STUDIES ON THE PHYSIOLOGY OF THE HUMAN EPIDIDYMIS AND VAS DEFERENS I. BIOCHEMICAL CONSTITUENTS M. Rajalakshmi, Ph.D. P.R.K. Reddy, Ph.D. M.R.N. Prasad, Ph.D. (Mysore), Ph.D. (Wisconsin)
Department of Zoology University of Delhi Delhi-110007, India and P. Upadhyaya, M.D. V.R. Minocha, M.B.B.S. Department of Paedlatric Surgery All India Institute of Medical Sciences New Delhi-110016, India
ABSTRACT The epldldymls and vas deferens from healthy men of various ages who died in road accidents were analyzed for the levels of total llplds, phosphollpidst slallc acidt RNA, DNA and protein. The concentration of total liplds and total phospholipld phosphorus showed little variation in the caput and cauda epldidymldes of young and old age groups. Total PLP was higher in the cauda than in the caput or corpus epidldymldes of both age groups. The major phospholipids in all segments of epldldymis and vas deferens were phosphafidyl choline and phosphatldyl ethanolamine. Concentration of siallc acid was similar in all the regions of the epldidymls but was lower in the vas deferens. DNA and RNA were maximal in the cauda epldldymides while protein was nearly the same in all the regions of the epldldymls. The significance of these changes is discussed. Accepted for publication
AUGUST 1975
June I I ,
VOL. 12 NO. 2
1975
175
CONTRACEPTION
I NTRODUCTION In attempts to evolve newer approaches to contraception in the male, considerable attention has been focused in recent years on the epididymis as an extragonadal site for control of fertility. In this context, studies on the physiology of the human epldidymis are of great relevance; however, our knowledge of the biochemical constituents of the human epididymis and vas deferens is meagre (1, 2, 3). The levels of a number of biochemical contltuents in the different segments of the human epididymis and vas deferens reported here constitute the first of a series of investigations on the physiology of the human epididymis. MATERIALS AND METHODS The epididymls and the vas deferens of healthy men of various ages who died in road accidents, without sustalnlng any apparent damage to the testes and accessory organs, were collected from the post-mortem room of the hospitals in Delhi within 4-8 hours after death. The epldidymis was divided arbitrarily into the caput, corpus and cauda epididymldes. The tissues were weighed on a torsion balance to the nearest 0.2 mg. One side of the epidldymis was used for the estimation of llplds and phospholiplds and the contralateral side for the estimation of siallc acid. In the second study, the epidldymls from two healthy men (50 years) who died in road accidents was used for the estimation of RNA, DNA and protein. Epldldymis from one patient suffering from benign prostatic hyperplasia (BPH) and from another patient with a unilateral cryptorchld testis were obtained following surgery and were analyzed for RNA, DNA and protein. Pieces of vas deferens obtained following surgical vasectomy of healthy men were analyzed for total liplds, total phosphollplds, DNA, RNA and slallc acid. Analysis of Lipids: Liplds were extracted from the tissues using the chloroform-methanol (2:l,v/v) solvent system as described by Folch et al. (4). Total phosphollpld phosphorus was estimated by the modified method of Marlnettl (5). Individual phospholipids were separated by thln-layer chromatography using the solvent system chloroform-methanol-7M ammonia (115:45:7.5, v / v / v ) (6), exposed to iodine vapour (7) and identified by referring the unknown samples to authentic standards. Total lipid was determined gravlmetrical ly.
176
AUGUST 1975
VOL. 12 NO. 2
CONTRACEPTION Analysis of Sialic Acid: The tissues were homogenized in 0.1N H2SO4 and hydrolyzed at 80°C for one hour and used for the estimation of bound sialic acid by the thiobarblturlc acid assay procedure of Warren (8) using appropriate controls. Anal)sis of RNA r DNA and protein: Tissueswere homogenized in 0.SN perchloric acid and RNA t DNA and protein were extracted following the procedure outlined by Rendlna (9). RNAt DNA and protein were estimated according to the procedures of Schneider (10), Burton (11) and Lowry et al. (12), respectively. The data were analyzed statistically using the students' 't' test. RESULTS AND DISCUSSION In order to determine the effects of age on the biochemical constltuents, the epldidymls was classified into two age groups: 1) young age group varying in age from 17-22 years, and 2) old age group from 65-68 years. Lipids and phospholipld phosphorus: The concentration of total Iipids and total phosphollpid phosphorus (PLP) showed little variation in the caput and cauda epidldymides and was similar in the two age groups; in the corpus epldidymldes, the concentration of total lipids was numerically higher but statistically insignificant (p > 0.05) in the young than in the old age groups (Table I). Total llpids were numerically maximal in the corpus epididymldes of the young age group; however, in the old age group, they were nearly the same in all the regions of the epldldymis. Total PLP was uniformly high in the cauda epldldymides in both the age groups though the differences between the segments were only marginal. This is in contrast to the observations of Riar, Setty and Kar (2) who reported that lipid phosphorus was maximal in the proximal corpus epididymides of a single healthy 35-year-old victim of a road accident. In both the age groups, the major phospholipids in all the segments of the epidldymis were phosphatldyl choline (PC) and phosphatidyl ethanolamine (PE) whereas polyglycerol phosphatide (PGP) and phosphatidlc acid (PA) were almost negllgible (Table II). Poulos and White (13) have also recorded high concentrations of PC and PE in ejaculated human spermatozoa. Phosphatidyl inositol (PI) + phosphatldyl serine (PS), lysophosphatldyl choline (LPC) + lysophosphatidyl ethanolamine (LPE) and sphlngomyelln occurred in nearly the same amounts in the different regions of the epididymis of both the age groups with minor fluctuations. High concentrations of phosphatidyl serlne and sphlngomyelln have been reported in
AUGUST 1975 VOL. 12 NO. 2
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CONTRACEPTION the human seminal plasmd; ejaculated human spermatozoa contain 21.4% sphlngomyelln, 28.8% phosphatidyl choline and 21.6% phosphatidyl ethanolamine (13) whereas the cauda epldldymldes of human (young age group) with its contained mature spermatozoa had 43.1% PC, 31.2% PE and 9.6% sphlngomyelin. It is therefore likely that the higher amounts of sphlngomyelin in ejaculated spermatozoa may be due to its acquisition by the spermatozoa during their transit through the vas deferens or the rest of the male reproductive tract during ejaculation. Alternatively, it is likely that a low amount of sphingomyelin in the tissue fraction of the epididymls may result in the decreased concentration of sphlngomyelln when the cauda epidldymides was analyzed in total. In the human epldldymis, sphlngomyelin expressed as percentage of total phosphorus did not show any significant change from the caput to the cauda epldidymldes as in the monkey (14). In the Rhesus monkey, the concentration of phosphatidyl choline, phosphatidyl ethanolamlne and sphingomyelln in the spermatozoa decrease progressively as they migrate from the caput to the cauda epidldymldes and remain at nearly the same level in the ejaculate (15). Though Poulos and White (13) have reported the percentage of different phospholiplds in ejaculated human spermatozoa, the ~:hanges, if any, in these constituents during the transit of the human spermatozoa through the epldldymis and vas deferens have not been investigated. Further, the role of these phosphollpids in the maturation of the spermatozoa and in their metabolic activity is still not clear. The phospholipids may not serve as a source of energy for human spermatozoa in the absence of oxidiz~able carbohydrates after eiaculatlon (13). Glycerylphosphoryl choline (GPC) is found in the different regions of the human epididymls with the highest concentration in the corpus epidldymides (2); it has been suggested that epldldymal phosphatidyl choline may serve as a source of GPC (16). In the patient with BPH, a higher concentration of total llpids and PLP was observed in the caput epldldymldes than in the epldldymls of normal men of either age group; however, the concentration of total lipids and PLP in the cauda epid|dymldes of the patient with BPH was lower than in the two age groups of normal men (Table I). Since these biochemical estimations were conducted on tissues obtained from only one patient, no conclusions can be drawn regarding their significance. Siallc acid: The concentration of slallc acid was numerically maxlmal in the cauda epldidymides of both the age groups but the differences were not statistically significant (1o • 0.05; Table III). Similar results
180
AUGUST 1975 VOL. 12 NO. 2
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CONTRACEPTION spermatozoa. In the human vas deferens, phosphatidyl choline (46.33%), phosphafidyl ethanolamlne (27.13%) and sphingomyelin (19.15%) represented the highest percentage of total phosphollplds resembling the condition in the cauda epldldymldes. The content of protein in the vas deferens was higher than in any other segment of tl~q epidldymis; similar data hove been reported by Riar, Setty and Kar (1). Since spermatozoa are retained in the human vas deferens in a viable state for 2-3 months after vasectomy, for a period of time much longer than that in the epididymls of any other species, the vas deferens with all its biochemical constituents and its ability to concentrate high amounts of androgens (19) should therefore be considered as a dynamic organ rather than as a passive tube for the passage of spermatozoa during ejaculation. In the case of epididymal occlusions, successful pregnancies have been reported by epldldymovasostomy (20), indicating the possible role of the vas deferens in the maturation of spermatozoa at least in the human. Further, recanallzafion of vas deferens after vasectomy results in the emission of motile spermatozoa in the ejaculate which are apparently infertile. The question of whether the physiology of the vas deferens was in some way markedly affected by these surgical procedures which provided a passage for the spermatozoa but did not provide the ideal conditions for them to be fertile needs to be examined. ACKNOWLEDGEMENTS This work was supported by grants from the Indian Council of Medical Research, Ministry of Health and Family Planning, Government of India, University Grants Commission and the Ford Foundation. The authors thank Mr. S.K. Jain for his technical assistance. REFERENCES 1.
Riar, S.S., Setty, B.S. and Kar, A.B.: Biochemical composition of primate epididymls. Curr. Sci. 41:453-455 (1972).
2.
Riar, S.S., Setty, B.S. and Kar, A.B.: Studies on the physiology and biochemistry of mammalian epididymls. Biochemical composition of epididymis. A comparative study. Ferfil. Steril. 24:355-363 (1973).
3.
Setty, B.S., Chowdhury, S.R. and Jehan, Q.: Biochemical composition of the human vas deferens. Contraception 9:601-609 (1974).
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CONTRACEPTION
.
Folch, J., Lees, M. and Sloane-Stanley, G.H.: A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509 (1957).
.
Marinettl, G.V.: Chromatographic separation, identification and analysis of phosphatldes. J. Lipid Res. 3:1-20 (1962).
.
Abramson, D. and Blecher, M.: Quantitative two-dlmenslonal thinlayer chromatography of naturally occurring phospholiplds. J. Lipid Res. 5:628-631 (1964).
.
Barret, G.C.: Iodine as a "non-destructive" colour reagent in paper and thln-layer chromatography. Nature 194:1171 (1962).
.
9.
Warren, L.: The thlobarbiturlc acid assay of siallc acids. Chem. 234:1971-1975 (1959). Rendlna, G.: Experimental methods in modem biochemistry. Saunders Co., Philadelphia (1971).
J. Biol. W.B.
10.
Schnelder, W.C.: Determination of nuclelc aclds in tissues by pentose analysis. In: Methods in Enzymology. Vol. III, Eds. S.P. Colowlck and N . O . Kaplan. Academic Press, New York, p. 680, (1957).
11.
Burton, K.: A study of the conditions and mechanism of the dlphenylamine reaction for the colorlmetric estimation of deoxyribonucleic acid. Biochem. J. 62:315-323 (1965).
12.
Lowry, C).H., Rosenbrough, A . L . , Farr, A.L. and Randall, A.J.: Protein measurement in Folln phenol reagent. J. Biol. Chem. 193:265-275 (1951).
13.
Poulos, A. and White, I . G . : The phosphollpid composition of human spermatozoa and seminal plasma. J. Reprod. Fert. 35:265-272 (1973).
14.
Dinakar, N . , Arora, R. and Prasad, M.R.N.: Effects of mlcroquantities of testosterone on the epldidymis and accessory glands of the castrated Rhesus monkey, Macaca mulatta. J. Endocr. 60:399408 (1974).
186
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15.
Arora, R., Dinakar, N. and Prasad, M.R.N.: Biochemical changes in the spermatozoa and luminal contents of different regions of the epldldymls of the Rhesus monkeyt Macaca mulatta. Contraception 11:689-699 (1975).
16.
Scott, T.W., Dawson, R.M.C. and Rowlands, I.W.: Phosphollpld interrelationships in rat epldidymal tissue and spermatozoa. Biochem. J. 87:507-512 (1963).
17.
Rajalakshml, M. and Prasad, M.R.N.: Changes in the epididymis of adult rats following experimentally induced cryptorchldlsm. Andrologla 6:293-302 (1974).
18.
Baumgarten, H.G., Holstein, A.F. and Rosengren, E.: Arrangement, ultrastructure and adrenerglc innervatlon of smooth musculature of the ductull efferentes, ductus epldidymides and ductus deferens of man. Z. Zellforsch. 120:37-79 (1971).
19.
Rajalakshmit M.t Arora, R., Bo6et T.K., Dinakar, N., Gupta, G., Thampan, T.N.R.V., Prasad, M.R.N., Anand Kurnart T.C. and Moudgal, N.R.: Physiology of the epldldymls and induction of functional sterility in the male. J. Reprod. Fert. Suppl. (in press, 1975).
20.
Bedford, J.M., Calvint H. and Cooper, G.W.: The maturation of spermatozoa in the human epidldymls. J. Reprod. Fert. Suppl. 18:199-213 (1973).
AUGUST ]975
VOL. ]2 NO. 2
187
Department of Zoology University of Delhi Delhi-110007, India and P. Upadhyaya, M.D. V.R. Minocha, M.B.B.S. Department of Paedlatric Surgery All India Institute of Medical Sciences New Delhi-110016, India
ABSTRACT The epldldymls and vas deferens from healthy men of various ages who died in road accidents were analyzed for the levels of total llplds, phosphollpidst slallc acidt RNA, DNA and protein. The concentration of total liplds and total phospholipld phosphorus showed little variation in the caput and cauda epldidymldes of young and old age groups. Total PLP was higher in the cauda than in the caput or corpus epidldymldes of both age groups. The major phospholipids in all segments of epldldymis and vas deferens were phosphafidyl choline and phosphatldyl ethanolamine. Concentration of siallc acid was similar in all the regions of the epldidymls but was lower in the vas deferens. DNA and RNA were maximal in the cauda epldldymides while protein was nearly the same in all the regions of the epldldymls. The significance of these changes is discussed. Accepted for publication
AUGUST 1975
June I I ,
VOL. 12 NO. 2
1975
175
CONTRACEPTION
I NTRODUCTION In attempts to evolve newer approaches to contraception in the male, considerable attention has been focused in recent years on the epididymis as an extragonadal site for control of fertility. In this context, studies on the physiology of the human epldidymis are of great relevance; however, our knowledge of the biochemical constituents of the human epididymis and vas deferens is meagre (1, 2, 3). The levels of a number of biochemical contltuents in the different segments of the human epididymis and vas deferens reported here constitute the first of a series of investigations on the physiology of the human epididymis. MATERIALS AND METHODS The epididymls and the vas deferens of healthy men of various ages who died in road accidents, without sustalnlng any apparent damage to the testes and accessory organs, were collected from the post-mortem room of the hospitals in Delhi within 4-8 hours after death. The epldidymis was divided arbitrarily into the caput, corpus and cauda epididymldes. The tissues were weighed on a torsion balance to the nearest 0.2 mg. One side of the epidldymis was used for the estimation of llplds and phospholiplds and the contralateral side for the estimation of siallc acid. In the second study, the epidldymls from two healthy men (50 years) who died in road accidents was used for the estimation of RNA, DNA and protein. Epldldymis from one patient suffering from benign prostatic hyperplasia (BPH) and from another patient with a unilateral cryptorchld testis were obtained following surgery and were analyzed for RNA, DNA and protein. Pieces of vas deferens obtained following surgical vasectomy of healthy men were analyzed for total liplds, total phosphollplds, DNA, RNA and slallc acid. Analysis of Lipids: Liplds were extracted from the tissues using the chloroform-methanol (2:l,v/v) solvent system as described by Folch et al. (4). Total phosphollpld phosphorus was estimated by the modified method of Marlnettl (5). Individual phospholipids were separated by thln-layer chromatography using the solvent system chloroform-methanol-7M ammonia (115:45:7.5, v / v / v ) (6), exposed to iodine vapour (7) and identified by referring the unknown samples to authentic standards. Total lipid was determined gravlmetrical ly.
176
AUGUST 1975
VOL. 12 NO. 2
CONTRACEPTION Analysis of Sialic Acid: The tissues were homogenized in 0.1N H2SO4 and hydrolyzed at 80°C for one hour and used for the estimation of bound sialic acid by the thiobarblturlc acid assay procedure of Warren (8) using appropriate controls. Anal)sis of RNA r DNA and protein: Tissueswere homogenized in 0.SN perchloric acid and RNA t DNA and protein were extracted following the procedure outlined by Rendlna (9). RNAt DNA and protein were estimated according to the procedures of Schneider (10), Burton (11) and Lowry et al. (12), respectively. The data were analyzed statistically using the students' 't' test. RESULTS AND DISCUSSION In order to determine the effects of age on the biochemical constltuents, the epldidymls was classified into two age groups: 1) young age group varying in age from 17-22 years, and 2) old age group from 65-68 years. Lipids and phospholipld phosphorus: The concentration of total Iipids and total phosphollpid phosphorus (PLP) showed little variation in the caput and cauda epidldymides and was similar in the two age groups; in the corpus epldidymldes, the concentration of total lipids was numerically higher but statistically insignificant (p > 0.05) in the young than in the old age groups (Table I). Total llpids were numerically maximal in the corpus epididymldes of the young age group; however, in the old age group, they were nearly the same in all the regions of the epldldymis. Total PLP was uniformly high in the cauda epldldymides in both the age groups though the differences between the segments were only marginal. This is in contrast to the observations of Riar, Setty and Kar (2) who reported that lipid phosphorus was maximal in the proximal corpus epididymides of a single healthy 35-year-old victim of a road accident. In both the age groups, the major phospholipids in all the segments of the epidldymis were phosphatldyl choline (PC) and phosphatidyl ethanolamine (PE) whereas polyglycerol phosphatide (PGP) and phosphatidlc acid (PA) were almost negllgible (Table II). Poulos and White (13) have also recorded high concentrations of PC and PE in ejaculated human spermatozoa. Phosphatidyl inositol (PI) + phosphatldyl serine (PS), lysophosphatldyl choline (LPC) + lysophosphatidyl ethanolamine (LPE) and sphlngomyelln occurred in nearly the same amounts in the different regions of the epididymis of both the age groups with minor fluctuations. High concentrations of phosphatidyl serlne and sphlngomyelln have been reported in
AUGUST 1975 VOL. 12 NO. 2
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CONTRACEPTION the human seminal plasmd; ejaculated human spermatozoa contain 21.4% sphlngomyelln, 28.8% phosphatidyl choline and 21.6% phosphatidyl ethanolamine (13) whereas the cauda epldldymldes of human (young age group) with its contained mature spermatozoa had 43.1% PC, 31.2% PE and 9.6% sphlngomyelin. It is therefore likely that the higher amounts of sphlngomyelin in ejaculated spermatozoa may be due to its acquisition by the spermatozoa during their transit through the vas deferens or the rest of the male reproductive tract during ejaculation. Alternatively, it is likely that a low amount of sphingomyelin in the tissue fraction of the epididymls may result in the decreased concentration of sphlngomyelln when the cauda epidldymides was analyzed in total. In the human epldldymis, sphlngomyelin expressed as percentage of total phosphorus did not show any significant change from the caput to the cauda epldidymldes as in the monkey (14). In the Rhesus monkey, the concentration of phosphatidyl choline, phosphatidyl ethanolamlne and sphingomyelln in the spermatozoa decrease progressively as they migrate from the caput to the cauda epidldymldes and remain at nearly the same level in the ejaculate (15). Though Poulos and White (13) have reported the percentage of different phospholiplds in ejaculated human spermatozoa, the ~:hanges, if any, in these constituents during the transit of the human spermatozoa through the epldldymis and vas deferens have not been investigated. Further, the role of these phosphollpids in the maturation of the spermatozoa and in their metabolic activity is still not clear. The phospholipids may not serve as a source of energy for human spermatozoa in the absence of oxidiz~able carbohydrates after eiaculatlon (13). Glycerylphosphoryl choline (GPC) is found in the different regions of the human epididymls with the highest concentration in the corpus epidldymides (2); it has been suggested that epldldymal phosphatidyl choline may serve as a source of GPC (16). In the patient with BPH, a higher concentration of total llpids and PLP was observed in the caput epldldymldes than in the epldldymls of normal men of either age group; however, the concentration of total lipids and PLP in the cauda epid|dymldes of the patient with BPH was lower than in the two age groups of normal men (Table I). Since these biochemical estimations were conducted on tissues obtained from only one patient, no conclusions can be drawn regarding their significance. Siallc acid: The concentration of slallc acid was numerically maxlmal in the cauda epldidymides of both the age groups but the differences were not statistically significant (1o • 0.05; Table III). Similar results
180
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A U G U S T 1975
VOL. 12 NO. 2
CONTRACEPTION spermatozoa. In the human vas deferens, phosphatidyl choline (46.33%), phosphafidyl ethanolamlne (27.13%) and sphingomyelin (19.15%) represented the highest percentage of total phosphollplds resembling the condition in the cauda epldldymldes. The content of protein in the vas deferens was higher than in any other segment of tl~q epidldymis; similar data hove been reported by Riar, Setty and Kar (1). Since spermatozoa are retained in the human vas deferens in a viable state for 2-3 months after vasectomy, for a period of time much longer than that in the epididymls of any other species, the vas deferens with all its biochemical constituents and its ability to concentrate high amounts of androgens (19) should therefore be considered as a dynamic organ rather than as a passive tube for the passage of spermatozoa during ejaculation. In the case of epididymal occlusions, successful pregnancies have been reported by epldldymovasostomy (20), indicating the possible role of the vas deferens in the maturation of spermatozoa at least in the human. Further, recanallzafion of vas deferens after vasectomy results in the emission of motile spermatozoa in the ejaculate which are apparently infertile. The question of whether the physiology of the vas deferens was in some way markedly affected by these surgical procedures which provided a passage for the spermatozoa but did not provide the ideal conditions for them to be fertile needs to be examined. ACKNOWLEDGEMENTS This work was supported by grants from the Indian Council of Medical Research, Ministry of Health and Family Planning, Government of India, University Grants Commission and the Ford Foundation. The authors thank Mr. S.K. Jain for his technical assistance. REFERENCES 1.
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