Biochemical and Biophysical Research Communications 288, 619–625 (2001)
doi:10.1006/bbrc.2001.5810, available online at http://www.idealibrary.com on
ossible Involvement of Aquaporin-7 and -8 in Rat Testisevelopment and Spermatogenesis
iuseppe Calamita,*,1 Amelia Mazzone,* Antonella Bizzoca,† and Maria Svelto*Department of General and Environmental Physiology and †Department of Pharmacology and Human Physiology,niversity of Bari, Bari, Italy
eceived September 17, 2001
niferous tubule fluid serves as a vehicle for spermtshdt(tsnahrotttiI(ptp
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Fluid secretion and reabsorption are of central im-ortance in male reproductive (MR) physiology. How-ver, the related molecular mechanisms are poorlynown. Here, potential roles for AQP7 and AQP8, twoquaporin water channels abundantly expressed inhe MR tract, were investigated by studying their ex-ression and distribution in the developing testis ofhe Wistar rat. By semiquantitative RT-PCR and im-unoblotting, first expression of AQP7 was noted at
ostnatal day 45 (P45), with levels increasing substan-ially at P90 and remaining at high levels thereafter.QP8 began to be expressed at P15, rapidly increasedntil P20, and remained fairly stable thereafter. Im-unohistochemical analyses demonstrated AQP7 in
longated spermatids, testicular spermatozoa, and re-idual bodies at P45 with increased signal intensityhereafter. AQP8 was observed in primary spermato-ytes from P20 to P30 and, in elongated spermatids,esidual bodies and Sertoli cells at P30 and thereafter.he ontogeny and distribution of AQP7 and AQP8 inat testis suggest involvement in major physiologichanges in testis development and spermatogenesis.2001 Academic Press
Fluid homeostasis is of major importance in a num-er of processes in the male reproductive physiology,ncluding testis development, spermatogenesis, sperm
aturation and storage, secretion of seminal liquidnd egg fertilization. During postnatal testis develop-ent, Sertoli cells secrete fluid to form a fluid-filled
ubular lumen (1) which is one of the first morpholog-cal events characterizing the formation of the blood-estis barrier (Sertoli cell barrier) and the beginning ofpermatogenesis (2, 3). In the adult animal, the semi-
1 To whom correspondence and reprint requests should be ad-ressed at Dipartimento di Fisiologia Generale ed Ambientale, Uni-ersita degli Studi di Bari, via Amendola 165/A, 70126 Bari, Italy.ax: 39 0805443388. E-mail: [email protected].
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ransportation and possible further maturation ofperm (4). A remarkable efflux of water from the cellas been evoked to explain the striking cytoplasm con-ensation needed for differentiation of round sperma-ids into elongating spermatids during spermiogenesis5, 6). The seminiferous fluid is mostly reabsorbed inhe efferent ducts (7) while fluids rich in nutrients areecreted by seminal vesicles and prostate and areeeded for sperm to survive and fertilize eggs. Alter-tions in fluid balance in the male reproductive tractave been already reported to result in long-term at-ophy of testes (8) and it is very likely that other formsf male infertility will be discovered. However, whilehe transporters accounting for the salt movements inhe male reproductive physiology begin to be charac-erized (9), the molecular mechanisms by which waters transported in the MR tract remain poorly defined.dentification of multiple aquaporin water channelssee Ref. 10 for review on aquaporins), variously ex-ressed in the secretory and absorptive portions ofhe MR tract, suggests roles for these proteins in MRhysiology.The cDNAs of two aquaporins, AQP7 and AQP8,ere recently reported in the MR and gastrointestinal
racts (11 and 12, 13, and 14, respectively). AQP7 haseen found to be permeable to small neutral solutes,uch as glycerol and urea in addition to water (11).QP8 has been shown to transport water (12–15) and
in mouse, but not in rat or human) also urea (14). Inhe MR tract of adult rat, AQP7 was observed in sper-atids and testicular (16) and epididymal (17) sperma-
ozoa while abundant AQP8 was noted to be variouslyistributed in testis (17, 18). The AQP7 and 8 genesave been cloned and characterized structurally in hu-an (19) and mouse (20) and human (21), respectively.
n spite of their strong expression in MR tract, nonformation is available regarding the regulation andunction of AQP7 and AQP8 in reproductive biology.
This study was undertaken to determine the devel-pmental expression and distribution of AQP7 and
AQP8 in Wistar rat testis, in order to evaluate possiblermdSciAwdt
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oles in developing testis, spermatogenesis and spermaturation. As this work was nearing completion, the
evelopmental expression of AQP7 and AQP8 inprague–Dawley rats was reported (22); however, dis-repancies with our studies were observed in the tim-ng, cellular expression and relative abundance ofQP7 and AQP8. Consequently, additional studiesere carried out to clarify these differences and furtherefine the expression of AQP7 and AQP8 in developingestis.
ATERIALS AND METHODS
Animals. Male Wistar rats of varying ages were obtained fromorini sas (S. Polo D’Enza, Italy), either with their actual mothers orith foster mothers. Animals, if not sacrificed earlier, were weanedt 21 days of age and fed and watered ad libitum. For all thexperiments rats were decapitated after anesthesia and the tissuesere collected as described in the following sections.
RNA extraction and RT-PCR experiments. Tissues were removedrom the testis of rats of different ages and frozen in liquid nitrogen.otal RNAs were isolated by the TRIzol extraction method (TRIzoleagent, Life Technologies, Gaithersburg, MD). As previously de-cribed (17), RT-PCR analysis was carried out by employing theeneAmp RNA PCR Core kit (Perkin–Elmer, Branchburg, NJ) using
he rat AQP7 or AQP8 specific primers RSA7-start (59-ATGGCC-GTTCTGTGCTG-39) and RSA7-stop (59-TCTAAGAACCCTGTGG-GG) and RSA8-start (59-CGGGATCCATGGCTGACAGTTACCAT-9) and RSA8-stop (59-CGGAATTCACCTCGACTTTAGAAT-39), re-pectively. This led to the amplification of expected 810- and 732-bpragments of the rat AQP7 and AQP8 coding regions, respectively.he cDNAs amplified were then cloned into the pCR2.1 vector (TAloning kit, Invitrogen, San Diego, CA) and the identity of the in-erted DNA fragments was verified by sequencing. RT-PCRs wereormalized against the b-actin expression using a pair of b-actinrimers, BAF (59-CAGATCATGTTTGAGACCTT-39) and BAR (59-GGATGTCMACGTCACACTT-39; M 5 A 1 C) which lead to themplification of a 509-bp DNA fragment.
Preparation of testis homogenate and immunoblotting. Testesrom P2 through P15 developing rats were pooled by litter in order toave adequate samples for processing; specimens from older animalsere processed individually. Homogenates of pooled testes were pre-ared as previously reported (17). For the immunoblotting analyses,omogenates (60 mg of proteins) were mixed with the Laemmli buffernd heated at 90°C for 4 min before being submitted to SDS–PAGE.he immunoblotting experiments were carried out as previouslyescribed (17) by using commercially available rabbit anti-rat AQP8ffinity-purified antibodies (Alpha Diagnostic International, San An-onio, TX) or anti-rat AQP7 affinity purified antibodies (kindly pro-ided by Drs. G. P. Nicchia and A. Frigeri) at final concentrations of00 or 200 ng/ml, respectively.
Immunohistochemical experiments. After sacrifice, testes weresolated and immersed overnight at 4°C in 4% paraformaldehyde inBS. The tissues were then washed in PBS (3 3 10 min) and finallyryoprotected by soaking in PBS containing 25% sucrose. Cryostatections (8 mm) were prepared and placed on silanized microscopelides. The immunohistochemical experiments were performed asreviously described (23). Briefly, after blocking the endogenouseroxidases, the sections were treated with blocking solution for 1 h,ncubated overnight with the anti-rat AQP8 or anti-rat AQP7 affinityurified antibodies (1 or 0.5 mg/ml, respectively), washed with thelocking buffer and incubated for 1 h with a biotinylated goat anti-abbit IgG (Vector Laboratories, Burlingame, CA) diluted at a finaloncentration of 10 mg/ml in blocking buffer. After several washes,
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ections were incubated for 1 h with horseradish peroxidase conju-ated streptavidin (Vector Laboratories) at a concentration of 5 mg/l. The immunohistochemical reaction was visualized by incubationith 3-amino 9-ethylcarbazole (AEC peroxidase substrate kit, Vectoraboratories) for 10 min. Sections were then counterstained withematoxylin. Slides were cover-slipped with an aqueous mountingedium and viewed on a Leica DMRXA photomicroscope. Control
mmunostaining was performed with the preadsorbed AQP8 anti-odies or by omitting the anti-AQP8 antibodies.
ESULTS
evelopmental Expression of AQP7 and AQP8in Rat Testis
By semiquantitative RT-PCR, the AQP7 mRNA wasetected beginning from postnatal day 45 (P45) whilehe signal intensity reached the maximal extent at P90nd thereafter (Fig. 1A). AQP8 was found to be ex-ressed earlier than AQP7 as its transcript was de-ected beginning from P15 and was fairly stable at itsaximal intensity at P20 (Fig. 1B). This pattern of
evelopmental expression was fully consistent withmmunoblots of testis homogenates prepared from de-eloping Wistar rats and incubated with affinity puri-ed anti-rat AQP7 or rat AQP8 antibodies (Figs. 2And 2B). A 23- to 24-kDa band corresponding to the
FIG. 1. Expression of the AQP7 and AQP8 mRNAs in the devel-ping testis of Wistar rat. Semiquantitative RT-PCR of total RNAamples from indicated developing rat testes from postnatal day 2P2) to adult (P180). (A) AQP7 expression (810-bp band) is noted at45 and becomes high and stable at P90 and thereafter. (B) AQP8
732-bp band) is expressed at P15 and reaches high signal intensityt P20 and thereafter. RT-PCR of b-actin (509-bp band) was used toormalize the expression of AQP7 and AQP8.
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QP7 protein (17) was weakly detected at P45 and theelated immunoreactivity steadily increased until P60hen the signal became strong and fairly stable (Fig.A). AQP8 was detected at P30 as a 24- to 25-kDaoublet, a 32- to 40-kDa diffuse component and a high-olecular-mass band (Fig. 2B) that, as previously re-
orted (17), corresponded to the core protein, the gly-osylated AQP8 (glyAQP8) and, likely, a multimericorm of AQP8, respectively. Interestingly, the strongmmunoreactivity of the 24-kDa band (lower band ofhe AQP8 doublet) was strong at P30, while it steadilyecreased over the following sixty days and disap-eared at P180 (Fig. 2B). No bands were noted inontrol immunoblots with anti-AQP7 or AQP8 antibod-es preadsorbed with a 20:1 molar excess of the immu-izing peptides (data not shown).
istribution of AQP7 and AQP8 in DevelopingRat Testis
AQP7 or AQP8 cellular and subcellular distributionn developing rat testis was assessed by immunohisto-hemical analysis of postnatal (P2 through P120)istar rat testis.
FIG. 2. Immunoblotting analyses of AQP7 and AQP8 in pooledomogenates of developing rat testis. Blots incubated with affinityurified anti-rat AQP7 (A) or rat AQP8 (B) antibodies. AQP7 isetected as a 23- to 24-kDa band whereas AQP8 appears as a 24–25oublet (double arrows), a 32- to 40-kDa diffuse componentglyAQP8), and a high-molecular-weight band representing the corerotein, the glycosylated form, and, likely, a multimeric form (arrow-ead) of AQP8, respectively. AQP7 is weakly detected at P45 while atrong immunoreactivity is noted thereafter (P60 to P180). AQP8 isetected at P30 and reaches a high and stable intensity at P45.
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QP7 immunoreactivity was first observed at P45 inhe luminal aspect of the seminiferous epithelium ofome tubules. Itsteadily increased until P75 and there-fter when all seminiferous tubules showed immuno-eactivity (Figs. 3A, 3C, 3E, 3G, and 3I). AQP7 labelingas restricted to the plasma membrane and cytoplas-ic mass of elongated spermatids, testicular sperma-
ozoa and residual bodies (Figs. 3E, 3G, and 3I; seensets). At high magnification, in the cytoplasmic massf elongated spermatids, strong labeling was seen overGolgi-like apparatus (Figs. 3E and 3I, insets). No
eactivity was observed in control experiments wherehe anti-AQP7 antibodies were omitted (Figs. 3B, 3D,F, 3H, and 3J). This pattern of cellular and subcellu-ar distribution of AQP7 was consistent with that pre-iously found in the testis of Wistar adult rat (16, 17).y contrast, it was different from that reported in theeveloping testis of Sprague–Dawley rat where AQP7as detected considerably earlier, at P28, and found in
ound spermatids and in the plasma membrane of sec-ndary spermatocytes (22).A transient but distinct AQP8 immunoreactivity was
estricted to the cytoplasmic mass of primary sper-atocytes of rat testis from P20 to P25-30 (Figs. 4C
nd 4D). Starting from P30, AQP8 immunostainingas observed in the cytoplasmic mass of elongated
permatids, residual bodies and, apparently, over theytoplasmic ramifications of Sertoli cells (Figs. 4F, 4G,I, 4J, 4L, and 4M). The signal intensity of this patternteadily increased until P90 and thereafter. Due to thelose association of the Sertoli cells to the germ ele-ents (Fig. 4M), the presence of AQP8 in spermato-
ytes could not be completely excluded. This may ex-lain the apparent discrepancy with previous worksescribing the expression in rat testis (17, 22). Noabeling was observed in control experiments omittinghe anti-AQP8 antibodies (Figs. 4B, 4E, 4H, 4K, and 4N).
ISCUSSION
Definition of the aquaporin water channels ex-ressed in the male reproductive tract will undoubt-dly provide important information in understandingentral processes such as the secretion of fluid to formhe lumen of seminiferous tubules occurring duringestis development and the fluid movements duringpermatogenesis and sperm concentration and matu-ation. Our studies of postnatal developing rat testisocument that AQP7 expression first appears at post-atal day 45, increases steadily until P90, and is sus-ained at high levels in adult animals. Overall, AQP8egins to be expressed at P15, increases rapidly until20 and remains fairly stable during testis develop-ent and, thereafter, in adult animals.The specific appearance of AQP7 in elongated sper-atids parallels the beginning of spermiogenesis, a
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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) and90 (I, J). Cryostat sections were incubated with affinity purified anti-AQP7 antibodies (A, C, E, G, I); immunolabeling controls in B, D, F, H, andwere performed by omitting the anti-AQP7 antibodies (negative control). (A, C) At P20 and P30, virtually no labeling is observed in testis. (E,, I) At P45 and thereafter, strong AQP7 immunolabeling is seen at the luminal rim of seminiferous tubules, where staining is observed withinlongated spermatids (see insets in E and I), testicular spermatozoa (G, inset; double arrows) and residual bodies (E, right inset; arrowheads). Inlongated spermatids, a Golgi-like apparatus (E, left insets; I, inset; arrows) shows stronger immunolabeling than the surrounding cytoplasm. Nommunoreactivity is observed in any negative control sections (B, D, F, H). Magnification: (A, B) 3200, (C–J) 3100, (insets in E, G, I) 3400.
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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).ections 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, andwere performed by omitting the anti-AQP8 antibodies (negative control). (A) At P10, no AQP8 immunoreactivity is observed in testis. (C,
) At P20, the AQP8 protein is seen in primary spermatocytes (arrowheads). (F, G, I, L) At P30 and thereafter, the luminal portion of alleminiferous tubules shows AQP8 immunoreactivity although the signal intensity is variable among the tubules, depending on the specifictage of spermatogenesis. (J, M) At higher magnification, labeling is observed in the elongated spermatids (M, J, arrows) and in the ramifiedytoplasm of Sertoli cells (M, double arrows). No immunoreactivity is observed in any negative control sections (B, E, H, K, N). l.c., Leydigell. Magnification: (A–C, E, G) 3200, (D, J, M) 3400, (F–I, K, L, N) 310.
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process in which spermatids progress to mature sper-mrttssbwAmKwaowKcabo
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atozoa. AQP7 may therefore relate to the cell volumeeduction of spermatids by mediating the efflux of wa-er from spermatids during spermiogenesis. However,his role of AQP7 in spermiogenesis may not be sotraightforward both because AQP7 is permeable tomall neutral solutes in addition to water (11) andecause rat spermatids express at least one additionalater channel, AQP8 (12, 17, this study). A role forQP7 in the morphological change of secondary sper-atocytes to spermatids has been suggested byageyama and co-workers (22). However, in this studyith developing testes of Wistar rats and in others ondult rats (16, 17), no AQP7 immunoreactivity wasbserved in secondary spermatocytes. Hence, the veryeak and transient expression of AQP7 observed byageyama and co-workers (22) in secondary spermato-
ytes of Sprague–Dawley rat at P28 raises questionsbout the physiological relevance, although the possi-ility of a strain-to-strain variability should not beverlooked in explaining this.Interestingly, the appearance of AQP8 in testes of
5-day-old rats coincides with central processes suchs the formation of the Sertoli cell barrier (3) and fluidecretion by Sertoli and germ cells to form the lumen ofhe seminiferous tubules (1–3, 24). This may suggestnvolvement of AQP8 in the secretion of water to formfluid-filled tubular lumen, one of the first morpholog-
cal events announcing the formation of the blood-estis barrier and the beginning of spermatogenesis.QP8 is presumed to have additional roles in the sper-atogenic cycle as it is expressed in elongated sperma-
ids and in Sertoli cells at P45 and thereafter. In Ser-oli cells, AQP8 may play a role in the secretion ofubular fluid or, more in general, in the secretion of theilieu for germ cell transportation and maturation.oreover, like AQP7, AQP8 may be involved in the
ytoplasmic condensation occurring during differenti-tion of spermatids into spermatozoa. Because FSHnd the synergistic actions of FSH and LH (indirectlyy testosterone) are critically important both in pu-erty and adult spermatogenesis (25, 26) it is temptingo speculate that the expression of AQP8 in testis coulde under hormonal control, as also suggested byageyama and co-workers (22). Interestingly, the de-elopmental expression and cellular distribution ofQP8 in the rat seminiferous tubule is very similar to
hat of the cystic fibrosis transmembrane conductanceegulator (27–29), CFTR, evidence that may suggest aunctional correlation between AQP8 and CFTR. Aunctional link between CFTR and another aquaporinxpressed in rat spermatids, AQP7, has been recentlyypothesized by Gong and co-workers (29). Moreover,FTR has already been reported to activate an aqua-orin, AQP3, in the airway epithelium (30). Thus, aslso speculated by Gong and co-workers (29), underFTR activation AQP8 and/or AQP7 (or unknown
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permatids differentiate and remodel into testicularpermatozoa. This attractive hypothesis is corrobo-ated by the evidence that testicular biopsies fromystic fibrosis patients and infertile men carrying mu-ations in the CFTR gene evidence a reduced numberf mature spermatids and many malformed testicularpermatozoa (31).While the ontogeny and cellular distribution ofQP8 suggest appealing roles for AQP8 in developing
estis and portend physiologic and pathophysiologicunctions in later life, the functional meaning of thearge intracellular localization featured by this aqua-orin in rat testis remains unclear. Speculatively, theubcellular distribution of AQP8 might relate to itsecycling between the intracellular compartment andhe plasmamembrane (hormonal control?) or an in-olvement in the osmoregulation of the cytoplasm andts vesicle content. Definition of the regulatory mean-ng of the subcellular distribution of AQP8 may be a
atter of further study.In summary, this work describes the expression ofQP7 and AQP8 in developing testis of Wistar rat.oles for AQP8 are suggested in the secretion of water
o form a fluid-filled seminiferous tubular lumen, anvent that triggers the beginning of spermatogenesis,nd in the secretion of the tubular fluid serving as aehicle for sperm transportation and possible furtheraturation of sperm. AQP8 and/or AQP7 are pre-
umed to be responsible for most of the cell volumeeduction by which spermatids differentiate in sperma-ozoa during spermiogenesis. Interesting questions re-ain to be answered about the functional and regula-
ory meaning of the subcellular distribution of AQP8.evertheless, AQP7 and AQP8 may prove central tooth normal physiology and pathophysiology of theeproductive tract, and may ultimately provide poten-ial targets for contraceptive strategies.
CKNOWLEDGMENTS
This work is dedicated to the memory of Professor L. D. Russell, anxemplary man and scientist whose stimulating, passionate, andelpful discussions in understanding the distribution of AQP7 andQP8 in rat testis we very much appreciated. We thank Drs. An-
onella Bizzoca and G. P. Nicchia and A. Frigeri for their helpfulontributions and for providing the anti-AQP7 antibodies, respec-ively. Support of the European Community (EU-TMR Networkrant ERB 4061 PL 97-0406) is gratefully acknowledged.
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