Molecular Study of mitochondrial cytochrome oxidase subunit 1 gene in the oocyte in vitro maturation in the Iraqi ewes

This study was conducted on 668 ovary from abattoir of adult ewes. Three types oocytes aspirated from mature follicles (Oocyte with cumulus cells , partial surrounded by cumulus cells ,and oocytes without cumulus cells ) it is incubated in the Co 2 incubator to maturation in TCM199 .The step of oocyte maturation previously the in vitro fertilization, therefore the investigation of oocyte in the molecular level very important for detection the oocyte quality ,so the defect in the mitochondrial gene Cytochrome Oxidase subunit 1 (CO1) may take place through the oocyte cultured in the tissue culture media. Subsequent by genomic DNA extraction by (Bioneer Kit), to investigation the goal gene, samples yielded evidence of amplified CO1 gene on the agarose gel electrophoresis. No mutation in the CO1 sequence compared with the Al-naemi sheep breed and Assaf sheep breed . The conclusion , via PCR and sequencing of CO1 gene, no differences in the oocytes through the oocyte in vitro maturation in the age 3-6 old of ewes. Introduction The oocyte is surrounded by layers of cumulus cells and attached to the granuloma cells by a stalk of cumulus cells, it is made up of an oocyte enclosed by a glycoprotein coat, the zona pellucida, and is surrounded by tight, compact layers of cumulus cells (1-2). Eukaryotic cells contain two distinct genomes, one is located in the nucleus (nDNA) and is transmitted in a mendelian fashion, whereas the other is located in mitochondria (mtDNA) and is transmitted by maternal inheritance (3). A group of mitochondrial diseases are caused by mutations of the mtDNA and inherited (4). Human oocytes and early embryos from aging, clinically infertile women contain mitochondria with ultra structural features similar to those senescing or degenerating somatic cells including vaculation swelling and mineral deposition (5). Mitochondria are organelles that play an essential role in cellular energetic metabolism, homeostasis, and death (6). ATP depletion causes a series of cell injuries including plasma membrane permeabilization, lipid degradation, phospholipase A2 activation, intracellular calcium elevation, calpain activation, loss of membranecytoskeleton linkage and alteration of function aggregation of membrane proteins. Among these pathological changes plasma membrane become permeable to macromolecules the injury of the cells become irreversible (7). Mitochondrial genome very important for fertility in the ewes, the mitochondria energy house of cell, therefore any defect in the mitochondrial genomes led to low quality of oocyte with the infertility of ewes. Mitochondrial DNA (mtDNA) has been used to study the domestication history of many species (8), and the complete mtDNA sequence of sheep has been determined 660 nucleotides base pairs (bp) (9). Mitochondrial DNA content is critical to fertilization outcome and serves as an important marker of oocyte quality (10-11), The mean mtDNA copy number for the fertilized oocytes was 250,454, whereas for the unfertilized group it was 163, 698. There were significant differences for mtDNA copy number between the male factor infertility female factor infertility unfertilized oocytes and between unexplained infertility and female factor infertility group (12). Mitochondrial DNA copy number is an important indicator of oocyte developmental competence (13) COXI (NADH uniquinone oxidoreductase), this complex consist of 42 or 43 sub unit (14). Complex 1 of the respiratory chain consists of 41 subunits, 7 of which are encoded by mtDNA (15). Materials and Methods Ewes’ genitalia collected from abattoir, the age of ewes between 3-6-years old. The best age of oocyte quality between 3-6 years (16), the study included oocyte recovery from 668 ovaries, the ovaries transmitted by cooler container at 22 C washed with preserved by sterile normal saline. Oocyte aspirated by needle gage 18. The oocyte recovery mixed with TCM 199 in pitry dish. The procedure was done in humidified incubators maintained at 38.5 C in air with 5% CO2 and incubated 24 hour in the Co2 incubator. In the Biotechnology of Baghdad Center of Al– Nahrian University, oocytes recovered from tissue culture media in the petri dish and washing with normal saline, mechanically rid out the cumulus cells many times aspiration until to denude the oocyte. Subsequent by Genomic DNA extracted from oocytes after oocytes maturation. Genomic DNA was amplified using the polymerase chain reaction (PCR) (17-18-19). Detection of cytochrome oxidase subunit 1 gene was conducted by using primers for amplification of Ovis CO1 gene. A fragment 660 bp of Ovis CO1 ( 20) was amplified using a forward primer (Ovis CO1-F: 5-GCTGGTATCACAATACTACT-3 and a reverse primer (Ovis CO1-R: 5-TAGTCCTAGGAAATGCTGTG-3. These primer sets were supplied by IDT (Integrated DNA Technologies) company, Canada. Sequencing of CO1 gene was performed by national instrumentation center for environmental management (nicem) online at (http://nicem.snu. ac.kr/main/?en_skin=index.html), biotechnology lab, machine is DNA sequencer 3730XL, Applied Biosystem), Homology search was conducted using Basic Local Alignment Search Tool (BLAST) program which is available at the National Center Biotechnology Information (NCBI) online at (http:// www.ncbi.nlm.nih.gov) and BioEdit program. Result and Discussion Oocyte Collected from ewes ovaries obtained from abattoir, it is aspirated from mature follicle (21). There are

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