Biological and immunological roles of proteins in the sperm of domestic animals (review)

Animal Reproduction Science, 8 (1985) 1--40

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Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

BIOLOGICAL AND IMMUNOLOGICAL ROLES OF PROTEINS IN THE SPERM OF DOMESTIC ANIMALS (REVIEW)

JOSEF MATOU~EK

Institute of Animal Physiology and Genetics, Czechoslovak Academy of Sciences, Lib~chov (Czechoslovakia) (Accepted 25 April 1984)

ABSTRACT

Matou~fek, J., 1985. Biological and immunological roles of proteins in the sperm of domestic animals (review). Anita. Reprod. Sci., 8: 1--40. This review aims at giving the up-to-date results in studies of proteins in spermatozoa and seminal fluids, mainly in domestic animals. Attention was given to protein and antigenic substances appearing during spermatogenesis and spermiogenesis in the testes. The problem of proteosynthesis of haploid testicular cells was also noted. Peptide hormones in the testes have an important role for the function of the testicle and the synthesis of sperm and testicular fluid proteins. Epididymal proteins are very important for the fertility and motility of spermatozoa in all mammalian species, but only some of them have been isolated and chemically and functionally characterized. It looks as though the specific accessory sex gland proteins are not necessary for actual fertilizing capacity, but this is probably not the case. Many proteins have been isolated from seminal plasma and studied. Some of them are important because they enable spermatozoa to survive in the female genital tract. The physiological function of a series of other proteins/ enzymes is still unknown, however, although their significance is demonstrated by studies showing that many of them are bound to the sperm cells. The sperm cell proteins are of both primary and secondary origin. From the primitive spermatogonium to u n i o n of the spermatozoon with the egg, the male germ cell undergoes continual metabolic, protein synthetic, protein destructive and, of course, morphological changes. The primary spermatozoa-specific proteins are mainly formed during meiotic processes and during spermiogenesis in the Sertoli cells. There are experimental and clinical results which show that specific sperm cell and seminal plasma proteins can induce immune processes in males and in females.

INTRODUCTION

The isolation and chemical characterization of the spermatozoal and seminal plasma proteins of domestic animals is mainly the o u t c o m e of recent scientific research. Twenty years ago most work was carried o u t with only partially purified and -- from the immunological point of view -complex proteins, and hence with complex antigens. Apart from their histochemical aspects, such studies were naturally of little biochemical significance and could hardly be utilized physiologically. 0378-4320[85/$03.30

© 1985 Elsevier Science Publishers B.V.

Complete purification and chemical and immunological characterization of these proteins made it possible to trace their genetic control in relation to ontogenetic development and hormone levels in the blood stream. The surface of sperm cells has likewise been studied intensively only in recent years; the findings show that the structure of the surface of mammalian sperm cells undergoes molecular changes during the passage of the cells through the epididymis and as a result of capacitation and that these changes are essential for the spermatozoa t o be capable of fertilization. Leaving aside the basic c o m p o n e n t of sperm cells -- the gigantic information system coded in the head of the sperm cell -- a sperm cell has two main systems decisive for its motility and fertility. The first is associated with the midpiece of the sperm cell and with the tail and it comprises the enzymes assuring membrane transport, glycolysis, the citric acid cycle, oxidative phosphorylation and other metabolic activities. These processes furnish the energy required for the function of the axonemal microtubules, i.e., for the motility of the spermatozoa. The other enzyme systems are associated with the acrosome and plasma membranes and t h e y are important for the contact of the spermatozoon with the ovum. We shall now discuss the various known enzymes and proteins needed for correct function and protection of the sperm cell. TESTICULAR PROTEINS Let us start with the proteins and antigens which begin to appear during spermatogenesis and spermiogenesis. The first specific antigen capable of inducing auto-immune disease and subsequent aspermatogenesis in the male guinea-pig seems to be manifested during sexual maturation (Katsh, 1960), at the time when secondary spermatocytes appear in the testicular tissue. When Menge (1965) injected adult bulls with homogenized homologous testes whose seminiferous tubules contained only primary spermatocytes, he failed to induce any significant changes in the production and quality of the spermatozoa, while the injection of testicular tissue from sexually mature bulls seriously impaired spermatogenesis. The progressive increase in the number of specific antigens accompanying development of the germ cells is due partly to biochemical changes within these cells and partly to the adsorption of soluble proteins present in the testicular fluids in which the germ cells move about. Testicular fluid pro reins

Soluble specific proteins have already been found in the fluid of rat seminiferous tubules and rete testis (Koskimies and Kormano, 1973), bull seminiferous tubules (Pavlovi~ et al., 1980) and bull rete testis (Stan~k and Dost~l, 1974). Bull rete testis fluid was also shown to contain an androgenbinding protein (ABP) (Le Cacheux et al., 1981) which binds 5 a-dihydro-

testosterone and is produced by the Sertoli cells (Fritz et al., 1974). The finding that this protein was also present in bull seminal plasma led Le Cacheux et al. (1981) to consider the possibility of using its presence in this fluid as a marker of activity of the Sertoli cells. ABP has likewise been demonstrated in ram and rabbit testes (Carreau et al., 1979). Jegon et al. (1978) demonstrated that the ABP concentration in ram fete testis fluid was significantly greater during the mating period than in the phase of sexual inactivity.

Testicular cell proteins During spermatogenesis germ cells go through series of changes including mitosis, meiosis and formation of sperm. Several differentiation antigens have been recognized on germ cells during this process. In connection with biochemical changes in guinea-pig testicular cells, Baum (1959) demonstrated that nucleohistone was converted to protamine in the germ cells in the terminal phase of spermatogenesis. Protamines stabilize the DNA of the sperm cell nucleus in an extremely compact configuration (Chevalllier, 1983). Lactate dehydrogenase X (LDH X) is present on testicular spermatogenic cells and later on the spermatozoa themselves. This isoenzyme is important for the course of metabolic processes responsible for the motility and viability of the sperm cells. It is adsorbed to the surface of the sperm cells, from which it can be released into the seminal plasma (Gavella et al., 1982). Its synthesis is genetically controlled by a locus which is activated already at the outset of sexual maturation, in the primary spermatocyte phase. LDH X has not yet been demonstrated on somatic cells (Goldberg, 1974; Li, 1974). Valenta et al. (1967) and Hyldgaard-Jensen et al. (1970) described individual differences in the molecular structure of the C sub-unit of LDH X. Immunization with mouse LDH X significantly reduced the fertility of female rabbits; the experimental results showed a block of conception after immunization (Goldberg, 1974). Yasuzumi et al. (1983) demonstrated dispersion of fibronectin, another differentiation, on early rabbit spermatids, but not on mature spermatozoa. Similarly, Radu and Voisin (1975) demonstrated the presence of a number of auto-antigens on haploid spermatids in guinea-pigs, but not on the spermatogonia and spermatocytes. Sorbitol dehydrogenase is another enzyme characteristic of testicular germ cells (Bishop et al., 1967). Further testicular enzymes (but bound in the ram until sperm cells appear in the testes) include the isoenzyme phosphodiesterase, with a molecular weight of 165 000, whose sy,nthesis does not start until the time of sexual maturation (Tash, 1976). Zilcov (1974) demonstrated that ram testis and epididymis contained alkaline phosphatase. The glycosidase beta-galactosidase, which hydrolyses a number of glycoproteins and other saccharide compounds, has been isolated from bull testes (Distler and Jourdian, 1973). Two differentiation antigens XT-1 and XT-2 of spermatogenesis were

defined by monoclonal antibodies in mice (Bechtol et al., 1979). Autoimmunological protein from rabbit spermatozoa membrane was isolated by Zhi-quan et al. (1979). The molecular weight of this membrane autoantigen was approximately 28 000. Histological study revealed severe degeneration of seminiferous tubules in the testis of the immunized rabbit. Spermatogenesis was arrested at the early spermatocyte stage of development. H - - Y antigen on testicular cells

Mtiller et al. (1978) demonstrated that rat testes secreted H--Y antigen, which plays an important role in the cellular differentiation of the male sex organs (for a review see Ohno, 1977). It is controlled from an autosomal gene responsible for the formation of a precursor substance which, in mammals, is subsequently modified b y a gene bound to the Y chromosome (Polani and Adinolfi, 1983). Its presence in the embryo's somatic cells is decisive for development in a male direction. In birds we have the opposite situation, i.e., H--W antigen, under similar genetic control from the W chromosome, predisposes to female development (Gilmour, 1967). H--Y and H--W antigens are phylogenetically very close together and are very constant. Antibodies from female mice immunized with mouse sperm cells (Goldberg et al., 1971) can detect H--Y antigen on the cells of most mammals, including man. Only a functionally very important protein can be maintained phylogenetically in this way. The results of Akram and Weniger (1968) also indicate that H--Y and H--W antigens are functionally equivalent. These authors demonstrated that chick embryonic testes, genetically initially ZZ, could be transformed to ovaries if cultivated together with embryonic XY bull testes. This transformation is evidence not only of the equivalence, but also of the organogenetic significance of these two antigens. H--Y antigen from bull embryonic testicnlar cells which reached a chick testicular cell began to function as ovarian, organ-organizing H--W antigen. Ohno (1977) further mentioned unpublished results obtained by Engel, showing that if 5.5-day chick ovaries, genetically initially ZW, were cultivated for four days in the presence of iso-immune H--Y mouse antiserum, their ovarian differentiation stopped and changed to testicular differentiation. This is further evidence of the equivalent and organogenetic function of H--Y and H--W antigens. In association with H--Y antigen, however, Wachtel (1977) showed that the mere presence of this antigen in the embryonic cell did not guarantee testicular differentiation and considered that at least three genes were necessary for this differentiation. Apart from the structural gene for H--Y antigen synthesis, a gene activating the H--Y locus and another gene coding the cell membrane receptor for H--Y antigen ought to participate in differentiation.

Proteosynthesis of haploid testicular ceils The first hypotheses of the possible gene activity of haploid testicular cells arose from studies of blood groups and histocompatibility antigens on sperm cells (Vojtls~kov~ et al., 1969; Fellous and Dausset, 1970; Bennett and Boyse, 1973). Any haploid expression, and hence synthesis of the relevant protein, could reliably be detected, however, only if the proteins determined by the diploid genes were broken down during or after meiosis and were reformed under the control of the haploid gene. Some authors (Beatty, 1970, 1974: Beatty and Gluecksohn-Waelsh, 1972; Kiddy and Hafs, 1971) raised the question of whether the interval between termination of reduction division and spermiogenesis in the Sertoli cells would be long enough for such biochemically significant changes. Further limitation of haploid activity is indicated by the relatively low RNA content of the spermatids and the resultant limited proteosynthesis (Balinsky, 1970). When considering possible post-meiotic synthetic processes, we must likewise not forget that sperm cells, from the spermatid to the ejaculated spermatozoon, are exposed to various protein molecules present in the testis and the epididymis and to the proteins of the accessory sex glands. Some of these proteins are unquestionably adsorbed to the sperm cell surface and enrich the cell's antigenic spectrum (Weft, 1961; Weft and Rodenburg, 1962; Matou~ek, 1964c; Hunter and Nornes, 1969; Hunter, 1969; Vukoti~" et al., 1978; for a review see Weft, 1967); they may possibly even mask other, primary antigens. On the other hand, several studies can be adduced in support of the existence of haploid gene activity. Bennett and Boyse (1973) incubated mouse spermatozoa with H--Y antigen antibodies and then used them for insemination. They achieved a slight, but significant, shift of the sex ratio indicative of the exclusion of some spermatozoa bearing a Y chromosome owing to the cytotoxic effect of the serunL (These results were made problematic by the findings Hoppe and Koo, 1984.) Another team (D'Agostino et al., 1978) found that spermatids, as well as pachytene spermatocytes, synthesized mRNA and hence protein also. Similar results obtained by Monesi et al. (1978) demonstrated gene activity during the haploid phase of spermatogenesis in mice. Among others, one reason may be that spermatids are joined together in a syncytium by cytoplasm bridges, via which mRNA transcribed by the haploid genome, or its protein products, can be diffused freely from one spermatid to another. The results of experiments with kinetic isotope labelling further demonstrate that the proportion of ribosomal and polyadenyl RNA produced during meiosis is maintained during development of the spermatids and show that this stable polyadenyl RNA is present in the polysomes and that it is therefore probably the mRNA linked up in protein synthesis. These experiments thus imply that the differentiation of sperm cells, and hence the synthesis of given proteins, is regulated by both meiotic and post-meiotic transcription. That does not mean that the haploid genotype is expressed phenotypically in the ejaculated

gametes, however. A review by Erickson et al. (1981) submits further evidence of RNA and protein synthesis in the post-meiotic phase of spermatogenesis. The studies of Yasuzumi et al. (1983) and particularly of Radu and Voisin (1975), who demonstrated a few auto-antigens which were present on haploid spermatids, but not on spermatogonia or spermatocytes, likewise furnish evidence of the gene and proteosynthetic activity of haploid cells, especially spermatids, and very important results were reported by Hedge et al. (1983). These authors observed that phytohaemagglutinin (PHA) was specifically bound to the equatorial region of the Y human spermatozoa. This seems to indicate the presence of a specific glycoprotein receptor for PHA in Y gametes. At all events, the antigenic structure of sperm cell precursor cells alters during spermatogenesis and spermiogenesis. It is assumed that these antigens can induce cell-mediated immunity in aspermatogenesis of auto-immune origin. A number of experiments have been carried out with the aim of isolating and purifying aspermatogenic antigens (for a review see Tung, 1977). Although in most cases the antigens were not completely purified, many of their fractions displayed aspermatogenic activity and it was found that these fractions were localized both on the surface plasma membrane of the sperm cell and on the acrosome. Peptide hormones in the testes

We cannot discuss proteins in the testes without touching on the protein hormones, i.e., in particular gonadotropins and their receptors in the testes, because they are directly associated with the synthesis of androgens and these in turn with the synthesis of the spermatozoal and seminal plasma forming proteins. The gonadotropic hormones LH and FSH (together with the releasing hormones) are generally held to be responsible for initiation of the processes whose end purpose is to assure sexual maturity and normal spermatogenesis. The LH concentration is relatively constant from birth up to sexual maturity, however, while the FSH level is low at birth and rises during sexual maturation (Karg et al., 1976). Schaunbacher (1979) also demonstrated that receptors for FSH, as well as for LH, were present in bull testicular tissue from birth up to sexual adulthood. Despite this, sensitivity to gonadotropins is not acquired until the time of sexual maturation. However, adequate findings for explaining induction and the functions safeguarding spermatogenesis are not yet available. Also very interesting are the findings of Sharpe and Fraser (1980) and Sharpe et al. (1981). They detected an LHRH-like factor in the interstitial fluid surrounding the Leydig cells and in seminiferous tubules. They have also shown that this factor is secreted in vitro by cultured rat Sertoli cells. The authors think that this Sertoli cell hormone may have, in combination with gonadotropins, a central regulatory role in the maintenance of normal testicular function and fertility.

Antigenicity of FSH and combined hormones of FSH and LH in PMSG (pregnant mare serum gonadotropins) seems to be very weak (Schams et al., 1978; Saumande and Chupin, 1981; J. Matou§ek and J. ~ a , 1984, unpublished results). This situation is very advantageous to superovulation in cows which are selected as donors of embryos. The decreasing n u m b e r of ova ovulated in the repeated superovulation (Donaldson and Perry, 1983) is probably caused by the exhaustion of primary follicules in the ovary, or by factors other than antibodies. We still know very little about the testicular fluid and cell proteins. The above-mentioned research only represents a fragment of the real state of affairs. There are, without doubt, m a n y other differentiation proteins in various spermatogenic cells beginning from spermatogonia to spermatozoa. These proteins are surely very important for the normal spermatogenesis and the fertility functions of adult germ cells. We known nothing about specific proteins in Leydig and other somatic testicle cells. I am convinced that the investigation in this direction will bring not only theoretical, but also clinical news, which can help to solve various practical problems (to induce reversible sterility in men and male animals used for the determination of oestrous stage in females and excitation of their sexual activity, to block Leydig cell secretion in sexually-deviated men, etc.). These purposes are joined not only with the immunological characterization of the testicle proteins by normal and monoclonal antibodies, but also with their isolation and chemical and functional determination. EPIDIDYMAL PROTEINS The epididymis is an important organ, since it is there that the sperm cells acquire motility and fertilizing capacity. It is the site of synthesis of a series of proteins which are either adsorbed to the spermatozoa or form a suitable medium for them, while on the other hand proteins and the superficial parts of the sperm cells (e.g., the cytoplasm droplets) are also destroyed there (for a review see Orgebin-Christ et al., 1975; Hamilton, 1975). For maturation processes (the acquisition of fertilizing properties), the caput and corpus epididymidis are the most important, while the part for the acquisition of motility and preservation of the sperm cells is the cauda epididymidis (Gibbsons, 1983; Katz, 1983). Foumier-Delpech et al. (1983) demonstrated that spermatozoa from ram rete testis and caput epididymidis were not capable of fusion with the zona pellucida of a ewe ovum, and that t h e y did not acquire this property until t h e y had passed through the corpus epididymidis. The ability of ram spermatozoa to fuse with the zone pellucida of a ewe ovum was not associated in any way with motility. The reaction of the sperm cell with the zona pellucida is evidently associated with the binding to the sperm cell acrosome or plasma membrane of a specific protein-(s) secreted proximally to the caput epididymidis. Tung et al. (1980) and Kope~'n~ et al. (1983) demonstrated in guinea-pig epididymis the

secretion of glycoproteins which were adsorbed to the head of the sperm cells and modified the surface structures of the acrosome and the spermatozoal auto-antigens. Fournier-Delpech et al. (1979), by inseminating the uterine horns of sheep with ram spermatozoa from different segments of the epididymis, found that sperm cells from the middle of the corpus epididymidis were incapable of ensuring fertilization that would lead to the birth of a lamb. Sperm cells from the distal part of the corpus epididymidis and from the cauda led to normal embryogenesis in over 70% of the cases. On the other hand, Hunter et al. (1978) inseminated sows oviducts with boar spermatozoa from the upper part of the corpus epididymidis, together with medium TCM 199, and demonstrated fertilization of 87% of the eggs in 4--6 h. The differences between these results may be due to interspecies differences and to differences in local synthesis of the proteins responsible for the ability of the sperm cell to fuse with the zona pellucida of the egg. The proteins responsible for this fusion have not yet been isolated from the epididymis.

Identification of the epididymal proteins Up to now, only a few substances have been identified in the epididymal fluids. Acott and Hoskins (1978, 1981), Acott et al. (1979) and Brandt et al. (1978) demonstrated that a glycoprotein, which they termed a "forward mobility protein", was synthesized in the bull's epididymis. This protein -- which the authors also found in bull seminal plasma -- changes immobile sperms from the caput epididymidis, in the presence of a raised a m o u n t of cyclic AMP, to motile sperm. Setchell and Hinton (1981) likewise confirmed in male rats (and in rams and boars) that the lumen fluids of the epididymis, especially in the proximal part, dramatically evoke motility. They attributed the major influence to carnitine, but assumed that other substances also had an effect. The presence of carbonic anhydrase in the testicular and epididymal tubules of the bull has been studied histochemically (Goyal et al., 1980). Strong activity was demonstrated in the efferent ductules and the initial segments of the epididymis and weaker activity in other parts of the epididymis. It is interesting to note that carbonic anhydrase is not synthesized under the control of androgens. In bulls it is synthesized long after sexual adulthood (at a b o u t 50 weeks). It is assumed that it has an acidophilic effect in the epididymis and that it co-participates in the maturation and motility of the sperm cells. Androgen-binding protein (ABP) has been demonstrated in the bull epididymis as well as in the testes. Le Cacheux et al. (1981) were of the opinion that ABP could also play a physiological role in sperm cell maturation. Its presence in bull seminal plasma indicates that it is produced in large amounts. SedlSkov~ et al. (1968) isolated serum albumin from the epididymal fluids of boars. Schellpfeffer and Hunter (1976) isolated a protein with a molecular weight

of 133 000 from boar cauda epididymidis fluid, but without defining it chemically. Bull seminal ribonuclease, which was first studied and isolated from seminal vesicle fluid (Matou~ek, 1967, 1969; Dost~l and Matou~ek, 1973; Matou~ek and Grozdanovi~, 1973; Matou~ek et al., 1973a,b) is also synthesized in the caudal fluid (Matou~ek et al., 1981). Various proteins of a polymorphous character have been detected in the bull and boar cauda epididymidis (Dost~l and Matou~ek, 1972; Matou~ek, 1972b), but they have not yet been isolated or chemically defined. The form of genetic control (one locus with two alleles) has been determined in the case of one boar protein migrating to the anode (Matou~ek, 1972b). In boars and rabbits, 5'-nucleotidase is also synthesized in the epididymis (Mann and LutwakMann, 1981). The total protein content of the various parts of the epididymis makes an interesting study. In the lumen fluid at the commencement of the caput epididymidis, Schellpfeffer and Hunter (1976) put it at 0.30%, whereas at the transition to the corpus epididymidis it rises to 3.2% and the proximal part of the corpus actually contains 6.46%. The protein concentration thereafter steadily diminishes, and in the proximal part of the cauda epididymidis it amounts to only 3.3%. The drop in the protein content of the cauda may be due to degradation of the proteins in this part of the epididymis by proteolytic enzymes, or to greater fluid secretion. According to Schellpfeffer and Hunter (1976), six specific proteins are transported to the epididymis in the testicular fluid; two of these disappear after entering the caput, but the other four can be detected all the way along the epididymis. The initial part of the boar epididymis produces one specific antigen, which is adsorbed almost immediately to the sperm cells; another antigen produced by the epithelial cells of the caput epididymis is likewise bound to the sperm cells, in the distal part of the organ. Three further antigens augmenting the antigenic spectrum of the sperm cells are produced in the proximal part of the corpus epididymidis. In bulls also, this part of the epididymis is the site of the initial production of a specific protein which is still chemically unidentified (Stane'*k and Dost~l, 1974). It is in this phase of passage of the spermatozoa through the epididymis that their maturation is completed. It is thus in the cauda epididymidis that the greatest adsorption of the proteins synthesized in the preceding parts of the epididymis to the migrating sperm cells takes place, but it is probably also the site of the synthesis of enzymes which destroy unwanted or undesirable components of the spermatozoa. In bulls it is the site of initial synthesis of seminal ribonuclease (Matou~ek et al., 1981), which apparently, by adsorption, inactivates undesirable RNA on the sperm cell head (J. Klaudy and J. Dost~l, 1983, personal communication) and on the cytoplasm droplets (Matou~ek et al., 1980a). Unwanted components already collect in the cytoplasm droplets from the testis, where formation of the droplet begins.

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Protein synthesis in the epididymis and hormones Protein synthesis in the epididymis and other parts of the genital tract is a genetic business in which the gene effectors are in most cases androgenic hormones and their metabolites. Blaquier (1975) discussed the dependence of specific protein synthesis in the epididymal epithelium in rats and Ka~ka and Kope~n~ (1977) described it in mice. Jones et al. (1980), using the incorporation of 3sS methionine, showed in rats that the rate of synthesis in different parts of the epididymis varied. After castration, incorporation was less than 10% of normal, in all the parts of the epididymis. When testosterone was administered to castrated animals, protein synthesis returned to normal in five days. The same authors, however, demonstrated that protein synthesis in the initial segments of the epididymis was also dependent on factors present in the testicular fluid. Pujol and Bayard (1979) showed that the maximum androgen receptor concentration in the rat was in the caput epididymidis and the smallest in the corpus epididymidis. Jones and Dott (1980), five weeks after castrating rabbits, not only observed disappearance of almost all the sperm cells from the epididymis, but they also demonstrated, by electrophoresis, loss of synthesis of the proteins typical of a normal epididymis and the appearance of blood serum proteins. The administration of testosterone blocked these changes in the composition of the epididymal fluids. Prasad et al. (1973), Gupta et al. (1974) and Rajalakshmi et al. (1976) likewise demonstrated a correlation between proteosynthesis in the epididymis and testosterone levels. Reduced testosterone synthesis in orchitic bulls with damaged Leydig cells led to loss of seminal ribonuclease synthesis in the cauda epididymidis (Matou~ek et al., 1980a). Orgebin-Crist and Jahad (1978) demonstrated that 5~lihydrotestosterone (an active testosterone metabolite) induced the synthesis of RNA in a cell culture prepared from the proximal segment of the rabbit corpus epididymidis and proteins capable of transforming infertile sperm cells into fertile cells. This is direct evidence of the function of the cells of the epididymal epithelium in the maturation of the sperm cells. To sum up our knowledge about proteosynthesis in the epididymis, sperm cells acquire fertilizing activity as a result of metabolic changes which begin in the ductus efferentes, but take place mainly in the caput and corpus epididymidis. According to Dacheux and Paquignon (1980), motility changes are associated chiefly with an increase in the cyclic 3'-5' AMP concentration due either to activation of adenyl-cyclase or to inhibition of the nucleotides of phosphodiesterase (for a review see Gibbons, 1983). In addition to a raised cAMP concentration, forward movement of the sperm cells requires a specific protein medium containing motility factor --forward mobility protein (Brandt et al., 1978). For the spermatozoa to be able to find, penetrate and fertilize the female germ cell, however, specific conditions in the plasma membrane and the acrosome of the sperm

11 cell, as well as motility, are needed. Dacheux and Paquignon (1980) termed the substances which cause these changes "fertilizing factors"; they are likewise synthesized in the epididymis. These findings draw attention to the importance of the epididymis and to its proteosynthetic activity. As regards proteosynthesis in the epididymis we know a little more in relation with proteosynthesis in testes, b u t n o t yet enough for a clear understanding of processes that are responsible for the maturation and long conservation of spermatozoa in the epididymis. The ability of the cauda epididymidis to store the fertile sperm cells for a long time is surprising. If we were able to apply the cauda storage conditions to in vitro surroundings it would be a useful aid for the organization of artificial insemination in sows, sheep and other animal species. Many cases of sperm infertility are due to errors in the secretory and metabolic functions in the epididymis. If we knew more a b o u t the normal function of the epididymis, we would then be able to correct these errors. We know that some epididymal proteins are polymorphic, but we do not know the reason for it. Similarly to the testicle proteins, an inhibition (chemical or b y monoclonal antibodies) of the metabolically important protein synthesized in the epididymis could irreversibly or perhaps reversibly sterilize males. PROTEINS OF THE ACCESSORY

SEX GLANDS

It looks as though the accessory sex glands do not produce the specific proteins necessary for the actual fertilizing capacity, since the eggs of the majority of mammals can be fertilized by spermatozoa from the terminal part of the cauda epididymidis. It seems incomprehensible, however, that nature should maintain such powerful protein secretory activity in the seminal vesicles of boars and bulls, over such a long period of evolution, without a reason. If Schellpfeffer and Hunter (1976), using starch gel electrophoresis, demonstrated seven cathodal and six anodal protein components in boar seminal plasma, of which only albumin and the slow alpha2 component were serum proteins, then specific proteosynthesis in the accessory sex glands is remarkable, to say the least. Most of these proteins (all of the cathodal and three of the anodal proteins) were synthesized and secreted by the seminal vesicles. Two anodal protein components were shown to be in prostato-urethral fluid. It was proved by means of antibodies to boar ejaculated spermatozoa and seminal plasma that a number of these antigens were adsorbed onto sperm cells. It is n o w known (Mann and Lutwak-Mann, 1981) that some of these primarily soluble antigens b o u n d secondarily to sperm cells can intervene biochemically in sperm cell metabolism - - i n particular in the biochemistry and configuration of the spermatozoid membrane.

12 Proteins o f the individual accessory sex glands

In the case of bulls, the significance of proteins secondarily bound to the sperm cells is confirmed, for instance, by the high nucleolytic enzyme (mainly 5'-nucleotidase and cyclic nucleotide phosphodiesterase) values in the seminal vesicle fluid. Similar enzymes include nucleotide pyrophosphatase, nucleoside phosphorylase, pyridine nucleosidase, adenosine 3'-5'monophosphate phosphodiesterase and nonspecific phosphodiesterase (Mann and Lutwak-Mann, 1981). Pyridine nucleosidase is synthesized in bull seminal vesicles; it has been purified and its molecular weight is 36 000. 5'-nucleotidase has already been isolated by Heppel and Hilmoe (1951) and later by Strze~ek and Wolos (1974). The latter authors also studied its immunological properties and assumed that is was bound to the sperm cells. Fini et al. (1983) found that 5'-nucleotidase is present in bull seminal plasma in three different molecular forms. One is particulate and the other two forms are soluble. The partially-purified particulate enzyme showed a molecular weight of 190 000. Seminal ribonuclease is another nucleolytic enzyme present in the fluids of the bull's reproductive systel~ Apart from a small amount in the cauda epididymidis, it is synthesized in the seminal vesicles and the ampullae of the ductns deferentes (Matou~ek et al., 1980a, 1981). This enzyme has been isolated from bull seminal plasma (Floridi and D'Alessio, 1967), seminal vesicle tissue (Hosokawa and Irie, 1971; Stane"k et al., 1975) and seminal vesicle fluid (DostA1 and Matou'~ek, 1973). Since it degrades all types of RNA, which it breaks down in pyrimidine sequences, and has been classified as D-pyrimidine 2'-nucleotidyltransferase (Hol:~ and Grozdanovi~, 1972), it is supposed that it inactivates RNA residues on the sperm cells. It also has an immuno-suppressive effect, however, and it acts both on homologous lymphocytes (Stane"k et al., 1980) and heterologous lymphocytes (Stan~k et al., 1978; Sou~ek and Matou~ek, 1979; Matoursek et al., 1979; Sou~ek et al., 1981, 1983). It may also act as a defence for the sperm cells, and it probably inhibits the activation of immune cells which might attack the sperm cells after coitus or insemination in the female genital tract. Beer and Neaves (1978), Prakash et al. (1976), Lord et al. (1977) and Marcus et al. (1979) presumed that a similar substance was present on human sperm cells and in human seminal plasma. Alongside its important functions in the reproduction of cattle, bull seminal ribonuclease has an aspermatogenic effect (Matou~ek and Grozdanovi~, 1973; Leone et al., 1973; Matou~ek et al., 1973b, 1978; H l i ~ k et al., 1981), an embryotoxic effect (Matou~ek and Grozdanovi~, 1973, Matou~ek et al., 1973a) and a cancerostatic effect (Matou~ek, 1973; Stane"k and Matou~ek, 1976; Cinatl et al., 1977; Vescia et al., 1980) in heterologous species. In bulls, synthesis of seminal ribonuclease begins during sexual maturation. It is synthesized first of all in the seminal vesicles at about 20 weeks, in the following weeks in the deferent duct ampullae and at about 30 weeks

13 in the cauda epididymidis. The synthesis of this protein depends on the blood plasma testosterone concentration (Matou~ek et al., 1980a, 1981). Compared with the other body fluids, nucleolytic enzyme activities in the seminal plasma of domestic animals are very high. In bulls, 5'-nucleotidase and cyclic nucleotide phosphodiesterase activity are the highest (Mann and Lutwak-Mann, 1981). Another substance isolated from bull seminal plasma is phospholipidbinding protein (PBP), which causes haemolysis of both homologous and heterologous erythrocytes (Kysilka, 1973) and releases the cytoplasm droplets from epididymal sperm cells (Matou~ek and Kysilka, 1980). It was isolated by Kysilka (1973), who also described its ability to bind phospholipids, especially those of bovine erythrocytes (Kysilka, 1975). This protein, which has a molecular weight of 80 000, is synthesized in the seminal vesicles and the deferent duct ampullae (Matou~ek and Kysilka, 1980). Its synthesis starts during sexual maturation, but a few weeks later than seminal ribonuclease, and is likewise dependent on the blood plasma testosterone concentration (Matou~ek et al., 1980b). Functionally, bull seminal plasma PBP is not species-specific, it also releases the cytoplasm droplets from ram, boar and rabbit sperm cells. A similar protein, giving a crossreaction with PBP, is synthesized in the seminal vesicles and deferent duct ampullae of the ram (Matou~ek and Kysilka, 1984). Hofmann and Unsicker (1982) isolated a very rich protein fraction promoting nerve growth from bull seminal vesicles. Boar seminal vesicles synthesize two strongly basic proteins (isoelectric points 8.8. and 9.4) and a haemagglutinin (Boursnell and Coombs, 1966; BoursneU, 1967; Sedl~kov~ et al., 1977). Dost~l et al. (1978) isolated from boar prostate fluid a specific protein with the three phenotypes S, SF and F. They also studied this protein from its biochemical aspects. The authors isolated an S variant with a 0.74 frequency and an F variant with a 0.26 frequency and found that both had a molecular weight of 13 000. An amino acid analysis showed that the only difference between them was that the S variant had one aspartic acid more than the F variant and the latter one glycine more than the S variant. A lecithinase, which according to Cortel (1980) is of significance for sperm cell fertility, has been isolated from Cowper's glands. Proteins of a polymorphous character are also present in the fluids of animals' accessory sex glands. Genetic control of polymorphism was demonstrated in the case of two chemically unidentified proteins in boar seminal vesicle fluid, the K! zone with three phenotypes being determined genetically by two alleles and the KII zone with six phenotypes by three alleles (Dost~il, 1970). An extensive polymorphous protein zone has also been demonstrated in the fluids of bull seminal vesicles and deferent duct ampullae (Valenta et al., 1965) and in seminal vesicle fluid collected from the domestic cock's vas deferens (Stratil, 1970). Veselsk~ and Kdbek (1970) detected individual immunological esterase differences in the fluids of the accessory sex glands of bulls. Serum polymorphous transferrins, as well as specific

14 proteins, have been demonstrated in bull seminal vesicle fluid (Stane"k and Dost~l, 1972). Proteins demonstrated in seminal plasma A number of workers (for a review see Weil, 1967) have studied the seminal plasma proteins, which include the proteins not only of all the accessory sex glands, but also of the epididymis (Pavlovi~ et al., 1980) and possibly of the testes too. It is no doubt easier to obtain seminal plasma from ejaculate collected with an artificial vagina than to collect the fluids from the individual sex organs; this is done mostly at the abattoir or by cannulation from the living animals. Gel disc electrophoresis and isoelectric focusing on polyacrylamide gel (Lavon, 1972) have proved to be very helpful in the separation of seminal plasma proteins. The characteristic attribute of seminal plasma is its enzymatic activity. For instance, it contains enzymatic substances responsible in human and boar semen for coagulation, gelatinization and liquefaction of the semen. Phospholipase activity, has been demonstrated in human, bull and ram sperm. Mann and Lutwak-Mann (1981) discuss these and further proteins and enzymes in their book. Many of them also possess proteolytic, and other activities. Lactoferrin, and iron carrier, is likewise present in animal seminal plasma; Roberts and Boursnell (1975) demonstrated it in bulls. Some of the seminal plasma proteins bind other elements, such as calcium (calcium-binding protein) in bull seminal plasma and seminal vesicle fluid (Luka~ et al., 1976), or zinc in boar seminal plasma (Roberts et al., 1974). Alongside the enzymes enumerated in the preceding section, the seminal plasma of domestic animals also contains acid and alkaline phosphatase, pyrophosphatase, phosphodiesterase and ATPase, etc. (for a review see Mann and Lutwak-Mann, 1981). The most important phosphatase in domestic animals is alkaline phosphatase, which hydrolyses phosphoric acid esters best at pH 9. Strze~ek and Glogowski (1979a) isolated two molecular forms of alkaline phosphatase from bull seminal plasma. The two forms differed in respect of their substrate affinity and their antigenic determinants (Strze~ek and Glogowski, 1979b). The same authors studied the properties of alkaline phosphatase in frozen bull semen (Glogowski and Strze~ek, 1979). From ram seminal plasma they isolated three forms of alkaline phosphatase (Glogowski and Strze~ek, 1981) and demonstrated a decrease in the amount of this enzyme in seminal plasma after experimentally induced inflammation of the reproductive organs. As regards phosphodiesterase, ram seminal plasma contains a mixture of at least three isoenzymes, all of which differ from spermatozoal phosphodiesterase, but resemble the isoenzymes of the male accessory sex organs (Tash, 1976). In bulls, as in rams, phosphodiesterase is partly soluble and partly bound to the sperm cells. The soluble fraction contains four or five different forms (Stephens et al., 1979). Georgiev (1974) demonstrated sorbitol dehydrogenase

15 in bull and ram seminal plasma and three types of non-specific esterases -arylesterase, alylesterase and cholinesterase -- in bull seminal plasma (Georgiev et al., 1976a). Balbierz et al. (1975, 1976) identified non-specific esterases, alkaline and acid phosphatase and (among nitrogen bases) glyceryl phosphorylcholine and ergothionine in horse seminal plasma. They attempted to correlate the amount of these enzymes in the seminal plasma with anomalies in the stallions' spermiograms and with possible disorders of the accessory glands and testes, but the results so far have been too variable (Balbierz et al., 1976). Glutamic oxalacetate transaminase (GOT) is also of seminal plasma origin. It is bound to the spermatozoa immediately after ejaculation and its release during the freezing of bull sperm cells is an indicator of cryogenic changes in the cellular structure of the spermatozoa and hence of damage to the plasma membrane and the mitochondrial spiral filament (Graham and Pace, 1967; Pace and Graham, 1970; Strze~ek et al., 1981). GOT is readily released from sperm cells incubated at 37°C and the amount of enzyme released is an indicator of the resistance or destabilization of the plasma membrane and hence of the suitability of the sperm for glycerol dilution and freezing (Strze~ek et al., 1981). Pyridine nucleosidase (NAD nucleosidase) is an enzyme whose quantity, though very variable, is genetically fixed. The male offspring of bulls whose seminal plasma is devoid of seminal nucleosidase likewise do not possess this enzyme (or only very little). Conversely, the male offspring of bulls with high activity likewise possess high activity (Macmillan et al., 1975). Cyclic AMP phosphodiesterase activity is present both on the sperm cells and in the seminal plasma (Tash, 1976). Deoxyribonuclease activity (alkaline and acid) in the seminal plasma of domestic animals is relatively low (Mann and Lutwak-Mann, 1981). Using disc electrophoresis on polyacrylamide gel containing DNA as the substrate, Georgiev et al. (1976b) demonstrated four different deoxyribonucleases in bull seminal plasma. Reddy and Bhargava (1979) isolated from bull seminal plasma a protein with antimicrobial activity, which they called seminal plasmin. It has a molecular weight of 17 000, its isoelectric point is 9.8 and it inhibits ribosomal RNA inEscherichia coli. It takes effect via inhibition of RNA polymerase. Seminal plasmin also inhibits growth of Gram-positive and Gramnegative bacteria. It does not significantly affect the fertility of cows, however. Inhibin, which inhibits the activity of the gonadotropic hormones in both the male and the female organism, was isolated from bull seminal plasma by Chari et al. (1978). The acrosin inhibitors in seminal plasma are proteins with a high affinity for acrosin and trypsin. Fritz et al. (1975a,b) described the purification and properties of these inhibitors and their biological function. The main role of inhibitors in seminal plasma is evidently to inactivate any free acrosin released from dying spermatozoa in the vicinity of the egg which might damage the egg and its capacity for fertilization. Zelezn~ et al. (1980)

16 came to the conclusion that acrosin inhibitors in seminal plasma in general, and the bull inhibitor BUSI I in particular, inhibit acrosin by binding it if the enzyme appears in the active form prematurely, i.e., in the male genital tract. According to Zaneveld et al. (1973), acrosin inhibitors present in the plasma are adsorbed to the surface of the spermatozoal membrane immediately after ejaculation. During the freezing of bull sperm, acrosin inhibitor activity in the extraceUular medium increases. Zaneveld et al. (1973) and Strze~ek et al. (1981) assumed that this occurred at the expense of the spermatozoal acrosome, probably as a consequence of destruction of the lipoprotein of the spermatozoa during freezing. Both natural and synthetic inhibitors of acrosomal enzymes are discussed in a review by Zaneveld (1982). ~echovfi et al. (1979a) isolated three proteinase inhibitors with low isoelectric points and molecular weights in the region of 9000 from bull seminal plasma; all three displayed the same inhibitory properties and inhibited acrosin, trypsin and chymotrypsin; inhibition of kallikrein was very slight. ~echov~ et al. (1979b) isolated a further basic acrosin inhibitor (BUSI II) from bull seminal plasma. It was likewise not very acrosin specific, since it also inhibited trypsin and partly inhibited chymotrypsin. Veselsk~ and ~echov~ (1980) found that this inhibitor was synthesized in the bull cauda epididymidis, deferent duct ampullae and seminal vesicles, while the inhibitor BUSI I was synthesized only in the ampullae and the seminal vesicles. There are also other seminal plasma proteins with binding properties in relation to acrosin, however, like the glycoprotein causing sperm cell decapacitation (Mann and Lutwak-Mann, 1981). Some workers have tried to evaluate the functional activity of the individual organs or cells of the reproductive system from the level of certain proteins and other substances in the seminal plasma. In addition to the above androgen-binding protein (Le Cacheux et al., 1981) and the function of the Sertoli cells, Mann et al. (1957), Mann (1975) and Tischner et al. (1974) claimed that the amount of the nitrogen base glyceryl-phosphorylcholine (GPC) in stallion seminal plasma was an indicator of epididymal activity, while ergothionine (EGT) reflected the activity of the deferent duct ampullae, because that was precisely where it was synthesized. Kosiniak (1979), however, demonstrated that GPC, in the stallion, was synthesized in the deferent duct ampullae as well as in the epididymis. Kosiniak and Bittmar (1981) demonstrated that the GPC and EGT level, together with the total protein and fructose level, varied with the time of year and hence in relation to the mating season. Berndtson et al. (1974) found that the quantitative movements of these substances were correlated to the blood plasma testosterone concentration. Kulangara (1969, 1972) demonstrated polymorphous proteins in ram seminal plasma and on ram spermatozoa and Madeyska-Lewandowska (1976) demonstrated them in bull seminal plasma.

17

Significance of hormones for protein synthesis in the accessory sex glands The androgen level is an important factor for protein synthesis both in the epididymis and in the accessory sex glands. Higgins et al. (1976) demonstrated that the weight and RNA content of rat seminal vesicles fell during the first 14 days after castration to 15--20% and the DNA content to 40%. DNA losses correlated well with the loss of cells observed in the epithelium of the seminal vesicles. The administration of testosterone evoked immediate RNA synthesis in the gland, which attained the values in the control group within 80 h. The weight of the seminal vesicles attained control values in a further 30 h, the DNA content in 40 h and proliferation of the epithelium in 40--50 h. The stroma cells of the seminal vesicles were unaffected. An animal's androgenic status thus influences epithelial cell proliferation in the accessory sex glands. Testosterone seems to control protein synthesis in these glands indirectly, by influencing the number of cells in the epithelium. Testosterone is first of all converted to active 5a-dihydrotestosterone by means of 5~-reductase. 5a~lihydrotestosterone has a marked affinity for steroid-binding cytoplasmic receptor proteins present in the accessory sex glands and in the testis (Hansson et al., 1976a,b). Many of these proteins are bound to the nuclear chromatin fractions of the target cells and it is therefore supposed that they act in these cases directly on given regulative processes of gene transcription (Ohno, 1971; Tymoczko and Liao, 1976). Another possibility under consideration is that androgen-receptor complexes may play an important role in the utilization of certain nuclear ribonucleic acids. A steroid receptor and further specific proteins could be bound to the RNA product and in this way influence both RNA and protein synthesis in different cells of the genital system. Few studies have so far been published from the aspect of androgenic control of synthesis of the proteins of the accessory sex glands of domestic animals. Matou~ek et al. (1980a, 1981) published data on testosterone levels from the point of view of seminal ribonuclease synthesis during ontogenesis for normal and orchitic bulls, respectively. Among other proteins synthesized in the seminal vesicles and deferent duct ampullae they gave data for phospholipid-binding protein (Matou~ek et al., 1980b). Specific male proteins in the serum of boars (Matou~ek and SchrSffel, 1965) and rats (Roy and Neuhaus, 1966) are also influenced by androgens. Nothing is known about their physiological role, however, and their relationship to the seminal plasma proteins is likewise obscure. Some doubts were raised about the regulative capacity of gene expression by hormones, mainly by androgens, including specific transcription and translation processes, were raised by Kistler et al. (1981), who described a discrepancy between synthesis of the rat seminal vesicle secretory protein known as SVS IV and the level of SVS IV mRNA capable of translation. Between the ages of 25 and 60 days, synthesis of this protein rose in rats from 0.8% to 20%, but the amount SVS IV mRNA capable of translation

18 only quadrupled. The authors concluded that synthesis of this protein was regulated by other factors as well as by translation mRNA, but did not indicate what these factors might be. They introduced another u n k n o w n into the discrepancy between protein SVB IV synthesis and the androgen level in pubescent rats, by speculating upon the possible synergistic effect of prolactin, whose level rises in pubescent rats 10--15 days sooner than the androgen level. Goyal et al. (1980) also demonstrated that carbonic anhydrase synthesis in the testicular and epididymal tubules of bulls was not controlled by androgens. In concluding, we can state that the accessory sex glands of domestic animals produce large quantities of proteins, some of which have already been isolated and thoroughly studied chemically, and in some cases biologically, while some are known only immunologically as antigens and others may still be unknown. Their significance for the ability of spermatozoa to survive in the female genital tract, to travel along it and to get rid of their useless portion seems to be indisputable (e.g., immuno-suppressive substances in the seminal plasma and on the spermatozoa which protect the latter from attack by female immune cells, the inhibition of acrosin, the removal of cytoplasm droplets by phospholipid-binding protein, etc.). The physiological function of a series of other enzymes is still unknown, however, although their significance is demonstrated by studies showing that many of them are bound to the sperm cells (for a review see Weil, 1967) or that t h e y adhere to other germ and somatic cells (Vukoti~ and Pavlovi~, 1981). Some proteins, however, also have other biochemical functions not associated with the sperm cells (inhibin, seminal plasmin, some nucleotidases, the factor acting on the growth of nerve cells, etc.). Isolation and study of the proteins in the fluids of the male sex organs is most desirable. Each of these proteins should also be studied individually from the aspect of the function of the androgens which do or do not act on its synthesis. There is not much hope, at present, of discovering the correlation between some seminal plasma proteins and the fertility of spermatozoa. Firstly we have to isolate and chemically determine these proteins and to study their biological functions in sperm cells. We should also k n o w which proteins are immuno-suppressive, auto-immunogenic and iso-immunogenic in female genital organs. PROTEINS ON THE SPERM CELLS

In the section on testicular antigens, we discussed results demonstrating the presence of specific protein antigens on sperm precursor cells. Understandably, during spermiogenesis in the Sertoli cells and passage of the sperm cells through the seminiferous tubules, the rete testis, the epididymis and other parts of the genital tract, the antigenicity of the cells increases and ejaculated spermatozoa are the richest in specific proteins. The very first studies of sperm antigens were actually carried out on

19 ejaculated spermatozoa (Metalnikoff, 1900; Landsteiner and Levine, 1926), using immunological methods. Barker and Amann (1970) demonstrated four specific antigens on bull epididymal spermatozoa by means of immune antibodies and Kulangara (1969) demonstrated 5--10 antigens on ejaculated ram spermatozoa. Matou]ek (1964b), Mittal et al. (1965) and Ackerman and Gonzalez-Enders (1969) demonstrated antibody activity against bull and rabbit sperm antigens, which caused significant inhibition of metabolic processes when the antibodies came into contact with sperm cells. The presence of blood group antigens on bun and boar sperm cells has also been studied (Docton et al., 1952; Menge et al., 1962a,b; Schmid et al., 1964; Matou~ek, 1964a, 1970; Matou~ek et al., 1967; Podliachouk and Dikov, 1970; Padma, 1969, 1972; Bezenko and Novikov, 1974; Meyer, 1969, 1972; Thiele et al., 1974); it was found that only soluble blood group substances were adsorbed to them secondarily. The only domestic animals in which histocompatibility antigens have been studied are pigs and cattle. These antigens were found to be only weakly expressed on boar sperm cells (Jilek and Veselsk:~, 1972; Vaiman et al., 1978) and not at all on bovine sperm cells (Chardon et al., 1983). In recent years, sperm cell antigens have been unmasked by means of monoclonal antibodies. In this way, Schmell et al. (1982) unmasked on mouse sperm cells one protein with a molecular weight of 200 000 daltons (on the acrosome) and others with a molecular weight of 60 000 and 40 000 daltons, respectively (on the tail). All three antigens were species-specific. The tail proteins were sperm cell specific, while the acrosomal protein was also present on other mouse tissues. Monoclonal antibodies to the sperm membrane proteins, but especially antibodies to the acrosomal protein, blocked penetration of the spermatozoa into the egg. Antibodies to the tail proteins immobilized the sperm cells and partly impaired contact with the egg. Feuchter et al. (1981), using monoclonal antibodies, demonstrated three antigenic determinants on the head of mouse epididymal sperm cells and one specific for the tail. These antigens made their first appearance on the sperm cells in the corpus epididymidis and are thus characteristic of maturation processes which take place in the epididymis. The authors assumed that they could be formed on the surface of the sperm cells as a result of the release of components from the sperm cell cytoplasm, by modification of pre-existent surface parts, or by the adsorption of new proteins from the epididymal fluid. Primakoff and Myles (1983) demonstrated five different zones on the surface of guinea-pig sperm cells by means of monoclonal antibodies.

Specific sperm cell proteins Modern biochemical techniques have extended and intensified the study of sperm cell antigens. For instance, Vierula and Rajaniemi (1980), when studying the surface of bull spermatozoa, used a lactoperoxidase-catalysed

20 radioiodine technique to label proteins and glycoproteins containing tyrosine. Electronoptic autoradiography showed that labelling was disturbed evenly throughout all parts of the bull sperm cell, in the plasma membrane. Electrophoresis of soluble radioactive proteins demonstrated the presence of five radioactive fractions with a molecular weight of 140 000 to 15 000 daltons. The first three of which were sperm cell proteins and the other two (26 000 and 15 000 daltons) came from the seminal plasma. Russel et al., 1983 used two-dimensional silver-stained polyacrylamide gel electrophoresis for their studies, and detected, as has been already noted, more than 250 polypeptides on the ejaculated boar spermatozoa. These results confirmed a series of earlier immunological studies showing that the surface of sperm cells and seminal plasma have a number of components in common and that the spermatozoal superficial proteins a n d seminal plasma proteins interact (Weft, 1961; Hunter and Hafs, 1964; Matou~ek, 1964c; Ishibashi and Yasunda, 1968; Hunter and Nornes, 1969; Li and Behrman, 1970; Kope~n~, 1971; Bratanov et al., 1973; Kononov and Houbanova, 1975; Orgebin-Crist et al., 1975; Ka~ka and Kope~n~, 1977; Voglmayr et al., 1979). Proteins can also migrate from sperm cells into the surrounding fluid (Hunter and Hafs, 1964; Kulangara, 1969; Barker and Amann, 1970; Matou~ek, 1972a). The substances which sperm cells are able to concentrate on their surface include a series of enzymes which form specific receptors capable of binding various substances by both primary and secondary adsorption. ATPase and other phosphatases are bound to rabbit sperm cells in this way (Gordon and Dandekar, 1977). Carboglutelin, a further specific receptor, binds glucose and sugars in boar sperm cells (Van der Horst, 1972). The nuclear basic proteins, which, together with DNA, form the chief part of the sperm cell, i.e., the nucleus, are of no small significance for differentiating between the sperm cells of different animals. At present, we know little of their structure, their binding to DNA and their functional activity, although they are presumed to play a role in gene expression. At the junction of the tail and the midpiece of the mammalian sperm cell, the mitochondrial sheath and the plasma membrane contain hexokinase, lactate dehydrogenase and all the other glycolytic enzymes needed for the metabolism of fructose and other hexoses to lactic acid. The motility and structure of the sperm cell are assured by two different classes of proteins which put the cytoskeleton -- the micro filaments and the microtubules -together. The microfftaments are composed of actin, the microtubules mainly of tubulin and the arms attached to the outer doublet microtubules consist of dynein, an ATPase protein (Dustin, 1978; Mann and LutwakMann, 1981). These 9 + 2 flagellar tubules and a fairly complex array of associated accessory structures are responsible for motility of spermatozoa (Gibbons, 1983). One characteristic of spermatozoal mitochondria, in all species, is the presence of a cytochrome--cytochrome oxidation respiratory system and of various dehydrogenases and other enzymes participating

21 in sperm cell metabolism. The spermatozoal mitochondria contain mechanisms mainly for control of the energy relationships of the sperm cell, i.e., ATP, ADP and AMP. ATP was isolated first from ram spermatozoa. Together with ATPase it acts directly on the mobility and metabolism of the sperm cells. In addition to adenosine phosphates, bull, ram, boar and stallion sperm cells contain guanosine triphosphate (GTP), guanosine diphosphate (GDP) and nicotinamide-adenine dinucleotide (NAD) (for a review see Mann and Lutwak-Mann, 1981). AMP
1982). The lysosomal enzymes contained in the cytoplasm droplets on bull and ram spermatozoa include acid phosphatase, acid protease, beta-glucuronidase, aryl sulphatase, RNAase and DNAase (Dott and Dingle, 1968). A crosomal proteins The enzymes of the spermatozoal acrosome play an important part in the interaction of the sperm cell and the egg. The most important and the most thoroughly studied is the proteolytic enzyme acrosin. Harrison (1983), F15rke et al. (1983) demonstrated it on spermatids. Like trypsin, acrosin splits the carboxyl bonds of arginine and lysine. In addition to acrosin, the head of the sperm cell contains an acrosin inhibitor, which is not identical

22 with the acrosin inhibitors present in seminal plasma (Mann and LutwakMann, 1981; Harrison, 1983). Among its sperm cell functions, acrosin also acts on the kinin--kallikrein system, which may participate in stimulation of sperm cell motility and improved penetration of the spermatozoa across the cervical mucosa. Acrosin is bound to the inner acrosomal membrane as proacrosin. The mechanism by which proacrosin is converted to acrosin is still unknown. Huneau et al. (1983) studied this phenomenon in rams. Proacrosin and acrosin can both occur in several forms on the sperm cells of the same species. For instance, two forms of proacrosin (molecular weights 55 000 and 53 000) and three types of acrosin (MW 49 000, 34 000 and 25 000) were isolated from boar spermatozoa (Parrish and Polakoski, 1978). ~elezn~ and ~echov~ (1982) isolated two forms of acrosin from ejaculated boar sperm. Beta-acrosin (MW 35 000) is formed from alphaacrosin, which has a molecular weight of 49 000 and is probably identical with the ma-acrosin whose isolation was reported by Kennedy et al. (1981) and with the acrosin with a molecular weight of 49 000 isolated by Parrish and Polakoski (1978). ~elezn~ and ~echov~ (1982) expressed the view that two forms of boar acrosin were formed from proacrosin by auto-digestion. This would mean that several active forms of acrosin can be formed from proacrosin. The beta form is probably free and not bound to the acrosomal membrane, while proacrosin and alpha-acrosin are bound to the acrosome. Beta-acrosin is relatively stable in a neutral medium and in the presence of calcium ions it is probably the active form of acrosin in vivo, when it most probably takes effect in the free state. Berruti (1981) purified three forms of acrosin (alpha, beta and gamma) from bull spermatozoa; their molecular weights were approximately 36 000, 34 000 and 25 000. The amino acid composition of the alpha form is similar to the molecular structure of human and rabbit spermatozoal acrosin. Acrosin is an important enzyme and according to the majority of authors, it participates in the fertilization of the egg, by helping the sperm cell to cross the zona pellucida. After the sperm head has penetrated the egg, acrosin, either bound or free, participates in dispersion of the nuclear chromatin (Mann and Lutwak-Mann, 1981). Doubts as to the significance of acrosin for the above functions were expressed by Bedford and Cross (1978) and particularly by Taylor (1983), who demonstrated that rabbit spermatozoa stripped of their acrosomes remained fully fertile. Brown (1982), from investigations on sheep, pig and gerbil eggs came to the conclusion that acrosin has only a synergistic effect. Glycosidases also participate in contact of the sperm cell and the egg (Mann and Lutwak-Mann, 1981). One of them is neumminldase, which breaks down the glycoside bonds in sialopyranosylacetylgalactosamine and may thus play a role in penetration of the sialoglycoprotein-rich zona pellucida. Hyaluronidase can likewise play a given role. Other acrosomal enzymes contributing to penetration of the egg include arylsulphatase and acid phosphatase, the latter of which, according to Harrison (1983), is

23 localized outside the acrosome. Fl~chon (1979a,b) demonstrated glycoproteins on bull, boar, ram and rabbit sperm cells. Klint et al. (1983) likewise isolated two membrane giycoproteins (MW 43 000 and 36 000) from boar spermatozoa. They were present on the head of both ejaculated and epididymal cells. The authors assumed that membrane proteins could participate in initial contacts of the sperm cells with the egg. Kope~n:~ et al. (1980) detected a substance with esterase activity on the acrosome of rabbit sperm cells, while Meizel (1970), Bryan and Unnithan (1972) and Georgiev and Stan~k (1981) demonstrated non-specific esterase activity on bull spermatozoa. In the post-acrosomal zone of human spermatozoa, Clarke and Yanagimachi (1978) described the presence of an actin-like substance which could likewise be of significance in the interaction of the sperm with the egg. Georgiev (1980) demonstrated polymorphism of the proteins extracted from the acrosome of ejaculated bull spermatozoa. Electrophoresis in a cathodal system with a pH of 4.3 clearly showed three types of individual proteins which the author termed A, B and C. Kulangara and Beatty (1971) demonstrated polymorphous spermatozoal proteins determined in rabbits by the albino locus. In concluding the section on sperm cell proteins, it is necessary to recall that a number of these proteins are not of primary origin. They are mostly formed in the superficial parts of the sperm cells by the mutual interaction of the spermatozoids with the fluids surrounding them. These processes pass from the seminiferous tubules to actual ejaculate. The changes in the protein spectrum still continue in the female reproductive tract. From the primitive spermatogonium to the union of the spermatozoon with the egg, the m~le germ cell undergoes continual metabolic and, from the outset, morphological changes. As far as the nuclear proteins are concerned, these changes continue after fertilization until the embryo begins to produce its own proteins. The primary (mainly organ-specific) proteins are likewise formed during meiotic processes and during spermiogenesis in the Sertoli cells. It is probable that new proteins can also be formed after meiosis under genetic control of the haploid genome. Some of the sperm cell proteins -- especially those of secondary origin -- may be released during dilution of the sperm or freezing of the spermatozoa by time factors, which take effect after ejaculation and act as markers of given biochemical processes (Strze~ek et al., 1981). Others, such as spermatozoal androgenbinding protein, symbolize the activity of the Sertoli cells (Le Cacheux et al., 1981). The detection of over 250 polypeptides in the plasma membrane of the ejaculated boar spermatozoa (Russel et al., 1983) is remarkable. To perform the similar investigation and to isolate and characterize (chemically and functionally) spermatozoan proteins in other species of animals is a task for the future. The determination of sperm proteins which induce orchitic changes in the testes would also be useful both in animals and in man. Also, we have little knowledge about the constituents released from

24 dead spermatozoa and about their influence on the viability of sperm not only during in vitro, but also in in vivo conditions. The nuclear basic protein investigations will be very important from the genetical point of view and mainly for future genetic engineering alms. To discover the proteins specific for Y and X spermatozoa would be a fascinating program. There are some papers which try to prove such possibilities (Erickson et al., 1981; Hedge et al., 1983). SPERM CELL PROTEINS AS IMMUNOGENIC COMPONENTS Induction o f immune processes by sperm cell antigens in males The spermatozoon, as a cell which develops long after birth and, morover, adsorbs to its surface a series of specific proteins is immunologically a foreign cell to its bearer. The animal organism consequently has well constructed blood-testicular barriers thoroughly isolating the entire ejaculatory system. Damage to these barriers in general and to the testicular barrier in particular, either b y mechanical injury or by inflammation, leads to the release of sperm precursor cells and sperm cells from the convoluted tubules into the blood bed. The result is an auto-immunization b y these cells, antibody formation and activation of the cellular immune system. The o u t c o m e may be aspermatogenesis of varying extent, in many cases of an irreversible character, and temporary or permanent infertility of the male. Antibodies can be produced either locally, in the genital tract, or outside it and infiltrate from the blood into the seminal fluids (for a review see Riimke, 1974). In most cases, the seminal plasma of infertile men was found to contain only IgG and IgA immunoglobulins, and even these in only very low concentrations. A high serum sperm-agglutinating, immobilizing and cytotoxic antibody titre was not necessarily always a sign of infertility (Rfimke, 1974). When antibodies were present in the seminal plasma in a titre of over 16, such men were always infertile, irrespective of the state of spermatogenesis in their testes (Riimke, 1974; Quinlivan and Sullivan, 1977; Friberg and Tilly-Friberg, 1977). Auto-antigenic sites occur on various parts of the sperm cell. Agglutinins can be adsorbed to the head of the sperm cell and different parts of the tail (Riimke, 1974). If the fluorescence technique is used, fluorescence can be seen on the front of the head, the equatorial segment, on the neck, the midpiece and the tail. The chemical determination of these auto-antigens on different parts of the sperm cell is still in its infancy. All that we know so far is that they are of both a membrane and a nuclear character. The auto-immune character of aspermatogenesis has also been demonstrated in domestic animals. Perez and Quellar Carrasco (1964) described this phenomenon in bulls. Losos et al. (1966) induced testicular degeneration in bulls by means of iso-immunization with spermatozoa and testicular homogenate. Menge (1965), using the same antigenic stimuli, reduced the

25 number and the motility of ejaculated bull spermatozoa, and in later experiments Menge and Christian (1971) induced complete suppression of spermatogenesis. Negative results with the auto-immunization of bulls with sperm were reported (from the aspect of spermatogenesis) by Johnston et al. (1964). No correlation between classification as an unsatisfactory potential breeder and the presence of sperm-agglutinating antibodies in the serum and seminal plasma of bulls has been found (Purswell et al., 1983). Also antibodies against seminal plasma proteins (in man and rabbit) cause agglutination or immobilization of sperm cells (Chen et al., 1971; Yantomo et al., 1971; Shigeta et al., 1980). Koyama et al. (1983) proved human seminal plasma no. 7 antigen (Ferrisplan) was at its highest concentration in the seminal plasma of azoospermic patients, whereas in oligospermic and normospermic men it gradually decreased. Monoclonal antibody to this antigen has sperm-immobilizing activity (Shigeta et al., 1980). It seems that the negative results as regards the damage of sperm motility or spermatogenic processes in bull, ram, boar and rabbit by seminal plasma antibodies (Weft and Roberts, 1965; Menge and Protzman, 1967; Veselsk:~ and Matou]~ek, 1973) are misleading. On the contrary, as already mentioned, certain substances in the accessory sex glands -- immunosuppressive factors (Prakash et al., 1976; Lord et al., 1977; Beer and Neaves, 1978; Marcus et al., 1979) -- seem to give the sperm cells immune protection in the female genital tract. The main immuno-suppressant in bulls is seminal ribonuclease (Stan~k et al., 1978, 1980; Sou~ek and Matou~ek, 1979; Matou~ek et al., 1979; Sou~ek et al., 1981, 1983). Beer and Billingham (1971) also assumed that the above substances in seminal plasma had anti-antigenic function, because they demonstrated that ejaculated spermatozoa did not immunize the female after coitus, whereas epididymal sperm cells were immunogenic.

Induction of immune processes by spermatozoal antigens in females In domestic animals in which coitus or insemination takes place comparatively rarely, the danger of iso-immunization of the female by spermatozoal antigens is substantially smaller than in man. This view is contradicted by the results of Soviet authors (Malinovsky and Ivanov, 1975; Polyantsev and Maximov, 1975; Ivanov and Malinovsky, 1975) who demonstrated not only an increase in the anti-sperm antibody titre, but also reduced fertility in cows after a larger than usual number of inseminations. Experimental immunization also evokes antibody synthesis against sperm antigens in domestic animals and lowers their fertility, as demonstrated in heifers (Menge, 1967, 19.69; Omram and Hulka, 1971) and rabbits (Menge, 1971; Menge et al., 1972, 1975; Bell, 1969). Some authors, however {Kiddy et al., 1959; Goel et al., 1967), failed to demonstrate a decrease in the fertility of heifers and goats immunized with homologous sperm. This question is therefore still an open problem. It is my personal opinion that the possibility of female domestic animals being immunized by homologous sperm ejaculate or insemination into their sex organs is extremely small.

26

Damage to the mucosa of the female sex organs b y inflammation or a venereal disease, followed by insemination with sperm, led to elevation of the spermagglutinin titre in cows (Ilinskij and Salnikov, 1970; Cira~.dinov, 1972; Strze~ek and Bunko, 1973; Marchuk, 1975; Pavlichenko et al., 1975). Some authors believe that natural antibodies -- if they are present in the serum in a high titre and find their way into the cervical secretion -- could also inhibit conception in cows (Zharkin and Osipova, 1975; Bratanov et al., 1975). Futher experiments in this area are still needed, however. CONCLUSION

The previous sections of this review show that both the sperm cells and the fluids of the male genital tract contain many proteins which are functionally very important for reproduction. Some of these proteins are present primarily in the spermatozoa, the others are adsorbed onto sperm secondarily from the fluids surrounding the sperm cells from testes until they reach the female oviduct. These fluids contain a large number of protein (and hence antigenic) substances of significance for the induction and maintenance of the viability, motility, immunological protection and fertility of the spermatozoa. Any interference with the structure and concentration of these proteins can have adverse consequences for the fertilizing capacity of the sexual cells. Some enzyme activities, or physiological activity of non-enzymatic proteins in the spermatozoa and fluids forming the seminal plasma can characterize some cell activity and pathological changes in the reproductive tract. Many studies have elucidated biochemical processes in the sperm which are important for the ability of the spermatozoa to migrate along the female tract and to fertilize the egg. Conversely, we still have a limited knowledge of the protein substances responsible for the function of reproductive cells and organs. It would be useful to do more in the isolation and chemical and functional characterization of these proteins. Most of them are organ-specific substances which are not present in other somatic cells, or other b o d y fluids. They have a special function in reproduction, but can have yet a broader sense in nature.

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