Search

The main goal of this study was to characterize the expression patterns of genes which play a role in mitochondrial DNA biogenesis and metabolism during the maturation of bovine oocytes with different meiotic competence and health. Meiotically more and less competent oocytes were obtained separately either from medium (MF) or small (SF) follicles and categorized according to oocyte morphology into healthy and light-atretic. The four oocyte categories were matured and collected after 0, 3, 7, 16 and 24 h of maturation. Either total RNA or poly(A) RNA were extracted from oocytes and the expression of selected mitochondrial translational factors (TFAM, TFB1M, and TFB2M), MATER, and Luciferase as external standard was assessed using a real-time RT-PCR. The level of TFAM, TFB1M and MATER poly(A) RNA transcripts significantly decreased during maturation in both healthy and light-atretic MF and SF oocytes. On the other hand, the level of TFB2M poly(A) increased during maturation in healthy and light-atretic SF oocytes, in contrast to MF oocytes. The abundance of TFAM total RNA was significantly higher after maturation than that before maturation in all oocyte categories. However, no differences in TFB1M and TFB2M total RNA were found in any oocyte categories. It can be concluded that the gene expression patterns differ in maturing bovine oocytes in dependence on their meiotic competence and health. The TFAM and TFB1M poly(A) RNAs are actively deadenylated at different meiotic stages but TFB2M poly(A) RNA remains elevated in light-atretic less competent oocytes until the completion of meiosis., (Copyright © 2019. Published by Elsevier Inc.)

Background: The Y chromosome microdeletions belong to the frequent genetical causes of male infertility. The aim of our study was to introduce reliable molecular genetic diagnosis of Y chromosomal microdeletions and to determine the prevalence of Y chromosomal microdeletions in Czech males with serious reproductive disorders., Methods and Results: The Y chromosome microdeletions were screened in 198 Czech men with serious reproductive disorders with decreased sperm count. The Y chromosome microdeletions were disclosed in 8/198 (4.0%) examined males. The AZFc deletion type was revealed in 62.5% (5/8) and the combined AZFc + b microdeletion in 37.5% (3/8) of cases. Neither isolated AZFb nor AZFa microdeletion were found in any subject of the investigated group., Conclusions: Incidence of individual types of Y chromosomal microdeletions in Czech males with serious reproductive disorders was assessed. The standardised molecular genetic diagnosis of Y chromosomal microdeletions was introduced into the practice.

Reproductive genetics (RG) is another new field of medical genetics, integrated with reproductive medicine, assisted reproduction and developmental genetic. RG is closely linked to the perioconceptional prevention, perinatology, ultrasound and biochemical screening in the end of the first and beginning of the second trimesters. RG is based on the system of specialized genetic counseling, clinical cytogenetics, molecular cytogenetics and molecular genetics to provide prefertilization, preimplantation and classical prenatal diagnosis in the Ist to IIIrd trimesters. Thus, RG is part of the fetal medicine and therapy. The six years experience with RG is summarized. A system of the specialized health care, organized, if possible in one integrated center of RG and reproductive medicine (RM) is presented. Reproductive medicine provides all necessary clinical gynecological and andrological surveillance, with assisted reproduction and further obstetrical ultrasound examinations, including nuchal translucency measurements and 2D, 3D ultrasound, echocardiography examinations, if indicated, as well as the invasive method of prenatal diagnosis and perinatology care. Specialized genetic counseling and cytogenetic analysis, if indicated, should be offered to all partners with reproductive disorders as well as to oocyte donors. Chromosome anomalies are disclosed in 6% of men with abnormal sperm analysis as well as in women with severe reproductive disorders. In males with severe oligo, azoospermia, the sperm aneuploidy analysis by molecular cytogenetic methods is recommended. Advised is also the molecular genetic detection of Y chromosome microdeletions, which is detected in 9% of our azoospermic men with deletions in AZFb region. CFTR gene mutations and intron 8 and 10 polymorphism examination is provided not only in men with obstructive azoospermia (CBAVD), but also if severe oligospermy with less than 1 x 10(6) sperm/ml is detected. Molecular genetic analysis of thrombophilic mutations of factor II., V. (Leiden) and MTHFR gene in unexplained recurrent abortions and in cases with unsuccessful IVF is part of the diagnostic strategy. The population frequencies of carriers of mutations of factor II. (2.3%), factor V.-Leiden (5.7%) and MTHFR gene (38%) were determined. The laser biopsy of the first polar body and of blastomeres was introduced for FISH analysis of chromosome aneuploidies. Quantitative fluorescent PCR (QFPCR) detection is used for testing of the most frequent delta F508 CFTR gene mutation and the most frequent aneuploidies of chromosome 13, 18, 21, X and Y. QFPCR was successfully tested for male fetal sex examination from partially purified fetal cells in the maternal blood. The first trimester ultrasound and biochemical screening is recommended to all successful pregnancies after different IVF methods. If borderline levels of first trimester biochemical screening of PAPP-A protein and beta hCG are detected without pathological ultrasound findings, classical triple test of biochemical screening in 16th week of gestation is recommended. If pathological results of ultrasound and biochemical screening are disclosed, invasive prenatal genetic diagnosis is indicated as well as in pregnancies after ICSL, if there is not any obstetrical contraindication.

The effects of administration of an established, inactivated IBR vaccine were studied in 30 cows from two herds (one seropositive and one seronegative). All acquired immunity which, after subsequent intratracheal infection with IBR virus, prevented development of symptoms in the cows and protected their foetuses against viral infection in utero. The calves were all healthy and were born at normal term. Ten non-vaccinated cows from the seronegative herd responded to the experimental infection with mild respiratory disease and abortion of four out of 10 foetuses. Organs from the aborted foetuses were found to have IBR virus. In a breeding herd, without clinical signs of disease but with 40% of cows tested as seropositive, a 2-year disease-control programme was initiated. A total of 234 newborn calves were examined and it was shown that immunization of their dams with an inactivated vaccine conferred full in utero protection against IBR-virus infection. When such calves are reared in isolation they can be used as the nucleus for a seronegative breeding herd.

The intrauterine infection of four- to nine-month-old bovine foetuses with the PI-3, BVD-MD viruses, performed 7 to 72 days prior to their delivery, did not exert any significant influence upon the susceptibility of primary cell cultures from foetal organs and tissues to further viral infection in vitro. The BVD-MD and IBR viruses multiplied in the primary cell cultures from the organs of a foetus infected with the PI-3 virus seven days before delivery even in the presence of endogenous PI-3 virus. Persisting infection with the PI-3 virus also failed to influence the susceptibility of foetal organ cultures to infection with the IBR and PI-3 viruses in vitro. The IBR virus and endogenous PI-3 virus multiplied simultaneously to high titres in the organ cultures of thymus and lungs whereas in the organ cultures of kidneys, spleen and testes the multiplication of endogenous PI-3 virus was suppressed.

A trial was performed with heifers at the age of six to seven months. The animals were experimentally infected with the lymphocytes of a virus-productive donor. Infection was produced in all the nine cases, as demonstrated by means of the positive syncytial test. As indicated by the results of the trial, the antibodies to the enzootic bovine leucosis virus (BLV) were produced soon after experimental infection. A high sensitivity of the serum-neutralization test and the ELISA method was demonstrated in this connection: by these methods, the antibodies were identified already two to three weeks after experimental infection whereas by the immunodiffusion test they could be detected only after five weeks. Twenty-four animals were exposed to natural contact infection. Within 270 days of the trial, the disease after contact was recorded only in one heifer out of the four that were in close contact with the experimentally infected animals. In this case, as compared with experimental infection, the antibodies were produced much later--after 85 to 93 days. Leucosis was recorded in none of the remaining animals. The reasons why such a favourable result was obtained were the thorough disinfection of the stables after blood collections and the strict observance of the aseptic conditions. The results of experimental infection in three cows were identical with those obtained in young cattle. In the experimentally infected dairy cows, antibodies in milk were determined by the ELISA method. As found, in milk the antibodies to BLV appear two to three weeks later than they do in serum. The ELISA method of BLV antibody detection can be used for the identification of infected animals in herds where enzootic bovine leucosis occurs.

Growing organ cultures prepared from foetuses experimentally infected in utero by the viruses PI-3 and BVD-MD or bovine parvovirus proved to be suitable in tests with the re-isolation of these viruses 7, 14, 21, 35, 42 and 70 days after infection. The attempts at demonstrating them by the traditional method of the inoculation of the primary cell culture of foetal kidneys were successful only at the re-isolation of the PI-3 virus seven days after infection. The PI-3 virus without cytopathic effect was demonstrated in the primary cell culture prepared from the spleen, lungs, kidneys and testes of foetuses delivered seven days from infection. The cytopathic effect manifested itself after cell transplantation in the first passage when further multiplication of the virus occurred.

The continual cell lines of bovine kidneys MDBK and AUBEK, and porcine kidneys RPD and IBRS, spontaneously infected with Mycoplasma arginini and Acholeplasma laidlawii, were decontaminated by the method of selective elimination. Two elimination procedures were modified to be used for the decontamination: one based on the reduction of infection by the light treatment of the cultures, the other based on the selection of mycoplasma-free cell population through cell clonation. On the basis of a long-continued control of the cell clones a methodical procedure of the preparation of mycoplasma-free cell lines was worked out.

BLV detection by the syncytial test was performed in 27 heifers experimentally and naturally infected by the enzootic bovine leukosis virus (BLV). The presence of BLV was demonstrated in 94.7% of the animals. The bovine foetal spleen cells (FBS) were found to be suitable for the syncytial test. Positive animals not reacting to infection by the production of anti-BLV antibodies were identified during the syncytial-test investigation. The importance of this finding for the programme of controlling enzootic bovine leukosis on farms is discussed. As suggested by the results, temporary occurrence of anti-BLV antibodies followed by their disappearance can be observed together with a negative result of the syncytial test in some circumstances. The discussion deals with the problems of the determination of anti-BLV antibodies in milk, and/or milk secretion, by the ELISA method.

The ultra-thin section method, the method of negative staining, and immunoelectron microscopy were used for detecting BLV and for determining its morphological characteristics in the FLS continual cell line used as a virus antigen producer for the ELISA test. Particles of C type, about 110 nm in size, having a structure corresponding to BLV, were detected in the FLS cells on the ultra-thin sections. The viruses were located extracellularly, in cytoplasmic vacuoles, and in different stages of maturation by budding from cell plasma membrane. BLV presence was also demonstrated by immunoelectron microscopy.

The continual cellular lines of bovine kidneys MDBK and AUBEK and of monkey kidneys VERO were infected spontaneously in the course of long-range cultivation by the mycoplasmas M. arginini and A. laidlawii. The mycoplasmic infection reduced significantly the growth activity of the cells and influenced negatively their morphology. The method of an indirect proof of mycoplasmas by bisbenzimid Hoechst suited well to demonstrate the infection; it had been modified to a simple and rapid test. The starting infection could be demonstrated already at the time when the amount of infected cells in the culture was relatively low.