Effects of fetal bovine serum, FSH and 17beta-estradiol on the culture of bovine preantral follicles.

We describe a 7-d culture in droplets of collagen gel of isolated small bovine preantral follicles in medium with or without 10% fetal bovine serum (FBS). In addition, the effect of human recombinant FSH and 17beta-estradiol on the morphology and growth of the preantral follicles was investigated in medium without FBS. After culture in medium with 10% FBS, the increase in follicle diameter was 13.1 +/- 8.4 microm, the percentage of BrdU-labeled cells was 49.9 +/- 11.3 and the number of cells per area granulosa was 11.1 +/- 1.8. Omission of serum from the culture medium had no effect on the percentage of labeled cells, but the diameter increase was lower and the cells were smaller. Apparently, serum affects the size of the granulosa cells from small preantral follicles rather than the stimulation of cell proliferation. Addition of human recombinant FSH and/or 17beta-estradiol to serum-free medium resulted in a larger diameter increase during culture compared with that of the control. With FSH, this was due to an increase in cell proliferation, while with estradiol this was caused by an increase in granulosa cell size. The effects of simultaneous treatment with FSH and estradiol was simply the combination of their individual effects. In conclusion, small bovine preantral follicles can be cultured for 7 d in the absence of serum and hormones. The follicles increase in diameter and react to FSH with enhanced cell proliferation and to estradiol with an increase in cell size.

Cytogenetic analysis of day-7 bovine embryos by cytophotometric DNA measurements.

A Feulgen staining procedure that stains the DNA of individual fixed nuclei stoichiometrically was used to analyse cytophotometrically the incidence of total ploidy and mixoploidy in 28 day-7 bovine embryos that had been fixed after collection ('non-cultured' embryos). The influence of culture on the incidence of chromosome abnormalities was further studied in another group of 24 embryos ('cultured' embryos) by culturing them for 24 h in Whittingham's medium. Of the total 52 embryos studied, two appeared to be entirely abnormal: one embryo was completely haploid, whereas the other embryo was completely triploid. Individual hyperdiploid nuclei and hypodiploid nuclei were frequently observed in the otherwise diploid embryos. As haploid polar bodies can still be present in morulae and blastocysts (to a maximum of three), only embryos with more than three hypodiploid nuclei were considered as abnormal. Of the 'non-cultured' embryos, 33.3% had one or more hyperdiploid nuclei, whereas 51.9% had more than three hypodiploid nuclei. In this latter group, 35.7% of the embryos also had hyperdiploid nuclei. The results also showed that day-7 bovine embryos that are completely haploid, completely triploid or mixoploid cannot be detected only by examining their morphology. It is concluded that the incidence of, especially, mixoploidy in embryos can be better studied by measuring the DNA content of the individual nuclei of an embryo rather than by analysing chromosomes, as in the latter method only dividing cells can be analysed. The presence of hyperdiploid and hypodiploid nuclei may indicate the frequent occurrence of mitotic segregation failures during mitosis in bovine embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Survival and proliferation of rat gonocytes in vitro.

Quiescent gonocytes were isolated from fetal testes of rat 18-day post coitum and cultured alone or on monolayers of somatic cells from different origins. The gonocytes specifically adhered to Sertoli cells, isolated from 21 to 23-day-old rat testes; this adherence was necessary for their survival in vitro. Addition of follicle-stimulating hormone and testosterone to these cultures did not increase the viability of the gonocytes. Serum was found to be deleterious to the germ cells. Electron-microscopic examination of Sertoli-cell-gonocyte co-cultures revealed the presence of numerous adhesion plaques between these cells, indicating that Sertoli cells and gonocytes are able to communicate in vitro. Gonocytes, in co-culture with Sertoli cells, were viable for at least 9 days. The gonocytes did not spontaneously resume proliferation. The simple culture system described in the present paper should be useful in studying the nature of the factors that are responsible for sending the quiescent gonocytes into the cell cycle and for stimulating the formation of A spermatogonia, a process characterizing the start of spermatogenesis.

A monoclonal antibody recognizing a differentiation marker on rat gonocytes.

Monoclonal antibodies (MAb) were raised against a testicular membrane fraction from 18-day post coitum (p.c.) rat testes. One antibody, designated 4B6.3E10 (mu, kappa), was obtained which specifically reacted with gonocytes in the fetal testis. No significant cross-reactivity with other tissues from the 18-day p.c. embryo was found. MAb 4B6.3E10 was reactive with rat gonocytes from 17-day p.c. until the day of birth. Germ cells at later stages of testis development did not show any labelling. The epitope recognized by 4B6.3E10 is a carbohydrate as periodate treatment leads to a loss of reactivity of the antibody. By SDS-PAGE and Western blotting of proteins extracted from a testicular membrane fraction from 18-day p.c. testes, MAb 4B6.3E10 was found to recognize at least 3 protein moieties with apparent molecular weights in the ranges of 80-100, 120, 160-180 kDa (either under reducing- or non-reducing conditions). The results suggest that MAb 4B6.3E10 recognizes a specific differentiation marker for fetal rat gonocytes.

Cell-cell interaction between rat Sertoli cells and mouse germ cells in vitro.

An antiserum against rat germ cell membranes was prepared, and after absorption with protein extracts of rat liver and kidney and mouse testis, this antiserum reacted only with rat germ cell membranes and juxtanuclear vesicles in rat Sertoli cells. Germ cell-free rat Sertoli cell monolayers were cultured in vitro. Freshly isolated mouse germ cells adhered to these monolayers within 1 h. After a minimum of 3 days of such a co-culture, immunofluorescence and immunoblotting revealed that the mouse germ cells had obtained rat-antigenic determinants in their membranes. Our results indicate that this appearance of rat-specific antigens on mouse germ cells is specific and inducible.

Humoral immune response of dogs after vaccination against leptospirosis measured by an IgM- and IgG-specific ELISA.

An IgM- and IgG-specific ELISA was used to measure the antibody response stimulated in dogs by vaccination with a leptospiral bacterin containing chemically inactivated Leptospira interrogans serotype icterohaemorrhagiae and serotype canicola leptospires. All dogs produced anti-leptospiral IgM and IgG. The IgM production was of the primary response type after each vaccination (primary vaccination, booster vaccination and annual revaccination). A substantial anti-leptospiral IgG response could be demonstrated only after the first booster vaccination and the annual revaccination. Annual revaccination resulted in a higher and much longer persisting IgG response than did the first booster vaccination. A revision of the vaccination scheme is suggested.

Serodiagnosis of canine leptospirosis by solid-phase enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Leptospira interrogans serotype canicola in dogs was developed and evaluated. Comparison of the ELISA with the microscopic agglutination test (MAT) showed that, during the first two weeks after an experimental infection with serotype canicola, the ELISA detected antibody at higher dilutions than the MAT. After the second week post-infection both tests detected antibody at almost equal titres (r = 0.89). The outer envelope (OE) antigen of serotypes icterohaemorrhagiae, copenhageni and canicola was fairly serotype-specific, whereas the pellet (P) antigen showed more cross-reactivity. Both OE and P antigen of Leptospira biflexa strain Patoc I could be used as cross-reacting antigen in the ELISA. Compared to the MAT, the ELISA has some technical advantages. It is suggested that the ELISA would be useful as a screening test.

Determination of specific anti-leptospiral immunoglobulins M and G in sera of experimentally infected dogs by solid-phase enzyme-linked immunosorbent assay.

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) to detect specific anti-leptospiral IgM and IgG in sera of dogs experimentally infected with Leptospira interrogans serotype canicola are reported. In all dogs specific anti-leptospiral IgM was detected from the second half of the first week after infection, the maximum being attained during the second week. Subsequently the IgM titre gradually decreased. Specific anti-leptospiral IgG was detected later and increased gradually to reach almost the same level as the IgM titre after two to three months. During the initial stage of the infection, when the microscopic agglutination titre was still negative or very low, a high IgM titre was accompanied by a negative or very low IgG titre in every case. After the initial stage a substantial IgG titre was also detectable. It is suggested that the test is suitable for serodiagnostic purposes, particularly for the diagnosis of a current infection in an individual.

The effect of oral immunization on the population of lymphocytes migrating to the mammary gland of the sow.

Sows were immunized orally with live Escherichia coli according to various immunization schedules. Six pregnant gilts were used; 4 immunized at various intervals during the last month of gestation, 1 control immunized after parturition following suppression of lactation by weaning and 1 non-immunized control. The effect of oral vaccination on cell populations from lymphoid organs was studied. The in vitro proliferative responses of the cell populations to K88 antigen, anti-Ig sera and mitogens were used to demonstrate the distribution of sensitized lymphocytes over different lymphoid organs. The capacity of these cells to produce antigen-specific Ig was determined by in ovo translation of their mRNA. Oral administration of antigen resulted in the appearance of K88-positive cells in lymphoid organs. In lactating sows, sensitized cells preferentially occurred in the mammary lymph nodes, whereas after suppression of lactation such a distribution was not seen. A possible route of migration of sensitized lymphocytes is discussed in relation to the local immune response. The antibody isotype produced by sensitized lymphocytes seemed to depend on the immunization schedule. The most effective schedule was one starting early in gestation and comprising frequent administration of antigen. This caused an optimal distribution of sensitized lymphocytes capable of IgA production.

Induction and effector phases of the immune response: physiological and pathophysiological features.

A review is given on the current state of knowledge about the induction and effector phases of the immune response. Physiological and pathophysiological features of the regulation of the immune response are discussed comparatively for both man and animals.

The histocompatibility complex GLA in the goat.

The current status of research on the major histocompatibility complex of the goat (GLA) is discussed, as well as its similarities to other species, although some of the results are preliminary.

Skin grafting and graft rejection in goats.

As part of a study to define the major histocompatibility complex of the goat and to show certain aspects of its biological function, a skin-grafting technique is described which might also be applicable for similar studies in other ruminant species.

Quantification of turkey immunoglobulins IgA, IgM and IgG in serum and secretions.

The immunoglobulins IgA, IgM and IgG of the turkey were quantitated in individual serum samples as well as in pooled sera. The variability of Ig levels in normal, healthy birds was quite high: IgA: mean value 0.633 mg/ml (4.0 -x - 2.5 -x); IgM: mean value 4.37 mg/ml (0.5 -x - 1.4 -x) and IgG: mean value 8.92 mg/ml (0.6 -x - 1.7 -x). Immunoglobulin levels in egg-yolk, egg-white, bile and some intraocular tissues were quantitated as well. An interesting finding was, that the forementioned variability was by far not so high with respect to IgG levels in 20 egg-yolk samples: mean value 5.1 mg/ml (0.86 x- - 1.17 -x). Though IgG and IgM could be detected in pooled turkey bile, IgA predominated in this secretion. In aqueous humor, iris tissue and vitreous body only IgG could be detected.

Protein A reactivity of various mammalian immunoglobulins.

Serum samples and immunoglobulin fractions of eight mammalian species were applied to a Sepharose--protein A column. As with the human immunoglobulin subclasses IgG1, IgG2 and IgG4, all examined IgG classes and subclasses were bound to a greater or lesser extent to protein A. However, the binding of IgG1 of ruminants was very poor. Polyclonal IgM and IgA of the pig, the dog and the cat may be separated in protein A reactive and protein A non-reactive fractions. In addition, monoclonal canine IgM and IgA partially reacted with protein A. In combination with methods such as ammonium sulphate precipitation, ion exchange chromatography and gel-filtration, affinity chromatography with protein A is recommended for the rapid purification of certain Ig (sub)classes of a number of mammalian species.

Common precursor molecule as origin for the ectopic-hormone-producing-tumour syndrome.

When messenger R.N.A. (m-R.N.A.) extracted from various hormone-secreting tumours was injected into Xenopus eggs, the translation products in all cases proved to be a protein of molecular weight 65 00. Analysis by polyacrylamide-gel electrophoresis and specific precipitation reactions with antibodies showed a striking similarity between the various proteins. When translation products of m-R.N.A. from calcitonin-secreting medullary thyroid carcinoma (M.T.C( and the non-secreting anaplastic form of M.T.C. were incubating with specific enzyme systems (the microsomal fraction) from both types of tumour, enzymes from anaplastic M.T.C. had no effect on the translation products, whereas enzymes from differentiated M.T.C. degraded the translation products from both differentiated and anaplastic M.T.C. The results support the hypothesis that the primary gene product of all the different types of carcinoma cell studied is a single large protein (a hormone precursor or prohormone) containing different specificities. The specific enzyme system in each carcinoma cell probably selects the specific hormone liberated from this primary protein.

In vitro stimulation of goat peripheral blood lymphocytes: optimization and kinetics of the response to mitogens and to allogeneic lymphocytes.

Experiments were performed to establish the optimal concentrations for specific and non-specific lymphocyte transformation in vitro, in cultures containing 10(5)-10(6) caprine lymphocytes. A microculture system was used in conjunction with a semiautomatic sample harvester. The assay was optimized for mitogen concentration (PHA-P, Con A, PWM and LPS) and allogeneic cell number, number of responding cells, incubation time and amount of tracer. The effect of addition of serum and the cytotoxic effect of phytomitogens on cultured cells is discussed.

The regulation of pollen tube growth in Douglas-fir following high doses of ionizing radiation.

Acute irradiation of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) pollen by X-rays enhances pollen tube elongation. To determine whether this effect was due to physical damage of the pollen membranes or to metabolic processes, irradiated and control pollen were grown under conditions of anoxia. Thus, it was determined that physical damage only could be of minor importance. As parameters for metabolic processes RNA- and protein synthesis were studied. The only difference between irradiated and control pollen with respect to RNA synthesis was the time at which it started. Protein synthesis in vitro revealed that polyribosomes from irradiated pollen were more active than those from control pollen at lower concentrations. These results were confirmed by in ovo experiments with both polysomal preparations. The results indicate that X-ray irradiation is interfering with the main regulatory mechanism of pollen tube growth, which is de- and re-masking of, mainly, presynthesized mRNA.

Histocompatibility in ruminants. The production and evaluation of allo-antibodies for GL-A typing in goats.

The production of goat lymphocytotoxic allo-antisera by immunization by lymphocytes or allografting is described. Analysis of typing results of several goat families and the correlation between the lengthening of allograft survival and graft exchange between compatible and incompatible animals, suggest the existence of a major histocompatibility system (GL-A) in goats.