Expression Of Zona Pellucida Genes Research Articles

Expression of genes encoding zona pellucida glycoproteins and cortical granule distribution in porcine oocytes isolated from small and medium follicles in relation to puberty status of donors

The zona pellucida proteins belong to a group of proteins that regulate the processes of gamete recognition, interaction, and fusion, since they are recognized as primary and secondary sperm receptors. It is suggested that cortical granule distribution is significantly associated with the zona pellucida structure and membrane preparation to cortical and acrosome reaction. Therefore, this study investigated the zona pellucida marker gene expression (ZP2 and ZP4 protein) in relation to cortical granules distribution in oocytes and puberty status of donors. Oocytes were collected from adult cyclic sows (isolated from medium and small follicles) and juvenile gilts (isolated only from small follicles). The oocytes were examined by RT-qPCR and by confocal microscopy. The expression of genes for ZP2 and ZP4 protein in oocytes collected from small follicles from cycling sows was higher in comparison to oocytes from medium follicles or oocytes collected from small follicles from juvenile gilts (P < 0.001). We also observed increased expression of both ZP2 and ZP4 mRNA in oocytes collected from small follicles (juvenile gilts) as compared to medium follicles (cycling sows) (P < 0.001). Moreover, we found a difference in the distribution of cortical granules. In oocytes from medium follicles, the peripheral localization of cortical granules was twice higher than their central concentration. However, in oocytes derived from small follicles (cyclic sows) cortical granules in comparison to oocytes from medium follicles were more centrally localized (P < 0.05). It has been suggested that the donor puberty status and the size of follicles significantly influenced the zona pellucida gene expression and cortical granule localization in porcine oocytes. This is accompanied by fertilization specificity and fertilizability of porcine oocytes in vitro.

6 CLONING AND EXPRESSION OF BOVINE FACTOR IN THE GERMLINE ALPHA (FIGLA) IN OOCYTES AND EARLY EMBRYOS: A POTENTIAL TARGET OF microRNA-212

Factor In the GermLine Alpha (FIGLA), a basic helix-loop-helix transcription factor, was first identified in regulating coordinate expression of zona pellucida genes in mice. It plays a crucial role in the formation of primordial follicles and lack of FIGLA in mice alters the expression of many oocyte specific genes that are required for fertilization and early embryonic survival. The objective of this study was to characterise the expression and regulation of bovine FIGLA during early embryogenesis. The cloned bovine FIGLA cDNA is 660 bp in length, which encodes a protein of 165 amino acids. Expression of bovine FIGLA mRNA is restricted to ovarian tissue and can be detected in fetal ovaries harvested as early as 90 days of gestation when primordial follicles start to form. Expression analysis demonstrated that FIGLA mRNA is abundant in germinal vesicle and metaphase II stage oocytes, as well as in embryos from pronuclear to 8-cell stage, but barely detectable in embryos collected at morula and blastocyst stages, suggesting that FIGLA might be a maternal effect gene. Recent studies in zebrafish have highlighted the importance of non-coding small RNAs (microRNAs) as key regulatory molecules targeting maternal mRNAs for degradation during embryonic development. We hypothesised that FIGLA, as a maternal transcript, is regulated by microRNAs during early embryogenesis. Using microInspector, an algorithm for detection of possible interactions between microRNAs and target mRNA sequences, a microRNA binding site (miR-212) was identified in the 3′-UTR of the bovine FIGLA mRNA. Alignment of the 3′-UTR of FIGLA mRNAs from bovine, human and mouse shows complete conservation of the ‘seed’ region indicating that miR-212 might be a post-transcriptional regulator of FIGLA and the microRNA: mRNA interaction is evolutionary conserved. Expression analysis indicates that bovine miR-212 is expressed in oocytes and tends to increase at the 4-cell and 8-cell stage embryos followed by a decline at morula and blastocyst stages, indicating that miR-212 is presumably of maternal origin and potentially involved in maternal transcript degradation during the maternal-to-embryonic transition. To validate the role of miR-212 in silencing FIGLA, a luciferase reporter assay was performed using HeLa cells. The luciferase activity in cells expressing a luciferase construct containing the entire 3′ UTR of bovine FIGLA was suppressed by ∼40% in the presence of miR-212. We also investigated the stability of FIGLA mRNA in cells transfected with bovine FIGLA expression plasmid in the presence or absence of miR-212. Expression of bovine FIGLA mRNA was significantly reduced in the presence of mir-212 compared to control cells transfected with FIGLA construct alone. In summary, our data establish miR-212 as a potential post-transcriptional regulator of FIGLA during the maternal-to-embryonic transition in bovine embryos. Future studies aim to determine if miR-212 mimic can inhibit endogenous FIGLA expression in bovine embryos and its effect on subsequent embryonic development.

Ovarian gene expression in the absence of FIGLA, an oocyte-specific transcription factor

BackgroundOvarian folliculogenesis in mammals is a complex process involving interactions between germ and somatic cells. Carefully orchestrated expression of transcription factors, cell adhesion molecules and growth factors are required for success. We have identified a germ-cell specific, basic helix-loop-helix transcription factor, FIGLA (Factor In the GermLine, Alpha) and demonstrated its involvement in two independent developmental processes: formation of the primordial follicle and coordinate expression of zona pellucida genes.ResultsTaking advantage of Figla null mouse lines, we have used a combined approach of microarray and Serial Analysis of Gene Expression (SAGE) to identify potential downstream target genes. Using high stringent cutoffs, we find that FIGLA functions as a key regulatory molecule in coordinating expression of the NALP family of genes, genes of known oocyte-specific expression and a set of functionally un-annotated genes. FIGLA also inhibits expression of male germ cell specific genes that might otherwise disrupt normal oogenesis.ConclusionThese data implicate FIGLA as a central regulator of oocyte-specific genes that play roles in folliculogenesis, fertilization and early development.

Effect of Follicle-stimulating Hormone and Luteinizing Hormone on Zona Pellucida Gene Expression during Bovine Oocyte Maturation In Vitro.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in bovine oocyte maturation. This study tested the effect of FSH and LH on bovine ZP gene expression during oocyte maturation in vitro with RNase protection assay. These gonadotropins had no effect on ZP gene expression in bovine oocytes cultured for 5 or 15 hours, but significant suppression of germinal vesicle breakdown (GVB) and metaphase II (MII) generation were observed with FSH. These data suggest that neither FSH nor LH has a significant effect on ZP gene expression in bovine oocytes. And the ZP gene expression could be independent of the mechanism of oocyte nuclear maturation in cattle.

Cluster of genes encoding the major egg envelope protein of zebrafish

The proteins of fish egg envelopes are encoded by genes that are closely related to the genes for human zona pellucida proteins. A cluster of three genes coding for an egg envelope protein was isolated from the zebrafish, Danio rerio. The three genes, zp2a, zp2b, and zp2c, are located within an 11 kb region and are each comprised of eight exons spanning 1.85 kb. The exon-intron structures of the genes are nearly identical; however, their deduced amino acid sequences diverge at exon 7 (zp2b and zp2c from zp2a) and exon 8 (zp2c from zp2b). Exons 2-7 have a structural organization similar to exons in the carboxy-terminal half of the human zona pellucida ZP1, ZP2, and ZPB genes, suggesting they arose from a common ancestral gene. Sequence comparisons indicate that the deduced zebrafish proteins are most closely related to human ZPB. Zebrafish mRNAs coding for each of the three ZP2 variants have been found either as full-length cDNAs or expressed sequence tags. Distinct from the wf(female) gene of winter flounder which we first reported (Lyons et al., 1993: J Biol Chem 268:21351-21358), expression of the zebrafish zp2 genes was found to be ovary-specific, instead of liver-specific, and the promoter regions of zp2a and zp2b, while different, both contained E-box sequences (CANNTG) that have been demonstrated to be essential for coordination of zona pellucida gene expression in mammalian oocytes. Mixed peptide sequence analysis was used to identify the major polypeptide component of isolated zebrafish egg envelopes as the zp2 gene product.

Inhibition of Zona Pellucida Gene Expression by Antisense Oligonucleotides Injected Into Mouse Oocytes

During murine oogenesis, the zona pellucida proteins (ZP1, ZP2, and ZP3) are synthesized and secreted to form an extracellular matrix that surrounds the oocyte and mediates specific biological functions essential to mammalian fertilization and early development. To investigate the relationship among the zona proteins during zona matrix assembly, we have undertaken to inhibit de novo biosynthesis of specific zona proteins with antisense oligonucleotides complementary to the 5'-ends of ZP2 (nucleotide position 19-42) and ZP3 (nucleotide 21-44) mRNAs. When injected into the cytoplasm of growing mouse oocytes, the antisense oligonucleotides targeted specific zona mRNAs for degradation, as confirmed by a RNase protection assay. Individual zona pellucida protein synthesis was followed by immunoprecipitation with ZP2- and ZP3-specific monoclonal antibodies. New zona protein synthesis from the targeted mRNA was abolished, but nontargeted zona protein continued to be synthesized. Interestingly, abolishment of either ZP2 or ZP3 protein synthesis prevented the incorporation of the other protein into the extracellular zona matrix. These results suggest that ZP2 and ZP3 proteins are independent of each other in their biosynthesis but are dependent upon each other for their incorporation into the zona pellucida matrix. This study provides an experimental system in which destruction of a targeted mRNA generates a transient loss-of-expression phenotype during mouse oocyte growth.

ZAP-1 DNA Binding Activity Is First Detected at the Onset of Zona Pellucida Gene Expression in Embryonic Mouse Oocytes

ZAP-1 (zona pellucida gene activating protein-1) is a putative transcription factor controlling the oocyte-specific expression of mouse and human zona pellucida genes. The DNA binding activity of ZAP-1 first appears in oocytes from 19-day-old mouse embryos and reaches a maximum level at 10 days after birth. This developmental profile closely parallels that of mouse zona pellucida gene transcription, which is detected in oocytes at 19 days of fetal life using a sensitive RT-PCR method and is maximal in 10-day-old animals. DNA binding activity similar to that of ZAP-1 is present in ovarian extracts from rat, human, and opossum, suggesting that the ZAP-1 protein may be conserved among mammals.