Serum allotypes in ovarian follicular fluids of pigs

Summary. Sera and ovarian follicular fluids of 158 sows were tested with 27 allotype reagents. Immunodiffusion in agar gel (microtest) and haemagglutination inhibition were used as detection methods.
Out of eight 'individual' (Lpb 1,-2,-3,-4,-5,-6,-7,-9) and four 'common' (Lpb 12,-13,-14,-16) specificities of serum beta-lipoproteins (LDL), 11 were present in sera, but none in follicular fluids. On the other hand, Lpr 1 and Lpr (x) allotypes of the VHDL + VLDL beta-lipoprotein system were detected both in sera and in follicular fluids. Of four antigens of the Gp system (Gp A,-a, -B,-b), only the 'dominant' characters, Gp A and Gp B, occurred in the follicular fluid. The typing of polymorphic IgG immunoglobulins (IgG-a or IgG-b system) showed that B1 or A2, B2 or A1 and B3 or A(x) allotypes could be detected both in serum and follicular fluid. Among allotypes that were not yet genetically classified, only the P3 specificity was not found in the population tested. The G1 allotype (preliminarily described as an alpha-globulin) was present in sera only, and the remaining allotypes, G9, P1, P16 and P23 (alpha- or beta-globulins) were present both in sera and follicular fluids.
The mechanism of the transmission of serum proteins into ovarian follicles and their possible importance is discussed.     

Immunogenetic Polymorphism of Lipoproteins in Swine: Genetic, Immunological and Physiochemical Characterization of the Two Allotypes Lpr1 and Lpr2

Results of immunogenetic, immunochemical and physicochemical investigations on two serum allotypes of swine are reported. The allotypes, designated Lpr1 and Lpr2, have been identified by specific alloprecipitins in agar gel. Genetic studies indicate that the allotypes are specified by two codominant autosomal allelic genes, Lpr1 and Lpr2. All pigs 3 months of age or older were classified as belonging to one of three phenotypes, Lpr1, Lpr2 or Lpr1,2, each corresponding to one of three genotypes Lpr1/1, Lpr2/2 or Lpr1/2, respectively. The Lpr1 gene was absent or was found at low frequency in the breeds tested. The allotypes were found to occur in two physicochemical forms; in association with chylomicrons and very low density lipoproteins (VLDL) and, primarily, as a Lpr multimer free of the major lipoproteins showing very high density (VHD), d greater than 1.21 g/ml, and MW +/- 190,000. Gel-electrophoretic mobility for VHD-Lpr is different for each of the three Lpr genotypes residing in gamma-fast and beta-slow regions, but is identical for VLD-Lpr in which Lpr was found complexed with apo-B, migrating as VLDL in the alpha-2 slow (pre-beta) region. Serum levels of Lpr varied during the lifetime and between individuals and, especially, between sera of homozygous pigs being higher in Lpr1/1 than Lpr2/2. A linear relationship for Lpr1 and an atypical, inverse relationship for Lpr2 have been observed between the gene dosage, heterozygous vs. homozygous, and the Lpr serum level.     

Identification of new apolipoprotein B epitopes and haplotypes and their distribution in swine populations

Results from comparative immunogenetic studies on inheritance and identification of four new apolipoprotein B (apoB) allotypes and three additional apoB haplotypes and their distribution in miniature and domestic swine are presented. Immunological surveys on the four new and 16 previously described Lpb allotypes and genetic analysis of their segregation in progenies, of miniature and domestic swine and their crosses, indicate that three new allotypes designated Lpb9, Lpb10 and Lpb101 are individual (mutant) apoB epitopes, each representing a discriminating marker for one of the new apoB haplotypes specified by three new apoB alleles designated Lpb⁹, Lpb¹⁰ and Lpb¹⁰¹. The fourth allotype, Lpb20, is one of the common epitopes forming the alternative epitope pair with Lpb10, and is a constituent of each of the eight previously described and two new apoB haplotypes. The new apoB alleles have so far been found only in miniature swine, with Lpb¹⁰ being the most frequent in the Göttingen, Vietnamese Potbelly and Japanese Miniature, Lpb⁹ was detected only in Minnesota Miniature and Lpb101 only in Vietnamese Potbelly. The common allotype, Lpb20, shares immunological similarities with human apoB indicating its ancestral origin, whereas none of the alloreagents detecting the three individual apoB variants, Lpb9, Lpb10 or Lpb101, showed cross-reactivity with human apoB, suggesting their exclusive swine origin and evolvement during speciation through mutations.     

The results of international comparative testing of reagents to swine serum protein allotypes conducted in 1987-1988

Antisera to 15 allotypes of pig serum protein were raised and compared with those prepared elsewhere, within the framework of International Comparison Test of pig blood groups and polymorphic proteins and enzymes in 1987-1988. Of 15 allotypes tested 8 were found to coincide with the known GpB, GpD, GpA, Gpa, IGH3 C1, Lpb3, Lpb12, Lbr1. For 7 allotypes left, no analogues were found out. They seem very likely to be new allotypes of pig serum proteins.     

Characteristics of some breeds of swine and populations of Georgian and Western Siberian wild boar by immunogenetic systems of blood serum proteins]

A bank of reagents for hog serum protein allotypes has been created. All of these allotypes passed international comparison tests in 1987-1988. The bank can be used for typing the animals for four generally accepted (Gp, LpB, Lpr, and IgGH) and several experimental systems. In this study, immunogenetic characteristics of some pig breeds (Large White, Lithuanian White, Swedish Landrace, Kakhetinskaya, and Svanetskaya) bred in Georgia are compared with those of western Siberian breeds (Large White, Kemerovskaya, and Northern Siberian), and some foreign breeds, as well as with European, Caucasian, and Siberian subspecies of the wild boar.     

Separation of swine plasma LDL from Lpb2/3 heterozygotes into two apoB allelic haplotypes, Lpb2 and Lpb3, with apoB epitope specific antibodies

Studies were performed to investigate the separation of Lpb (lipoprotein B) species present in plasma of heterozygous swine bearing the Lpb2 and Lpb3 apoB mutant genes. Low density lipoprotein (LDL) fractions from Lpb2/2 and Lpb3/3 homozygotes were coupled to a matrix and used to isolate affinity-purified antibodies anti-Lpb2 and anti-Lpb3 from swine alloimmune sera, one with specificity for the Lpb2 epitope(s) and the other for Lpb3. These antibodies in turn were used to construct two immunosorbers, anti-Lpb2 and anti-Lpb3 Sepharose columns. To separate the two Lpb haplotype populations present in LDL, a density gradient ultracentrifuge subfraction (d 1.032-1.043 g/ml) obtained from Lpb2/3 heterozygous pigs was applied to the specific immunosorbers. The retained fraction from the anti-Lpb2 column reacted in the double immunodiffusion test with anti-Lpb2 and anti-Lpb13 immune sera but not with either anti-Lpb3 or anti-Lpb12, while the unretained fractions reacted with anti-Lpb3 and anti-Lpb12 but not with either anti-Lpb2 or anti-Lpb13. The reaction patterns obtained with the two sets of alloimmune sera indicate the existence of two separate lipoprotein populations in LDL: one lipoprotein carrying the Lpb2 and Lpb13 epitopes corresponding to the Lpb2 apoB allele, and the other carrying the Lpb3 and Lpb12 allotypes specified by the Lpb2 gene. Immunoblotting with anti-Lpb2 and anti-Lpb3 and silver staining showed that the epitopes of both isolated LDL subpopulations are associated with apoB-100. Neutral lipid analyses showed no differences between the isolated Lpb2 and Lpb3 lipoprotein species from the Lpb2/3 heterozygotes. These studies demonstrate that plasma LDL subfractions from Lpb heterozygous swine can be separated into two haplotype populations, each corresponding to the product of one apoB gene, and reveal a new insight into the phenotypic expression of plasma LDL, and the LDL phenotype-genotype relationship. Furthermore, this approach will facilitate studies on metabolic differences of two structurally distinct LDL, unaffected by in vitro manipulation, exposed to the metabolic milieu of one individual.     

Allotype polymorphism of serum globulins (Gp system) in pigs

By alloimmunization with whole serum reagents were obtained which revealed two new allotypes, GS and P30, in pigs. Evidence is presented that the allotypes G8, P30 and previously described G4 and G6 are controlled by three complex autoso-mal allelic genes forming a closed system. For this new allotype system we propose the designation Gp (globulin. pig). For alternative G4-GS and G6-P30, forming pairs of mutually exclusive alloantigens. it is proposed to use a standard designation GpA-Gpa and GpB-Gpb respectively.     

Characteristics of Immunogenetic Systems and Blood Serum Proteins in Some Pig Breeds and Georgian and Western Siberian Wild Boar Populations

A bank of reagents for hog serum protein allotypes has been created. All of these allotypes passed international comparison tests in 1987–1988. The bank can be used for typing the animals for four generally accepted (Gp, LpB, Lpr, and IgGH) and several experimental systems. In this study, immunogenetic characteristics of some pig breeds (Large White, Lithuanian White, Swedish Landrace, Kakhetinskaya, and Svanetskaya) bred in Georgia are compared with those of western Siberian breeds (Large White, Kemerovskaya, and Northern Siberian), and some foreign breeds, as well as with European, Caucasian, and Siberian subspecies of the wild boar.     

Homology between the Lpm system of allotypes in the American mink and the Gp system of allotypes in the domestic pig]

The 5 alpha-macroglobulin allotypes alpha M1, alpha M2, alpha M3, alpha M4 and alpha M5 were identified in pig. The alpha M1 allotype was reported as a marker of pig alpha-macroglobulin, the latter being homologous to alpha 2-macroglobulins in human and in mink. The allotypes alpha M2-alpha M5 were specified as markers of the second isotypical variant of pig alpha-macroglobulins, which was homologous to mink Lpm macroglobulin (alpha 1M). As seen from data obtained by International Comparative Test ISABR 87-88, alpha M1 is a new allotype, while allotypes alpha M2--alpha M5 correspond to four allotypes in the Gp system (Janik et al.). Based on these data, a conclusion was made on the homology between the Lpm system in american mink and the Gp system in pig. Since the allotypes studied are the part of alpha-macroglobulins, a locus controlling them was designated the AM locus. We also find it more advantageous to apply the same name to the homologous locus in mink, instead of the Lpm used earlier. Genetic control of 5 allotypes was studied and the structure of the AM locus in pig analysed in detail. Comparative study of organization of the above locus and the homologous locus in mink was carried out.     

Rodgers (Rg) and Chido (Ch) determinants on human C4: characterization of two C4 B5 subtypes, one of which contains Rg and Ch determinants

The genetically determined polymorphism of the fourth component of human complement was further extended with the aid of a panel of human allo-anti-C4 sera, anti-Rodgers and anti-Chido. These antisera were found previously to react with the alpha-chains of the C4 molecules controlled by the C4A and C4B loci, respectively. We analyzed a number of new and rare C4 allotypes, and found that they generally followed the expected pattern. Some interesting exceptions, however, were found. The alpha-chain of the allotype C4A1 was found to react with anti-Chido, unlike all other C4A allotypes. Also the C4B5 allotype could be subdivided into two subtypes on the basis of their reaction with anti-Rodgers. They were tentatively named B5Rg+ and B5Rg-. Moreover, the B5Rg+ subtype reacted not only with anti-Rodgers but also with some anti-Chido sera, indicating for the first time that Chido and Rodgers determinants are present on the same allotype.     

A new lipoprotein allotype Lprs and allele Lpr1,3 in the pig

Specific alloprecipitins were found in blood plasma of pigs, immunized by sera of Lpr1 positive donors. These precipitins detected a new allotype of the lipoprotein Lpr system which was designated Lpr3. Genetic studies confirmed its codominant inheritance and subgroup character. This linear subgroup of allotype Lprl is controlled by the allele Lpr^1,3. Investigations in populations of 14 pig breeds showed significant interbreed differences in the frequencies of alleles Lpr¹, Lpr² and Lpr^1,3.     

Genetic chasing of T helper cell idiotype and allotype genes

The present experiments were performed to study whether the genes responsible for the expression of T-cell idiotypes and allotypes could be mapped in relation to immunoglobulin (Ig) heavy chain V- and C-genes. Use was made of our antiserum 5936, which detects idiotypes in B6 anti-B10.BR sera and on Lyt-1⁺, 2.3^–B6 anti-B10.BR T-cell populations, and antiserum 6036, which detects allotypes on Lyt-1⁺, 2.3^–B6 T cells, but which does not react against Ig. The reactivity of these antisera with T cells from (B6 x C3H.OH) x C3H.OH backcross mice and CBA-allotype congenic B6 mice was investigated because 5936 idiotypes and 6036 allotypes appeared to be associated with Igh-1 ^b genes (B6) and not with Igh-1 ^b genes (C3H.OH, CBA). Our results will show, first, that 5936 idiotypes on Lyt-1⁺, 2.3^–B6 anti-B10.BR T cells are synthesized by genes linked to Igh-1 ^b allotype genes and they are situated either within Ig heavy chain V-genes or centromeric to them. Second, our results will show that 6036 allotypes on Lyt-1⁺, 2.3^–B6 T cells are produced by genes also linked to Igh-1 ^b -allotype genes, and the 6036 allotype genes are situated between Ig-VH and prealbumin genes.Abbreviations used in this paper BCGF B cell growth factor - B6 C57B1/6 - C_H constant region of immunoglobulin heavy chain - Con A concanavalin A - FCS fetal calf serum - Id idiotype - Ig immunoglobulin - LPS lipopolysaccharide - M mouse - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MRBC mouse red blood cells - NMS normal mouse serum - NP nitrophenacetyl - NRS normal rabbit serum - PFC plaque forming cell - R rabbit - Tcf T cell factor - Tcr T cell receptor - TNP Trinitrophenol - V_H variable region of Ig heavy chain Definitions of terms used in this paper: T-cell idiotypes, structures on T-cell membranes or released T-cell molecules detected by an anti-idiotypic antiserum (5936) produced against specific immunoglobulin idiotypes. The 5936 T-cell idiotypes are related to the specific binding of IA^k gene products by certain Igh-1^b T cells. T cell allotypes, structures on T-cell membranes or released T-cell molecules detected by an antiserum (6036) produced against 5936 idiotype-bearing T-cell molecules. The 6036 T-cell allotypes are related to the binding by Igh-1^b T cells of all Ia gene products tested, and they are non-cross-reactive with immunoglobulin allotypes.     

Allotype suppression in the chicken. V. Abnormal isotype ratios of chronically suppressed IgM and IgG allotypes

Chronic allotype suppression was generated in M-1 (C mu), G-1 (C gamma) heterozygous B14 line chickens either by embryonal injection or by maternal transfer of anti-M-1b allotype antibodies. Using a sensitive radioimmunoassay to detect the suppressed IgM-1b, a striking disparity was observed between the degree of suppression of the IgM-1b allotype and that of the genetically linked IgG-1i allotype. The amount of suppressed IgM-1b ranged between 0.2 and 3% of total serum IgM in chronically suppressed birds. The levels of the genetically linked IgG-1i, however, comprised 5 to 25% of total serum IgG in such birds. The allotypes measured in suppressed chickens were qualitatively identical to those in normal, nonsuppressed birds by serological criteria. These results are discussed within the context of isotype regulation in the chicken.     

Preferential assignment of allotype a1 globulin for the production of early IgM anti-para-azobenzenearsonate antibodies in heterozygous a1a3 rabbits.

Rabbits of allotype a1a3 were injected on days 0, 2, and 4 with mixtures containing equal amounts of pigeon erythrocytes (Prbc) coupled to para-azobenzenearsonate (AA) and to para-azobenzene-N-trimethylammonium (TMA). On day 6, the allotypes of antibody from plaque-forming cells (PFC) of the blood were determined by observing the inhibition of plaque formation by anti-allotype sera. Anti-AA PFC appeared to consist for the most part of cells making antibody of allotype a1 since 65% of them were inhibited by anti-a1 serum and only 8% by anti-a3. Anti-TMA PFC, on the other hand, appeared to consist mostly of cells making antibody of allotype a3, since less than 1% of them were inhibited by anti-a1 but 47% by anti-a3. Antibody allotype for spleen PFC was also determined on day 6 and was similar to that found for blood PFC. Anti-AA PFC were inhibited 74% by anti-a1 serum and 15% by anti-a3 whereas anti-TMA PFC were inhibited 19% by anti-a1 and 43% by anti-a3. Serum hemolysin specific for AA hapten from a1a3 animals was also strongly inhibited by anti-a1 serum but not by anti-a3 whereas the converse was true for hemolysin against TMA hapten. The a1a3 rabbits, in whcih the anti-AA was restricted to allotype a1, were mated to produced homozygous a3a3 animals. When the PFC and serum antibodies of these a3a3 offspring were examined by specific inhibition, the anti-AA activity was found to be of allotype a3 rather than being a-negative. The number of anti-AA PFC in the blood of a3a3 rabbits was lower than that in blood of a1a3 or a1a1 animals. In addition, the TMA hapten appeared to inhibit the response to the AA hapten. Thus a1a3 rabbits immunized with AA-Prbc alone had 14-fold more anti-AA PFC or 18-fold higher anti-AA hemolysin titer than a3a3 animals immunized with both AA-Prbc and TMA-Prbc. Our results are discussed in relation to various explanations which have been offered for an imbalance of allotypes in a given antibody.     

Significant differences in capillary electrophoretic patterns of follicular fluids and sera from women pre-treated for in vitro fertilization

Human ovarian follicular fluids and sera obtained from women pre-treated for in vitro fertilization (IVF) were investigated by capillary zone electrophoresis. Comparison of the matching physiological liquids showed substantial differences in the electrophoretic patterns. Significant decrease in the alpha(1)- and gamma-fractions of follicular fluids of every woman were observed, whereas other fractions of the samples did not show such alterations. Since follicular fluid is a product of both, secretion by granulosa cells and diffusion from the theca capillaries, we can assume that the forced production of follicular fluid upon hormone stimulation (with gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH) and corionic gonadotroph hormone (hCG)) may play role in the uneven presence of the proteins.     

Serologic and structural characterization of immunoglobulin chains secreted by rabbit-mouse hybridomas

Serologic and primary structural analyses of Ig chains secreted by several rabbit-mouse hybridomas have shown that these hybrid cells produce heavy (H) or light (L) chains identical to those isolated from rabbit sera. Two of the cell lines (7D2, 7D6) secreted rabbit H chains with a m.w. of 55,000 each of which expressed a full complement of variable and constant region allotypes (a3, d11, e15). These apparently normal rabbit H chains were secreted in a complex with a m.w. about 130,000, and serologic studies indicated that this complex contained a covalently linked mouse kappa L chain. Two other cell lines (4C1, 12F2) produced allotype b4 L chains with m.w. of 23,000 and 25,000, and a third (1D4P5) produced an allotype b5 L chain with a m.w. of 23,000. Serologic analyses indicated that the allotypes on these chains are equivalent to those expressed by normal rabbit Ig molecules. Partial amino acid sequence data obtained for the L chain products showed them to be typical of rabbit L chains, and to be significantly different from mouse L chains.     

Immunogenetic study on the polymorphism of serum α2-lipoproteins in mink. IV. Diallelism at the Ld locus of low-density lipoprotein

Antibodies against a new allotype, Ld2, of mink low-density lipoprotein (LDL) were obtained by alloimmunization with a preparation of this lipoprotein. The two known allotypes of LDL, designated Ld1 and Ld2, are coded for by codominant alleles of the autosomal Ld locus. This locus is probably involved in the genetic control of the whole serum pool of LDL molecules. In Ld1/Ld2 heterozygotes, LDL is represented by two homozygous types of molecules, Ld1 and Ld2; it has no hybrid molecules bearing both allotypic specificities together. The results suggest that the Ld locus has, presumably, only two alleles in the mink populations studies. Mink LDL having allotypes Ld1 and Ld2 was found to be homologous to human and pig LDLs. Antigenic specificity of Ld1 allotype was established in the sera of a wide phylogenetic range of mammals and in the human LDL. The parallelism between the phylogenetic antiquity of the Ld1 gene and its high frequency in mink and other species may be attributed to the selective value of this gene, which has been retained unaltered during macroevolution.     

Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.     

Growth hormone but not prolactin concentrations in the fluid of bovine ovarian cysts are related to the cystic stage of luteinization

Regulation of follicular growth and ovulation as well as steroid production by the ovary depends principally on gonadotropins. However nonsteroid systemic hormones and autocrine and paracrine factors contribute to the regulation of ovarian function. The objectives of the present work were 1) to asses the presence of growth hormone (GH) and prolactin (PRL) in fluid drawn from normal bovine ovarian follicles, cysts or cystic corpora lutea; 2) to relate the stage of luteinization of the cyst with the GH and PRL concentrations in fluids; and 3) to asses the feasibility of providing a defined nonsteroid hormone marker to distinguish between normal and pathological ovarian structures. Cysts were classified according to histological and morphological appearance as follicular or luteal. Concentrations of GH, PRL, estrogens (E2), progesterone (P4) and testosterone (T) were measured in follicular and cystic fluids. On the basis of the E2 to P4 ratio, ovarian formation classes were further divided into two subclasses (E2 dominant and P4 dominant). The results provide evidence of 1) the presence of immunoreactive GH and PRL in all the follicular and cystic fluids assayed, 2) an increasing concentration of GH correlated to the stage of luteinization of the cyst and a direct correlation between GH and P4 concentrations, 3) a significant variability of intraovarian fluid PRL concentration not related to the histological class of the cyst nor to the concentrations of steroid hormones examined, and 4) the possibility of distinguishing 6 different ovarian formation classes by merely measuring GH, P4, E2 and T concentrations in fluids. These data contribute to a better understanding of the endocrine milieu of bovine ovarian cystic degeneration.     

Structural polymorphism of murine complement factor H controlled by a locus located between the Hc and the beta 2M locus on the second chromosome of the mouse

Structural polymorphism of murine factor H protein was demonstrated by using three different methods. 1) By prolonged agarose electrophoresis and immunofixation, factor H protein was visualized in the beta region as a single, distinct protein band in freshly bled EDTA-plasmas from many laboratory and wild mice. Two variants were detected among a large number of tested strains; one, referred to as H.1, moved faster to the anodal region (type strain, BALB/c), and the other, referred to as H.2, moved more slowly to the anodal region (type strain, STR). The F1 hybrid between BALB/c and STR exhibited a combining type of factor H protein, which was observed in each parent. 2) Two-dimensional peptide mapping analysis was carried out with tryptic peptides of these two factor H allotypes. Almost all of the spots in the maps of tryptic peptides were common to both allotypes. However, three distinct spots among the 57 spots detected in the map of tryptic peptides of the H.1 allotypes were not detected in that of H.2 allotype, whereas two spots among the 56 spots in the map of H.2 allotype were unique for this allotype. The F1 hybrid between BALB/c and STR showed a combining type of the map of parent. 3) Alloantisera against each of H allotypes were successfully produced in BALB/c or BALB/c-H.2 (a congenic strain with H.2 allotype) by repeated injection of each purified factor H protein either from the BALB/c or the STR strain. These findings indicated that the observed variants of factor H represent antigenically and structurally distinguishable allotypes. The allotypes of murine factor H protein are controlled by a single codominant locus located between the Hc locus and the beta 2M locus on the second chromosome of the mouse. This was shown by phenotyping the Hc locus and H locus with backcross progenies between A/J (one of strain with H.1) and MoA (one of strain with H.2). The recombination frequency between these two loci was 0.17 +/- 0.046.