Changes in sub-cellular localisation of trophoblast and inner cell mass specific transcription factors during bovine preimplantation development.

BACKGROUND: Preimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle. To gain an insight into these processes we have studied protein and mRNA distribution during the crucial stages of bovine development. Protein distribution of lineage specific markers OCT4, NANOG, CDX2 were analysed in 5-cell, 8-16 cell, morula and blastocyst stage embryos. ICM/TE mRNA levels were compared in hatched blastocysts and included: OCT4, NANOG, FN-1, KLF4, c-MYC, REX1, CDX2, KRT-18 and GATA6. RESULTS: At the mRNA level the observed distribution patterns agree with the mouse model. CDX2 and OCT4 proteins were first detected in 5-cell stage embryos. NANOG appeared at the morula stage and was located in the cytoplasm forming characteristic rings around the nuclei. Changes in sub-cellular localisation of OCT4, NANOG and CDX2 were noted from the 8-16 cell onwards. CDX2 initially co-localised with OCT4, but at the blastocyst stage a clear lineage segregation could be observed. Interestingly, we have observed in a small proportion of embryos (2%) that CDX2 immunolabelling overlapped with mitotic chromosomes. CONCLUSIONS: Cell fate specification in cattle become evident earlier than presently anticipated - around the time of bovine embryonic genome activation. There is an intriguing possibility that for proper lineage determination certain transcription factors (such as CDX2) may need to occupy specific regions of chromatin prior to its activation in the interphase nucleus. Our observation suggests a possible role of CDX2 in the process of epigenetic regulation of embryonic cell fate.

Gilts and sows produce similar rate of diploid oocytes in vitro whereas the incidence of aneuploidy differs significantly.

Oocytes derived from prepubertal gilts show reduced developmental competence when compared to oocytes collected from adult sows. Therefore, the aim of the study was to investigate whether gilts (4-5 months old) and adult sows (average age 3.5 years) of the same breed (Polish Landrace x Polish Large White crossbred) differ with regard to the rate of chromosomally unbalanced oocytes after IVM. COCs derived from individual pairs of slaughterhouse ovaries were matured in vitro and analyzed cytogenetically by conventional staining (Giemsa) and FISH methods (probes corresponding to centromeric regions of pig chromosomes 1 and 10). Altogether, 72 females (31 sows, 41 gilts) and 430 secondary oocytes (194 and 236 oocytes of sows and gilts, respectively) were investigated. Cytogenetic analysis revealed diploid (Giemsa, FISH) and aneuploid (FISH) spreads. The incidence of diploid oocytes was similar for sows (26.0%) and gilts (24.5%) whereas the rate of aneuploid oocytes (nullisomic/disomic) was eight times higher in gilts (10.8%) than in sows (1.3%). Diploid and aneuploid oocytes were observed in 64% of investigated females. Pig chromosome 10 was more frequently disomic/nullisomic compared to chromosome 1 suggesting, that like in human, small porcine chromosomes are often involved in the nondisjunction process. In conclusion, chromosomal imbalance significantly contributes to in vitro embryo production in the pig, since over 60% of females produced diploid or aneuploid gametes. The significantly higher rate of aneuploidy among oocytes derived from gilt ovaries may contribute to the reduced developmental competence of gametes collected from nonmature female pigs.

Use of somatic cell nuclear transfer to study meiosis in female cattle carrying a sex-dependent fertility-impairing X-chromosome abnormality.

Animal models have played an important part in establishing our knowledge base on reproduction, development, and the occurrence and impact of chromosome abnormalities. Translocations involving the X chromosome and an autosome are unique in that they elicit sex-dependent infertility, with male carriers rendered sterile by synaptic anomalies during meiosis, whereas female carriers conceive but repeatedly abort. Until now the limited access to relevant fetal oocytes has precluded direct study of meiotic events in female carriers. Because somatic cell nuclear transfer (SCNT) circumvents meiotic problems associated with fertility disturbances in translocation carriers, we used SCNT to generate embryos, fetuses, and calves from a cell line derived from a deceased subfertile X-autosome translocation carrier cow to study the meiotic configurations in carrier oocytes. Data from 33 replicates involving 2470 oocyte-donor-cell complexes were assessed for blastocyst development and of these, 42 blastocysts were transferred to 21 recipients. Fourteen pregnancies were detected on day 35 of gestation. One of these was sacrificed for ovary retrieval on day 94 and three went to term. Features of oocytes from the fetal ovary and from the newborn ovaries were examined. Of the pachytene spreads analyzed, 16%, 82%, and 1.5% exhibited quadrivalent, trivalent/univalent, and bivalent/univalent/univalent structures, respectively, whereas among the diakinesis/metaphase I spreads, 16% ring, 75% chain, and 8.3% bivalent/bivalent configurations were noted, suggesting that the low fertility among female carriers may be related to synaptic errors in a predominant proportion of oocytes. Our results indicate that fibroblasts carrying the X-autosome translocation can be used for SCNT to produce embryos, fetuses, and newborn clones to study such basic aspects of development as meiosis and to generate carriers that cannot easily be reproduced by conventional breeding.

Genetical and biotechnological methods of utilization of female reproductive potential in mammals.

The investigations included: 1/ Establishment of culture systems that would maintain the three-dimensional structure of bovine intact early antral follicles (EAF) or isolated cumulus-oocyte-granulosa complexes (COCGs) and increase the resulting portion of cumulus-oocyte complexes (COCs) with normal morphology for subsequent in vitro maturation (IVM) and in vitro fertilization (IVF), 2/ Quality assessment of IVM bovine oocytes and resulting day-8 blastocysts produced in TCM199 medium supplemented with fetal bovine serum (FBS), fatty acid free bovine serum albumin (fafBSA) or polyvinylpyrrolidone (PVP40), 3/ Testing the polymorphism of the genes: retinol binding protein (RBP4), epidermal growth factor (EGF), prostaglandin-endoperoxide synthase 2 (PTGS2), insulin-like growth factor 2 (IGF2), amphiregulin (AREG) and prolactin (PRL), and their effects on reproductive traits in swine. Isolated COCGs created in culture follicle-like structures and their oocytes achieved meiotic competence and matured to metaphase II at a higher rate than did oocytes from smaller diameter follicles which were cultured intact. The proportion of COCs with normal morphology significantly increased when isolated COCGs were embedded in microdrops of collagen gel or cultured on inserts covered with gel rather than in large gel volumes. No significant effect of maturation media composition on meiotic spindle morphology and the rate of apoptotic bovine oocytes was observed. Among day-8 embryos derived from oocytes matured with PVP40 a reduced blastocyst rate and elevated apoptotic index were found, whereas total cell count was not affected. Gene expression study also revealed a decrease in relative abundance for IGF2 and its receptor (IGF2R), and heat shock protein 70 (Hsp70) genes in PVP40 group and a significant elevation in fafBSA derived embryos. The significant effect of reproduction traits of swine (litter size and litter weight) was found for RBP4, EGF, IGF2 and AREG genes. A new polymorphism was also revealed within a promoter region of PRL gene.

Chromosome abnormalities in secondary pig oocytes matured in vitro.

Abnormalities of chromosome segregation during in vitro maturation of oocytes cause failure of in vitro fertilization. Oocytes collected from pig ovaries after slaughter were matured in vitro (IVM) for 30-48 h. In total, 1144 secondary oocytes were studied cytogenetically. An unreduced (diploid) chromosome set was identified in 146 spreads (12.8 %). A higher proportion of diploidy was noticed in secondary oocytes matured for 40 h and longer (15.0 %) than in the groups matured for 30 and 36 h (9.0 %). Among 998 secondary oocytes with the reduced chromosome number, 612 could be analyzed in detail. Hypohaploidy (n=19-1) was identified in 22 cells (3.59 %) and a hyperhaploid (n=19+1) set of chromosomes was identified in 15 cells (2.45 %). The rate of aneuploidy, estimated by doubling the rate of hyperhaploidy was 4.9 %. It was also found that aneuploid spreads occurred more frequently in the group of oocytes matured for 40 h and longer. Small acrocentrics were mostly found as an extra chromosome in the hyperhaploid spreads. Our study indicates that to avoid an excess of chromosomally abnormal secondary oocytes, IVM duration of pig oocytes should not exceed 40 h.