Balkan Journal of Medical Genetics

THE RELATIONSHIP BETWEEN TRANSCRIPT EXPRESSION LEVELS OF NUCLEAR ENCODED (TFAM, NRF1) AND MITOCHONDRIAL ENCODED (MT-CO1) GENES IN SINGLE HUMAN OOCYTES DURING OOCYTE MATURATION
Ghaffari Novin M, Allahveisi A, Noruzinia M, Farhadifar F, Yousefian E, Dehghani Fard A, Salimi M
*Corresponding Author: Dr. Azra Allahveisi, Department of Anatomical Sciences, Faculty of Medicine, Kurdistan University of Medical Sciences, Pasdaran Street, Sanandaj, Iran. Tel: +98-873-664-673. Fax: +98-873-364- 674. E-mail: allavaisie@gmail.com
page: 39

MATERIALS AND METHODS

Sample Collection. Oocytes in various stages of human oocyte maturation were obtained from nine normal women (20-35 years old), undergoing intracytoplasmic sperm injection (ICSI) treatment because of male factor infertility. There was no evidence of ovarian pathology for any of the patients donating oocytes. Examination proceeded according to the following criteria: 1) medical history, clinical examination and common hormonal tests; 2) Rotterdam criteria to ensure polycystic ovary syndrome; 3) diseases related to the endocrine system, such as hyperprolactinemia, thyroid dysfunction; 4) possible surgical history of the reproductive system; 5) normal ovulatory period (25-35 days); 6) body mass index (BMI) between 18.3-22.2 kg/m2; 7) day 3 basal ultrasonography and hormone profile with folliclestimulating hormone (FSH) <10.0 mIU/mL, estradiol <40.0 pg/mL and antral follicle count (AFC) >6; and 8) no history of smoking. Treatment and Donation. Informed consent were obtained from the patients at the Infertility and Reproductive Health Research Center (IRHRC) at Taleghani Hospital of Shahid Beheshti University of Medical Sciences and Sarem Women’s Hospital, Tehran, Iran. The study protocol was approved by the Ethical Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran. All nine patients were treated according to the agonist protocol. The gonadotropin-releasing hormone (GnRH) agonist was begun in mid-luteal phase of the previous cycle (day 21), as determined by serum E2 and P concentrations. Stimulation of the follicular growth, performed by FSH recombination, was started after sufficient down-regulation. This continued by daily injections according to individual endocrine and ovarian ultrasonic responses until at least one 18 mm diameter follicle showed. Ovulation was induced by human chorionic gonadotropin (HCG). Thirty-six hours later, vaginal puncture was performed under ultrasound-guided in vitro fertilization (IVF) and ICSI protocols using transvaginal probes for follow-up of the cumulusoocyte complex (COC). First, mechanical procedures used to remove cumulus granulose cells by intermittent pipetting and then followed by enzymatic hyaluronidase treatment. Oocytes were subsequently washed in culture media (global media) before use. Collected oocytes were studied for nuclear maturity under a stereo microscope (Olympus, Tokyo, Japan). Morphological characteristics of the collected oocytes were classified into three categories based on the nuclear condition: 1) mature oocytes (MII) with first polar body (n = 8); 2) immature oocytes GV (n = 10); and 3) immature oocytes [metaphase I (MI)] without original polar body (n = 9). Sample Processing for Gene Expression. Oocytes were processed using Ambion Single Cell-to- CT® Kit (Life Technologies, Foster City, CA, USA) according to the manufacturer’s protocol. Sibling oocytes were rapidly transferred to sterile RNasefree microtubes to minimize any changes in gene expression. Then, oocytes were transferred to lysis solution containing DNase I. The enzyme was inactivated using stop solution following 5 min. of incubation at room temperature and DNA digestion. Then, Single Cell VILO® RT Mix and Single Cell SuperScript® RT (Life Technologies, Carlsbad City, CA, USA) were added to each sample reaction. Reverse transcription-PCR (RT-PCR), was performed using 15.5 μL sample volume per reaction. After completing RT-PCR, PreAmp® reaction mixtures were prepared by adding 0.2 × pooled TaqMan® Gene expression assay to Single Cell PreAmp® Mix (Life Technologies) for each gene. Then, cDNA of each gene was specifically amplified through the following thermal cycling condition: 95 °C for 10 min. and then 17 cycles that included 15 seconds at 95 °C and 4 min. at 60 °C and a final 10 min. heat at 99 °C. Gene Expression Analysis. Quantitative RTPCR was used to quantify mRNA transcript levels of TFAM, MT-CO1 and NRF1 genes using hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA transcripts as endogenous reference. Relative mRNA expression levels of the studied genes were quantified using TaqMan® qRT-PCR analysis (Applied Biosystems, Foster City, CA, USA) in 48-well plates. Relative gene expression was calculated using the 2-ΔΔCT formula. The Ct value of each transcript was calculated via TaqMan® (Applied Biosystems) gene expression assay for transcripts. Designing Primers and Probes. The specifications of the forward and reverse primers and Taq- Man® (Applied Biosystems) probes are listed in Table 1. Statistical Analysis. Analysis of variance (ANOVA) with the post-hoc Tukey test and the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) software, version 16.0, were used to determine differences between the mean values.

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