S S GURAYA and ARBANS J KAUR Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana , India

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1 Proc. Indian Acad, Sci. (Anim. Sci.), Vol. 90, Number 5, SeptcIl1ber 1981, pp. 49S-soi. o Printed in India. Effect of castration on some hydrolytic enzymes in the rat vas deferens: A histochemical study S S GURAYA and ARBANS J KAUR Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana , India MS received 1 April 1980; revised 10 April 1981 Abstract. A histochemical study bas been made of alterations in some hydrolytic enzymes such as alkaline phosphatase, acid phosphatase and S'-nucleotidase of the vas deferens of sexually mature albino rats following bilateral testeetomy. Three weeks after operation, vas deferens shows a significant decrease in the activities of acid phosphatase, alkaline phosphatase and S'-nucleotidase indicating conspicuous metabolic alterations and reduction in transport mechanisms following removal of the source of androgens. Keywords. Rodent; vas deferens; histochemistry: enzymes; castration albino rat. 1. Introduction The male accessory sex organs are highly dependent on androgenic hormones to maintain their normal structure and functions. To date, the epididymes and pros. tate gland have usually been chosen as the type organs for studying the mechanism of action of androgenic hormones (Dawson and Rowlands 1959; Glover 1960; Lubicz-Nawrocki and Glover 1970; Mainwaring 1977). In these tissues, activities of a number of hydrolytic and glycolytic enzymes have been shown to be androgen dependent (Allen and Slater 1957, 1958; Sholl and Leathem 1973; Brooks 1976, 1978). In contrast, the vas deferens has received scant attention as an androgen target tissue, inspite of the current interest in the increasing use of vasectomy as an approach to contraception. Keeping in view the importance of vas deferens and relatively very little previous histochemical work on it, the present study was undertaken to study some hydrolytic enzymes, viz., alkalinephosphatase, acid phosphatase and 5'-nucleotidase in the vas deferens of intact and castrated rats. Z, Materials and methods Bilateral testectomies were performed in ten sexually mature albino (Wistar strain) rats. The operations were performed under ether anaesthesia. The instruments were thoroughly cleaned and sterilized. A longitudinal median cut was given P.(B)-l 495

2 496 S S Guraya and Arba113 J Kaur on the scrotum. Both the testes were removed leaving epididymes and vasa deferentia in situ. Three weeks after the operation, their vasa deferentia Were removed and processed for the study of some hydrolytic enzymes as shown in table 1. Some rats of the same age and might were kept as controls. 3. Re5Qlts 3.1: Alkaline phosphatase 3.1a. Normal (table I, figure 1): The intensity of alkaline phosphatase (ALP) reaction was stronger in the lamina propria than in the basal portions of the epithelium. Fine-scattered granules due- to ALP activity were also present in the apical portions of the epithelium. A very weak reaction of ALP was seen in the stereocilia. 3.lb. Castrated: A marked decrease in the ALP activity was seen throughout the vas deferens following castration (figure 2). Compared to the normal rata. the reection was more pronounced in the basal portions of the epithelium than in the.lamina propria Acidphosphatase (AcP) Normal: The vas deferens of intact rat showed relatively less AcP as compared to ALP activity. AcP activity was particularly more in the apical than in the basal portions of the epithelium (figure 3). The principal cells showed the highest AcP activity particularly towards their luminal side. 3.2b. Castrated: A significant decrease in the AcP activity was seen throughouj the vas deferens following castration (figure 4), only the apical portions of the epithelium showed a moderate reaction '-nucleotidase 3.3a. Normal: Intense 5'-nucleotidase activity was present in the lamina propria and apical border of the epithelium along with its stereocilia, whereas the basal portions of the epithelium showed a moderate enzyme activity (figure 5). 3.3b. Castrated: Following castration a marked decrease in 5'-nucleotidase activity was observed (figure 6). 4. Disc"on This histochemical study has revealed that alkaline phosphatase activity is relatively more than that of acid phosphatase in the rat vas deferens. This is in contrast to the observations of Gregoire et al (1973) on human vas deferens as they have reported more AcP activity. Niemi (1965) has reported the absence of ALP activity in rat vas deferens which has been clearly demonstrated here. Allen and Slater (1957) have reported that ALP in the epididymis may be involved in the transport of sugar molecules and related substances. ALP activity is usually associated with the cell surface extensions involved in the transport ofsubstances

3 Effect oj castration in vas deferens 497 Figure Alkaline phosphatase activity of normal rat vas deferens. x Decrease in alkaline phosphatase activity in the castrated rat vas deferens. X Acid phosphatase activity in the vas deferens of normal rat. x Decrease in acid phosphatase activity in the rat vas deferens following castration. ~, '-nucleotidase activity in the intact rat vas deferens. x Marked decrease in 5'-nucleotidase activity following castration in the rat vas deferens. x 100.

4 Table 1. Histochemical techniques employed (Pearse 1968) and the reactions obtained in various portions of the rat vas deferens.. Normal Castrated very strong reaction; ++ = strong reaction; +... modorate reaction i ± = weak reaction ; - = negative reaction. cited in Pearse (968). ~

5 500 S S Guraya and Arbans JKaur (Gregoire et al 1973),its presence in the vas deferens also suggests active transport of organic substances in the duct epithelium. The enzyme AcP is believed to be a secondary sex characteristic in the male reproductive system. It occurs in large quantities in semen and exhibits variations with various conditions of infertility (Nun et of 1972). Its presence in the apical portions of the epithelium along with stereocilia as reported earlier (Niemi 1965; Gregoire et a/ 1973; Hoffer 1976) suggests that it may be a secretory product of the vas epithelium itself (Martan 1969). Hoffer (1976) interpreted a large number of AcP positive dense bodies in the human vasa as phagelysosomes, which may be involved in the digestion of proteins of testicular origin absorbed by the vas deferens. Flickinger (1(3) also reported that the apical region of rat vas epithelium contains a large number of coated vesicles, vacuoles and Iysosomes, These structural features also suggest that this part of vas deferens epithelium is functional in absorption and secretion of substances. 5'-nucleotidase activity of the vas deferens has not been investigated previously. Its localization in the lamina propria and stereocilia suggests that it may also be involved in some transport processes. The overall decrease in ALP, AcP and 5'-nucleotidase activities of "as deferens following castration appears to indicate a decrease in its various metabolic activities related to synthesis and transport of substances. This also suggests that the activities of these enzymes are under androgenic control. The strong ALP reaction of the basal portions of the epithelium in castrated rats may be related to the hypertrophy of basal cells following castration. It thus appears that these basal cells are probably activated due to a decrease in the distention forces and I08S of sperms, as has also been shown in the case of epididymis (Martan and Risley 1963). In the principal cells of the epididymis cnly Golgi area has been shown to be AcP positive (Martan and Risley 1963). Thus, ifthe similar situation prevails for the vas deferens, then the loss of its AcP activity indicates a reduction of Golgi complex and its probable disintegration leading to the loss of synthetic capacities of the vas deferens. The evidences presented here strongly suggest that only in the presence of testes (testicular androgens) the vas deferens can maintain its structural and functional attributes. 5. CODCIQSions Following castration. the vas deferens shows significant decrease in the activities of acid phosphatase, alkaline phosphatase and 5'-nucleotidase indicating conspi.. euous metabolic alterations, reduction in transport mechanisms and dependency of these enzymes on testicular androgens. References AIleD J M and Slater J A chemical and histochemical study of alkaline phosphatase and aliesterase iii the epididymis of normal and castrated mice; Anat. Rec AlleD J M and Slater J J 1958 A chemical and histochemical study of acid phosphatase in the epididymis of normal, castrate and hormone replaced castrate mice; Anat. Rec

6 Effect of castration in vas deferens 501 Brooks D E 1976 Activity and androgenic control of glycolytic enzymes in the epididymis and epididymal spermatozoa of rat; Biochem. J Brooks D E 1978 Activity and androgenic oontrol of enzymes associated with the tricarboxylic acid cycle. lipid oxidation and mitochondrial shuttles in the epididymis and epididymal spermatozoa of the rat; Biochem, J Dawson R M C and Rowlands I W 1959 Glycerophosphorycholine in the male reproductive organs of rats and guinea pigs; Q. J. Exptl, Physiol Flickinger C J 1973 Regional variations in endoplasmic reticulum in the vas deferens of normal and vasectomized rats; Anat, he Glover T D 1960 Spermatozoa from the isolated cauda epididymides of rabbits and rams ; effect of artificial cryptorchidism; J. Reprod. Fertil, Gregoire A T. Moran M J and Cuadros A 1973 Activity and localization of isocitric dehydrogenase. aspartate amino-transferese, alanine aminotransferase, alkaline and acid phosphatase of the human vas deferens; Fertility Sterility Hoffer A P 1976 The ultrastructure of the ductus deferens in man; Bioi. Reprod Lubicz-Nawrocki C and Glover T D 1970 Effect of gonadectomy and testosterone replacement on the viability of epididymal spermatozoa in the golden hamster (Mesacriceus auratus); J. Endocrinol, 48 xxli Mainwaring WI P 1977 The mechanism of action of androgens; in monographs on endocrinology (eds.) F Gross, M M Grumbach, A Labhart, M B Lipsett, T Mann, L T Samuels and J Zander (New York: Springer-Verlag) Marian J and Risley P L C 1963 Holocrine secretory cells of the rat epididymis; Anat, Rec, Martan J 1969 Epididymal histochemistry and physiology; Blol, Reprod, Suppl Niemi M 1965 The fine structure and histochemistry of the epithelial cells of the rat vas deferens; Acta Anat Nun S, Musacchio I and Epstein J A 1972 Variations in seminal plasma constituents from fertile, subfertile and vasectomized azoospermic men; Fertil. Steril, Pearse AGE 1968 Histochemistry, theoretical and applied (London: J and A Churchill Ltd.) pp, Sholl S A and Leathem J H 1973 Effects of postnatal maturation and castration on rat epididymal carbohydrate metabolism (37083); Proe. Soc. Exp, BioI. Med