprague–Dawley rats was reported (22); however, discrepancies with our studies were observed in the timing, cellular expression and relative abundance of AQP7 and AQP8. Consequently, additional studies were carried out to clarify these differences and further define the expression of AQP7 and AQP8 in developing testis. MATERIALS AND METHODS Animals. Male Wistar rats of varying ages were obtained from Morini sas (S. Polo D’Enza, Italy), either with their actual mothers or with foster mothers. Animals, if not sacrificed earlier, were weaned at 21 days of age and fed and watered ad libitum. For all the experiments rats were decapitated after anesthesia and the tissues were collected as described in the following sections. RNA extraction and RT-PCR experiments. Tissues were removed from the testis of rats of different ages and frozen in liquid nitrogen. Total RNAs were isolated by the TRIzol extraction method (TRIzol reagent, Life Technologies, Gaithersburg, MD). As previously described (17), RT-PCR analysis was carried out by employing the GeneAmp RNA PCR Core kit (Perkin–Elmer, Branchburg, NJ) using the rat AQP7 or AQP8 specific primers RSA7-start (5⬘-ATGGCCGGTTCTGTGCTG-3⬘) and RSA7-stop (5⬘-TCTAAGAACCCTGTGGTGG) and RSA8-start (5⬘-CGGGATCCATGGCTGACAGTTACCAT3⬘) and RSA8-stop (5⬘-CGGAATTCACCTCGACTTTAGAAT-3⬘), respectively. This led to the amplification of expected 810- and 732-bp fragments of the rat AQP7 and AQP8 coding regions, respectively. The cDNAs amplified were then cloned into the pCR2.1 vector (TA cloning kit, Invitrogen, San Diego, CA) and the identity of the inserted DNA fragments was verified by sequencing. RT-PCRs were normalized against the -actin expression using a pair of -actin primers, BAF (5⬘-CAGATCATGTTTGAGACCTT-3⬘) and BAR (5⬘CGGATGTCMACGTCACACTT-3⬘; M ⫽ A ⫹ C) which lead to the amplification of a 509-bp DNA fragment. Preparation of testis homogenate and immunoblotting. Testes from P2 through P15 developing rats were pooled by litter in order to have adequate samples for processing; specimens from older animals were processed individually. Homogenates of pooled testes were prepared as previously reported (17). For the immunoblotting analyses, homogenates (60 g of proteins) were mixed with the Laemmli buffer and heated at 90°C for 4 min before being submitted to SDS–PAGE. The immunoblotting experiments were carried out as previously described (17) by using commercially available rabbit anti-rat AQP8 affinity-purified antibodies (Alpha Diagnostic International, San Antonio, TX) or anti-rat AQP7 affinity purified antibodies (kindly provided by Drs. G. P. Nicchia and A. Frigeri) at final concentrations of 400 or 200 ng/ml, respectively. Immunohistochemical experiments. After sacrifice, testes were isolated and immersed overnight at 4°C in 4% paraformaldehyde in PBS. The tissues were then washed in PBS (3 ⫻ 10 min) and finally cryoprotected by soaking in PBS containing 25% sucrose. Cryostat sections (8 m) were prepared and placed on silanized microscope slides. The immunohistochemical experiments were performed as previously described (23). Briefly, after blocking the endogenous peroxidases, the sections were treated with blocking solution for 1 h, incubated overnight with the anti-rat AQP8 or anti-rat AQP7 affinity purified antibodies (1 or 0.5 g/ml, respectively), washed with the blocking buffer and incubated for 1 h with a biotinylated goat antirabbit IgG (Vector Laboratories, Burlingame, CA) diluted at a final concentration of 10 g/ml in blocking buffer. After several washes,
FIG. 1. Expression of the AQP7 and AQP8 mRNAs in the developing testis of Wistar rat. Semiquantitative RT-PCR of total RNA samples from indicated developing rat testes from postnatal day 2 (P2) to adult (P180). (A) AQP7 expression (810-bp band) is noted at P45 and becomes high and stable at P90 and thereafter. (B) AQP8 (732-bp band) is expressed at P15 and reaches high signal intensity at P20 and thereafter. RT-PCR of -actin (509-bp band) was used to normalize the expression of AQP7 and AQP8.
sections were incubated for 1 h with horseradish peroxidase conjugated streptavidin (Vector Laboratories) at a concentration of 5 g/ ml. The immunohistochemical reaction was visualized by incubation with 3-amino 9-ethylcarbazole (AEC peroxidase substrate kit, Vector Laboratories) for 10 min. Sections were then counterstained with hematoxylin. Slides were cover-slipped with an aqueous mounting medium and viewed on a Leica DMRXA photomicroscope. Control immunostaining was performed with the preadsorbed AQP8 antibodies or by omitting the anti-AQP8 antibodies.
RESULTS Developmental Expression of AQP7 and AQP8 in Rat Testis By semiquantitative RT-PCR, the AQP7 mRNA was detected beginning from postnatal day 45 (P45) while the signal intensity reached the maximal extent at P90 and thereafter (Fig. 1A). AQP8 was found to be expressed earlier than AQP7 as its transcript was detected beginning from P15 and was fairly stable at its maximal intensity at P20 (Fig. 1B). This pattern of developmental expression was fully consistent with immunoblots of testis homogenates prepared from developing Wistar rats and incubated with affinity purified anti-rat AQP7 or rat AQP8 antibodies (Figs. 2A and 2B). A 23- to 24-kDa band corresponding to the
620
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Immunoblotting analyses of AQP7 and AQP8 in pooled homogenates of developing rat testis. Blots incubated with affinity purified anti-rat AQP7 (A) or rat AQP8 (B) antibodies. AQP7 is detected as a 23- to 24-kDa band whereas AQP8 appears as a 24 –25 doublet (double arrows), a 32- to 40-kDa diffuse component (glyAQP8), and a high-molecular-weight band representing the core protein, the glycosylated form, and, likely, a multimeric form (arrowhead) of AQP8, respectively. AQP7 is weakly detected at P45 while a strong immunoreactivity is noted thereafter (P60 to P180). AQP8 is detected at P30 and reaches a high and stable intensity at P45.
AQP7 protein (17) was weakly detected at P45 and the related immunoreactivity steadily increased until P60 when the signal became strong and fairly stable (Fig. 2A). AQP8 was detected at P30 as a 24- to 25-kDa doublet, a 32- to 40-kDa diffuse component and a highmolecular-mass band (Fig. 2B) that, as previously reported (17), corresponded to the core protein, the glycosylated AQP8 (glyAQP8) and, likely, a multimeric form of AQP8, respectively. Interestingly, the strong immunoreactivity of the 24-kDa band (lower band of the AQP8 doublet) was strong at P30, while it steadily decreased over the following sixty days and disappeared at P180 (Fig. 2B). No bands were noted in control immunoblots with anti-AQP7 or AQP8 antibodies preadsorbed with a 20:1 molar excess of the immunizing peptides (data not shown). Distribution of AQP7 and AQP8 in Developing Rat Testis AQP7 or AQP8 cellular and subcellular distribution in developing rat testis was assessed by immunohistochemical analysis of postnatal (P2 through P120) Wistar rat testis.
In line with the above immunoblotting studies, AQP7 immunoreactivity was first observed at P45 in the luminal aspect of the seminiferous epithelium of some tubules. Itsteadily increased until P75 and thereafter when all seminiferous tubules showed immunoreactivity (Figs. 3A, 3C, 3E, 3G, and 3I). AQP7 labeling was restricted to the plasma membrane and cytoplasmic mass of elongated spermatids, testicular spermatozoa and residual bodies (Figs. 3E, 3G, and 3I; see insets). At high magnification, in the cytoplasmic mass of elongated spermatids, strong labeling was seen over a Golgi-like apparatus (Figs. 3E and 3I, insets). No reactivity was observed in control experiments where the anti-AQP7 antibodies were omitted (Figs. 3B, 3D, 3F, 3H, and 3J). This pattern of cellular and subcellular distribution of AQP7 was consistent with that previously found in the testis of Wistar adult rat (16, 17). By contrast, it was different from that reported in the developing testis of Sprague–Dawley rat where AQP7 was detected considerably earlier, at P28, and found in round spermatids and in the plasma membrane of secondary spermatocytes (22). A transient but distinct AQP8 immunoreactivity was restricted to the cytoplasmic mass of primary spermatocytes of rat testis from P20 to P25-30 (Figs. 4C and 4D). Starting from P30, AQP8 immunostaining was observed in the cytoplasmic mass of elongated spermatids, residual bodies and, apparently, over the cytoplasmic ramifications of Sertoli cells (Figs. 4F, 4G, 4I, 4J, 4L, and 4M). The signal intensity of this pattern steadily increased until P90 and thereafter. Due to the close association of the Sertoli cells to the germ elements (Fig. 4M), the presence of AQP8 in spermatocytes could not be completely excluded. This may explain the apparent discrepancy with previous works describing the expression in rat testis (17, 22). No labeling was observed in control experiments omitting the anti-AQP8 antibodies (Figs. 4B, 4E, 4H, 4K, and 4N). DISCUSSION Definition of the aquaporin water channels expressed in the male reproductive tract will undoubtedly provide important information in understanding central processes such as the secretion of fluid to form the lumen of seminiferous tubules occurring during testis development and the fluid movements during spermatogenesis and sperm concentration and maturation. Our studies of postnatal developing rat testis document that AQP7 expression first appears at postnatal day 45, increases steadily until P90, and is sustained at high levels in adult animals. Overall, AQP8 begins to be expressed at P15, increases rapidly until P20 and remains fairly stable during testis development and, thereafter, in adult animals. The specific appearance of AQP7 in elongated spermatids parallels the beginning of spermiogenesis, a
621
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Immunohistochemical localization of AQP7 in Wistar rat testis at postnatal days P20 (A, B), P30 (C, D), P45 (E, F), P75 (G, H) and P90 (I, J). Cryostat sections were incubated with affinity purified anti-AQP7 antibodies (A, C, E, G, I); immunolabeling controls in B, D, F, H, and J were performed by omitting the anti-AQP7 antibodies (negative control). (A, C) At P20 and P30, virtually no labeling is observed in testis. (E, G, I) At P45 and thereafter, strong AQP7 immunolabeling is seen at the luminal rim of seminiferous tubules, where staining is observed within elongated spermatids (see insets in E and I), testicular spermatozoa (G, inset; double arrows) and residual bodies (E, right inset; arrowheads). In elongated spermatids, a Golgi-like apparatus (E, left insets; I, inset; arrows) shows stronger immunolabeling than the surrounding cytoplasm. No immunoreactivity is observed in any negative control sections (B, D, F, H). Magnification: (A, B) ⫻200, (C–J) ⫻100, (insets in E, G, I) ⫻400.
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Immunohistochemical localization of AQP8 in Wistar rat testis at P10 (A, B), P20 (C–E), P30 (F–H), P75 (I–K), and P120 (L–N). Sections were incubated with affinity purified anti-rat AQP8 antibodies (A, C, D, F, G, I, J, L, M); immunolabeling controls in B, E, H, K, and N were performed by omitting the anti-AQP8 antibodies (negative control). (A) At P10, no AQP8 immunoreactivity is observed in testis. (C, D) At P20, the AQP8 protein is seen in primary spermatocytes (arrowheads). (F, G, I, L) At P30 and thereafter, the luminal portion of all seminiferous tubules shows AQP8 immunoreactivity although the signal intensity is variable among the tubules, depending on the specific stage of spermatogenesis. (J, M) At higher magnification, labeling is observed in the elongated spermatids (M, J, arrows) and in the ramified cytoplasm of Sertoli cells (M, double arrows). No immunoreactivity is observed in any negative control sections (B, E, H, K, N). l.c., Leydig cell. Magnification: (A–C, E, G) ⫻200, (D, J, M) ⫻400, (F–I, K, L, N) ⫻10. 623
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
process in which spermatids progress to mature spermatozoa. AQP7 may therefore relate to the cell volume reduction of spermatids by mediating the efflux of water from spermatids during spermiogenesis. However, this role of AQP7 in spermiogenesis may not be so straightforward both because AQP7 is permeable to small neutral solutes in addition to water (11) and because rat spermatids express at least one additional water channel, AQP8 (12, 17, this study). A role for AQP7 in the morphological change of secondary spermatocytes to spermatids has been suggested by Kageyama and co-workers (22). However, in this study with developing testes of Wistar rats and in others on adult rats (16, 17), no AQP7 immunoreactivity was observed in secondary spermatocytes. Hence, the very weak and transient expression of AQP7 observed by Kageyama and co-workers (22) in secondary spermatocytes of Sprague–Dawley rat at P28 raises questions about the physiological relevance, although the possibility of a strain-to-strain variability should not be overlooked in explaining this. Interestingly, the appearance of AQP8 in testes of 15-day-old rats coincides with central processes such as the formation of the Sertoli cell barrier (3) and fluid secretion by Sertoli and germ cells to form the lumen of the seminiferous tubules (1–3, 24). This may suggest involvement of AQP8 in the secretion of water to form a fluid-filled tubular lumen, one of the first morphological events announcing the formation of the bloodtestis barrier and the beginning of spermatogenesis. AQP8 is presumed to have additional roles in the spermatogenic cycle as it is expressed in elongated spermatids and in Sertoli cells at P45 and thereafter. In Sertoli cells, AQP8 may play a role in the secretion of tubular fluid or, more in general, in the secretion of the milieu for germ cell transportation and maturation. Moreover, like AQP7, AQP8 may be involved in the cytoplasmic condensation occurring during differentiation of spermatids into spermatozoa. Because FSH and the synergistic actions of FSH and LH (indirectly by testosterone) are critically important both in puberty and adult spermatogenesis (25, 26) it is tempting to speculate that the expression of AQP8 in testis could be under hormonal control, as also suggested by Kageyama and co-workers (22). Interestingly, the developmental expression and cellular distribution of AQP8 in the rat seminiferous tubule is very similar to that of the cystic fibrosis transmembrane conductance regulator (27–29), CFTR, evidence that may suggest a functional correlation between AQP8 and CFTR. A functional link between CFTR and another aquaporin expressed in rat spermatids, AQP7, has been recently hypothesized by Gong and co-workers (29). Moreover, CFTR has already been reported to activate an aquaporin, AQP3, in the airway epithelium (30). Thus, as also speculated by Gong and co-workers (29), under CFTR activation AQP8 and/or AQP7 (or unknown
aquaporins) might mediate the efflux of water by which spermatids differentiate and remodel into testicular spermatozoa. This attractive hypothesis is corroborated by the evidence that testicular biopsies from cystic fibrosis patients and infertile men carrying mutations in the CFTR gene evidence a reduced number of mature spermatids and many malformed testicular spermatozoa (31). While the ontogeny and cellular distribution of AQP8 suggest appealing roles for AQP8 in developing testis and portend physiologic and pathophysiologic functions in later life, the functional meaning of the large intracellular localization featured by this aquaporin in rat testis remains unclear. Speculatively, the subcellular distribution of AQP8 might relate to its recycling between the intracellular compartment and the plasmamembrane (hormonal control?) or an involvement in the osmoregulation of the cytoplasm and its vesicle content. Definition of the regulatory meaning of the subcellular distribution of AQP8 may be a matter of further study. In summary, this work describes the expression of AQP7 and AQP8 in developing testis of Wistar rat. Roles for AQP8 are suggested in the secretion of water to form a fluid-filled seminiferous tubular lumen, an event that triggers the beginning of spermatogenesis, and in the secretion of the tubular fluid serving as a vehicle for sperm transportation and possible further maturation of sperm. AQP8 and/or AQP7 are presumed to be responsible for most of the cell volume reduction by which spermatids differentiate in spermatozoa during spermiogenesis. Interesting questions remain to be answered about the functional and regulatory meaning of the subcellular distribution of AQP8. Nevertheless, AQP7 and AQP8 may prove central to both normal physiology and pathophysiology of the reproductive tract, and may ultimately provide potential targets for contraceptive strategies. ACKNOWLEDGMENTS This work is dedicated to the memory of Professor L. D. Russell, an exemplary man and scientist whose stimulating, passionate, and helpful discussions in understanding the distribution of AQP7 and AQP8 in rat testis we very much appreciated. We thank Drs. Antonella Bizzoca and G. P. Nicchia and A. Frigeri for their helpful contributions and for providing the anti-AQP7 antibodies, respectively. Support of the European Community (EU-TMR Network Grant ERB 4061 PL 97-0406) is gratefully acknowledged.
REFERENCES
624
1. Fawcett, D. W. (1975) Ultrastructure and function of the Sertoli cell. In Handbook of Physiology (Greep, R. O., and Atwood, E. B., Eds.), Vol. 5, pp. 21–53, Am. Physiol. Soc., Washington, DC. 2. Vitale, R., Fawcett, D. W., and Dym, M. (1973) The normal development of the blood–testis barrier and the effects of clomiphene and estrogen treatment. Anat. Rec. 176, 331–344. 3. Russell, L. D., Bartke, A., and Goh, J. C. (1989) Postnatal devel-
Vol. 288, No. 3, 2001
4.
5.
6.
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
17.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
opment of the Sertoli cell barrier, tubular lumen, and cytoskeleton of Sertoli and myoid cells in the rat, and their relationship to tubular fluid secretion and flow. Am. J. Anat. 184, 179 –189. Hinton, B. T., and Setchell, B. P. (1993) Fluid secretion and movement. In The Sertoli Cell (Russell, L. D., and Griswold, M. D., Eds.), pp. 249 –267, Cache River Press, Clearwater, FL. Russell, L. D. (1979) Spermatid–Sertoli tubulobulbar complexes as devices for elimination of cytoplasm from the head region of late spermatids of the rat. Anat. Rec. 194, 233–246. Sprando, R. L., and Russell, L. D. (1987) Comparative study of cytoplasmic elimination in spermatids of selected mammalian species. Am. J. Anat. 178, 72– 80. Clulow, J., Jones, R. C., and Hansen, L. A. (1994) Micropuncture and cannulation studies of fluid composition and transport in the ductuli efferentes testis of the rat: Comparison with the homologous metanephric proximal tubule. Exp. Physiol. 79, 915–928. Hess, R. A., Bunick, D., Lee, K. H., Bahr, J., Taylor, J. A., Korach, K. S., and Lubahn, D. B. (1997) A role for estrogens in the male reproductive system. Nature 390, 509 –512. Darszon, A., Labarca, P., Nishigaki, T., and Espinosa, F. (1999) Ion channels in sperm physiology. Physiol. Rev. 79, 481–510. Borgnia, M. J., Nielsen, S., Engel, A., and Agre, P. (1999) Cellular and molecular biology of the aquaporin water channels. Annu. Rev. Biochem. 68, 425– 458. Ishibashi, K., Kuwahara, Y., Gu Y., Kageyama, A., Tohsaka, F., Suzuki, F., Marumo, F., and Sasaki, S. (1997) Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea. J. Biol. Chem. 272, 20782–20786. Ishibashi, K., Kuwahara, Y., Kageyama, A., Tohsaka, F., Marumo, F., and Sasaki, S. (1997) Cloning and functional expression of a second new aquaporin abundantly expressed in testis. Biochem. Biophys. Res. Commun. 237, 714 –718. Koyama, Y., Yamamoto, T., Kondo, D., Funaki, H., Yaoita, E., Kawasaki, K., Sato, N., Hatakeyama, K., and Kihara, I. (1997) Molecular cloning of a new aquaporin from rat pancreas and liver. J. Biol. Chem. 272, 30329 –30333. Ma, T., Yang, B., and Verkman, A. S. (1997) Cloning of a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys. Res. Commun. 240, 324 –328. Koyama, Y., Ishibashi, K., Kuwahara, Y., Inase, N., Ichioka, M., Sasaki, S., and Marumo, F. (1998) Cloning and functional expression of human aquaporin-8 cDNA and analysis of its gene. Genomics 54, 169 –172. Suzuki-Toyota, F., Ishibashi, K., and Yuasa, S. (1999) Immunohistochemical localization of a water channel, aquaporin-7 (AQP7), in the rat testis. Cell. Tissue Res. 295, 279 –285. Calamita, G., Mazzone, A., Cho, Y. S., Valenti, G., and Svelto, M. (2001) Expression and localization of the aquaporin-8 water channel in rat testis. Biol. Reprod. 64, 1660 –1666.
18. Tani, T., Koyama, Y., Nihei, K., Hatakeyama, S., Ohshiro, K., Yoshida, Y., Yaoita, E., Sakai, Y., Hatakeyama, K., and Yamamoto, T. (2001) Immunolocalization of aquaporin-8 in rat digestive organs and testis. Arch. Histol. Cytol. 64, 159 –168. 19. Ishibashi, K., Yamauchi, K., Kageyama, Y., Saito-Ohara, F., Ikeuchi, T., Marumo, F., and Sasaki, S. (1998) Molecular characterization of human aquaporin-7 gene and its chromosomal mapping. Biochim. Biophys. Acta 1399, 62– 66. 20. Calamita, G., Spalluto, C., Mazzone, A., Rocchi, M., and Svelto, M. (1999) Cloning, structural organization and chromosomal localization of the mouse aquaporin-8 water channel gene. Cytogenet. Cell Genet. 185, 237–241. 21. Viggiano, L., Rocchi, M., Svelto, M., and Calamita, G. (1999) Assignment of the aquaporin-8 water channel gene (AQP8) to human chromosome 16p12. Cytogenet. Cell Genet. 84, 208 –210. 22. Kageyama, Y., Ishibashi, K., Hayashi, T., Xia, G., Sasaki, S., and Kihara, K. (2001) Expression of aquaporins 7 and 8 in the developing testis. Andrologia 33, 165–169. 23. Calamita, G., Mazzone, A., Bizzoca, A., Cavalier, A., Cassano, G., Thomas, D., and Svelto, M. (2001) Expression and immunolocalization of aquaporin-8 water channel in rat gastrointestinal tract. Eur. J. Cell Biol., in press. 24. Russell, L. D. (1993) Role in spermiation. In The Sertoli Cell (Russell, L. D., and Griswold, M. D., Eds.), pp. 269 –303, Cache River Press, Clearwater, FL. 25. Russell, L. D., Alger, L. E., and Nequin, L. G. (1987) Hormonal control of pubertal spermatogenesis. Endocrinology 120, 1615– 1632. 26. Griffin, J. E., and Wilson, J. D. (1994) Disorders of the testes. In Harrison’s Principles of Internal Medicine (Isselbacher, K. J., et al., Eds.), 13th ed., pp. 2006 –2017, McGraw-Hill, New York. 27. Trezise, A. E., Linder, C. C., Grieger, D., Thompson, E. W., Meunier, H., Griswold, M. D., and Buchwald, M. (1993) CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents. Nat. Genet. 3, 157–164. 28. Boockfor, F. R., Morris, R. A., DeSimone, D. C., Hunt, D. M., and Walsh, K. B. (1998) Sertoli cell expression of the cystic fibrosis transmembrane conductance regulator. Am. J. Physiol. 274, C992–C930. 29. Gong, X. D., Li, J. C., Cheung, K. H., Leung, G. P., Chew, S. B., and Wong, P. Y. (2001) Expression of the cystic fibrosis transmembrane conductance regulator in rat spermatids: Implication for the site of action of antispermatogenic agents. Mol. Hum. Reprod. 7, 705–713. 30. Schreiber, R., Nitschke, R., Greger, R., and Kunzelmann, K. (1999) The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells. J. Biol. Chem. 274, 11811–11816. 31. Van der Ven, K., Messer, L., Van der Ven, H., Jeyendran, R. S., and Ober, C. (1996) Cystic fibrosis mutation screening in healthy men with reduced sperm quality. Hum. Reprod. 11, 513–517.
625
Possible Involvement of Aquaporin-7 and -8 in Rat Testis Development and Spermatogenesis Giuseppe Calamita,* ,1 Amelia Mazzone,* Antonella Bizzoca,† and Maria Svelto* *Department of General and Environmental Physiology and †Department of Pharmacology and Human Physiology, University of Bari, Bari, Italy
Received September 17, 2001
Fluid secretion and reabsorption are of central importance in male reproductive (MR) physiology. However, the related molecular mechanisms are poorly known. Here, potential roles for AQP7 and AQP8, two aquaporin water channels abundantly expressed in the MR tract, were investigated by studying their expression and distribution in the developing testis of the Wistar rat. By semiquantitative RT-PCR and immunoblotting, first expression of AQP7 was noted at postnatal day 45 (P45), with levels increasing substantially at P90 and remaining at high levels thereafter. AQP8 began to be expressed at P15, rapidly increased until P20, and remained fairly stable thereafter. Immunohistochemical analyses demonstrated AQP7 in elongated spermatids, testicular spermatozoa, and residual bodies at P45 with increased signal intensity thereafter. AQP8 was observed in primary spermatocytes from P20 to P30 and, in elongated spermatids, residual bodies and Sertoli cells at P30 and thereafter. The ontogeny and distribution of AQP7 and AQP8 in rat testis suggest involvement in major physiologic changes in testis development and spermatogenesis. © 2001 Academic Press
Key Words: AQP7; AQP8; aquaporin water channel; fluid; spermatid; Sertoli cell; spermatogenesis; testis.
Fluid homeostasis is of major importance in a number of processes in the male reproductive physiology, including testis development, spermatogenesis, sperm maturation and storage, secretion of seminal liquid and egg fertilization. During postnatal testis development, Sertoli cells secrete fluid to form a fluid-filled tubular lumen (1) which is one of the first morphological events characterizing the formation of the bloodtestis barrier (Sertoli cell barrier) and the beginning of spermatogenesis (2, 3). In the adult animal, the semi1 To whom correspondence and reprint requests should be addressed at Dipartimento di Fisiologia Generale ed Ambientale, Universita` degli Studi di Bari, via Amendola 165/A, 70126 Bari, Italy. Fax: 39 0805443388. E-mail: [email protected].
niferous tubule fluid serves as a vehicle for sperm transportation and possible further maturation of sperm (4). A remarkable efflux of water from the cell has been evoked to explain the striking cytoplasm condensation needed for differentiation of round spermatids into elongating spermatids during spermiogenesis (5, 6). The seminiferous fluid is mostly reabsorbed in the efferent ducts (7) while fluids rich in nutrients are secreted by seminal vesicles and prostate and are needed for sperm to survive and fertilize eggs. Alterations in fluid balance in the male reproductive tract have been already reported to result in long-term atrophy of testes (8) and it is very likely that other forms of male infertility will be discovered. However, while the transporters accounting for the salt movements in the male reproductive physiology begin to be characterized (9), the molecular mechanisms by which water is transported in the MR tract remain poorly defined. Identification of multiple aquaporin water channels (see Ref. 10 for review on aquaporins), variously expressed in the secretory and absorptive portions of the MR tract, suggests roles for these proteins in MR physiology. The cDNAs of two aquaporins, AQP7 and AQP8, were recently reported in the MR and gastrointestinal tracts (11 and 12, 13, and 14, respectively). AQP7 has been found to be permeable to small neutral solutes, such as glycerol and urea in addition to water (11). AQP8 has been shown to transport water (12–15) and (in mouse, but not in rat or human) also urea (14). In the MR tract of adult rat, AQP7 was observed in spermatids and testicular (16) and epididymal (17) spermatozoa while abundant AQP8 was noted to be variously distributed in testis (17, 18). The AQP7 and 8 genes have been cloned and characterized structurally in human (19) and mouse (20) and human (21), respectively. In spite of their strong expression in MR tract, no information is available regarding the regulation and function of AQP7 and AQP8 in reproductive biology. This study was undertaken to determine the developmental expression and distribution of AQP7 and
619
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AQP8 in Wistar rat testis, in order to evaluate possible roles in developing testis, spermatogenesis and sperm maturation. As this work was nearing completion, the developmental expression of AQP7 and AQP8 in Sprague–Dawley rats was reported (22); however, discrepancies with our studies were observed in the timing, cellular expression and relative abundance of AQP7 and AQP8. Consequently, additional studies were carried out to clarify these differences and further define the expression of AQP7 and AQP8 in developing testis. MATERIALS AND METHODS Animals. Male Wistar rats of varying ages were obtained from Morini sas (S. Polo D’Enza, Italy), either with their actual mothers or with foster mothers. Animals, if not sacrificed earlier, were weaned at 21 days of age and fed and watered ad libitum. For all the experiments rats were decapitated after anesthesia and the tissues were collected as described in the following sections. RNA extraction and RT-PCR experiments. Tissues were removed from the testis of rats of different ages and frozen in liquid nitrogen. Total RNAs were isolated by the TRIzol extraction method (TRIzol reagent, Life Technologies, Gaithersburg, MD). As previously described (17), RT-PCR analysis was carried out by employing the GeneAmp RNA PCR Core kit (Perkin–Elmer, Branchburg, NJ) using the rat AQP7 or AQP8 specific primers RSA7-start (5⬘-ATGGCCGGTTCTGTGCTG-3⬘) and RSA7-stop (5⬘-TCTAAGAACCCTGTGGTGG) and RSA8-start (5⬘-CGGGATCCATGGCTGACAGTTACCAT3⬘) and RSA8-stop (5⬘-CGGAATTCACCTCGACTTTAGAAT-3⬘), respectively. This led to the amplification of expected 810- and 732-bp fragments of the rat AQP7 and AQP8 coding regions, respectively. The cDNAs amplified were then cloned into the pCR2.1 vector (TA cloning kit, Invitrogen, San Diego, CA) and the identity of the inserted DNA fragments was verified by sequencing. RT-PCRs were normalized against the -actin expression using a pair of -actin primers, BAF (5⬘-CAGATCATGTTTGAGACCTT-3⬘) and BAR (5⬘CGGATGTCMACGTCACACTT-3⬘; M ⫽ A ⫹ C) which lead to the amplification of a 509-bp DNA fragment. Preparation of testis homogenate and immunoblotting. Testes from P2 through P15 developing rats were pooled by litter in order to have adequate samples for processing; specimens from older animals were processed individually. Homogenates of pooled testes were prepared as previously reported (17). For the immunoblotting analyses, homogenates (60 g of proteins) were mixed with the Laemmli buffer and heated at 90°C for 4 min before being submitted to SDS–PAGE. The immunoblotting experiments were carried out as previously described (17) by using commercially available rabbit anti-rat AQP8 affinity-purified antibodies (Alpha Diagnostic International, San Antonio, TX) or anti-rat AQP7 affinity purified antibodies (kindly provided by Drs. G. P. Nicchia and A. Frigeri) at final concentrations of 400 or 200 ng/ml, respectively. Immunohistochemical experiments. After sacrifice, testes were isolated and immersed overnight at 4°C in 4% paraformaldehyde in PBS. The tissues were then washed in PBS (3 ⫻ 10 min) and finally cryoprotected by soaking in PBS containing 25% sucrose. Cryostat sections (8 m) were prepared and placed on silanized microscope slides. The immunohistochemical experiments were performed as previously described (23). Briefly, after blocking the endogenous peroxidases, the sections were treated with blocking solution for 1 h, incubated overnight with the anti-rat AQP8 or anti-rat AQP7 affinity purified antibodies (1 or 0.5 g/ml, respectively), washed with the blocking buffer and incubated for 1 h with a biotinylated goat antirabbit IgG (Vector Laboratories, Burlingame, CA) diluted at a final concentration of 10 g/ml in blocking buffer. After several washes,
FIG. 1. Expression of the AQP7 and AQP8 mRNAs in the developing testis of Wistar rat. Semiquantitative RT-PCR of total RNA samples from indicated developing rat testes from postnatal day 2 (P2) to adult (P180). (A) AQP7 expression (810-bp band) is noted at P45 and becomes high and stable at P90 and thereafter. (B) AQP8 (732-bp band) is expressed at P15 and reaches high signal intensity at P20 and thereafter. RT-PCR of -actin (509-bp band) was used to normalize the expression of AQP7 and AQP8.
sections were incubated for 1 h with horseradish peroxidase conjugated streptavidin (Vector Laboratories) at a concentration of 5 g/ ml. The immunohistochemical reaction was visualized by incubation with 3-amino 9-ethylcarbazole (AEC peroxidase substrate kit, Vector Laboratories) for 10 min. Sections were then counterstained with hematoxylin. Slides were cover-slipped with an aqueous mounting medium and viewed on a Leica DMRXA photomicroscope. Control immunostaining was performed with the preadsorbed AQP8 antibodies or by omitting the anti-AQP8 antibodies.
RESULTS Developmental Expression of AQP7 and AQP8 in Rat Testis By semiquantitative RT-PCR, the AQP7 mRNA was detected beginning from postnatal day 45 (P45) while the signal intensity reached the maximal extent at P90 and thereafter (Fig. 1A). AQP8 was found to be expressed earlier than AQP7 as its transcript was detected beginning from P15 and was fairly stable at its maximal intensity at P20 (Fig. 1B). This pattern of developmental expression was fully consistent with immunoblots of testis homogenates prepared from developing Wistar rats and incubated with affinity purified anti-rat AQP7 or rat AQP8 antibodies (Figs. 2A and 2B). A 23- to 24-kDa band corresponding to the
620
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Immunoblotting analyses of AQP7 and AQP8 in pooled homogenates of developing rat testis. Blots incubated with affinity purified anti-rat AQP7 (A) or rat AQP8 (B) antibodies. AQP7 is detected as a 23- to 24-kDa band whereas AQP8 appears as a 24 –25 doublet (double arrows), a 32- to 40-kDa diffuse component (glyAQP8), and a high-molecular-weight band representing the core protein, the glycosylated form, and, likely, a multimeric form (arrowhead) of AQP8, respectively. AQP7 is weakly detected at P45 while a strong immunoreactivity is noted thereafter (P60 to P180). AQP8 is detected at P30 and reaches a high and stable intensity at P45.
AQP7 protein (17) was weakly detected at P45 and the related immunoreactivity steadily increased until P60 when the signal became strong and fairly stable (Fig. 2A). AQP8 was detected at P30 as a 24- to 25-kDa doublet, a 32- to 40-kDa diffuse component and a highmolecular-mass band (Fig. 2B) that, as previously reported (17), corresponded to the core protein, the glycosylated AQP8 (glyAQP8) and, likely, a multimeric form of AQP8, respectively. Interestingly, the strong immunoreactivity of the 24-kDa band (lower band of the AQP8 doublet) was strong at P30, while it steadily decreased over the following sixty days and disappeared at P180 (Fig. 2B). No bands were noted in control immunoblots with anti-AQP7 or AQP8 antibodies preadsorbed with a 20:1 molar excess of the immunizing peptides (data not shown). Distribution of AQP7 and AQP8 in Developing Rat Testis AQP7 or AQP8 cellular and subcellular distribution in developing rat testis was assessed by immunohistochemical analysis of postnatal (P2 through P120) Wistar rat testis.
In line with the above immunoblotting studies, AQP7 immunoreactivity was first observed at P45 in the luminal aspect of the seminiferous epithelium of some tubules. Itsteadily increased until P75 and thereafter when all seminiferous tubules showed immunoreactivity (Figs. 3A, 3C, 3E, 3G, and 3I). AQP7 labeling was restricted to the plasma membrane and cytoplasmic mass of elongated spermatids, testicular spermatozoa and residual bodies (Figs. 3E, 3G, and 3I; see insets). At high magnification, in the cytoplasmic mass of elongated spermatids, strong labeling was seen over a Golgi-like apparatus (Figs. 3E and 3I, insets). No reactivity was observed in control experiments where the anti-AQP7 antibodies were omitted (Figs. 3B, 3D, 3F, 3H, and 3J). This pattern of cellular and subcellular distribution of AQP7 was consistent with that previously found in the testis of Wistar adult rat (16, 17). By contrast, it was different from that reported in the developing testis of Sprague–Dawley rat where AQP7 was detected considerably earlier, at P28, and found in round spermatids and in the plasma membrane of secondary spermatocytes (22). A transient but distinct AQP8 immunoreactivity was restricted to the cytoplasmic mass of primary spermatocytes of rat testis from P20 to P25-30 (Figs. 4C and 4D). Starting from P30, AQP8 immunostaining was observed in the cytoplasmic mass of elongated spermatids, residual bodies and, apparently, over the cytoplasmic ramifications of Sertoli cells (Figs. 4F, 4G, 4I, 4J, 4L, and 4M). The signal intensity of this pattern steadily increased until P90 and thereafter. Due to the close association of the Sertoli cells to the germ elements (Fig. 4M), the presence of AQP8 in spermatocytes could not be completely excluded. This may explain the apparent discrepancy with previous works describing the expression in rat testis (17, 22). No labeling was observed in control experiments omitting the anti-AQP8 antibodies (Figs. 4B, 4E, 4H, 4K, and 4N). DISCUSSION Definition of the aquaporin water channels expressed in the male reproductive tract will undoubtedly provide important information in understanding central processes such as the secretion of fluid to form the lumen of seminiferous tubules occurring during testis development and the fluid movements during spermatogenesis and sperm concentration and maturation. Our studies of postnatal developing rat testis document that AQP7 expression first appears at postnatal day 45, increases steadily until P90, and is sustained at high levels in adult animals. Overall, AQP8 begins to be expressed at P15, increases rapidly until P20 and remains fairly stable during testis development and, thereafter, in adult animals. The specific appearance of AQP7 in elongated spermatids parallels the beginning of spermiogenesis, a
621
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Immunohistochemical localization of AQP7 in Wistar rat testis at postnatal days P20 (A, B), P30 (C, D), P45 (E, F), P75 (G, H) and P90 (I, J). Cryostat sections were incubated with affinity purified anti-AQP7 antibodies (A, C, E, G, I); immunolabeling controls in B, D, F, H, and J were performed by omitting the anti-AQP7 antibodies (negative control). (A, C) At P20 and P30, virtually no labeling is observed in testis. (E, G, I) At P45 and thereafter, strong AQP7 immunolabeling is seen at the luminal rim of seminiferous tubules, where staining is observed within elongated spermatids (see insets in E and I), testicular spermatozoa (G, inset; double arrows) and residual bodies (E, right inset; arrowheads). In elongated spermatids, a Golgi-like apparatus (E, left insets; I, inset; arrows) shows stronger immunolabeling than the surrounding cytoplasm. No immunoreactivity is observed in any negative control sections (B, D, F, H). Magnification: (A, B) ⫻200, (C–J) ⫻100, (insets in E, G, I) ⫻400.
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Immunohistochemical localization of AQP8 in Wistar rat testis at P10 (A, B), P20 (C–E), P30 (F–H), P75 (I–K), and P120 (L–N). Sections were incubated with affinity purified anti-rat AQP8 antibodies (A, C, D, F, G, I, J, L, M); immunolabeling controls in B, E, H, K, and N were performed by omitting the anti-AQP8 antibodies (negative control). (A) At P10, no AQP8 immunoreactivity is observed in testis. (C, D) At P20, the AQP8 protein is seen in primary spermatocytes (arrowheads). (F, G, I, L) At P30 and thereafter, the luminal portion of all seminiferous tubules shows AQP8 immunoreactivity although the signal intensity is variable among the tubules, depending on the specific stage of spermatogenesis. (J, M) At higher magnification, labeling is observed in the elongated spermatids (M, J, arrows) and in the ramified cytoplasm of Sertoli cells (M, double arrows). No immunoreactivity is observed in any negative control sections (B, E, H, K, N). l.c., Leydig cell. Magnification: (A–C, E, G) ⫻200, (D, J, M) ⫻400, (F–I, K, L, N) ⫻10. 623
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
process in which spermatids progress to mature spermatozoa. AQP7 may therefore relate to the cell volume reduction of spermatids by mediating the efflux of water from spermatids during spermiogenesis. However, this role of AQP7 in spermiogenesis may not be so straightforward both because AQP7 is permeable to small neutral solutes in addition to water (11) and because rat spermatids express at least one additional water channel, AQP8 (12, 17, this study). A role for AQP7 in the morphological change of secondary spermatocytes to spermatids has been suggested by Kageyama and co-workers (22). However, in this study with developing testes of Wistar rats and in others on adult rats (16, 17), no AQP7 immunoreactivity was observed in secondary spermatocytes. Hence, the very weak and transient expression of AQP7 observed by Kageyama and co-workers (22) in secondary spermatocytes of Sprague–Dawley rat at P28 raises questions about the physiological relevance, although the possibility of a strain-to-strain variability should not be overlooked in explaining this. Interestingly, the appearance of AQP8 in testes of 15-day-old rats coincides with central processes such as the formation of the Sertoli cell barrier (3) and fluid secretion by Sertoli and germ cells to form the lumen of the seminiferous tubules (1–3, 24). This may suggest involvement of AQP8 in the secretion of water to form a fluid-filled tubular lumen, one of the first morphological events announcing the formation of the bloodtestis barrier and the beginning of spermatogenesis. AQP8 is presumed to have additional roles in the spermatogenic cycle as it is expressed in elongated spermatids and in Sertoli cells at P45 and thereafter. In Sertoli cells, AQP8 may play a role in the secretion of tubular fluid or, more in general, in the secretion of the milieu for germ cell transportation and maturation. Moreover, like AQP7, AQP8 may be involved in the cytoplasmic condensation occurring during differentiation of spermatids into spermatozoa. Because FSH and the synergistic actions of FSH and LH (indirectly by testosterone) are critically important both in puberty and adult spermatogenesis (25, 26) it is tempting to speculate that the expression of AQP8 in testis could be under hormonal control, as also suggested by Kageyama and co-workers (22). Interestingly, the developmental expression and cellular distribution of AQP8 in the rat seminiferous tubule is very similar to that of the cystic fibrosis transmembrane conductance regulator (27–29), CFTR, evidence that may suggest a functional correlation between AQP8 and CFTR. A functional link between CFTR and another aquaporin expressed in rat spermatids, AQP7, has been recently hypothesized by Gong and co-workers (29). Moreover, CFTR has already been reported to activate an aquaporin, AQP3, in the airway epithelium (30). Thus, as also speculated by Gong and co-workers (29), under CFTR activation AQP8 and/or AQP7 (or unknown
aquaporins) might mediate the efflux of water by which spermatids differentiate and remodel into testicular spermatozoa. This attractive hypothesis is corroborated by the evidence that testicular biopsies from cystic fibrosis patients and infertile men carrying mutations in the CFTR gene evidence a reduced number of mature spermatids and many malformed testicular spermatozoa (31). While the ontogeny and cellular distribution of AQP8 suggest appealing roles for AQP8 in developing testis and portend physiologic and pathophysiologic functions in later life, the functional meaning of the large intracellular localization featured by this aquaporin in rat testis remains unclear. Speculatively, the subcellular distribution of AQP8 might relate to its recycling between the intracellular compartment and the plasmamembrane (hormonal control?) or an involvement in the osmoregulation of the cytoplasm and its vesicle content. Definition of the regulatory meaning of the subcellular distribution of AQP8 may be a matter of further study. In summary, this work describes the expression of AQP7 and AQP8 in developing testis of Wistar rat. Roles for AQP8 are suggested in the secretion of water to form a fluid-filled seminiferous tubular lumen, an event that triggers the beginning of spermatogenesis, and in the secretion of the tubular fluid serving as a vehicle for sperm transportation and possible further maturation of sperm. AQP8 and/or AQP7 are presumed to be responsible for most of the cell volume reduction by which spermatids differentiate in spermatozoa during spermiogenesis. Interesting questions remain to be answered about the functional and regulatory meaning of the subcellular distribution of AQP8. Nevertheless, AQP7 and AQP8 may prove central to both normal physiology and pathophysiology of the reproductive tract, and may ultimately provide potential targets for contraceptive strategies. ACKNOWLEDGMENTS This work is dedicated to the memory of Professor L. D. Russell, an exemplary man and scientist whose stimulating, passionate, and helpful discussions in understanding the distribution of AQP7 and AQP8 in rat testis we very much appreciated. We thank Drs. Antonella Bizzoca and G. P. Nicchia and A. Frigeri for their helpful contributions and for providing the anti-AQP7 antibodies, respectively. Support of the European Community (EU-TMR Network Grant ERB 4061 PL 97-0406) is gratefully acknowledged.
REFERENCES
624
1. Fawcett, D. W. (1975) Ultrastructure and function of the Sertoli cell. In Handbook of Physiology (Greep, R. O., and Atwood, E. B., Eds.), Vol. 5, pp. 21–53, Am. Physiol. Soc., Washington, DC. 2. Vitale, R., Fawcett, D. W., and Dym, M. (1973) The normal development of the blood–testis barrier and the effects of clomiphene and estrogen treatment. Anat. Rec. 176, 331–344. 3. Russell, L. D., Bartke, A., and Goh, J. C. (1989) Postnatal devel-
Vol. 288, No. 3, 2001
4.
5.
6.
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
17.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
opment of the Sertoli cell barrier, tubular lumen, and cytoskeleton of Sertoli and myoid cells in the rat, and their relationship to tubular fluid secretion and flow. Am. J. Anat. 184, 179 –189. Hinton, B. T., and Setchell, B. P. (1993) Fluid secretion and movement. In The Sertoli Cell (Russell, L. D., and Griswold, M. D., Eds.), pp. 249 –267, Cache River Press, Clearwater, FL. Russell, L. D. (1979) Spermatid–Sertoli tubulobulbar complexes as devices for elimination of cytoplasm from the head region of late spermatids of the rat. Anat. Rec. 194, 233–246. Sprando, R. L., and Russell, L. D. (1987) Comparative study of cytoplasmic elimination in spermatids of selected mammalian species. Am. J. Anat. 178, 72– 80. Clulow, J., Jones, R. C., and Hansen, L. A. (1994) Micropuncture and cannulation studies of fluid composition and transport in the ductuli efferentes testis of the rat: Comparison with the homologous metanephric proximal tubule. Exp. Physiol. 79, 915–928. Hess, R. A., Bunick, D., Lee, K. H., Bahr, J., Taylor, J. A., Korach, K. S., and Lubahn, D. B. (1997) A role for estrogens in the male reproductive system. Nature 390, 509 –512. Darszon, A., Labarca, P., Nishigaki, T., and Espinosa, F. (1999) Ion channels in sperm physiology. Physiol. Rev. 79, 481–510. Borgnia, M. J., Nielsen, S., Engel, A., and Agre, P. (1999) Cellular and molecular biology of the aquaporin water channels. Annu. Rev. Biochem. 68, 425– 458. Ishibashi, K., Kuwahara, Y., Gu Y., Kageyama, A., Tohsaka, F., Suzuki, F., Marumo, F., and Sasaki, S. (1997) Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea. J. Biol. Chem. 272, 20782–20786. Ishibashi, K., Kuwahara, Y., Kageyama, A., Tohsaka, F., Marumo, F., and Sasaki, S. (1997) Cloning and functional expression of a second new aquaporin abundantly expressed in testis. Biochem. Biophys. Res. Commun. 237, 714 –718. Koyama, Y., Yamamoto, T., Kondo, D., Funaki, H., Yaoita, E., Kawasaki, K., Sato, N., Hatakeyama, K., and Kihara, I. (1997) Molecular cloning of a new aquaporin from rat pancreas and liver. J. Biol. Chem. 272, 30329 –30333. Ma, T., Yang, B., and Verkman, A. S. (1997) Cloning of a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys. Res. Commun. 240, 324 –328. Koyama, Y., Ishibashi, K., Kuwahara, Y., Inase, N., Ichioka, M., Sasaki, S., and Marumo, F. (1998) Cloning and functional expression of human aquaporin-8 cDNA and analysis of its gene. Genomics 54, 169 –172. Suzuki-Toyota, F., Ishibashi, K., and Yuasa, S. (1999) Immunohistochemical localization of a water channel, aquaporin-7 (AQP7), in the rat testis. Cell. Tissue Res. 295, 279 –285. Calamita, G., Mazzone, A., Cho, Y. S., Valenti, G., and Svelto, M. (2001) Expression and localization of the aquaporin-8 water channel in rat testis. Biol. Reprod. 64, 1660 –1666.
18. Tani, T., Koyama, Y., Nihei, K., Hatakeyama, S., Ohshiro, K., Yoshida, Y., Yaoita, E., Sakai, Y., Hatakeyama, K., and Yamamoto, T. (2001) Immunolocalization of aquaporin-8 in rat digestive organs and testis. Arch. Histol. Cytol. 64, 159 –168. 19. Ishibashi, K., Yamauchi, K., Kageyama, Y., Saito-Ohara, F., Ikeuchi, T., Marumo, F., and Sasaki, S. (1998) Molecular characterization of human aquaporin-7 gene and its chromosomal mapping. Biochim. Biophys. Acta 1399, 62– 66. 20. Calamita, G., Spalluto, C., Mazzone, A., Rocchi, M., and Svelto, M. (1999) Cloning, structural organization and chromosomal localization of the mouse aquaporin-8 water channel gene. Cytogenet. Cell Genet. 185, 237–241. 21. Viggiano, L., Rocchi, M., Svelto, M., and Calamita, G. (1999) Assignment of the aquaporin-8 water channel gene (AQP8) to human chromosome 16p12. Cytogenet. Cell Genet. 84, 208 –210. 22. Kageyama, Y., Ishibashi, K., Hayashi, T., Xia, G., Sasaki, S., and Kihara, K. (2001) Expression of aquaporins 7 and 8 in the developing testis. Andrologia 33, 165–169. 23. Calamita, G., Mazzone, A., Bizzoca, A., Cavalier, A., Cassano, G., Thomas, D., and Svelto, M. (2001) Expression and immunolocalization of aquaporin-8 water channel in rat gastrointestinal tract. Eur. J. Cell Biol., in press. 24. Russell, L. D. (1993) Role in spermiation. In The Sertoli Cell (Russell, L. D., and Griswold, M. D., Eds.), pp. 269 –303, Cache River Press, Clearwater, FL. 25. Russell, L. D., Alger, L. E., and Nequin, L. G. (1987) Hormonal control of pubertal spermatogenesis. Endocrinology 120, 1615– 1632. 26. Griffin, J. E., and Wilson, J. D. (1994) Disorders of the testes. In Harrison’s Principles of Internal Medicine (Isselbacher, K. J., et al., Eds.), 13th ed., pp. 2006 –2017, McGraw-Hill, New York. 27. Trezise, A. E., Linder, C. C., Grieger, D., Thompson, E. W., Meunier, H., Griswold, M. D., and Buchwald, M. (1993) CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents. Nat. Genet. 3, 157–164. 28. Boockfor, F. R., Morris, R. A., DeSimone, D. C., Hunt, D. M., and Walsh, K. B. (1998) Sertoli cell expression of the cystic fibrosis transmembrane conductance regulator. Am. J. Physiol. 274, C992–C930. 29. Gong, X. D., Li, J. C., Cheung, K. H., Leung, G. P., Chew, S. B., and Wong, P. Y. (2001) Expression of the cystic fibrosis transmembrane conductance regulator in rat spermatids: Implication for the site of action of antispermatogenic agents. Mol. Hum. Reprod. 7, 705–713. 30. Schreiber, R., Nitschke, R., Greger, R., and Kunzelmann, K. (1999) The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells. J. Biol. Chem. 274, 11811–11816. 31. Van der Ven, K., Messer, L., Van der Ven, H., Jeyendran, R. S., and Ober, C. (1996) Cystic fibrosis mutation screening in healthy men with reduced sperm quality. Hum. Reprod. 11, 513–517.
625