Development Competence and Molecular Characterization of Quail Oocytes after Ovulation

Abstract

Natural fertilization in avian species occurs in the infundibulum shortly after oocyte is ovulated. Once the ovum moves beyond the infundibulum, the secretions from magnum hinder sperm to interact with the oocyte, but intracytoplasmic sperm injection (ICSI) enabling the fertilization of oocytes from magnum has been demonstrated in past studies. However, in these studies, birds must have been sacrificed for oocyte collection. If the oocyte in the shell gland can be used for ICSI studies, animal ethnic issue of sacrificing birds can be avoided and it is expected that the study can be applied to save the endangered avian species. Accordingly, the objective of the first study was to determine fertilizing capability of the oocytes with the time elapsed after ovulation. First, Japanese quail sperm and inositol 1,4,5-trisphosphate (IP3) were injected by ICSI technique to induce ex ovo fertilization. Injection was performed to the oocytes collected at (1) 0.5-1.5 (2) 3.5-4 (3) 5-6 (4) 9-10 and (5) 22-23 hr after ovulation. The injected oocytes were cultured in DMEM inside a CO2 incubator for 24 hr. In order to determine the fertilization and blastoderm development rates of the oocytes, the morphology of the blastoderms was observed under a stereo microscope and histological verification was conducted after staining with 4.6-diamidino-2-phenylindole (DAPI), respectively. The highest rate of fertilization (60%) was observed in oocytes collected at 0.5-1.5 hr post ovulation, whereas it ranged between 36.4-18.8% in oocytes collected at other hours. 16.7% of oocytes collected at 0.5-1.5 hr post ovulation were developed beyond Eyal-Giladi and Kochav (EG&K) stage IV. However, the development of the oocytes collected at later hours post ovulation ceased development before EG&K stage IV. In this study, the positive sign for fertilization was seen in oocytes collected at all hours post ovulation. This indicates that the fertilization capability is maintained until the egg is oviposited. However, the embryonic development of the eggs collected from isthmus and beyond ceases before EG&K stage IV. Therefore, an additional treatment to reverse the aging process of the oocytes can assist the embryonic development. In order to improve the fertilization rate and further embryonic development, it seems necessary to elucidate underlining mechanism of incompetency of oocyte development. Accordingly, the molecular changes in unfertilized oocytes were studied in relation to times after ovulation. Also, responsiveness of oocytes to IP3 injection was evaluated in terms of changes in mRNA expression. Here, quantitative RT-PCR analysis was employed to assess expression of several mRNA transcripts in the oocytes which may be associated with decay of fertilization and development. In order to observe the expression of the selected genes, the unfertilized oocytes were collected at 0.5-1.5 hrs post ovulation for negative control. For the IP3 injected group, the unfertilized oocytes collected at 0.5-1.5, 5-6 and 22-23 hrs post ovulation and injected with IP3 using ICSI method. Both groups of oocytes were incubated in DMEM for 3 hrs and each of the oocytes was subjected to total RNA extraction and cDNA synthesis for the Real time PCR analysis. ITPR3 mRNA expression was variable among the unfertilized and IP3-induced groups. Hence, there was no significant difference. On the other hand, as for mRNA expression of ITPR1 in the unfertilized oocyte and the IP3-injected oocytes, the up-regulated expression was observed at 0.5-1.5 hr post ovulation but much lower expression was at 5-6 hr and 22-23 hr post ovulation. The same expression pattern in ITPR2 mRNA was seen both in the unfertilized and IP3-injected oocytes. These correlated expression patterns of up-regulation at 0.5-1.5 hr and low expression at later hours post ovulation may indicate that IP3 receptors type 1 and type 2 are involved in the process of calcium release and oocyte activation. The same tendency of gene expression patterns were observed for BCL2L1, CDK1 and MAD2L2. The expression of BCL2L1, CDK1, MAPK1 and MAD2L2 was highly upregulated in the injected oocytes at 0.5-1.5 hr post ovulation when compared to oocytes at other time. This indicates that the oocytes 0.5-1.5 hr post ovulation showed the significantly high responsiveness to IP3 compared to other oocytes. Therefore, the low mRNA expression of ITPR1 and ITPR2 may be associated with the low fertilization rate in oocytes collected from shell gland and oviposited eggs. Since the fertilization competency is maintained until the egg is oviposited, if the reversal of the oocyte aging is possible, the oocytes collected from shell gland and oviposited eggs can be used for successful fertilization and embryonic development and finally hatch. The present study is the first step towards understanding the fertilization capability in the avian species and to apply the new findings to contribute towards saving birds from being sacrificed and protecting endangered avian species.