Document Type : Research Paper
Authors
1 Department of Animal Science, Sari Agricultural and Natural Resources University, Sari, Iran
2 Department of Animal Science, Faculty of Agriculture, University of Guilan, Rasht, Iran.
Abstract
Keywords
Main Subjects
Extended Abstract
Introduction
Oocyte maturation is a long and complicated process, and occurs when oocyte attains the necessary conditions for fertilization and reaching the blastocyst stage (Ferreira et al., 2009). The nuclear maturation of the oocyte occurs in the metaphase meiosis Π; this stage is confirmed by the appearance of the first polar body and oocyte at this stage has the ability to be fertilized by sperm. Cumulus cells share an intimate communication with the oocyte through gap junctions and paracrine factors, allowing a bidirectional supply of nutrients and signaling molecules that regulate the simultaneous development and maturation of both cell types (Lourenco et al., 2014). Zhang et al.,1995 Research showed that removing cumulus cells before maturation, significantly reduced the rate of oocyte maturation, fertilization, and in vitro development at all stages. Environmental factors play an important role in reproductive disorders (Peterson et al., 2010). Pesticide is a compound or a mixture of several chemical compounds, that it is use in order to stop, destroying or reduce the population of insects, rodents, nematodes, fungi, weeds, or any kind of plants, bacteria and microorganisms that known as pest (Talebi Jahromi, 2007). Exposure to pesticide causes ovarian failure in cow (Pocar et al., 2003) and may be one of the major causes of various dysfunction in human and animal reproductive system (Johari, et al., 2010). A feature of these chemicals is electrophilicity, which can affect cellular proteins, by changing cellular properties, oocyte and follicular cells lose their normal functionality (Mohseni kuchsefahani et al., 2008). In mice, Diazinon decreases the thickness of granulosa layers which leads to a decrease in estrogen production, which itself exacerbates the reduction of granulosa cells (Mohseni kuchsefahani et al., 2008). Therefore, this research was conducted to investigate the effect of different concentrations of Diazinon as a pesticide, on the oocytes maturation rate, the expression of steroidogenic genes and DNA methylation of promoter site of StAR gene, as a key gene in regulation of steroids gene expression, in in vitro maturation of goat oocytes.
Materials and Methods
Unless otherwise stated, all of the chemicals were purchased from Sigma-Aldrich and Gibco. In order to prepare goat oocytes, ovaries were prepared from a slaughterhouse and transported to the laboratory into the flask containing warm saline (30-34℃). Oocyte-cumulus complexes (COC) was removed from small antral follicles with the slicing method, only COCs with at least 3 layers of cumulus cells and homogeneous cytoplasm were selected. The collected COCs were washed 4 times with SOF-HEPES solution and once in maturation medium (TCM-199), then transferred to Medium 199. COCs were placed in a maturation medium for 24 hours, and were reached to metaphase stage Π (nuclear maturation). The experiment was examined in a completely randomized design with five treatments included: control, 75, 37.5, and 18.75 μM of Diazinon, and 0.5 μM of DMSO. mRNA was isolated from cumulus cells of oocytes by using RNeasy Micro Kit (QIAGEN, 74140) according to manufacturer’s instructions. The isolated mRNA was reverse-transcribed with the QuantiNova Reverse Transcription kit according to manufacturer’s instructions. The qPCR mixture (15 µL) consist of 1 µL cDNA, 7.5 µL SYBR Green Mastermix, 0.7 µL reverse primer, 0.7 µL forward primer, sterile ddH2O 5.1 µL (to 15 µL). Temperature profile included initial denaturation at 94°C for 3 min, followed by 40 cycles at 94°C for 10 second and at 60°C for 30 minute. YWHAZ Gene was used for normalization. Standard curves were diluted in water using serial 10-fold dilutions. The data of gene expression profile was analyzed using General Linear Model (GLM) of the Statistical Analysis System (SAS) software program. Genomic DNA was extracted from 150 cumulus cells with using the QIAamp DNA kit according to manufacturer’s instructions. The extracted DNA kept in -20°C until DNA bisulfitation. DNA was sodium-bisulfite modified with EpiTect Bisulfite Kit (QIAGEN). DNA bisulfite conversion was prepared by thermal cycling upon addition of the EpiTect bisulfite mix provided in the kit. The PCR reaction was carried out with amplification profile: 1 cycle at 94°C for 3 min (Initial denaturation), 34 cycles at (94°C for 40 sec (Denaturation), 60°C for 40 second (Annealing of primer), 72°C for 1 min (Extension)), 72°C for 10 min (Final extension), hold and cooling to 4°C.
To finding different GC percentage between treatments we used sequencing after the PCR. The percentage of methylation in promotor region of StAR gene was calculated by the following formula:
C / (C + T) * 100 (T is thymine and C is cytosine) (Kacevska et al., 2012).
Results and discussion
This study showed that increase in the concentrations of Diazinon insecticide significantly reduced the oocyte maturation rate compare the control treatment. The percentage of oocytes that reached MII-stage (nuclear maturation) in control treatment were %85±2.28, On the other side the highest concentrations of Diazinon (75 µm of Diazinon) caused almost 98% cell death. According to the results of other treatments, we can say that with the increase in Diazinon concentration, the percentage of oocytes that reach MII-stage steadily decreases. Treatment with 18.75 µm of Diazinon has shown a significant increase in StAR gene expression compared to control treatment and it can be related to decrease in DNA methylation in Cpg11 of prompter region (66.5% methylation in control treatment compare to 51.1% methylation in treatment with 18.75 µm of Diazinon). In relation to CYP11A1 gene expression we saw a significant decrease in 18.75 and 37.5 µm treatments, and also a significant decrease in 37.5 μm of 17βHSD. In other hand CYP19 gene expression has shown a remarkable reduction in 18.75 and 37.5 μm of Diazinon.
Conclusion
The addition of different concentrations of Diazinon in the goat maturation medium, significantly decreased the maturation rate of goat oocyte than the control group. Also the gene expression of CYP11A1, 17βHSD, CYP19 in cumulus cells were reduced, but the expression of StAR gene in cumulus cells was increased. So it can be concluded that Diazinon has disruptor effect in maturation rate of oocyte and it is able to interrupt the expression of steroidogenic genes in cumulus cells.
)