Expression of e NOS in rat testis from infancy to maturity 3

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1 49 (3) : , 2003 A cta Zoologica S inica 3 33 (, ) (enos),, 1 d 24 enos, : 1 d 2 enos ; 3 ; 1 18, ; 24 enos,, enos,, [ 49 (3) : , 2003 ] Expression of e NOS in rat testis from infancy to maturity 3 KAN G You2Min ZHAN G Jian L I Jian DUAN Xiang2Lin 33 ( College of L if e Science, Hebei Normal U niversity, S hijiaz huang , Chi na) Abstract In order to investigate change of endothelium nitric oxide synthase (enos) in rat spermatogenesis during de2 velopment, immunohistochemistry and morphometric analyses were used to examine the testes of postnatal day 1 to post2 natal 24 months old rats. Testicular tissue was removed from decapitated male Sprague2Dawley rats in 10 age groups : postnatal 1 day, postnatal 1 week, postnatal 2 weeks, postnatal 3 weeks, postnatal 1 month, postnatal 2 months, post2 natal 3 months, postnatal 12 months, postnatal 18 months and postnatal 24 months, and then placed immediately in a fixative consisting of 4 % paraformaldehyde and 011 M phosphate buffer ( PB, p H 714). Fixed testes were rinsed for at least 12 h at 4 in 011 M PB (p H 712) containing 30 % sucrose, and then cryostat serial sections were cut at 4 m thickness and mounted onto glass slides covered with 1 % gelatinum. Rabbit anti2rat enos antibody and SP kit were pur2 chased from the Beijing Zhongshan Biotechnical Company (product of ZYMED in America). Immunohistochemical stain2 ing for enos was performed using the SP technique with immunohistochemistry. The enos content of postive blood ves2 sel endothelium as well as Leydig cells were measured with Beihang analytic software. The results indicate that the cytoplasm of postive cells appeared homogeneously yellow and the nuclear counterstaining of hematoxylin were iodes. Specimens from postnatal day 1 to postnatal 2 weeks showed little immunoreactivity for enos. In contrast, blood vessel endothelium and Leydig cells from postnatal 3 weeks testes were strongly expressed enos immunoreactivity, while spermatids were immunostained appearing cresccentiform. Postnatal 1st month testes had strong positive substances in the blood vessel endothelium and Leydig cells, spermatids were heterogeneously stained ap2 pearing cresccentiform and long2bar2shaped. Immunoreactivity was found in blood vessel endothelium, Leydig cells and spermatids in postnatal 2nd month testes and the number of positive cells conspicuously increased. In postnatal 3rd month testes, the number of postive cells persistently increased. In postnatal 12th month testes the number of postive , (No ) [ This research was funded by grants from Hebei Provincial Natural Science Foundation (No ) ] 33 (Corresponding author). E2mail : net (1974 ),,, :, E2mail : com ν 2003 Acta Zoologica Sinica

2 Leydig cells persistently increased, whereas the number of blood vessels with positive endothelium decreased slightly ; In postnatal 18th month testes only the number of postive Leydig cells decreased slightly ; blood vessel endothelium and Ley2 dig cells usually expressed enos in 24th month testes, and some spermatocytes also displayed strong enos immunoreac2 tivity. In this age group, the testes were old so few spermatids showed immunoreactivity. Sertoli cells, spermatogenous cells and myoid cells in each group did not stain for enos. The above results suggests that : enos positive cells are dis2 tributed in the blood vessel endothelium, Leydig cells and spermatids ; positive cells were found in spermatids during differ2 ent stages of spermatogenesis, which suggests that NO might accelerate the process of spermatogenesis. The number of blood vessels with positive endothelium was increased sharply, which might relate to testicular aging and the reduction of spermatogenesis. NO possibly plays a role in the relaxation of seminiferous tubules and blood vessels, to modulate sperm transport and testicular blood flow respectively. The regulation of NO in blood vessels requires further study. The number of positive Leydig cells were increased with aging, and Leydig cells in 242month old testes appeared strongly positive. As a result, NO produced by Leydig cells might directly influence the secretion of testosterone. NO produced by Leydig cells may act as a messenger to mediate the action of numerous intracellular and extracellular neuroactive substances andgrowth factors, to regulate local blood flow and permeability, and to influence the contraction of peritubular myoblasts and the permeability of the lamina propria. We first observed spermatids that appeared to have positive reactivity to enos which were stained as crescentiform in the testes of post 3 week old rats. enos was strongly expressed at all stages of sper2 matogenesis in other groups except those more than 242month old, while the middle part of spermatids also displayed posi2 tive reactivity ; long2bar2shape positive spermatids. The expression of enos was significantly higher in the germ cells of adult than in those of aged animals. Therefore, spermatids appeared to be sites of NO production and activity at all stages of spermatogenesis, suggesting that NO might regulate the metamorphosis of spermatids. It was worth emphasising that some spermatocytes appeared positively reactive, which might, to some extent, be related to the reduction of sperm and the caducity of testes. By the postnatal 24th month, the blood vessel endothelium and Leydig cells expressed enos prolifi2 cally, while some spermatomeres also expressed enos, suggesting that NO might restrain the secretion of testosterone and influence testicular function. Although the function of NO in the testis requires further characterization, the expression of enos in blood vessel endotheliums, Leydig cells and regulation of enos by germ cells suggests that NO is involved in reg2 ulating spermatogenesis. The results suggests that the expression of enos may depend on the existence of germ cells and be associated with germ cell development, perhaps changes of the expression of enos in old age might be related to the se2 cretion of testosterone and the function of spermatoleosis [ Acta Zoologica Sinica 49 (3) : , 2003 ]. Key words Rat, Testis, Endothelial nitric oxide synthase, Immunocytochemistry (1998) enos (Nitric oxide synthase, NOS), Middendorff et ( Palmer et al., 1987 ; Nathan, 1992 ; al. (1997) Zini et al. (1996) Burnett et al., 1992), NOS, enos, enos, ( 2 5 s), NOS enos NOS : Fujisawa et al. (2001) NOS ( Nerve NOS, nnos) NOS enos (Endothelial NOS, enos) NOS ( In2 (2001) 30 d ducible NOS, inos), enos, enos, 60 d enos Ca enos,,, enos, ( Hellst rom et al., 1994 ; Weinberg et al., 1995 ; Davidoff et al., 1995) Burnett et 1 d 24 enos al. (1995) Ehren et al. (1994) NOS, O Bryan et al.

3 3 : ,, SD ( enos, ), 1 d ,, 7,,, 4 %, 4, 4 h, 30 %, 4 (: 4) ; 2, ( Reichert2J ung Model 975c) 6 m, (: 5) ; 3 1 % (: 6) ; , ABC, SP (: 7) ; 18 ZYMED, 1 % - 30 min, ; 24, (37 ) 30 min, enos ( 1 50,, (: 8), SAN TA CRUZ) 4, (: 9),, min,, 30 min, DAB2H 2 O min, 0101 mol/ L PBS, 1 3 min,, 212, enos PBS, 113 e NOS, 1 d 3, 20 40,, ( 1) ( ),,,, 3 3,,, 10 SPSS 1010, 18, 24,, ( 1), e NOS (: 2) ; 3,, (: 3) ; 1,, e NOS, 2, NOS, (2001),,, 3, PBS,, enos (: 1) 1 d, NO,, ; 1 2,,

4 enos ( / mm 2 ) Table 1 The number of blood vessels with enos postive blood endothlium and enos postive Leydig cells in rats of different ages Age Sample size (1) Number of postive blood endothlium (Mean SD) (2) Number of postive Leydig cells (Mean SD) (1 day postnatal) (1 week postnatal) (2 weeks postnatal) (3 weeks postnatal) (1 month postnatal) (2 months postnatal) (3 months postnatal) (12 months postnatal) (3 months postnatal) (24 months postnatal) (One2Way ANOVA) (1) : 3 P < ( Significantly different from postnatal 22week group) 33 P < (Significantly different from postnatal 22month group) 122month group) P < (Significantly different from postnatal 3333 P < ( Significantly different from postnatal 182month group) (2) : 3 P < (Significantly different from postnatal 22week group) (Significantly different from postnatal 12month group) month group) 33 P < (Significantly different from postnatal 32week group) 333 P < P < (Significantly different from postnatal 182month group) 3333 P < (Significantly different from postnatal 22, enos, ( Lissbrant et al., 1997 ; Fujisawa et al., 2001),, NOS,,,,, 313 e NOS 312 e NOS Chamness et al., 3 (1995) enos, NOS 3 ; 1 18,,, 24,,, NO

5 3 : 343, Fujisawa, M., K.,, (, 1993) ;, enos Zini et al. (1996) : 24,, ( References) Burnett, A. L., C. J. Lowenstein, D. S. Bredt, S. K. Chang and S. H. Snyder 1992 Nitric oxide : a physiological mediator of penile erection. Science 257 : Burnett, A. L., D. D. Richer, S. L. Chamness, M. P. Maguire, J. K. Crone, D. S. Bredt, S. H. Snyder and T. S. Chang 1995 Localization of nitric oxide synthase in the reproductive organs of the male rat. Bio. Reprod. 52 (1) : 1 7. Chamness, S. L., D. D. Ricker, J. K. Crone, C. L. Dembeck, M. P. Maguire, A. L. Burnett and T. S. Chang 1995 The effect of androgen on nitric oxide synthase in the male reproductive tract of the rat. Fertil. Steril. 63 (5) : Cheng, L. Z Histology. 2nd edn. Beijing : The Peoples Health Press, [ :, ] Davidoff, M. S., R. Middendorff, B. Mayer and A. F. Holstein 1995 Nitric oxide synthase (NOS21) in leydig cells of the human testis. A rch. Histo. Cytol. 58 (1) : Ehren, I., J. Adolfsson and and N. P. Wiklund 1994 Nitric oxide synthase activity in the human urogenital tract. U ro. Res. 22 : and S. Yamanaka, H. Tanaka, H. Okada, S. Arakawa Kamidono 2001 Expression of endothelial nitric oxide synthase in the sertoli cells of men with infertility of various causes. BJU International 87 (1) : Hellstrom, W. J. G., M. Bell, R. Wang and S. C. Sikka 1994 Effect of sodium nitroprusside on sperm motility, viability, and lipid peroxidation. Fertil. Steril. 61 : Lissbrant, E., U. Lofmark, O. Collin and A. Bergh 1997 Is nitric oxide involved in the regulation of the rat testicular vasculature? Biol. Reprod. 56 : Middendorff, R., D. Muller, S. Wichers, A. F. Hoistein and M. S. Davidoff 1997 Evidence for production and functional activity of nitric oxide in seminiferous tubules and blood vessels of the hu2 man testis. J. Cli n. Endocri nol. Metab. 82 : Nathan, C Nitric oxide as a secretory product of mammalian cell. The FA S EB Journal 6 (12) : O Bryan, M. K., A. Zini, C. Y. Cheng and P. N. Schlegel 1998 Human sperm endothelial nitric oxide synthase expression : correla2 tion with sperm motility. Fertil. Steril. 70 (6) : Palmer, R. M., A. G. Ferrige and S. Moncada 1987 Nitric oxide release accounts for the biological activity of endothelial relaxing factor. N at ure 327 (6122) : Wang, S. E., Y. Q. Chen and R. X. Zhou 2001 The expression and localization of nitric oxide synthaseis oenzymes in rat testis. Journal of Reproductive Medici ne 4 (10) : [,, (10) : ] Weinberg, J. B., E. Doty, J. Bonaventura and A. F. Hancy 1995 Nitric oxide inhibition of human sperm motility. Fertl. Steril. 64 : Zini, A., M. K. O Bryan, M. Magid and P. N. Schlegel 1996 Immunohistochemical localization of endothelial nitric oxide syn2 thase in human testis, epididymis, and vas deferens suggests a pos2 sible role for nitric oxide in spermatogensis, spermmaturation, and programmed cell death. Biol. Reprod. 55 :

6 ( Explanation of Plate) ( Plate ) 11 3 enos [enos negative comparison at postnatal 3 weeks] enos ( ) [Light micrograph showing enos positive blood vessel endothelium ( ) at postnatal 2 weeks] enos ( ) ( ) ( ) ( ) [ Light micrograph showing enos positive blood vessel endothelium ( ), Leydig cells ( ) and spermatids ( ) at postnatal 3 weeks] enos ( ) ( ) ( ) ( ) [ Light micrograph showing enos positive blood vessel endothelium ( ), Leydig cells ( ) and spermatids ( ) at postnatal 1 month ] enos ( ) ( ) ( ) ( ) [ Light micrograph showing enos positive blood vessel endothelium ( ), Leydig cells ( ) and spermatids ( ) at postnatal 2 months] enos ( ) ( ) ( ) ( ) [ Light micrograph showing enos positive blood vessel endothelium ( ), Leydig cells ( ) and spermatids ( ) at postnatal 3 months] enos ( ) ( ) ( ) ( ) [Light micrograph showing enos positive blood vessel endotheliums ( ), Leydig cells ( ) and spermatids ( ) at postnatal 12 months] enos ( ) ( ) [Light micrograph showing enos positive blood vessel endotheliums ( ) and Leydig cells ( ) at postnatal 24 months] enos ( ) [Light micrograph showing enos positive spermatomeres ( ) at postnatal 24 months] 400

7 : KAN G You2Min et al. : Expression of enos in rat testis from infancy to maturity Plate ( Explanation at the end of the text)