- Email: [email protected]
Studies on camel semen. II. Biochemical characteristics
AnimalReproduction Science, 12 (1986) 223-231
223
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
S t u d i e s on C a m e l S e m e n II. B i o c h e m i c a l C h a r a c t e r i s t i c s M.M. EL-MANNA 1, M.D. TINGARIe*, and A.K. AHMED 2
1Department of Physiology and Biochemistry and 2Department of Anatomy, College of Veterinary Medicine and Animal Resources, King Faisal University, P.O. Box 1757, Al-Ahsa 31982 (Saudi Arabia) *Present address: Department of Anatomy, Faculty of Veterinary Science, University of Khartoum, P.O. Box 32, Khartoum North (Sudan) (Accepted 24 April 1986 )
ABSTRACT El-Manna, M.M., Tingari, M.D. and Ahmed, A.K., 1986. Studies on camel semen. II. Biochemical characteristics. Anim. Reprod. Sci., 12: 223-231. The mean concentrations of fructose, citric acid, total proteins and lipids in the seminal plasma of camel were 23.5 ± 2.5, 9.8 ± 2.9, 775 __15 and 87 _+32 mg/ml respectively. The corpus prostatae seemed to be the principal source of fructose and citric acid, smaller amounts are contributed by the bulbourethral glands, ampulla ductus deferentis and pars disseminata of the prostate. Electrophoretic fractionation of seminal plasma proteins by capillary isotachophoresis revealed eight protein bands similar to those of blood serum. Electrofocusing in polyacrylamide gels on the other hand showed the presence of eighteen fractions with minor variations from fractions obtained from blood serum.
INTRODUCTION
Semen has been studied extensively in several mammalian and avian species (Mann, 1964, 1975). Its chemical make up varies not only from one species to another, but also among individuals belonging to the same species. The role of the secretions contributed to the seminal plasma by the various male accessory sex glands of reproduction has long been known, and so has the specialized chemical nature of these secretions. Among the most notable substances secreted in high concentrations are fructose and citric acid. The information available on semen composition in the camel is primarily limited to the work of Abdel-Raouf and E1-Naggar (1978) and E1-Naggar and Abdel-Raouf (1977). Our earlier report dealt with the biophysical characters of semen and sperm morphology (Tingari et al., 1986). The present investigation was designed to study the biochemical character0378-4320/86/$03.50
© 1986 Elsevier Science Publishers B.V.
224 istics of camel seminal plasma during the rutting season ( Tingari et al., 1984). An analysis of the accessory sex glands was also undertaken with the purpose of identifying the sources contributing to the secretion of fructose and citric acid. MATERIALSAND METHODS Materials
Four camels aged 6-9 years were used for weekly collections of semen and serum during the rutting season ( see Tingari et al., 1984). Semen was obtained by the method of electroejaculation as outlined elsewhere ( Tingari et al., 1986). The animals were apparently healthy and were kept in the University Farm in fenced yards with shaded areas. They had no contact with females a n d were fed on barley and straw and offered water ad libitum. Seminal plasma was obtained by centrifugation of freshly ejaculated semen at 4000 rpm for 15 min. Tissue samples from prostate, bulbourethral glands, urethra and ampulla ductus deferentis of adult camels (Ali et. al., 1978) were collected from AIHassa municipal slaughterhouse, They were dissected and transferred immediately to liquid nitrogen. Whenever possible, and depending on the tissue size available, the selectedgland was utilized for both fructose and citric acid determination; otherwise it was wholly devoted to one test or the other. A m i n i m u m of duplicate subsamples was analyzed from the selected gland of each animal. The gels used in electrofocusing ( P A G plates pH 3.6-9.5) and ampholine carrier ampholytes were obtained from LKB (LKB Produckter AB); other chemicals were purchased from BDH. Fructose determination
Weighed pieces of glands were homogenized in cold isotonic potassium chloride and then deproteinized using 2% zinc sulphate (7H20) and 0.1 N sodium hydroxide. T h e supernatant fluid was obtained after centrifugation at 4000 rpm for 15 min. Proteins were removed from t h e seminal plasma by zinc sulphate and sodium hydroxide precipitation. Fructose was measured in the supernatant fluid from t h e glands and seminal plasma by the resorcinol reaction (Lindner and Mann, 1960). Citric acid determination
Protein precipitation from the seminal plasma and supernatant fluid from the glands was effected utilizing 20% trichloroacetic acid. Citric acid was estimated by oxidation to the pentabromoacetone and subsequent treatment with sodium sulphide according to Lindner and M a n n (1960).
225
Protein analysis Total protein in seminal plasma was determined by the biuret method using bovine serum albumin as a standard (Gornall et al., 1949).
Electrophoretic separations Two methods of electrophoretic fractionation were used; capillary isotachophoresis and polyacrylamide gel electrofocusing. An LKB Tachophor 2127 was used for isotachophoresis according to the procedures specified for that instrument. A capillary tube 230 mm in length was used at a temperature of 10 oC. Protein absorption was monitored using a 280 nm U.V. detector; the current during detection was 40/~A and the analysis time 30 min. A spacer solution composed of amino acids and ampholine carrier ampholytes was added to the samples in order to resolve the different protein bands. The composition of the leading and terminating electrolytes, sample and spacer solution were similar to those reported by Delmotte (1977). The composition was as follows: leading electrolyte, 5 m M 2- (N-morpholino) -ethane sulphonic acid; 10 m M 2-amino-2-methyl-1, 3-propanediol; 0.1% Triton X-100, pH 9.0 - - terminating electrolyte, 11.4 mME-aminocaproic acid; 10 m M 2-amino-2methyl-l, 3 propanediol adjusted with barium hydroxide to pH 10.8 spacer solution, 10 ml solutions contained the following: 1.6 mg glycine, 1.6 mg valine, 1.44 mg fl-alanine, 0.18 ml 40% ampholine carrier ampholyte pH 7-9, 0.095 ml 40% ampholine carrier ampholyte pH 8-9.5, 0.3 ml 20% ampholine carrier ampholyte pH 9-11. Seminal plasma samples contained 6 ttl seminal plasma and 2/ll spacer solution, whereas the serum samples were composed of 2/A each of serum and spacer solution. LKB Multiphor 2117 was used for polyacrylamide gel electrofocusing according to the procedures recommended by the instruction manual. Samples of seminal plasma and serum were applied directly on the gel surface - - 3/11 samples of seminal plasma and 1 ,ul samples of serum using a micropipette at a distance of 10 mm from the cathode. The power supply (LKB 2197) was set at 25 W, 40 mA and 1300 V. The running time was 1.5 h during which the temperature was maintained at 10 °C by circulating water through the plate using an LKB Multitemp 2209 circulator. The gel used was an LKB PAG plate pH 3.5-9.5, with Coomassie Brilliant Blue R 250 as stain and ethanol : acetic acid aqueous solution for destaining. A Camag electrophoresis scanner fitted with an integrator was utilized for evaluation of peak areas.
Lipid analysis Total lipids in seminal plasma samples were extracted using chloroform :methanol mixture (2 : 1) according to Folch et al. (1957) as modified
1 2 3 4,
9.8~-0.7 21.6 _+1.4 34.0 ___2.1 41.2 _+2.5 10.7"-_0.8 24.2 _+3.2 27.0 _+5.6 22.3_ 1.4 20.3 +- 2.1
8
8
8 8 8 5 5 5 5
Mean ~- s.d.
17.0- 96.3 23.8-131.2 5.6- 25.5 17.0- 32.0 17.0- 40.0 15.0- 31.0 14.0- 25.0
8.8- 50.9
5.0- 23.8
Range
5 4 3 6 8 6 7
4
5
No. of samples
No. of samples
Concentration (mg/100 ml or 100 g tissue)
Citric acid
Fructose
36.0___ 3.1 24.9 +- 1.9 7.2___0.9 7.2 _+1.2 14.1 z 1.6 10.8__. 1.4 7.0 ÷ 0.9
35.3 z 2.2
15.8_+ 1.8
Mean +- s.d.
21.5-44.5 4.5-50.1 0-18.5 5.0-12.0 10.0-30.0 5.0-13.0 5.0-10.0
19.8-63.5
8.5-18.5
Range
Concentration (mg/100 ml or 100 g tissue )
Nos. 1-4 refer to camels of corresponding numbers. Whenever possible, both fructose and citric acid in the seminal plasma were assayed in the same sample.
Prostate gland ( pars disseminata) Prostate gland (corpus prostatae) Bulbourethral gland Ampulla Urethra Seminal plasma No. Seminal plasma No. Seminal plasma No. Seminal plasma No.
Tissue
Fructose and citric acid contents in seminal plasma and accessory glands
TABLE 1
t'~ bO
227 by Overturf and Dyer (1969). One millilitre samples of plasma were extracted with 20-fold chloroform : methanol mixture containing 0.2 volume of 0.05 M sodium chloride. An aliquot of the lipid extract dissolved in chloroform was utilized in qualitative TLC analyses using 20 × 20 cm precoated Silica Gel G plates. The plates were activated by heating at l l 0 ° C for 1 h. The plates containing spotted samples together with appropriate standards were developed using the solvent system petroleum e t h e r : d i e t h y l ether: acetic acid (80 : 20 : 1 ). The separated lipids were visualized under U.V. light at 366 nm after being sprayed with 2,7-dichlorofluoroscein (0.20% in absolute ethanol). RESULTS The results obtained for fructose, citric acid, total proteins and lipids in the seminal plasma and accessory glands are presented in Tables 1 and 2. The mean concentrations of the different constituents in the seminal plasma in mg/100 ml were as follows: fructose, 23.5 __2.5; citric acid 9.8 __2.9; total proteins, 775 + 15 and total lipids, 87 _+32. Correlation analyses showed that fructose and citric acid contents are closely related both within an individual animal and between animals. Correlation coefficients for the four animals were as follows: 0.73, 0.99, 0.95 and 0.98; that between the different animals was 0.85. Isotachophoretic analysis of seminal plasma proteins revealed a pattern similar to that of blood serum consisting mainly of eight peaks (Fig. 1 ). The average percentages of the protein bands as measured by the area under individual peaks are 2.34, 5.22, 21.85, 12.39, 16.57, 18.79, 9.92 and 12.92. Electrofocusing on the other hand showed about eighteen peaks qualitatively similar to those obtained from serum (Fig. 2 ). The percentage areas under these peaks as evaluated by scanning densitometry are given in Fig. 2. Qualitative analysis of lipids by TLC in non-polar solvents revealed four spotscorresponding to sterols and their esters, acylglycerols and phospholipids. DISCUSSION Semen analysis has shown that the concentrations of fructose, citric acid, total proteins and lipids in the seminal plasma were lower than those in semen collected by artificial vagina (E1-Naggar and Abdel-Raouf, 1977). Analysis of the accessory glands revealed that fructose is produced by the ampulla, bulbourethral glands together with the corpus prostatae, whereas citric acid is derived mainly from the corpus prostatae and bulbo-urethral glands with a minor contribution from the ampulla and pars disseminata of the prostate gland. On account of its comparatively much larger size than all others, the corpus prostatae appears to be the principal source of both fructose and citric acid. The positive correlation shown for these two components lends further support to this assumption.
228 TABLE 2 Total protein and lipid content in seminal plasma Source of seminal plasma
Total protein concentration (mg/100 ml) Mean___ s.d.
Total lipid concentration (rag/100 ml) Mean Jr s.d.
1 2 3 4
790 _+10 780_+ 6 780_+ 6 750_+ 8
98_+ 12 68_+ 3 135 q- 10 48___ 5
Samples of seminal plasma from a given animal remaining after fructose and citric acid determinations, were pooled together and utilized for protein and lipid contents in that animal. A minimum of duplicate subsamples was analyzed.
Electrophoretic analysis of plasma proteins as judged by isotachophoresis showed the presence of eight bands qualitatively similar to those of blood serum run under comparable conditions. E1-Naggar and Abdel-Raouf (1977) reported similar results for camel seminal plasma analyzed by paper electrophoresis but they indicated the presence of eleven protein bands. It is interesting to note the similarity in the overall pattern of the seminal plasma proteins of different domestic animals (Bennett, 1965; Garner and Ehlers, 1971; E1-Naggar and Abdel-Raouf, 1977) including the camel, a species devoid of seminal vesicles (All et al., 1978) which usually make a major contribution to the seminal plasma proteins composition (Dostal and Veslsky, 1972 ). It should be mentioned however, that Bennett (1965) comparing the seminal plasma proteins of bull, ram, boar and rabbit showed some species variation including number of components, their mobility, glycoprotein and lipoprotein content. Conflicting reports have accumulated concerning the similarity between seminal plasma proteins and blood serum proteins. Some authors, based on electrophoretic fractionation, claimed that seminal plasma proteins corresponded to blood serum glob÷ 3
C d
Fig. 1. Upper curve: isotachophoretic pattern of seminal plasma proteins. 1,2, prealbumin; 3, albumin; 4, ce-globulin; 5,6, fl-globulins; 7,8, ~-globulins: Lower curve: isotachophoretic pattern of blood serum proteins, a,b, prealbumin; c, albumin; d, a-globulin; e,f, fl-globulins; g,h, ~-globulins.
229 -
4-
4
11
7 56
8~1
16 12
17
1415 18
Fig. 2. Densitometer scanning tracings of electrofocusingpattern of seminal plasma proteins (upper curve ) and of blood serum proteins (lower curve). Note the presence of 18 protein components. The three" major bands have average areas of 19.2, 23.3 and 14.9; others are minor bands, the smallest being 0.3 in area.
ulins and albumin (Gray and Huggins, 1942; Ross et al., 1943; Ross, 1946); others indicated some quantitative differences. They observed that major components of seminal plasma exhibit mobilities similar to those of alpha globulins of blood serum but bands with serum albumin mobilities were either absent or very low in seminal plasma (Larson and Salisbury, 1954; Vesselinovitch, 1959). Larson et al. (1954), studying seminal plasma proteins immunologically, concluded that they are definite chemical entities not similar to any major protein components of blood or milk serum. Differences within the species have also been controversial. Vesselinovitch (1959), using paper and starch gel electrophoresis in the study of bovine seminal plasma, observed fluctuation in plasma proteins even in the same animal and concluded that these proteins are contributed by several glands not necessarily at the same secretory cycle. He also found no correlation between the protein pattern and age or fertility of bulls. Similar findings on human seminal plasma proteins were reported by Gray and Huggins (1942) and Ross (1946). Leslie and Quinlivan (1969), however, studying human seminal plasma by disc electrophoresis, observed that plasma from semen with low sperm counts showed an increase in the prealbumin and beta-1 globulin and a decrease in albumin, and concluded that this may prove of value in the study of infertile men. In the present study, although isotach-
230
ophoresis revealed close resemblance between seminal plasma proteins and blood serum proteins, electrofocusing, a technique with superior resolving power, showed both qualitative and quantitative differences in the protein patterns. Whether these differences are genuine or not awaits further biochemical characterization of the individual proteins. ACKNOWLEDGEMENTS
This work was supported by a grant AR-2-10, Saudi Arabian National Centre for Science and Technology (SANCST). We thank A.H. Saad and K. E1-Jouf for technical help.
REFERENCES Abdel-Raouf, M. and E1-Naggar, M., 1978. Studies on reproduction in camels (Camelus dromedarius). VI. Properties and constituents of ejaculated semen. VIIIth International Congress on Animal Reproduction and Artifical Insemination, Cracow. Ali, H.A., Tingari, M.D. and Moniem, K.A., 1978. On the morphology of the accessory male glands and histochemistry of the ampulla ductus deferentis of the camel (Camelus dromedarius). J. Anat., 125: 277-292. Bennett, J.P., 1965. Quantitative comparisons of the proteins of the seminal plasmas of bull, ram, rabbit and boar by agar gel electrophoresis. J. Reprod. Fertil., 9: 217-231. Delmotte, P., 1977. Analysis of complex p r o ~ i n mixtures by capillary isotachophoresis. Some qualitative and quantitative aspects. Science Tools, 24 (3). Dostal, J. and Veselsky, L., 1972. Proteins in the seminal plasma and accessory sexual gland fluids of the boar. J. Reprod. Fertil., 30: 255-267. EI-Naggar, M. and Abdel-Raouf, M., 1977. Studies on reproduction in camels (Camelus dromedarius). VIII, The electrophoretic pattern and the amino acid content of the seminal plasma protein. Indian Vet. J., 54: 239-243. Folch, J., Lees, M. and Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total Iipids from animal tissues. J. Biol. Chem., 266: 497-509. Garner, D.L. and Ehlers, M.H., 1971. Effects of storage at 5 ° C on the disc electrophoretic patterns of ovine and bovine seminal proteins. J. Reprod. Fertil., 27: 43-52. GornaU, A:G., Bardawill, C.J. and David, M.M., 1949. Determination on serum proteins by means of the biuret reagent. J. Biol. Chem., 177: 751-766. Gray, S.J. and Huggins, C., 1942. Electrophoretic analysis of human semen. Proc. Soc. Exp. Biol. Med., 50: 351-353. Larson, B.L. and Salisbury, G.W., 1954. The proteins of bovine seminal plasma. I. Preliminary and electrophoretic studies. J. Biol. Chem., 206: 741-749. Larson, B.L., Gray, R.S. and Salisbury, G.W., 1954. The proteins of bovine seminal plasma II. Ultracentrifugal and immunological studies and comparison with blood and milk serum. J. Biol. Chem., 211: 43-52. Leslie, W. and Quinlivan, G., 1969. Quantitative examinations of human seminal plasma proteins. Physiologist, 12: 334. Lindner, H.R. and Mann, T., 1960. Relationship between the content of androgenic steroids in the testes and the secretory activity of the seminal vesicles in the bull. II. Determination of fructose and citric acid in the seminal vesicles. J. Endocrinol., 21: 341-360. Mann, T., 1964. The Biochemistry of Semen and of the Male Reproductive Tract. Methuen, London, 493 pp.
231 Mann, T., 1975. Biochemistry of semen. In: D.W. Hamilton and R.O. Greep (Editors), Handbook of Physiology, Vol. V, Section 7. American Physiological Society, Washington, DC, pp. 461-471. 0verturf, 0. and Dyer, R.L., 1969. Experiments in biochemistry of animal lipids. In: Experiments in Physiology and Biochemistry, Vol. 2. Academic Press, London, pp. 89-163. Ross, V., 1946. Precipitin-reactions of human seminal plasma. J. Immunol. Vir. Res. Exp. Chemother., 52: 87-96. Ross, V., Miller, E.G., Moore, D.L. and Sikorski, H., 1943, Electrophoretic patterns of seminal plasma from some abnormal human semen. Proc. Soc. Exp. Biol. Med., 54: 179-181. Tingari, M.D., Ramos, A.S., Gaili, E.S.E., Rahma, B.A. and Saad, A.H., 1984. Morphology of the testis of the one-humped camel in relation to reproductive activity. J. Anat., 139: 133-143. Tingari, M.D., El-Manna, M.M., Rahim, A.T.A., Ahmed, A.K. and Hamad, M.H., 1986. Studies on camel semen. I. Electroejaculation and some aspects of semen characteristics. Anim. Reprod. Sci., 12: 213-222. Vasselinovitch, S.D., 1959. Electrophoresis of bovine semen. Part III. Characterization of the seminal plasma proteins. Can. J. Comp. Med., 23: 10-20.
223
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
S t u d i e s on C a m e l S e m e n II. B i o c h e m i c a l C h a r a c t e r i s t i c s M.M. EL-MANNA 1, M.D. TINGARIe*, and A.K. AHMED 2
1Department of Physiology and Biochemistry and 2Department of Anatomy, College of Veterinary Medicine and Animal Resources, King Faisal University, P.O. Box 1757, Al-Ahsa 31982 (Saudi Arabia) *Present address: Department of Anatomy, Faculty of Veterinary Science, University of Khartoum, P.O. Box 32, Khartoum North (Sudan) (Accepted 24 April 1986 )
ABSTRACT El-Manna, M.M., Tingari, M.D. and Ahmed, A.K., 1986. Studies on camel semen. II. Biochemical characteristics. Anim. Reprod. Sci., 12: 223-231. The mean concentrations of fructose, citric acid, total proteins and lipids in the seminal plasma of camel were 23.5 ± 2.5, 9.8 ± 2.9, 775 __15 and 87 _+32 mg/ml respectively. The corpus prostatae seemed to be the principal source of fructose and citric acid, smaller amounts are contributed by the bulbourethral glands, ampulla ductus deferentis and pars disseminata of the prostate. Electrophoretic fractionation of seminal plasma proteins by capillary isotachophoresis revealed eight protein bands similar to those of blood serum. Electrofocusing in polyacrylamide gels on the other hand showed the presence of eighteen fractions with minor variations from fractions obtained from blood serum.
INTRODUCTION
Semen has been studied extensively in several mammalian and avian species (Mann, 1964, 1975). Its chemical make up varies not only from one species to another, but also among individuals belonging to the same species. The role of the secretions contributed to the seminal plasma by the various male accessory sex glands of reproduction has long been known, and so has the specialized chemical nature of these secretions. Among the most notable substances secreted in high concentrations are fructose and citric acid. The information available on semen composition in the camel is primarily limited to the work of Abdel-Raouf and E1-Naggar (1978) and E1-Naggar and Abdel-Raouf (1977). Our earlier report dealt with the biophysical characters of semen and sperm morphology (Tingari et al., 1986). The present investigation was designed to study the biochemical character0378-4320/86/$03.50
© 1986 Elsevier Science Publishers B.V.
224 istics of camel seminal plasma during the rutting season ( Tingari et al., 1984). An analysis of the accessory sex glands was also undertaken with the purpose of identifying the sources contributing to the secretion of fructose and citric acid. MATERIALSAND METHODS Materials
Four camels aged 6-9 years were used for weekly collections of semen and serum during the rutting season ( see Tingari et al., 1984). Semen was obtained by the method of electroejaculation as outlined elsewhere ( Tingari et al., 1986). The animals were apparently healthy and were kept in the University Farm in fenced yards with shaded areas. They had no contact with females a n d were fed on barley and straw and offered water ad libitum. Seminal plasma was obtained by centrifugation of freshly ejaculated semen at 4000 rpm for 15 min. Tissue samples from prostate, bulbourethral glands, urethra and ampulla ductus deferentis of adult camels (Ali et. al., 1978) were collected from AIHassa municipal slaughterhouse, They were dissected and transferred immediately to liquid nitrogen. Whenever possible, and depending on the tissue size available, the selectedgland was utilized for both fructose and citric acid determination; otherwise it was wholly devoted to one test or the other. A m i n i m u m of duplicate subsamples was analyzed from the selected gland of each animal. The gels used in electrofocusing ( P A G plates pH 3.6-9.5) and ampholine carrier ampholytes were obtained from LKB (LKB Produckter AB); other chemicals were purchased from BDH. Fructose determination
Weighed pieces of glands were homogenized in cold isotonic potassium chloride and then deproteinized using 2% zinc sulphate (7H20) and 0.1 N sodium hydroxide. T h e supernatant fluid was obtained after centrifugation at 4000 rpm for 15 min. Proteins were removed from t h e seminal plasma by zinc sulphate and sodium hydroxide precipitation. Fructose was measured in the supernatant fluid from t h e glands and seminal plasma by the resorcinol reaction (Lindner and Mann, 1960). Citric acid determination
Protein precipitation from the seminal plasma and supernatant fluid from the glands was effected utilizing 20% trichloroacetic acid. Citric acid was estimated by oxidation to the pentabromoacetone and subsequent treatment with sodium sulphide according to Lindner and M a n n (1960).
225
Protein analysis Total protein in seminal plasma was determined by the biuret method using bovine serum albumin as a standard (Gornall et al., 1949).
Electrophoretic separations Two methods of electrophoretic fractionation were used; capillary isotachophoresis and polyacrylamide gel electrofocusing. An LKB Tachophor 2127 was used for isotachophoresis according to the procedures specified for that instrument. A capillary tube 230 mm in length was used at a temperature of 10 oC. Protein absorption was monitored using a 280 nm U.V. detector; the current during detection was 40/~A and the analysis time 30 min. A spacer solution composed of amino acids and ampholine carrier ampholytes was added to the samples in order to resolve the different protein bands. The composition of the leading and terminating electrolytes, sample and spacer solution were similar to those reported by Delmotte (1977). The composition was as follows: leading electrolyte, 5 m M 2- (N-morpholino) -ethane sulphonic acid; 10 m M 2-amino-2-methyl-1, 3-propanediol; 0.1% Triton X-100, pH 9.0 - - terminating electrolyte, 11.4 mME-aminocaproic acid; 10 m M 2-amino-2methyl-l, 3 propanediol adjusted with barium hydroxide to pH 10.8 spacer solution, 10 ml solutions contained the following: 1.6 mg glycine, 1.6 mg valine, 1.44 mg fl-alanine, 0.18 ml 40% ampholine carrier ampholyte pH 7-9, 0.095 ml 40% ampholine carrier ampholyte pH 8-9.5, 0.3 ml 20% ampholine carrier ampholyte pH 9-11. Seminal plasma samples contained 6 ttl seminal plasma and 2/ll spacer solution, whereas the serum samples were composed of 2/A each of serum and spacer solution. LKB Multiphor 2117 was used for polyacrylamide gel electrofocusing according to the procedures recommended by the instruction manual. Samples of seminal plasma and serum were applied directly on the gel surface - - 3/11 samples of seminal plasma and 1 ,ul samples of serum using a micropipette at a distance of 10 mm from the cathode. The power supply (LKB 2197) was set at 25 W, 40 mA and 1300 V. The running time was 1.5 h during which the temperature was maintained at 10 °C by circulating water through the plate using an LKB Multitemp 2209 circulator. The gel used was an LKB PAG plate pH 3.5-9.5, with Coomassie Brilliant Blue R 250 as stain and ethanol : acetic acid aqueous solution for destaining. A Camag electrophoresis scanner fitted with an integrator was utilized for evaluation of peak areas.
Lipid analysis Total lipids in seminal plasma samples were extracted using chloroform :methanol mixture (2 : 1) according to Folch et al. (1957) as modified
1 2 3 4,
9.8~-0.7 21.6 _+1.4 34.0 ___2.1 41.2 _+2.5 10.7"-_0.8 24.2 _+3.2 27.0 _+5.6 22.3_ 1.4 20.3 +- 2.1
8
8
8 8 8 5 5 5 5
Mean ~- s.d.
17.0- 96.3 23.8-131.2 5.6- 25.5 17.0- 32.0 17.0- 40.0 15.0- 31.0 14.0- 25.0
8.8- 50.9
5.0- 23.8
Range
5 4 3 6 8 6 7
4
5
No. of samples
No. of samples
Concentration (mg/100 ml or 100 g tissue)
Citric acid
Fructose
36.0___ 3.1 24.9 +- 1.9 7.2___0.9 7.2 _+1.2 14.1 z 1.6 10.8__. 1.4 7.0 ÷ 0.9
35.3 z 2.2
15.8_+ 1.8
Mean +- s.d.
21.5-44.5 4.5-50.1 0-18.5 5.0-12.0 10.0-30.0 5.0-13.0 5.0-10.0
19.8-63.5
8.5-18.5
Range
Concentration (mg/100 ml or 100 g tissue )
Nos. 1-4 refer to camels of corresponding numbers. Whenever possible, both fructose and citric acid in the seminal plasma were assayed in the same sample.
Prostate gland ( pars disseminata) Prostate gland (corpus prostatae) Bulbourethral gland Ampulla Urethra Seminal plasma No. Seminal plasma No. Seminal plasma No. Seminal plasma No.
Tissue
Fructose and citric acid contents in seminal plasma and accessory glands
TABLE 1
t'~ bO
227 by Overturf and Dyer (1969). One millilitre samples of plasma were extracted with 20-fold chloroform : methanol mixture containing 0.2 volume of 0.05 M sodium chloride. An aliquot of the lipid extract dissolved in chloroform was utilized in qualitative TLC analyses using 20 × 20 cm precoated Silica Gel G plates. The plates were activated by heating at l l 0 ° C for 1 h. The plates containing spotted samples together with appropriate standards were developed using the solvent system petroleum e t h e r : d i e t h y l ether: acetic acid (80 : 20 : 1 ). The separated lipids were visualized under U.V. light at 366 nm after being sprayed with 2,7-dichlorofluoroscein (0.20% in absolute ethanol). RESULTS The results obtained for fructose, citric acid, total proteins and lipids in the seminal plasma and accessory glands are presented in Tables 1 and 2. The mean concentrations of the different constituents in the seminal plasma in mg/100 ml were as follows: fructose, 23.5 __2.5; citric acid 9.8 __2.9; total proteins, 775 + 15 and total lipids, 87 _+32. Correlation analyses showed that fructose and citric acid contents are closely related both within an individual animal and between animals. Correlation coefficients for the four animals were as follows: 0.73, 0.99, 0.95 and 0.98; that between the different animals was 0.85. Isotachophoretic analysis of seminal plasma proteins revealed a pattern similar to that of blood serum consisting mainly of eight peaks (Fig. 1 ). The average percentages of the protein bands as measured by the area under individual peaks are 2.34, 5.22, 21.85, 12.39, 16.57, 18.79, 9.92 and 12.92. Electrofocusing on the other hand showed about eighteen peaks qualitatively similar to those obtained from serum (Fig. 2 ). The percentage areas under these peaks as evaluated by scanning densitometry are given in Fig. 2. Qualitative analysis of lipids by TLC in non-polar solvents revealed four spotscorresponding to sterols and their esters, acylglycerols and phospholipids. DISCUSSION Semen analysis has shown that the concentrations of fructose, citric acid, total proteins and lipids in the seminal plasma were lower than those in semen collected by artificial vagina (E1-Naggar and Abdel-Raouf, 1977). Analysis of the accessory glands revealed that fructose is produced by the ampulla, bulbourethral glands together with the corpus prostatae, whereas citric acid is derived mainly from the corpus prostatae and bulbo-urethral glands with a minor contribution from the ampulla and pars disseminata of the prostate gland. On account of its comparatively much larger size than all others, the corpus prostatae appears to be the principal source of both fructose and citric acid. The positive correlation shown for these two components lends further support to this assumption.
228 TABLE 2 Total protein and lipid content in seminal plasma Source of seminal plasma
Total protein concentration (mg/100 ml) Mean___ s.d.
Total lipid concentration (rag/100 ml) Mean Jr s.d.
1 2 3 4
790 _+10 780_+ 6 780_+ 6 750_+ 8
98_+ 12 68_+ 3 135 q- 10 48___ 5
Samples of seminal plasma from a given animal remaining after fructose and citric acid determinations, were pooled together and utilized for protein and lipid contents in that animal. A minimum of duplicate subsamples was analyzed.
Electrophoretic analysis of plasma proteins as judged by isotachophoresis showed the presence of eight bands qualitatively similar to those of blood serum run under comparable conditions. E1-Naggar and Abdel-Raouf (1977) reported similar results for camel seminal plasma analyzed by paper electrophoresis but they indicated the presence of eleven protein bands. It is interesting to note the similarity in the overall pattern of the seminal plasma proteins of different domestic animals (Bennett, 1965; Garner and Ehlers, 1971; E1-Naggar and Abdel-Raouf, 1977) including the camel, a species devoid of seminal vesicles (All et al., 1978) which usually make a major contribution to the seminal plasma proteins composition (Dostal and Veslsky, 1972 ). It should be mentioned however, that Bennett (1965) comparing the seminal plasma proteins of bull, ram, boar and rabbit showed some species variation including number of components, their mobility, glycoprotein and lipoprotein content. Conflicting reports have accumulated concerning the similarity between seminal plasma proteins and blood serum proteins. Some authors, based on electrophoretic fractionation, claimed that seminal plasma proteins corresponded to blood serum glob÷ 3
C d
Fig. 1. Upper curve: isotachophoretic pattern of seminal plasma proteins. 1,2, prealbumin; 3, albumin; 4, ce-globulin; 5,6, fl-globulins; 7,8, ~-globulins: Lower curve: isotachophoretic pattern of blood serum proteins, a,b, prealbumin; c, albumin; d, a-globulin; e,f, fl-globulins; g,h, ~-globulins.
229 -
4-
4
11
7 56
8~1
16 12
17
1415 18
Fig. 2. Densitometer scanning tracings of electrofocusingpattern of seminal plasma proteins (upper curve ) and of blood serum proteins (lower curve). Note the presence of 18 protein components. The three" major bands have average areas of 19.2, 23.3 and 14.9; others are minor bands, the smallest being 0.3 in area.
ulins and albumin (Gray and Huggins, 1942; Ross et al., 1943; Ross, 1946); others indicated some quantitative differences. They observed that major components of seminal plasma exhibit mobilities similar to those of alpha globulins of blood serum but bands with serum albumin mobilities were either absent or very low in seminal plasma (Larson and Salisbury, 1954; Vesselinovitch, 1959). Larson et al. (1954), studying seminal plasma proteins immunologically, concluded that they are definite chemical entities not similar to any major protein components of blood or milk serum. Differences within the species have also been controversial. Vesselinovitch (1959), using paper and starch gel electrophoresis in the study of bovine seminal plasma, observed fluctuation in plasma proteins even in the same animal and concluded that these proteins are contributed by several glands not necessarily at the same secretory cycle. He also found no correlation between the protein pattern and age or fertility of bulls. Similar findings on human seminal plasma proteins were reported by Gray and Huggins (1942) and Ross (1946). Leslie and Quinlivan (1969), however, studying human seminal plasma by disc electrophoresis, observed that plasma from semen with low sperm counts showed an increase in the prealbumin and beta-1 globulin and a decrease in albumin, and concluded that this may prove of value in the study of infertile men. In the present study, although isotach-
230
ophoresis revealed close resemblance between seminal plasma proteins and blood serum proteins, electrofocusing, a technique with superior resolving power, showed both qualitative and quantitative differences in the protein patterns. Whether these differences are genuine or not awaits further biochemical characterization of the individual proteins. ACKNOWLEDGEMENTS
This work was supported by a grant AR-2-10, Saudi Arabian National Centre for Science and Technology (SANCST). We thank A.H. Saad and K. E1-Jouf for technical help.
REFERENCES Abdel-Raouf, M. and E1-Naggar, M., 1978. Studies on reproduction in camels (Camelus dromedarius). VI. Properties and constituents of ejaculated semen. VIIIth International Congress on Animal Reproduction and Artifical Insemination, Cracow. Ali, H.A., Tingari, M.D. and Moniem, K.A., 1978. On the morphology of the accessory male glands and histochemistry of the ampulla ductus deferentis of the camel (Camelus dromedarius). J. Anat., 125: 277-292. Bennett, J.P., 1965. Quantitative comparisons of the proteins of the seminal plasmas of bull, ram, rabbit and boar by agar gel electrophoresis. J. Reprod. Fertil., 9: 217-231. Delmotte, P., 1977. Analysis of complex p r o ~ i n mixtures by capillary isotachophoresis. Some qualitative and quantitative aspects. Science Tools, 24 (3). Dostal, J. and Veselsky, L., 1972. Proteins in the seminal plasma and accessory sexual gland fluids of the boar. J. Reprod. Fertil., 30: 255-267. EI-Naggar, M. and Abdel-Raouf, M., 1977. Studies on reproduction in camels (Camelus dromedarius). VIII, The electrophoretic pattern and the amino acid content of the seminal plasma protein. Indian Vet. J., 54: 239-243. Folch, J., Lees, M. and Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total Iipids from animal tissues. J. Biol. Chem., 266: 497-509. Garner, D.L. and Ehlers, M.H., 1971. Effects of storage at 5 ° C on the disc electrophoretic patterns of ovine and bovine seminal proteins. J. Reprod. Fertil., 27: 43-52. GornaU, A:G., Bardawill, C.J. and David, M.M., 1949. Determination on serum proteins by means of the biuret reagent. J. Biol. Chem., 177: 751-766. Gray, S.J. and Huggins, C., 1942. Electrophoretic analysis of human semen. Proc. Soc. Exp. Biol. Med., 50: 351-353. Larson, B.L. and Salisbury, G.W., 1954. The proteins of bovine seminal plasma. I. Preliminary and electrophoretic studies. J. Biol. Chem., 206: 741-749. Larson, B.L., Gray, R.S. and Salisbury, G.W., 1954. The proteins of bovine seminal plasma II. Ultracentrifugal and immunological studies and comparison with blood and milk serum. J. Biol. Chem., 211: 43-52. Leslie, W. and Quinlivan, G., 1969. Quantitative examinations of human seminal plasma proteins. Physiologist, 12: 334. Lindner, H.R. and Mann, T., 1960. Relationship between the content of androgenic steroids in the testes and the secretory activity of the seminal vesicles in the bull. II. Determination of fructose and citric acid in the seminal vesicles. J. Endocrinol., 21: 341-360. Mann, T., 1964. The Biochemistry of Semen and of the Male Reproductive Tract. Methuen, London, 493 pp.
231 Mann, T., 1975. Biochemistry of semen. In: D.W. Hamilton and R.O. Greep (Editors), Handbook of Physiology, Vol. V, Section 7. American Physiological Society, Washington, DC, pp. 461-471. 0verturf, 0. and Dyer, R.L., 1969. Experiments in biochemistry of animal lipids. In: Experiments in Physiology and Biochemistry, Vol. 2. Academic Press, London, pp. 89-163. Ross, V., 1946. Precipitin-reactions of human seminal plasma. J. Immunol. Vir. Res. Exp. Chemother., 52: 87-96. Ross, V., Miller, E.G., Moore, D.L. and Sikorski, H., 1943, Electrophoretic patterns of seminal plasma from some abnormal human semen. Proc. Soc. Exp. Biol. Med., 54: 179-181. Tingari, M.D., Ramos, A.S., Gaili, E.S.E., Rahma, B.A. and Saad, A.H., 1984. Morphology of the testis of the one-humped camel in relation to reproductive activity. J. Anat., 139: 133-143. Tingari, M.D., El-Manna, M.M., Rahim, A.T.A., Ahmed, A.K. and Hamad, M.H., 1986. Studies on camel semen. I. Electroejaculation and some aspects of semen characteristics. Anim. Reprod. Sci., 12: 213-222. Vasselinovitch, S.D., 1959. Electrophoresis of bovine semen. Part III. Characterization of the seminal plasma proteins. Can. J. Comp. Med., 23: 10-20.