Seminal profiles of lysosomal enzymes in normal and infertile men.SP Dandekar, P Harikumar
Dept. of Biochemistry, Seth GS Medical College, Parel, Mumbai, India., India
Human seminal plasma is known to possess considerable proteolytic activity, much of which is associated with lysosomes. The activities of lysosomal hydrolases like alkaline proteinase, cathepsin-D, aryl-sulfatase and N-acetyl-beta-D-glucosaminidase in seminal plasma from randomly chosen infertile and vasectomised men have been compared. These enzymes have been implicated in the coagulation and liquefaction processes. The role of fructose and proteins in these processes has also been studied. The results indicate that cathepsin-D and aryl-sulfatase activity in infertile men were significantly lower than normo-spermic subjects. N-acetyl-beta-D-glucosaminidase was lowest in azoospermia suggesting that it could be used as a biochemical marker for azoospermia. Conversely, alkaline proteinase showed increased levels in all the infertile cases.
Keywords: Fructose, metabolism,Human, Infertility, Male, enzymology,pathology,Lysosomes, enzymology,Male, Proteins, metabolism,Reference Values, Semen, enzymology,Sperm Count,
Seminal plasma possesses considerable proteolytic activity which is implicated in the degradation of seminal proteins to proteoses and free amino acids.,, Several proteolytic agents secreted by the male accessory glands also participate in the two important processes which spontaneously occur in the ejaculated semen viz. semen coagulation and liquefaction,,. The enzymatic equipment of spermatozoa is similar to that of seminal plasma. A close relationship exists between the enzymatic equipment of spermatozoa and their fertilizing capacity.
Lysosomal hydrolases are considered to be important constituents of human seminal fluids. An extraordinary similarity between the enzymatic activities of the spermatozoa and those of the seminal plasma suggests that these enzymes need not all be of acrosomal origin. So much so, that the semen from azoospermics also shows lysosomal hydrolase activity. Considering the fact that the acrosome is a specialised lysosome, some hydrolases are also found to be present on the acrosome. A number of the hydrolases originate from accessory glands. Welker et al, have attempted to study the acrosomal proteinase activity of human spermatozoa, using gelatin. The aim of the present study is to investigate the interrelationship in infertile men possessed with the activities of lysosomal hydrolases such as cathepsin-D, aryl sulfatase, alkaline proteinase and N-acetyl-?-D-glucosaminidase (NAG) in male infertility.
A total of 87 normal, pathologic and vasectomised semen samples were studied.
Collection and preparation of semen : Ejaculates were obtained by masturbation, after a three days period of sexual abstinence; samples were collected in plastic jars and after liquefaction, analysis was performed according to WHO methods and standards.
The ejaculates comprising the normospermic group had a sperm count of >30 million per ml, >50% motile sperm with good forward motility. The oligospermic ejaculates had <30 million sperm per ml. The oligoaesthenozoospermic specimens comprised of low sperm counts (<30 million/ml), sperm motility decreased by more than 40% and had more than 20% morphological abnormalities.
Out of the 87 patients studied, 28 were normospermics; 11 azoospermics; 24 oligospermics and 14 oligoaesthenozoospermics. 10 semen samples from vasectomised men were also obtained. After liquefaction, the ejaculates were centrifuged at 800g for 10 mins to separate the spermatozoa from seminal plasma. The button of the spermatozoa was resuspended in Kreb’s Ringer solution, recentrifuged, and then resuspended in hypotonic buffer. The seminal plasma was recentrifuged at 3000g for 10 mins to eliminate any residual cells and debris. The samples were frozen and thawed six times using dry ice and acetone and centrifuged at 800g for 10 mins. The supernatant was frozen at -20°C until used as source of enzyme.
Activities of cathepsin D (EC.18.104.22.168), N-acetyl-?-D-glucosaminidase (EC.22.214.171.124) & arylsulfatase (EC.126.96.36.199), were determined using haemoglobin, p-nitrophenyl-N-acetyl-?-D-Glucosaminide & p-nitrocatechol sulphate respectively as substrates. Alkaline proteinase was assayed according to the method of Thakore & Harikumar at pH 8.5 using casein as substrate. Protein was measured by the method of Miller with bovine serum albumin as the standard.
Protein precipitation from seminal plasma before fructose estimation was done by using cadmium sulphate, according to Fujita & Iwatake. The fructose levels were determined using indol reagent.
Role of fructose and protein in coagulation and liquefaction :
Fructose in semen reflects the secretory function of the seminal vesicles and is used as a biochemical marker of seminal vesicles function. Since it is implicated in the coagulation and liquefaction process, fructose determination is also useful in the rare cases of ejaculatory duct obstruction. The results presented in [Table:I], shows that the levels of proteins in both oligospermic and azoospermic men were significantly (p<0.01 – 0.001) decreased.
Activities of lysosomal hydrolases :
The activities of cathepsin-D, aryl-sulfatase, N-acetyl-?-D-glucosaminidase and alkaline proteinase found in different groups of the ejaculate are presented in [Table:II].
Activity of cathepsin-D was noticed in all the five categories of ejaculates. Statistically significant (p<0.01-0.001) differences in activities were found among the vasectomised and azoospermic men. No significant differences were noted in the activities of NAG oligospermic samples as also in the samples with poor motility. However, significant alteration were seen once again in the vasectomised and azoospermic subjects. Significantly low values of aryl-sulfatase were noticed in all the four different categories as compared to the normospermic patients while alkaline proteinase levels were increased significantly in all the cases of infertility.
Male infertility is generally diagnosed in terms of the concentration of motility and morphology of spermatozoa. Often, these parameters do not truthfully reflect the fertilising capacity of the samples. For instance, some samples exhibiting normal spermiograms are often seen to be infertile. Therefore, there have been increasing efforts to device suitable biochemical parameters which could serve as effective markers for infertility. Parameters such as the levels of fructose, zinc; citric acid and marker enzymes like acid phosphatase; neutral ?-glucosidase.
Mann was the first to show the important role played by fructose, particularly as a marker of vesicular secretion. Montagnon et al have postulated that fructose is secreted in seminal vesicles along with specific proteins. The interrelationships between these two types of molecules is stronger than the simple Van der Waal’s forces. It is suggested that these complexes play a role in the phenomenon of coagulation by the mechanism of retention of fructose.
Our studies, presented in [Table - 1] showed a distinct decrease in the levels of proteins (p<0.01) in both oligospermic and azoospermic men, when compared with normospermic subjects. The decreased levels of proteins might be due to faulty fructose-protein complex formations. This might also explain the elevated levels of fructose apparently due to dissociation of fructose from proteins.
Lizana et al have shown that reactions involving serine proteinases may not be a part of the liquefaction process. In this context it was felt it would be interesting to evaluate the occurrence of various proteolytic enzymes in seminal plasma. Lysosomes form the major site of intracellular protein degradation. Guerin et al have attempted to correlate the activities of various hydrolytic enzymes with the fertility status of semen. Attempts have also been made by Garcia Diez et al to correlate enzymes and hormones in the differential diagnosis of azoospermia. They found that seminal determination of ?-glucosidase helps to differentiate between secretory azoospermia from the excretory type. The role of inhibin in spermatids has also been postulated. Allison & Hartree have analysed the role of lysosomal enzymes in the acrosome and their possible role in the fertilization from ram spermatozoa.
The results of the profiles of major lysosomal enzymes in normospermia, azoospermia and oligospermia are presented in [Table:II]. All lysosomal enzymes except alkaline proteinase showed a significant decrease in semen with less sperm count. However, individual enzymes showed marked differences in their response. Arylsulfatase, a mucopolysaccharidase was the most affected. This enzyme is involved in the stability and permeability of plasma membrane of spermatozoa. It is known to play a significant role of arylsulfatase in sperm capacitation, acrosome reaction and/or gamete interactions; the levels of arylsulfatase were determined in our study. All the four abnormal categories showed significantly low levels of the enzymes (47-79%), indicating that arylsulfatase could well serve as a biochemical marker for male infertility.
Many hydrolases have been detected in human sperm and seminal plasma,,. Among the glycosidases, NAG was found to be present in high concentrations, it is postulated that NAG might play a vital role in the penetration of sperms and interactions with the egg. It could also be involved in other processes such as liquefaction and sperm mucus contact.
It was found that human spermatozoa contain membrane-associated NAG-activity with an isoform distribution similar to that found in cauda epididymal fluid. The enzyme activity in this fluid was two-fold greater than in seminal plasma. This suggested that NAG activity becomes associated with human spermatozoa during epididymal transit. It is interesting to note that in our study, N-acetyl-?-D-glucosaminidase, which is implicated in cleaving glycoproteins, is markedly low (50%) in azoospermic men as well as in vasectomised men while it was nearly unaffected in oligospermic and asthenozoospermic samples. Thus, along with fructose, NAG could serve as a biochemical marker for azoospermia.
Cathepsin D, the major endopeptidase found in lysosomes has been implicated in a spectrum of physiopathological conditions in living cells. Although other aspartic proteinases have been studied extensively, cathepsin D has been isolated from rabbit testis by Srivastava and Ninjoor. It has been implicated in the activation of proacrosin. Disturbances of proteolytic potential of human semen may be the cause of sub-fertility or infertility. Though high concentration of aspartic proteinases like pepsinogen and gastricsin have been identified,,, not much work has been done on cathepsin-D. Our results indicate suppression of its activity by 20-40% in oligospermic and azoospermic men when compared with normospermic samples. The samples from vasectomised men also showed a significant decrease of these enzymes. Whether it reflects a decrease in de novo synthesis of lysosomal enzymes or the presence of endogenous inhibitors needs to be evaluated with purified systems.
Unlike cathepsin-D, arylsulfatase, and NAG alkaline proteinases showed a distinctly different pattern of response to sperm density. Alkaline proteinase increased 1.2 to 3 fold in azoospermic, oligospermic and asthenozoospermic and vasectomised samples as compared to normal samples. The different response of alkaline proteinase from other lysosomal enzymes may suggest a non-lysosomal location of this enzyme. However, the levels of alkaline proteinase and arylsulphatase seem to vary with the status of the semen.
The induction of acrosome reaction is found to be implicated by arylsulphatase, the level of which were found to be low in all abnormal categories in all semen samples. Whereas, reverse situation exists in the levels of alkaline proteinase, therefore, the ratio of the arylsulphatase / alkaline proteinase levels was calculated. The ratio was found to be significantly lower in abnormal groups (azoo, oligo, astheno and vasectomised) varying from 0.81 to 1.21. These findings suggest that calculation of the ratio will be better marker for infertility than only enzyme’s levels.
In conclusion, it is seen that fructose and protein may be involved in the phenomenon of coagulation and that the abnormal sample may be forming faulty fructose-protein complexes.
Also, the lysosomal enzymes and alkaline proteinase can be judiciously used as clinical markers for infertility. The precise role of these enzymes in the series of events leading to coagulation, liquefaction etc need to be evaluated using purified enzymes.
[Table - 1], [Table - 2]