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The induction of infertility by immunization with the sperm-specific lactate dehydrogenase, LDH-C4, suggests its use in a contraceptive vaccine. Development of an immunological contraceptive for human use, however, requires that there be no cross-reactions with somatic tissues. We have demonstrated, using enzyme-linked immunoabsorbence, solid-phase radioimmunoassay, and competitive inhibition radioimmunoassay, that antisera to LDH-C4 is specific and does not cross-react with the somatic isozymes, LDH-A4 and LDH-B4.

Immunization of female rabbits and mice with the sperm-specific isozyme of lactate dehydrogenase, LDH-C 4 , significantly reduced their fertility. Similar studies have been extended to nonhuman primates. Two female baboons, immunized with human LDH-C 4 , produced low antibody titers. These titers were markedly enhanced by booster injections of murine LDH-C 4 . An additional seven female baboons responded with relatively high antibody titers after receiving murine LDH-C 4 as both priming and booster dosages. All nine females received injections of murine LDH-C 4 at varying times determined by serum titer levels during fertility studies. These antisera reacted with human, mouse, and baboon LDH-C 4. In a series of breeding experiments, 22 of 30 matings, or 73%, were infertile as compared with 28% in control matings. This contraceptive effect of the vaccine containing LDH-C 4 was related to antibody titer and was reversible. Normal pregnancies ensued in animals in which the titer declined after termination of booster injections of vaccine.

Lactate dehydrogenase isozymes are inhibited when mercurial reagents are bound to cysteine-165 although no functional role is ascribed to this residue. Identical tryptic peptides containing this cysteine have been isolated from many LDH isozymes, including both A and B subunits. This report identifies an identical peptide from a third subunit type, C, of mouse. The rigorous conservation of this sequence implies an important functional role for this region of the molecule.

Known quantities of spermatozoa were placed directly into the oviducts, via the utero-tubal junction, of rabbits which had been immunized systemically with either Freund's complete adjuvant plus saline (control) or with adjuvant and saline containing sperm-specific lactate dehydrogenase (LDH-C 4 ; immune). Sperm transport through the oviducts was estimated by determining the percentage of sperm recovered from a flask connected to the fimbriae. An average of 26.1% of inseminated sperm was recovered from control oviducts while only 6.8% of such sperm were recovered from immune oviducts (the difference is significant; P 4 in the oviducal fluid of immune rabbits, and is probably a major factor contributing to the infertility of females immunized against LDH-C 4 .

Changes in total and soluble protein in giant foot-pad cells of Sarcophaga bullata pupae follow a biphasic curve with maxima at stages 3 and 9. These polytene cells undergo a sequence of well-defined morphological changes involving deposition of adult cuticle. Alterations in soluble protein species with increases and decreases in concentration, as well as gain and loss of individual components, are associated with developmental events. Molecular heterogeneity and developmental changes of DOPA oxidase, acid phosphatase, and malate dehydrogenase are evident in foot-pad cells. 14 C-leucine incorporation into foot-pad protein is high at stages 5 to 6. These data are discussed in terms of their relationship to cell morphology, function, and chromosome puffing activities.

Bovine spermatozoa aged for 6–7 days at room temperatures (18–30° C.) in a coconut milk extender retained their fertilizing capacity. However, their fertilizing ability appeared to be impaired by environmental hazards in the form of oxygen damage, exposure to visible light, and temperature shock. Protective measures developed and used in the preparation and in the storage of the sperm suspensions to minimize premature loss of sperm function involved the addition of catalase and egg yolk to the extender and the maintenance of cell suspensions in the dark in vials containing a minimal air space. The experimental evidence obtained with this improved coconut milk extender established its ability to support and sustain the fertilizing capacity of bovine sperm for 6–7 days at room temperatures at a level comparable to the fertilizing capacity of refrigerated sperm in milk-glycerol or egg yolk-citrate-glycine extenders. The economic importance of this room temperature diluent is suggested by the fact that it eliminates the costly refrigeration required to maintain stored and shipped bovine semen at 5° C.Coconut milk extender modified by the addition of 5% egg yolk also proved to be a satisfactory storage medium for sperm at low temperatures. It was found to be superior to both egg yolk-citrate and skim-milk extenders in conserving motility at 5° C. The fertilizing capacity of Ayrshire and Brown Swiss sperm stored at 5° C. in coconut milk extender which contained 15% coconut milk and 5% egg yolk compared favourably with the fertilizing capacity reported for sperm stored at 5° C. in other more widely used diluents. The number of motile sperm recovered from this extender after freezing to −79° C. was comparable to the number of motile sperm recovered from the egg yolk-citrate and skim-milk test diluents. The results of this study indicate that coconut milk extender is a versatile, easily prepared medium which can be used to preserve functional sperm over a wide temperature range.

Testes of sexually mature mice (Mus musculus) contain a unique lactate dehydrogenase isozyme associated with the spermatozoan cell. This isozyme differs in subunit composition and reactivity to the substrate DL-α-hydroxyvalerate when compared to the usual five isozymes found in other tissues. It has been termed LDH-X and has the same mobility as LDH-5 when subjected to electrophoresis on standard polyacrylamide gels. This enzyme first appears during development at the time spermatocytes first mature in the testes. From the time of birth until sexual maturity is reached the specific activity of LDH in testes extracts does not change appreciably. When the mice have reached the weight of 6.5 to 7.5 g with a testes weight of 20 mg, which is equivalent to an age of 15 to 17 days postpartum, LDH-X appears. It is at approximately this time that the first differentiative division of the primitive germ cell, the spermatogonium, occurs. For the next period of time, 15–20 days, the testes grow and differentiate at a rapid rate and during this same period the specific activity of the sperm LDH also increses. Histochemical evidence indicated that activity of LDH-1, 2, and 3 is highest in the interstitial cells while LDH-X activity is restricted to the cells that are close to the lumen of the testicular tubule.

Lactate (LDH), malate (MDH), and α-glycerophosphate (GDH) dehydrogenase were studied by polyacrylamide gel electrophoresis using various tissue extracts prepared from Leucophaea maderae. A single GDH was detected in muscle extract from various developmental stages while two forms of MDH were identified in all tissues studied. LDH-1, LDH-2, LDH-3, and LDH-5 were seen in the ovaries during all stages of the adult female's reproductive cycle. Larval ovaries and developing embryos displayed a similar pattern except for the absence of LDH-5, the slowest migrating isozyme. Sub-unit interaction and spectrophotometric experiments support this terminology. Active lactate dehydrogenase in ovaries undergoing vitellogenesis and developing embryos may be involved in supplying energy to these structures that may exist under partially anaerobic conditions.

Four electrophoretic zones of trout intestinal alkaline phosphatase are described on the basis of charge and molecular weight heterogeneity. Qualitative and quantitative differences in the intestinal phosphatase complement suggest three basic phenotypes, which are exemplified by the isozyme patterns of wild brook trout, hatchery brook trout, and lake trout. The partially purified and separated trout phosphatase zones have been characterized physically and chemically. The data presented clearly place these enzymes in the category of the non-specific alkaline phosphomonoesterases. In common with this group of enzymes, trout phosphatases catalyze the hydrolysis of a wide variety of phosphomonoesters and pyrophosphates, as well as transphosphorylation reactions.

1. 1. TTC was used under aerobic and anaerobic conditions to demonstrate succinic dehydrogenase activity in the cestode, Hymenolepis nana. It was observed that there was not equal reduction of the dye in all proglottids comprising the strobila of an intact worm. Reduction was observed in proglottids which were punctured with a surgical needle. Methylene blue was also used to verify the presence of this dehydrogenase. Preliminary studies conducted qualitatively indicated that sodium malonate inhibited succinate oxidation and TTC reduction. 2. 2. Apparent individuality of proglottids of the same strobila was observed and discussed. It was suggested that there are permeability differences along the strobila and that there may be an active and selective absorption of carbohydrates by proglottids, possibly involving phosphorylation processes.

Lactate dehydrogenase (LDH) X has been isolated in crystalline form from mouse testes. Homogeneity of the protein preparation was established by disc gel electrophoresis, analytical ultracentrifugation, and immunochemical analyses. LDH-X is clearly distinct in amino acid composition from LDH-1 and LDH-5 with respect to numbers of residues of leucine, glycine, threonine, and methionine. When 6 to 8 moles of p-hydroxymercuribenzoate are bound per mole of LDH-X substantial enzymic activity remains. A molecular weight of 140,000, comparable to that of beef heart LDH-1, was determined for LDH-X by zone velocity sedimentation in an isokinetic sucrose gradient. When LDH-X is incubated at 65° for 20 min it retains 65% of its original activity as compared to the virtually complete inactivation of LDH-1 and LDH-5.

Electrophoretic analysis of trout liver extracts indicates that an autosomal gene coding for hexose 6-phosphate dehydrogenase (H6P D) in lake trout, Hpd-L, is monomorphic. In brook trout, the gene is polymorphic, having at least three genetic variants termed Hpd-B 1, Hpd-B2, and Hpd-B3. F 1 hybrid splake exhibit the three expected phenotypes resulting from Hpd-L/Hpd-B 1, Hpd-L/Hpd-B2, and Hpd-L/Hpd-B 3 genotypes. Trout H6P D is ostensibly a dimer of mol wt 230,000. The characteristics of trout H6P D, including substrate specificity, genetic polymorphism, electrophoretic characteristics, subcellular localization, and tissue distribution are in close agreement with results obtained for mammalian H6P D. We suggest that trout and mammalian H6PDs, X-linked mammalian G6P D, and autosomal avian G6P D arose from a common ancestral type G6P D.

Peptide maps and amino acid composition of LDH crystallized from skeletal muscle extracts of brook trout (Salvelinus fontinalis) and lake trout (S. namaycush) are similar to those of the hybrid splake trout. The molecular weights are identical to mammalian LDH on the basis of sucrose density gradient centrifugation. The five A′–A isozymes are restricted to LDH from trout muscle and migrate very slowly toward the anode. The B′–B subunit series of isozymes are ubiquitous in trout tissues, although cell specific proportions of isozymes occur. While the A′–A subunit genes must be present in all trout cells, they are active only in the muscle where their products are responsible for essentially all of the LDH activity in this tissue. Isozyme patterns reveal that B subunit-containing tetramers (less anodal) predominate in LDH from liver, whereas B′ subunit molecules (more anodal) prevail in LDH from trout heart. Therefore, extracts from heart, muscle, and liver tissues were utilized to characterize and compare the duplicated subunit tetramers of LDH in terms of several physico-chemical and physiological parameters. Substrate kinetics show that trout B′ subunit tetramers are very sensitive to increases in pyruvate and lactate. These isozymes attain maximum initial velocity at about 0.8 μmole of pyruvate, while substrate inhibition begins at 7 μmoles. The B and the A′–A tetramers reach maximum initial velocity at 1.3 and 3.5 μmoles of pyruvate, respectively, and inhibition does not begin until substrate concentrations of about 20 μmoles are reached. Results are similar with lactate as substrate. Only the B′ tetramers exhibit substrate inhibition with lactate. An analysis of competitive pyruvate and oxamate inhibition reveals that the LDH active site(s) binds one molecule of inhibitor (or substrate). The inactivating temperature for trout B′ subunit tetramers is 70–75°C; for B subunit tetramers, 65–70°C; and for A′–A tetramers, 46–54°. The A′–A and the B subunit-containing tetramers possess the generally lower thermal stability characteristic associated with LDH-5 from most other animals, but thermolability is much more pronounced in the A′–A tetramers. Kinetics of the thermal inactivation process show that it is a first-order, unimolecular event consistent with protein denaturation. Homologies of trout LDH isozymes with those of higher vertebrates are discussed in relation to these data.

Unilateral cryptorchism produced by experimental retention of one testis in the abdomen of the mouse results in a rapid decrease in the weight of that testis. At the same time the specific activity of lactate dehydrogenase (LDH) decreases to 70% of the normal value. The sperm-specific isozyme, LDH-X, declines from 41% of the total activity to only 16% after eleven days in the abdomen. All LDH-X activity is lost by post-operative day 24. The relative proportion of the other isozymes of LDH increase following the operation. LDH-5, absent from the mature testis, is detectable in the cryptorchid testis at post-operative day 28 and represents 15% of the total LDH activity. These observations provide further support of the suggestion that LDH-X is only found in the testes when meiosis is occurring.

Regulation of the immune response is a complex phenomenon under the control of many genes. This process may be analyzed by comparing the immunogenicity of structurally similar but distinct antigens. Both synthetic immunogens with limited numbers of determinants (1) as well as antigenically complex proteins (2, 3) have been used to study immunoregulatory mechanisms. The LDH iso zymes are particularly interesting because they display a high degree of structural similarity but can be distinguished immunologically. Vertebrate LDH is a tetrameric enzyme. All tissues contain variable amounts of A (or M) and B (or H) subunits which randomly associate to produce five isozymes. The genetic control of the immune response to heterologous LDH-A and -B subunits has been studied in rabbits (4), rats (5), and inbred mice (6, 7). Antiserum to purified LDH-A4 or LDH-B4 reacts only with the isozymes containing the subunits used in immunization but not with the heterologous homotetramer (8, 9).

Mouse testes contain a unique form of cytochrome c. As demonstrated by the indirect immunofluorescence technique, the testis-specific cytochrome c is detectable in the primary spermatocyte and in cell types comprising the later stages of spermatogenesis. Interstitial cells, Sertoli cells, and spermatogonia contain the somatic form of cytochrome c, as does heart muscle.

Electrophoretic analysis of the developmental progression of isozymes provides a sensitive method for observing the expression of genes. Glucose-6-phosphate dehydrogenase (G-6-PD) activity profiles and isozyme patterns were examined in developing brook trout ( Salvelinus fontinalis ), lake trout ( Salvelinus namaycush ), and their viable hybrid, splake trout. In all crosses examined, the G-6-PD level of trout embryos dropped immediately upon fertilization and then steadily increased after completion of gastrulation. In the F 1 cross, brook ♀×lake ♂, the maternal isozyme pattern persisted until 1 week after completion of yolk resorption. In F 2 crosses, F 1 splake ♀×F 1 splake ♂, at hatching all embryos revealed either a brook or lake G-6-PD pattern. Thus, stable products from the oocyte (splake pattern) were no longer present, and the preferential expression of one parental gene (probably maternal) was demonstrated. In addition, the hybrid pattern was first detected 2 weeks before yolk resorption. This asynchrony in gene expression reflects incompatability between the maternal protein synthesizing machinery and the paternal genome. Perhaps it involves the recognition of more of the paternal genome than just the structural gene loci. Since the F 1 hybrid pattern was further delayed until after yolk resorption, suppression by a component in the yolk may also be involved.

Testis-specific lactate dehydrogenase, LDH-X, was localized spatially and temporally in the germinal epithelium of the mouse by the indirect fluorescent antibody technique. The enzyme was first detected in midpachytene primary spermatocytes and appeared to increase in concentration as spermatogenesis progressed to the spermatid. LDH-X synthesis, therefore, is initiated in the primary spermatocyte and continues at least until sometime during spermiogenesis. The uniform fluorescence of the cells containing LDH-X suggests that it is distributed throughout the cytoplasm rather than restricted to organelles such as mitochondria.

We have used specific antibodies against mouse phosphoglycerate kinase (PGK) isozymes in an immunohistochemical technique to determine which cells in testicular cross sections contain PGK-A and which contain PGK-B. Phosphoglycerate kinase-A is highly concentrated in interstitial cells and is weakly expressed in Sertoli cells and throughout the germinal epithelium. Phosphoglycerate kinase-B is highly concentrated in spermatids and spermatozoa, but it is not detectable in stage 8 spermatids or earlier gametogenic stages, or in somatic cells of the testis. These results confirm that PGK-B is a sperm-specific isozyme in mice.

The isozyme of lactate dehydrogenase (LDH) that is specific to testes, designated LDH-C4, is the predominant LDH isozyme in mammalian spermatozoa. Four high-affinity monoclonal antibodies have been developed to murine LDH-C4. These antibodies were tested for crossreactivity with LDH-C4 from rat, hamster, rabbit, and human by competitive binding radioimmunoassays. Monoclonal antibodies RG-1 and RG-2 are specific for adjacent or partially overlapping epitopes. The other two monoclonal antibodies each recognize separate and distinct determinants. One of these, designated RG-4, recognizes a sequential determinant that is contained in the coenzyme binding loop, residues 101-115 of the C subunit. Furthermore, RG-4 shows reduced binding affinity for rat LDH-C4 which differs in amino acid sequence at residue 108 and 111 in this region of the molecule. RG-4 also has reduced affinity for LDH-C4 of other species, which is consistent with substitutions in the amino acid sequences of the coenzyme binding loop. These differences between C4 of closely related species is in contrast to the high degree of conservation of this sequence in the LDH-A4 and LDH-B4 isozymes. These results provide useful information regarding homologies among species of LDH-C4 as well as the evolution of this isozyme.

Publisher Summary This chapter describes the assay method and properties of lactate dehydrogenase isozymes isolated from the mouse. Three homotetrameric isozymes of lactate dehydrogenase, designated as A 4 , B 4 , and C 4 or X, are found in mice. Each isozyme is expressed by a separate structural gene. All the lactate dehydrogenase isozymes require nicotinamide adenine dinucleotide kinase (NAD + ) or nicotinamide adenine dinucleotide dehydrogenase (NADH) as the coenzyme and catalyze the same chemical reactions. The purification of these enzymes is achieved by general ligand affinity chromatography in combination with ion-exchange chromatography to separate the multiple forms of lactate dehydrogenase. The activity of lactate dehydrogenase isozymes is determined at 25°C spectrophotometrically by following the changes in absorbance at 340 nm. The reaction mixture in a total volume of 1 ml contains 0.1 M Tris-HCl, pH 8.0, 1.0 m M pyruvate, 0.15 m M NADH, and a suitable amount of enzyme to produce a decrease in the absorbance of 0.05–0.l per minute at 340 nm. Lactate dehydrogenase isozymes are virtually inactive with α-ketoglutarate as the substrate. Thin-layer chromatography of the product is performed on cellulose plates with a fluorescent indicator.

Publisher Summary This chapter discusses the isolation of sperm specific lactate dehydrogenase (LDH) from mouse, rabbit, human testis, and human spermatozoa. The isolation of sperm-specific antigens facilitates the analysis of immunologically induced infertility and of the physiological role of these unique gene products of the male gamete. One such protein is the lactate dehydrogenase C4 isozyme (LDH-C4). This isozyme is distinct from the LDH isozymes found in other tissues. It is antigenic in both males and females, and the immune response to this isozyme can be manipulated to impair fertility. LDH-C4 has been purified from testes of the rat by the conventional methods of salt fractionation, ion-exchange chromatography, and gel filtration. A generalized procedure for the isolation of LDH-C4 can be based on an 8-(6-aminohexyl)-amino-5' AMP–Sepharose affinity gel and ion-exchange chromatography. The availability of this purified enzyme can facilitate studies of the role of the immune system in reproduction.

LDH-C4 is the form of lactate dehydrogenase unique to sperm and testes. Antibodies to LDH-C4 do not bind the somatic forms of this enzyme. We are making monoclonal antibodies specific for antigenic determinants of mouse LDH-C4 and are using these antibodies as probes to identify determinants is common among several species. Spleen cells from SJL/J mice immunized with mouse LDH-C4 were fused with P3-X63-Ag8 myeloma cells and four hybrid cell lines generated using cell fusion techniques. Scatchaitf analyses established that the antibodies have high affinities (Ka = 109 M-1) for LDH-C4 and are monoclonal. These antibodies were tested for cross-reactivity with rat, hamster, rabbit and human LDH-C4 using competitive binding assays. Monoclonal antibody (1)B-6:G1 detects a determinant common to all five species. Antibody (3)F-11:F4 is specific for a determinant unique to mouse LDH-C4. Antibody (3)F-12:G8 binds to mouse, rat rabbit and human LDH-C4 and antibody (3)A-8:H10 reacts with LDH-C4 from the mouse, rat, hamster, and rabbit. These results verify that each antibody is directed against a different determinant of LDH-C4 and, in addition, provide useful information for determining the amount of structural homology among different species of LDH-C4. Supported by NIH Grants R01 HD05863 and T32 HD07068.

The use of a well-defined, synthetic antigen is essential to progress in developing a vaccine for fertility control and to establish with certainty the utility of such technology. The studies with LDH-C4 are consistent with the feasibility of using a synthetic antigen in a vaccine to control fertility. LDH-C4 remains the best-developed candidate for future studies. Immunization with LDH-C4 does suppress fertility, and the complete biochemical characterization of this isozyme is well underway. These studies will provide a wealth of synthetic antigens to substitute for the natural product in experiments to determine whether immunologic contraception is appropriate and desirable for wide-scale application in human beings.

From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA. The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11. The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldh1, Ldh2, and Ldh3, respectively.

Development of an immunological contraceptive as an alternative birth control method requires an antigen that is gamete-specific, readily available, safe, and effective. With respect to these requirements, lactic dehydrogenase isoenzyme C4 (LDH-C4), found exclusively on sperm and in testis, is a promising candidate for a vaccine. This antigen has been extensively analyzed by immunological and biochemical methods. It has been found to reduce fertility when administered systematically to females in a number of animal models, including mice, rabbits, and baboons. Local immunization into the reproductive tract of female mice to produce IgA at the site of fertilization also results in marked fertility reduction. Rather than attempting to isolate large quantities of LDH-C4 for a vaccine, antibody binding sites on the LDH-C4 molecule are being studied to determine whether they can act as synthetic antigens to replace intact LDH-C4. One such synthetic peptide representing amino acids 5–15 of mouse LDH-C4 was attached to a macromolecular carrier, diphtheria toxoid, and used to immunize female baboons. These animals made antibody that bound to the native LDH-C4 molecule. Mating resulted in a 70% reduction in their fertility over that of nonimmune controls. This contraceptive effect was reversed when immunizations were discontinued. There were no apparent immunopathological side effects of the vaccine. These experiments confirm the feasibility of using a synthetic antigenic determinant of sperm-specific LDH-C4 as a vaccine to control fertility. Furthermore, specificity, availability, effectiveness, reversibility, and safety requirements for a successful immunological contraceptive technology can be satisfied by this approach.

Hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) was purified from mouse testes by ion-exchange chromatography, Sephadex G-200 filtration and Con A-agarose affinity chromatography. The final preparation had 94-fold purity and 12.2 units spec. act. of the enzyme (unit of specific activity = mumol N-acetylglucosamine released/h per mg protein at 37 degrees C and pH 4.5). Hyaluronidase is relatively heat stable and loses 10-20% of its activity at 50-55 degrees C for 10 min. Ea for eat denaturation of enzyme is 42-45 kcal between 45 an 63 degrees C. The Michaelis constant of mouse testicular hyaluronidase is 1.1 mg/ml hyaluronic acid. Antibodies to the purified enzyme were produced in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. Antiserum to hyaluronidase inhibited enzyme activity by 25%. Immunologically, mouse testicular hyaluronidase is species specific. Tissue extracts of mouse vital organs, except testes and epididymis did not react with the antisera, though nonspecific precipitation occurred between intestinal extracts and anti-hyaluronidase serum. Hyaluronidase was localized in testis sections by indirect immunofluorescence. A specific dark green fluorescence was localized on cell boundaries extending from spermatogonia to spermatids and appeared on the sperm acrosome. Cytoplasm of spermatogonia and spermatocytes showed light green fluorescence, whereas interstitial tissue was devoid of fluorescence.

Mature testes and spermatozoa of most mammalian and at least one avian species contain the C 4 isozyme of lactate dehydrogenase (LDH-X). This isozyme is unique in terms of the remarkable temporal and cellular specificity of its synthesis. It appears first in the primary spermatocyte, is found in no other tissue, and is completely absent from the female. Certain biochemical and enzymological properties of LDH-X correlate well with the requirements of sperm metabolism but the physiological rationale for a third subunit type remains unclear.

LDH-X, the sperm specific isozyme of lactate dehydrogenase was used as an antigen to induce infertility in female mice. Injection of rabbit anti-mouse LDH-X serum on Days 1-4 after coitus significantly reduced the number of pregnancies in these animals below the level observed in mice receiving serum from nonimmunized rabbits. Pregnancy frequency in mice injected with LDH-X in Freund’s Adjuvant was significantly decreased within 6-8 wk after the primary immunization. As a model to explain the mechanism of antibody action, we propose that supernumerary sperm within the perivitelline space of fertilized mouse ova provide specific recognition sites which are available to antibody to LDH-X. The antibody-antigen complex confers upon the embryo a heteroantigenicity which leads to its immunological rejection.

Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.

The sperm-specific isozyme of murine lactate dehydrogenase (LDH-C4) was injected into female mice of various strains. Two regulatory phenotypes characterize the resultant immunity to LDH-C4: one is manifested by high, intermediate or low levels of response, the other by the immediate or delayed maturation of peak titer. The response of several strains can be classified as high (SWR, SJL, BABL/c, C3H/He) and intermediate to low (A, CBA, DBA/2, DBA/1, C57BL/6) according to the level of antibody production and cell mediated immunity. BALB/c, SJL and SWR strains are immediate responders while DBA/2 and C3H/He mice are clearly delayed responders. Maturation and magnitude of response do not appear to be related. Both the antibody and cell mediated responses are T-dependent, but are not obviously associated with Ig allotype or H-2 regulation.

Female Swiss Webster mice were immunized intraperitoneally with mouse epididymal spermatozoa or with phosphate buffered saline (PBS) and their fertility was compared by (1) incidence and size of litters, (2) number of uterine implantation sites, and (3) incidence and number of fertilized eggs in the oviducts. Statistically significant reduction in fertility was noted following two courses of injections of spermatozoa; 12% of mice injected with spermatozoa had litters compared with 80% of mice injected with PBS. The infertility did not seem to be related to a failure in fertilization since the two groups of mice had a similar incidence and number of fertilized eggs in the oviducts. All female mice were found to have a ‘natural’ anti-acrosomal antibody. Following immunization with spermatozoa, antibodies to ‘postacrosomal’ region, the main piece and the midpiece of the tail, as well as cytotoxic antisperm antibodies, appeared. Anti-LDH-X antibody was not detected. However, correlation was not found between infertility and antisperm antibodies or sperm granulomata that developed in the peritoneal cavities. It is concluded that female mice receiving repeated i.p. injections of mouse spermatozoa become infertile and that the infertility is related to interference with events after fertilization.

A peptide bearing an antigenic determinant of the sperm-specific lactate dehydrogenase C4 isozyme (LDH-C4) has been isolated from a tryptic digest of the whole protein. This peptide, comprising residues 152-159 (MC152-159), reacts with rabbit anti-mouse LDH-C4. Immunization of rabbits with synthetic MC152-159 conjugated to bovine serum albumin induces an immune response which is specific for the peptide. Anti-MC152-159 IgG binds 125I-labeled mouse LDH-C4 and competition experiments demonstrate the specificity of this antigen antibody reaction.

Immunological regulation of fertility can be best achieved if a defined macromolecule is used to provide the antigenic stimulus. Proteins with known enzymatic properties are attractive molecules in this area if it can be demonstrated unequivocally that they are unique constituents of spermatozoa, and are completely foreign to the female. This paper describes immunosuppression of fertility by the sperm-specific isozyme of lactate dehydrogenase (LDH-X). In addition, evidence is reviewed which suggests that acrosomal proteinase and hyaluronidase may be unique to sperm. Data are presented which confirm the sperm specificity of LDH-X and the immunochemical homogeneity of purified preparations of this isozyme. Immunization of female rabbits with LDH-X significantly reduces the fertility of these animals. Experimental results indicate that the primary effect of immunization involves blockage of fertilization. While those ova which are fertilized in an immune environment develop and implant normally, there is a high rate of post-implantation embryo mortality. Rabbit blastocysts transferred to immune recipients implant at the same rate as in non-immune controls. These findings are consistent with the conclusion that the developing embryo must interact with antibody during the pre-implantation stages of pregnancy when antigenic sites conferred by the sperm LDH-X are available. Such interaction can be demonstrated directly by immunofluorescence techniques.

The natural response of the immune system to foreign proteins can be exploited in the development of a vaccine for control of fertility. Potential target antigens for such a vaccine include those that are found specifically on the surface of sperm and are absent from somatic tissues (reviewed by Aitken, 1982, Goldberg, 1983, Anderson and Alexander, 1983). Such antigens are uniquely suited to incorporation into a vaccine that may be applicable to both male and female use. They do not appear in the male until spermatogenesis begins, which is after the immune system has already become tolerant to “self-antigens”. Even after they appear, they are isolated from the male immune system by the blood-testes barrier. They are not found at all in female tissues. Thus these antigens can provoke antibody formation in either males or females. Furthermore, a vaccine would have the advantage over currently available contraceptive methodologies of not interfering with hormonal and metabolic functions.

An unusual pattern of LDH isozymes was observed by gel electrophoresis of an extract of a human testis. This isozyme composition is consistent with an allelic variant of Ldh-c.

1. 1. Spectroscopic analysis of intact larvae revealed the absorption bands in the approximate positions of cytochrome c, b, and a + a3. In addition the bands for hemoglobin and oxyhemoglobin were observed and appeared to be more intense in larvae which had been in the host muscle for at least 6 weeks. 2. 2. The catalytic activity of cytochrome oxidase and the cytochrome c reductases was determined spectrophotometrically in both larvae and adults. The enzymes appeared to be less active in the adult worms. Cytochrome oxidase was inhibited by cyanide, and by carbon monoxide in the dark. The latter inhibition was reversed by light. Succinic dehydrogenase activity was inhibited by malonate. 3. 3. The respiration of homogenates of the larvae is stimulated by succinic and α-ketoglutaric acids. Increased oxygen consumption in the presence of carbon monoxide was also observed. 4. 4. Enzymes of the tricarboxylic acid cycle, including aconitase, isocitric dehydrogenase, fumarase and malic dehydrogenase were measured in homogenates spectrophotometrically. Citrate formation from oxalacetic acid and acetate was demonstrated, utilizing paper partition chromatography. 5. 5. The evidence presented strongly indicates that the cytochrome system for electron transport and the tricarboxylic acid cycle for terminal oxidative processes occur in the tissues of the larvae of T. spiralis.

Speckled trout and lake trout contain five forms of lactate dehydrogenase, but a diflerent electrophoretic distribution of isozymes characterizes each species. The hybrid splake, which is produced artificially by fertilizing lake trout eggs with speckled trout sperm, contains nine isozymes. This comnplement of isozymeg In vivo could be produced in vitro by recombination of subunits from tissues of the parent species. In the splake trout, this complement is the result of heterozygosity at the gene locus responsible for synthesis of LDH-5. Extracts of trout eyes contain at least two additional forms of LDH which could not be demonstrated in other tissues.

Two fundamental physical properties of partially purified trout intestinal alkaline phosphatases which have been examined in detail are thermostability and activation energy. It has been clearly demonstrated that differences in thermostability exist between the trout isozymes, and that as a group they are more thermolabile than mammalian phosphatases. The activation energies of the two main trout phosphatase zones, however, are comparable to mammalian phosphatases. Experiments on cold-acclimation of hatchery brook trout suggest that a fundamental reorganization of the intestinal alkaline phosphatase complement occurs. There are drastic quantitative differences in the isozyme patterns of 4° C-acclimated fish and those acclimated to 10° C or 15° C. The 4° isozyme complement has a lower activation energy in the range of 2° C−10° C than does the 10° C isozyme complement. This finding strongly suggests that there is a reconstruction of the phosphatase isozyme complement with cold-acclimation. This is functionally significant in that it results in the production of enzymes better suited to the new thermal regime. Three mechanisms are discussed as possible regulators of this process.

Measurement of lactate dehydrogenase in reciprocal hybrids of trout during development revealed that a maternal effect was involved in the regulation of enzyme levels until resorption of the yolk sac was completed. Malate dehydrogenase specific activities were the same in these embryos and larvae. The more negatively charged B subunits of LDH predominated during early stages of embryogenesis in lake trout and brook trout with an increase in synthesis of A subunits evident as development progressed. Activation of the paternal A gene in reciprocal hybrids occurred at a relatively late stage with the LDH subunit specific to the retina appearing after hatching. Analysis of brook trout progeny from a cross of parental types with a variant and wild-type B subunit suggested nonrandom LDH tetramer assembly which may be genetically controlled.

Previous studies on the intermediary metabolism of Trichinella spiralis larvae have indicated that the conventional tricarboxylic acid cycle and cytochrome system are present in the tissues of this parasite (Goldberg, 1957a). To elucidate further the possible pathways of substrate-utilization by this organism, enzyme studies on the primary steps of carbohydrate-degradation have been carried out. The present communication is concerned with glycolysis in T. spiralis larvae. A preliminary report of these results has been presented elsewhere (Goldberg, 1957b).

The immobilizing and lethal effect of visible radiation on mammalian and avian sperm appears to be the result of a photosensitized oxidation (photodynamic action) initiated by an endogenous intracellular pigment acting as a photonacceptor. The action spectrum of the photoimmobilization of bovine sperm reveals maxima at 440, 480, and 550 mμ. Its approximate correspondence with the absorption spectrum of these cells implicates a heme-protein, possibly one of the cytochromes as the photosensitizing agent. Excitation of this intracellular chromophore by light probably results in a triplet sensitizer-oxygen complex, free radical formation and in the induction of a primary lesion presumably in the cytoplasmic portion of the cell. Further evidence for the cytoplasmic localization of the photosensitizer was obtained from the photoinactivation of decapitate but motile and metabolically active sperm tails. Observations on the decline in the fertilizing capacity of irradiated cock sperm and on the inability of the majority of eggs fertilized with irradiated sperm to develop normally suggest that light exposure may indirectly impair the nucleic determinants in the sperm nucleus. That the photodynamic action may involve an autocatalytic reaction is inferred by the time-course of the photoreaction in terms of the decline in motility of sperm during irradiation, and by the observation that even subimmobilizing exposures of sperm to light are sufficient to eventually cause loss of motility followed by cell death, in the dark. The rate of this “dark reaction” is related to the number of quanta captured by the sensitizer. Finally, using visible light as a stressor, and spermatozoa stored in vitro as the biological test sytem, it has been possible to obtain quantitative data which establish a positive correlation between sensitivity to stress (visible radiation) and cell age. This is discussed in reference to theories of cell senescence.

Exposure to light of bovine spermatozoa suspended in various media results in a progressive decline in metabolic activity followed by the premature death of the cells. The inhibitory and spermicidal effects of visible light resemble a photodynamic action; the photosensitizer is an intrinsic component of the cell.

There are five G6P-specific G6PD isozymes in both brook and lake trout. The most anodal isozyme in each species has the same electrophoretic mobility; however, the five lake trout isozymes are more widely spaced on polyacrylamide gels than are those from brook trout. In hybrids, i.e., splake trout, nine forms of G6PD can be resolved. These results can be explained by a model in which we assume that each isozyme is a tetramer and that two different subunit types are produced. In splake trout, three electrophoretically distinct subunits yield 15 tetramers. That only nine are detected is a consequence of coincident electrophoretic mobility of some of the possible subunit combinations. Our results indicate that the G6PD isozymes in these trout are the products of two codominant autosomal gene loci. The hypothesis that G6PD and H6PD arose from a common ancestral type G6PD is supported by microcomplement fixation data which show an immunochemical relatedness between these enzymes. The relationship of G6PD and H6PD in trout is discussed from an evolutionary standpoint.

Sperm-producing testes of mammals and some birds contain a unique form of lactate dehydrogenase (EC 1.1.1.27) which is a tetramer composed of C subunits, rather than the A and B subunits of lactate dehydrogenase isozymes 1-5. Immunochemical evidence is presented which confirms the hypothesis that the C subunit is encoded in a separate gene from the A and B polypeptides. The evolutionary relationships of these genes are discussed.

1. Brook trout hexose-6-phosphate dehydrogenase (H6PD) and lake trout H6PD are essentially identical in several parameters including pH optima, kinetic properties and activation energy. They differ in thermal stability. 2. The various oxidations catalyzed by trout H6PD differ in their response to the presence of Mg 2+ , ATP and several anions. 3. The apparent K m 's of all four substrates show a decrease with decreasing temperature. 4. Kinetics of substrate oxidation by H6PD suggest that the enzyme most likely functions as a G6PD.