Introduction
The main function of mitochondria is the production of adenosine triphosphate (ATP) by oxidative phosphorylation, and is performed by mitochondrial respiratory chain complexes located in the inner mitochondrial membrane (Wallace, Reference Wallace2005). Moreover, it plays an important role in ageing, calcium homeostasis, apoptosis and synthesis of many molecules such as proteins, nucleotides and steroids (Wolstenholme, Reference Wolstenholme1992; Pfeiffer et al., Reference Pfeiffer, Dirndorfer, Lang, Resenberger, Restelli, Hemion, Miesbauer and Frank2013). Because of these pleiotropic effects, mitochondrial defects have been related to several diseases such as obesity, diabetes, Alzheimer’s, Parkinson’s and cancer (Taylor and Turnbull, Reference Taylor and Turnbull2005; Wallace, Reference Wallace2005). In addition, mitochondrial dysfunction can be responsible for infertility in both males and females, as mitochondrial dysfunction affects not only sperm and oocyte quality but also the process of fertilization and early embryonic development (Van Blerkom et al., Reference Van Blerkom, Davis and Alexander2000; Sutarno et al., Reference Sutarno Cummins, Greeff and Lymbery2002).
Mitochondria have their own genome, which is mainly maternally inherited. The mitochondrial genome contains 13 genes that encode subunits of enzymes involved in the mitochondrial respiratory chain complexes to produce ATP. ND5 is one of the genes encoding subunit 5 of complex I (NADH dehydrogenase) (Wolstenholme, Reference Wolstenholme1992). Some authors have considered the effects of mtDNA polymorphisms on reproduction traits in livestock (Reicher et al., Reference Reicher, Seroussi, Weller, Rosov and Gootwine2012; Chen et al., Reference Chen, Wang, Xiang, Dun, Brahi, Yin and Zhao2017; Pradhan et al., Reference Pradhan, Pal, Samanta, Banerjee and Samanta2018). For example, Sutarno et al. (Reference Sutarno Cummins, Greeff and Lymbery2002) demonstrated that polymorphisms in the ND5 gene significantly affected the average calving rates in cattle.
In vitro embryo production (IVEP) is a useful tool for producing large numbers of embryos for research purposes to help understand factors affecting oocyte maturation and early embryonic development (Rizos et al., Reference Rizos, Clemente, Bermejo-Alvarez, de La Fuente, Lonergan and Gutiérrez-Adán2008). Although a few studies have examined the relationship between nuclear genes polymorphisms and IVEP (Cognie et al., Reference Cognie, Benoit, Poulin, Khatir and Driancourt1998; Lechniak et al., Reference Lechniak, Adamowicz, Stanisławski and Kaczmarek2002; Lechniak et al., Reference Lechniak, Szczepankiewicz, Kauss, Szulc and Szydłowski2005), no studies have investigated the effects of mtDNA polymorphisms on IVEP in livestock. Therefore, the aim of this study was to investigate the association between mitochondrial ND5 gene polymorphisms with in vitro maturation (IVM) of oocytes and subsequent embryonic development in Sanjabi ewes.
Materials and methods
In this study, blood and ovarian samples from Sanjabi ewes were obtained from a local slaughterhouse. For each ewe, 5 ml blood was obtained from the jugular vein before slaughter. The ovaries were collected immediately after slaughter and transported to the laboratory in a 0.9% saline solution supplemented with 100 mg/ml penicillin and streptomycin. For each ovarian sample, cumulus–oocyte complexes (COCs) with a diameter of more than 3 mm were recovered from follicles. In total, 100 ewes’ samples with more than two COCs were used for subsequent experiments.
COCs of each sample were matured in 50-μl droplets of IVM medium containing tissue culture medium 199 (TCM-199) supplemented with 10% fetal bovine serum (FBS), 0.5 μg/ml follicle-stimulating hormone (FSH), 5 μg/ml luteinizing hormone (LH), 1 μg/ml estradiol 17β, 50 μg/ml of gentamycin sulfate, 50 ng/ml epidermal growth factor and 2.5 mM sodium pyruvate. Petri dishes containing oocytes were incubated for 24 h at 39°C in an incubator with 5% CO2 and 90–95% relative humidity in air. After 24 h, the IVM rate for each sample was recorded.
For in vitro maturation (IVF), matured oocytes were washed three times with IVF medium and moved to IVF medium supplemented with 6 mg/ml bovine serum albumin and 2 µg/ml heparin. Insemination was performed by adding diluted fresh semen from a fertile ram and PHE (20 µmol/l penicillamine, 10 µmol/l hypotaurine and 1 µmol/l epinephrine). Finally, the inseminated oocytes were incubated at 39°C at 5% CO2 in air for 18 h.
After IVF, putative zygotes were cultured in synthetic oviduct fluid medium supplemented with 10% (v/v) FBS, 2× non-essential amino acid solution, 1.5 mM/l glucose and 0.1 mmol/l glutamine. The embryos were then incubated under conditions of 5% CO2, 39ºC and an humidified atmosphere for 48 h. The embryonic cleavage rate was recorded after 48 h of in vitro culture (IVC).
DNA samples were isolated from whole blood using a salt extraction method. A primer pair (forward: 5′-ACTAACCATACCCATCGCAGC-3′ and reverse: 5′-GCAGAGGGTAGTCATGGATGT-3′) was designed according to the reference sequence (accession number NC_001941.1) to amplify a 666-bp fragment of the ND5 gene using the polymerase chain reaction (PCR). The PCR protocol consisted of an initial denaturation step at 94°C for 5 min followed by 35 cycles of 94°C for 45 s, 64°C and 72°C for 60 s, with a final extension cycle at 72°C for 10 min. Each PCR reaction was carried out with in a 25 μl volume containing 100 ng of genomic template DNA, 10 pM of each primer, 250 mM of dNTP mix, 1 U Taq DNA polymerase, 2.5 μl of 10× PCR buffer, 1.5 μl of 25 mM MgCl2 and ultrapure water.
A modified single-stranded conformation polymorphism (SSCP) method was used to genotype the samples. In this method, 4 µl of each PCR product was mixed with 6 μl SSCP dye (80% formamide, 18% glycerol, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene–cyanol). DNA double strands were denatured at 94°C for 10 min, then an additional step at 60°C for 35 s was used to anneal the primers, after which the samples were cooled immediately on ice. Next, 5 μl of each sample were then loaded on a 6% acrylamide:bisacrylamide (29:1) gel. Electrophoresis was performed at 100 V for 6 h in 1× TBE and different conformations of the strands were visualized using a silver staining method (Bassam and Gresshoff, Reference Bassam and Gresshoff2007).
The relationship between different SSCP patterns and IVM and IVC rate was investigated using the following model:
${\rm Y_{ij} = \mu + B_i + e_{ij}}$
where Yij is the IVM or IVC rate; μ is the overall mean; Bi is the fixed effect of ith genotype; and eij is the residual error. As the IVM and IVC percentage showed non-normal distribution, differences between different SSCP pattern were analyzed using the Kruskal–Wallis non-parametric test by ranking the data, followed by Dunn’s post-hoc test. Analyses were performed in R programming language (v.3.5.2).
Results
Genotyping using the SSCP method showed six different banding patterns (named: A, B, C, D, E and F) (Fig. 1). The highest frequency was related to pattern B (47 %) and the lowest frequency was observed for patterns C and D (4%) (Table 1).
SSCP banding patterns were observed for the ND5 gene.
Mean ± standard deviation (SD), sum of rank scores, mean of rank scores and P-value observed for the IVM and IVC rates in different patterns of the ND5 gene
Results of the Kruskal–Wallis non-parametric test analysis of IVM and IVC data ranks in different SSCP band patterns are shown in Table 1. It is clear from this table that there were no significant differences between the SSCP patterns with regard to IVM rates (P > 0.05). The mean of IVM rank scores for patterns of A, B, C, D, E and F were 39.94, 49.42, 60.37, 58, 48.53 and 36.40, respectively. Similarly, no significant association was found between SSCP patterns and the mean of IVC rank scores. The mean of rank scores for patterns of A, B, C, D, E and F were 43.25, 47.72, 60.50, 60.50, 51.22 and 27.8, respectively.
Discussion
In this study, a modified SSCP method was chosen because few studies have reported information on polymorphisms of the ND5 gene in sheep, and this method does not require prior sequence information. One of the limitations of the SSCP method is its efficiency for small DNA fragments but, with our modification, we were able to detect the polymorphisms in a 666-bp fragment.
The results of PCR–SSCP showed the presence of polymorphisms in the ND5 gene. Although no studies have been performed to investigate polymorphisms of ND5 gene in this breed, these results were expected because mtDNA is more polymorphic than nuclear DNA (Wallace et al., Reference Wallace, Brown and Lott1999). Moreover previous studies on ND5 in sheep have reported polymorphisms in this region (Reicher et al., Reference Reicher, Seroussi, Weller, Rosov and Gootwine2012; Chen et al., Reference Chen, Wang, Xiang, Dun, Brahi, Yin and Zhao2017).
The results of this study showed that the ND5 gene does not affect IVM and IVC rates. One possible explanation for these results may be the lack of adequate sample per SSCP pattern. For example, with the exception of patterns B and E, the number of ewes was low in the other patterns (Table 1). In reviewing the literature, no data were found on the association between mtDNA polymorphisms and IVEP in livestock. However, several reports have shown a relationship between mitochondrial gene polymorphisms and reproduction traits of farm animals. Sutarno et al. (Reference Sutarno Cummins, Greeff and Lymbery2002) showed a significant association between the D-loop and ND5 polymorphisms and calving rates in cattle. Some authors have considered the effects of mtDNA polymorphisms and litter size in sheep (Reicher et al., Reference Reicher, Seroussi, Weller, Rosov and Gootwine2012; Chen et al., Reference Chen, Wang, Xiang, Dun, Brahi, Yin and Zhao2017). Some studies in pig have also suggested an association between mtDNA polymorphisms and reproductive traits such as numbers of oocytes, litter size, age at first farrowing and farrowing interval (Pradhan et al., Reference Pradhan, Pal, Samanta, Banerjee and Samanta2018; Liu et al., Reference Liu, Shi, Wang and Zhao2019).
Conclusion
Our study showed that our modified SSCP method could be used to genotype large fragments. This research also revealed the existence of polymorphisms in the ND5 gene of Sanjabi sheep. Moreover, there was no significant relationship between ND5 gene polymorphisms and IVM and IVC rates.
Acknowledgements
The authors thank Leila Soltani and Sara Samereh for their assistance in conducting IVEP.
Financial support
This work was supported by the Iran National Science Foundation (INSF) (grant number 95841263).
Conflict of interest
The authors declare none.
