Sequential histopathological and biochemical events in rat caput epididymis after alpha monochlorohydrin administration

Exp. Path., Bd. 12, S. 19-24 (1976) Department of Biophysics, Panjab University, Chandigarh, India

Sequential histopathological and biochemical events in rat caput epididymis after alpha monochlorohydrin administration By N. R. KALLA and K. S. CHOHAN With 7 figures (Received September 15, 1975) Key-words: testis; chlorohydrin; fertility; acid phosphatase; alkaline phosphatase; succinic dehydrogenase; nucleic acids; proteins; cholesterol; glycogen; rat; guinea pig; ram; pig; hamster.

Summary This paper presents sequential histological, histochemical and biochemical changes in rat caput epididymis after a single 100 mgJkg oral dose of alpha chlorohydrin. Desquamation of caput epithelium occurs as early as two days after drug treatment. The caput epididymis is blocked because of accumulation of testicular fluid containing exfoliated immature testicular cells and caput epithelium. There was no effect of drug on motility and morphology of spermatozoa examined from different segments of epididymis and vas deferens. Marked decrease in acid and alkaline phosphatases, nucleic acids and proteins have been registered after drug treatment. On the contrary increase in SDH, cholesterol and glycogen was observed after drug treatment. Decrease in phospholipids in initial stages has also been observed. Alpha chlorohydrin has been reported to inhibit the fertility of male rats, guinea pigs, rams, swines and hamsters (ERICSSON and BAKER 1970, JOHNSON and PURSEL 1970, KREIDER and DUTT 1970, BROWN and WHITE 1973, LUBICZ-NAWROCK and CHANG 1973). The precise modus operendi of this compound, as yet, is obscure. Findings emerged from studies on chlorohydrin indicate that physiological doses of drug cause changes in the integrity of the spermatoza by some unknown mechanisms rendering them infertile (ERICSSON and BAKER 1970, GUNN et a1.1970, LUBICZ-NAWROCK and CHANG 1973). Histological study of epididymis reveals that primarily the caput epididymis is effected by drug action after single high dose (ERICSSON 1970, GUNN et aI. 1970, SAMJOLIK and CHANG 1970). It has been suggested that alpha monochlorohydrin might be interferring with one of the vital functions of the epididymis i. e., the absorption of luminal fluid (GUNN et aI. 1970). COPPOLA (1969) on the other hand has suggested the possibility of chlorohydrin working as a metabolic antagonist to phospholipid synthesis. ERICSSON, however, has pointed out that chlorohydrin may produce changes in the vasculature of the epididymis which could hinder the sperm maturation (ERICSSON 1970). Since in all these studies, in response to high doses, the caput epididymis has been implicated as the target site of drug action, it was considered worthwhile to study in detail the histological, histochemical and biochemical changes in the caput epididymis region after single high dose in the hope that this study might throw some light on the mechanisms of drug action.

Material and methods Male albino rats, each weighing 200-225 gms were used in the present investigations. The rats were divided into 6 groups: group I served as control. Animals from groups 11- VI were sacrificed under chloroform anesthesia after 2, 5, 10, 20 and 40 days, respectively. Each animal was exposed to a single oral dose of 100 mgJkg body weight of alpha chlorohydrin in 1 ml of 0.25% methyl cellulose in sterile water. The control animals received 1 ml of 0.25% methyl cellulose alone. Epididymis. 2·

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weight was carefully noted after dissection. For gross histological study, the epididymis was fixed in Bouin's fluid and 8 micron thick paraffin sections were stained by haematoxylinJeosin. For simultaneous histochemical demonstration of DNA, carbohydrates (1: 2 glycol group) and proteins, triple staining of HIMES and MORIBER (1956) was employed. Alkaline and acid phosphatases activity was determined by the method of NATALSON (1961). KUN and BLOW (1949) procedure was employed for the estimation of SDH. Phospholipids, nucleic acids and proteins were extracted from wet tissue by the procedure of SCHNEIDER (1945). DNA and RNA were estimated by the procedure of DISCHE (1957) and CERROITTI (1955), respectively. The Biuret method of ROBINSON (1940) was applied for protein estimation. Phospholipids were estimated (as lipid phosphorus) by the method of MARINETTI (1962). ZAK (1957) procedure followed after modification of ZLATKIS was employed for cholesterol estimation. Glycogen contents were estimated by the anthrone procedure of FONG and others (1957). Spermatozoa collected from caput, corpus and cauda and vas deferens, after flushing with 1 ccm of oxygenated tyrode, were examined under olympus phase contrast microscope for the number. morphology and motility. Living and dead sperm ratio was estimated by using eosin technique,

Results A marked decrease (40 %) in caput acid and alkaline phosphatases was observed two days after drug administration followed by slender increase after 5 and 10 days (fig. 1 A- B). Sharp rise in SDH activity was observed up to five days (90 %) after treatment followed by decline in activity which was almost the same in 20 and 40 days treated groups (fig. lC). Phospholipids showed a tremendous fall in the initial stages (70 %), the slight increase in the subsequent group was not statistically significant (fig. 2A). Cholesterol and glycogen revealed increase in their content 2 days after drug administration followed by decrease in subsequent groups (fig. 2B-C), DNA, RNA and protein registered a continuous decrease after drug treatment(fig. 3A-C). Epididymis of the animals of treated groups showed marked edema revealing swelling of vas efferentia and initial segment of epididymis. No morphological changes in the corpus and caudal regions of the epididymis were observed in any of the groups after treatment. Exfoliation of epididymis epithelial cells was observed after 2 days of drug administration. At places where the epithelium was intact, the height of columnar cells was extremely reduced (fig. 5). The interductular connective tissue also showed marked edema: dilatation of this region of epididymis was apparent. In group III the exfoliation of lining epithelium increased and almost the whole of the epithelium was detached from the basement membrane (fig. 6). The whole of the duct of caput region was filled with cells of lining epithelium, spermatid, spermatozoa and sperm debris causing spermatocoele. At few places the epithelium was broken and sperms escaping in the interstitial connective tissue could be seen. The edema of connective tissue was more widespread. . In group IV (10 days) the histological picture of epididymis was almost the same as that of five days post-treated animals (groups IV and V) the spermatocoele and sperm granulomata (sperm in clumps with cells) occupied almost the whole of the epididymis (fig. 7). The lesion in interductular tissue was still present. This picture of caput epididymis persists even 40 days (group VI) after treatment. The concentration of spermatozoa in the epididymis, however, showed marked decrease after 20 days of drug treatment. In group VI, the epididymis was devoid of spermatozoa. However, in all the treated animals, groups II to IV, epididymal and vas deferens spermatozoa were morphologically normal and motile.

Discussion Although a good deal of information is available on the histopathology of epididymis after single heavy dose of chlorohydrin both at light microscopic (ERICSSON 1970, GUNN et al. 1970, SAMJOLIK and CHANG 1970) and electron microscopic level (HOFFER et al. 1973), this is the first account of sequential histological and biochemical changes in the testis-epididymis complex after 2 single high doses. Though the present observations have failed to resolve the exact mechanism of action, they do provide some meaningful information on some earlier explanations. 20

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ERICSSON (1970) has suggested that chlorohydrin specifically affects the endothelium of the blood vessels supplying the initial segment. Though direct evidence to support this hypothesis is lacking, the main bulk of evidence has been derived from (1) the studies of McMILLAN (1956) on epididymal lesion after ligation of the blood vessels to caput and (2) potentiated response of vasoconstrictor-norepinephrine to chlorohydrin (ERICSSON and NORLAND 1970a) - similarly a vasodilator apresoline inhibits the blood vessel mediated damage of serotonin on testis (BOCABELLA et al. 1962) and finally (3) the initial segment of caput epididymis is particularly vulnerable to sudden alteration in the blood supply. It is logical to believe that vulnerability of the caput plus the slow blood flow seems to cause chemical changes in vascular permeability. Chlorohydrin, therefore, produces a local ischemia in the caput epididymis. Ligation of blood vessels serving epididymis and chlorohydrin are identical in time of formation, location, type and final disposition of the epididymal lesion. However, in the light of studies of TURNER (1971) it is logical to believe that the drug may have its site of action in other than caput region, too. According to this author, in chlorohydrin treated animals ligature made around corpus region yields infertile spermatozoa from cauda epididymis 3 days after drug administration. Similar observations have also been reported in hamster (LUBICZ-NAWROCK and CHANG 1973). Functional integrity of the epididymis has been reported to be dependent upon androgens

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Figs. 4-7. Histology of the caput epididymis.

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and entry of the spermatozoa and/or testicular fluid (PRASAD et al. 1972). It has been suggested that caput SDH activity can be considered as a useful parameter in the functional integrity of the epididymis (PRASAD et al. 1972). Over all fall in SDH, except initial increase seems to be due to disintegration of the entire cellular system. As a normal consequence of drug action there should have been increase in the alkaline phosphatase activity because of fluid accumulation. Since drug effect starts as early as two hours after administration, involving the nucleus, the cell surface and endoplasmic reticulum (damaging both absorptive and secretory cells (HOFFER et al. 1973) severe depletion of alkaline phosphatase after 2 days of drug administration should not be surprising. Decrease in acid phosphatase in the initial stages after drug administration may be due to disorganization of the acid phosphatase synthesizing unit, the Golgi complex (HOFFER et al. 1973). Increased degree of cellular disorganization in any biological system increases acid phosphatase because of increased lysosomal activity. It has been suggested that alpha chlorohydrin structurally similar to glycerol, may act as a metabolic antagonist for phospholipid synthesis. As a consequence of this, naturally occurring phospholipids in epididymis tissue may be reduced and/or chlorinated phospholipids may be formed with the result that the total amount of phospholipids may reduce. In the present studies this argument seems more plausible to explain decrease in phospholipid in the epididymis but unaltered level of glycerol in testis and different segments of epididymis after chlorohydrin (HODGEN 1972) do not lend any support to this hypothesis. Partial increase in cholesterol after drug administration seems to be due to accumulation of testicular fluid which is rich in free and esterified sterols. Though direct evidence of androgen involvement in alpha chlorohydrin mediated damage is lacking the nonsensitivity of the drug in sperm free epididymis contrary to androgen dependent metabolically active epididymis suggests that androgen does play an important role in initiating the drug action (ERICSSON and NORLAND 1970b) if not in preventing or enhancing the damage. Increase in glycogen may be due to fluid accumulation (rat testicular fluid contains about 0.172 mgjcc of glycogen (PANDE et al. 1969) and exfoliated immature cells are also rich in glycogen) plus metabolically pooled glycogen, which seems to be due to lack of its utilization as secretory cells in the caput epididymis are also depleted after a single dose of chlorohydrin (HODGEN 1972). These cells utilize glycogen as a readily available source of energy. Exfoliation of epithelium, as an outcome of the drug action, inhibits cell activity including nucleic acids and therefore decrease in the activity of total proteins is a normal outcome of the sequence. Decreased DNA staining in epithelial cells after drug treatment and altered submicroscopic detail of the nucleus (including the nucleous) and endoplasmic reticulum (HOFFER et a1.1973) suggest an early sequence of changes in the DNA-RNA protein cycle.

Literature BOCCABELLA, A ,V., E. R. SALGADO and E. A. ALGER, Testicular function and histology following serotonin administration. Endocrinology 71, 827 -837 (1962). BRowr., C., and I. G. WHITE, Studies on the male antifertility drug 3-chloro-1, 2-propandioI. J. Reprod. Fert. 32, 337 -338 (1973). CERROITTI, G., Determination of nucleic acids in animal tissues. J. BioI. Chern. 214, 59 (1955). COPPOLA, J. A., An extragonadal male antifertility agent. Life Sci. 8, 43-49 (1969). DISCHE, Z., Methods in Enzymology, p.701, Vol. III. Eds. S. P. COLOWICK and N. O. KAPLAN, Academic Press, New York 1957. ERICSSON, R. J., Male antifertility compounds: U-5897 as a rat chemosterilant. J. Reprod. Fert. 22 213-222 (1970.) Fig. 4. Normal caput epididymis region. HE, x 100. Fig. 5. Caput epididymis after 2 days of drug administration. HE, x 60. Fig. 6. Caput epididymis after 5 days of drug administration. Arrows indicate the sites of exfoliation. HE, x 200. Fig. 7. Caput epididymis after 20 days of drug administration. HE, x 1000.

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ERICSSON, R. J., and V. F. BAKER, Male anti-fertility compounds: Biological properties of U-5897 and U-15646. J. Reprod. Fert. 21, 267 -273 (1970). and J. F. NORLAND, Potentiating response with norepinephrine to rat chemosterilant alpha chlorohydrin. Proc. Second Meeting Endocrine Soc. p. 243 (1970a). - Androgen involvement with male antifertility compound alpha chlorohydrin. Third Int. Cong Hormonal Steroids. p. 362 (1970b). . FONG, J., F. L. SCHAFFER and P. K. KIRK, In: Methods in Enzymology, p. 85, Eds. S. P. COLOWICK and N. O. KAPLAN. Academic Press, New York 1957. GUNN, S. A., T. C. GOULD and W. A. ANDERSON, Comparative mechanism of action of monochlorohydrin and cadmium induced necrosis of the caput epididymis of the rat. BioI. Reprod. 5, 35 to 42 (1970). HIMES, M., and L. MORIBER, A triple stain for deoxyribonucleic acid, polysaccharides and proteins. Stain Technol. 31, 67 (1956). HODGEN, G. D., Total glycerol in the excurrent ducts of the male rat. J. Reprod. Fert. 28, 277 to 280 (1970). HOFFER, A. P., D. W. HAMILTON and D. W. FAWCETT, The ultrastructural pathology of rat epididymis after administration of alpha chlorohydrin. U-5897. I. Effects of a single high dose. Anat. Rec. 175, 203-230 (1973). JOHNSON, L. A., and V. G. PURESL, Reversible sterility in swine fed on alpha chlorohydrin. J. Animal Sci. 31, 224 (1970). KREIDER, J. L., and R. H. DUTT, Temporary induction of infertility in rams with an orally administered chlorohydrin. J. Animal Sci. 31, 95 (1970). KUN, E., and L. G. BLOW, Colourimetric estimation of succinic dehydrogenase by triphenyl tetrazolium chloride. Science 109, 144 (1949). LUBICZ-NAWROCK, C. M., and M. C. CHANG, Effect of alpha chlorohydrin on epidimal spermatozoa in hamsters. BioI. Reprod. 9, 91 (1973). MARINETTI, G. V., Chromatographic separation, identification and analysis of phosphatides. J. Lipid Res. 3, 1 (1962). McMILLAN, E. W., The immediate effects of occlusion of superior epididymal arteries in the rat as demonstrated by the use of radio-opaque medium. Proc. Soc. Study Fert. 8, 67 (1956). NATALSON, S., Methods for estimating phosphatases. In: Microtechniques of clinical chemistry, p. 332. Charles C. Thomas, Springfield, 111., 1961. PANDE, J. K., P. DASSGUPTA and A. B. KAR, Comparative biochemistry of testicular fluid in some mammals. Gen. Compo Endocrinol. Suppl. 2,156-161 (1969). PRASAD, M. R. N., N. J. CHINOY and K. M. KADAM, Changes in succinic dehydrogenase levels in the rat epididymis under normal and altered physiological conditions. Fertil. Steril. 23, 186-190 (1972). ROBINSON, H. W., and C. G. HODGEN, The Biuret reaction in the determination of serum proteins. J. BioI. Chem. 135, 707 (1940). SAMOJLIK, E., and M. C. CHANG, Antifertility activity of 3-chloro-l,2-propanediol on male rats. BioI. Reprod. 2, 299-304 (1970). SCHNEIDER, W. C., Phosphorus compounds in animal tissues. J. BioI. Chem. 161, 293 (1945). TURNER, M. A., Effects of chlorohydrin upon the fertility of spermatozoa of the cauda epididymis of the rat. J. Reprod. Fert. 24, 267 - 269 (1971). ZAK, B., Simple rapid microtechnique for serum total cholesterol. Am. J. Clin. Pathol. 27, 583 (1957). Authors' addresses: N. R. KALLA, M. D. Building 10 - Room 10B-06, Reproduction Research Branch, National Institutes of Health. Bethesda, Maryland 20014 (U.S.A.). K. S. CHOHAN, Department of Physics, University of Calgery, Calgery (Canada).

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