Introduction
Parthenogenesis is defined as the development of an egg without contribution of the male gamete. Although parthenogenesis is a common mode of reproduction in some invertebrates, leading to live progeny, mammalian parthenotes are unable to develop to term in utero (Fukui et al., Reference Fukui, Sawai, Furudate, Sato, Iwazumi and Ohsaki1992). This event may be due to alterations in their genomic imprinting, an epigenetic mechanism that gives rise to different expression of the maternally and paternally inherited alleles of certain genes (John & Surani, Reference John and Surani1996; Feil et al., Reference Feil, Khosla, Cappai and Loi1998). In mammals, the maternal and paternal genomes are functionally different, therefore both genomes are required for normal embryonic development. For normal early embryonic development, different kinds of genes are involved such as developmentally related genes (Oct4, Nanog, Sox2, Stat3, Cx43, etc.), X-linked genes (HPRT1, Xist, Pgk1, etc.), or imprinting genes (IGF2R, Mest, H19, etc.). The reason for the limited developmental potential of parthenogenetic embryos seems to be an imbalance in the expression of embryonic developmental-related genes during different cell stages of embryos. Several reports have indicated that parthenogenetic mouse embryos (haploid and diploid) are small and die at early developmental stages due to the lack of paternally expressed genes (McGrath & Solter, Reference McGrath and Solter1984; Mann & Lovell-Badge, Reference Mann and Lovell-Badge1987). The paternal Mest/Peg1 and IGF2 genes are fully repressed in parthenogenetic mouse embryos (Szabo & Mann, Reference Szabo and Mann1996) and the absence of Peg1/Mest leads to a 20% reduction in fetal growth as reported by Feil et al. (Reference Feil, Khosla, Cappai and Loi1998). The IGF2R gene (that encodes the receptor for insulin-like growth factor type-2) is imprinted and expressed from the maternal allele after embryonic implantation. This gene was found to be responsible for regulation of growth and is associated with developmental competence (Bebbere et al., Reference Bebbere, Bogliolo, Ariu, Fois, Leoni, Succu, Berlinguer and Ledda2010).
Sox2 is one of the important early embryonic development-related genes (Li et al., Reference Li, Pan, Cui, Liu, Xu and Pei2007) and it has been observed that the overexpression of a dominant-negative form of this gene leads to the differentiation of ES cells into cells with a trophectoderm phenotype (Li et al., Reference Li, Pan, Cui, Liu, Xu and Pei2007; Masui et al., Reference Masui, Nakatake, Toyooka, Shimosato, Yagi, Takahashi, Okochi, Okuda, Matoba, Sharov, Ko and Niwa2007). Connexins (Cx43) and several gap junction proteins are a family of structurally related transmembrane proteins that are essential for many physiological processes and proper embryonic development. Mutations in connexin-encoding genes can lead to functional and developmental abnormalities of embryos (Laird, Reference Laird2006). It has been observed that human preimplantation embryos express predominantly Cx43 and that its level increases throughout development to the blastocyst stage (Hardy et al., Reference Hardy, Warner, Winston and Becker1996). The X-linked HPRT1 gene encodes the hypoxanthine–guanine phosphoribosyltransferase (HGPRT) enzyme that plays a central role in the generation of purine nucleotides through the purine salvage pathway (Finette et al., Reference Finette, Kendall and Vacek2002). The alleles of this X-linked gene show strong parental influences on the development of preimplantation mouse embryos before and after X inactivation (Sturm et al., Reference Sturm, Lafferty and Tam1999).
The aberrant expression of these genes has been detected in preimplantation embryos, mostly in mice and human parthenogenetic embryos (Park et al., Reference Park, Park and Kim2003; Han et al., Reference Han, Song, Uhum, Do, Kim, Chung and Lee2003). However, very little information is available for domestic animals. Therefore, the present study was conducted to compare the expression profile of these important genes during different stages of parthenogenetic (haploid and diploid) and IVF embryos in caprine to understand the development of parthenogenetic embryos in this species.
Materials and methods
Collection of the oocytes and in vitro maturation
Goat ovaries were collected from a local abattoir and carried to the laboratory in normal saline solution (NSS; 0.85%) containing antibiotics in a thermos flask at 35–37°C and within 2 h of slaughter. Cumulus–oocyte complexes (COCs) were aspirated from all visible non-atretic follicles by an 18-gauge needle attached to a 5-ml syringe that contained oocyte collection medium (OCM). Only excellent (i.e. more than five layers of cumulus cells and evenly granulated cytoplasm) and good (more than three layers of cumulus cells and evenly granulated cytoplasm) COCs were selected and drop-washed several times in OCM, and then further drop-washed 5–6 times with in vitro maturation (IVM) medium. Groups of 15–20 selected oocytes were placed in 60 μl droplets of IVM medium. The IVM medium used was TCM-199, supplemented with 10% fetal bovine serum (FBS), bovine serum albumin (BSA; 3 mg/ml), follicle-stimulating hormone (FSH; 0.5 μg/ml); leuteinizing hormone (LH; 10 IU/ml); goat follicular fluid (5%); sodium pyruvate 2 mg/ml; l-glutamine (0.1 mg/ml) and gentamycin (50 μg/ml). The droplets were covered with warm non-toxic mineral oil and cultured at 37°C, in an atmosphere of 5% CO2 and 20% oxygen under humidified air (95%) for 27 h in a CO2 incubator.
Production of parthenogenetic embryos
After maturation, the oocytes were treated with 0.1% hyaluronidase in TCM-199 followed by pipetting to remove the cumulus cells. Finally, cumulus-free oocytes were activated using 5 μm ionomycin in mSOF for 5 min followed by 4 h incubation with 2 mM 6-DMAP in mSOF. After activation, oocytes were taken out of the 6-DMAP drop, washed several times in modified synthetic oviductal fluid (mSOF) that was supplemented with 0.8% BSA and essential and non-essential amino acids and cultured in 100 μl of same medium in a CO2 incubator at 37°C, 5% CO2 and 95% air relative humidity until assessment to determine cleavage. For embryo development, cleaved oocytes were subsequently transferred to a fresh drop of mSOF and were further cultured.
Production of in vitro fertilized embryos
Fresh semen was collected from a buck using an artificial vagina. Fifty μl semen was added to 5 ml of quenched Sperm-TALP medium (Totey et al., Reference Totey, Singh, Taneja, Pawsche and Talwar1992) and centrifuged at 800–1000 rpm for 5 min. The pellet was dissolved in 5 ml fresh Sperm-TALP medium and centrifuged again as above. Finally 2 ml of Fert-TALP medium was added and cells were kept in a 5% CO2 in air incubator at 37°C for 1 h.
After maturation, the oocytes (with degree-1 and degree-2 cumulus expansion) were washed several times in Fert-TALP medium to remove expanded cumulus cell mass. After washing, oocytes were transferred to 50 μl of Fert-TALP medium and were inseminated with 50 μl of processed spermatozoa. The droplets were covered with warm paraffin oil and placed in a CO2 incubator at 37°C, 5% CO2 in air and 90% relative humidity. After 18 h of sperm–oocytes co-incubation, the oocytes (presumptive zygotes) were washed 10–15 times in embryo development medium, namely mSOF (Walker et al., Reference Walker, Hartwich and Seemark1996), and cultured in the same medium supplemented with 0.8% BSA. The cleavage rate was recorded at 48–72 h post insemination and further observations were made to monitor the development of the embryos.
RNA extraction and reverse transcription
For gene expression analysis, parthenogenetic (haploid and diploid) and IVF embryos of different cell stages from three independent experiments were picked up from the culture medium separately and washed with 1× PBS. Total RNA was isolated from 2–4-cell stage embryos (20 embryos), 8–16-cell stage embryos (six embryos) and morula-cell stage embryos (three embryos) using the Quick-RNA™ MicroPrep, Zymo Research Kit (catalogue no. R1050). cDNA was synthesized using the iScript™ Select cDNA Synthesis kit (Bio-Rad, catalogue no. 170–8897). After preparation of cDNA, the cDNA concentration was adjusted to 600 ng/μl and used for real-time PCR study.
Real-time PCR
Relative quantification was performed using real-time polymerase chain reaction (PCR) (Bio-Rad CFX Manager™ Software) and EvaGreen supermix (catalogue number 172–5200), that contained a double-stranded DNA-specific fluorescent dye. For amplification of the selected genes, primers were designed using Beacon Software. The sequences of primers for real-time PCR were as follows: β-actin F5′-TCATCACCATCGGCAATG-3′ and R5′-CCAATCCACACGGAGTAC-3′; Cx43 F5′-CATCATCAGTATCCTCTTCAAG-3′ and R5′-CAGTGGTAGTGTGGTAAGG-3′; HPRT1 F5′-CATTATGCTGAGGATTTGGAGAAG-3′ and R5′-GCCTGTTGACTGGTCGTTAC-3′; Sox2 F5′-CGGCAACCAGAAGAACAG-3′ and R5′-CGGCAGTGTGTACTTATCC-3′; Mest F5′-CCGACCTTCTGAGAGTGAG-3′ and R5′-CTGTGGATAGTGGCTAATGTG-3′; and IGF2R F5′-GAACTGTAAGCAGCAGAATCAC-3′ and R5′-GAGTCGTCCACCAAGTAAGC-3′. The real-time PCR thermocycling conditions were: an initial denaturation step at 95°C for 30 s followed by 45 cycles of 95°C for 5 s, annealing at 60°C for 15 s. The transcript levels of all five genes were quantified using the relative quantification method based on comparative threshold cycles values (Ct). The abundance of the gene was determined relative to the abundance of the housekeeping gene β-actin.
Analysis of data
For comparison of gene expression of parthenogenetic (haploid and diploid) embryos with IVF embryos, the relative expression of data was analysed using Rest 2009 software and a P-value <0.05 was considered to be significant.
Results
The results indicated that HPRT1 expression was upregulated (P < 0.05) only at the 2–4-cell stage of haploid embryos as compared with the control (Fig. 1). Cx43 was significantly (P < 0.05) downregulated in all the stages of haploid embryos compared with control, whereas it was upregulated (P < 0.05) in the 2–4-cell and morula stages of diploid embryos (Fig. 2). Sox2 was significantly (P < 0.05) downregulated in the morula-cell stage of haploid embryos compared with the control, whereas expression was upregulated (P < 0.05) in the 8–16-cell stage of diploid embryos (Fig. 3). Mest expression was upregulated (P < 0.05) in 2–4-cell stage of both haploid and diploid embryos, whereas it was downregulated in the 8–16-cell stage diploid embryos compared with the control (Fig. 4). IGF2R expression showed upregulation (P < 0.05) in the morula stage of parthenogenetic embryos as compared with the control (Fig. 5).
Relative expression of HPRT1 in different stages of parthenogenetic haploid and diploid embryos as compared with IVF-derived embryos in caprine. Asterisk indicates significant difference compared with the control.
Relative expression of Cx43 in different stages of parthenogenetic haploid and diploid embryos as compared with IVF-derived embryos in caprine. Asterisk indicates significant difference compared with the control.
Relative expression of Sox2 in different stages of parthenogenetic haploid and diploid embryos as compared with IVF-derived embryos in caprine. Asterisk indicates significant difference compared with the control.
Relative expression of Mest in different stages of parthenogenetic haploid and diploid embryos as compared with IVF-derived embryos in caprine. Asterisk indicates significant difference compared with the control.
Relative expression of IGF2R in different stages of parthenogenetic haploid and diploid embryos as compared with IVF-derived embryos in caprine. Asterisk indicates significant difference compared with the control.
Discussion
In the present study, all five genes showed significantly different patterns of expression at all the stages of parthenogenetic haploid and diploid embryos in comparison with similar stages of IVF embryos. This situation could be because haploid embryos have only one set of autosomes and X chromosome while diploid embryos have a double set of autosomes and two XX chromosomes that have different genomic imprinting, X inactivation and gene expression phenomena compared with in vitro fertilized (IVF) embryos. In vitro fertilized embryos have maternal and paternal genomes, exhibit balanced gene expression, methylation, gene imprinting and X inactivation and, therefore, survive easily. It has been suggested that assisted reproductive technology procedures affect the imprinting states of preimplanted embryos. Furthermore, environmental factors such as culture conditions and manipulation may influence methylation patterns, and thus affect the expression of genes in embryos at various developmental stages (Mann et al., Reference Mann, Lee, Doherty, Verona and Nolen2004). The disproportionate expression of early embryonic developmentally related as well as maternally and paternally imprinting genes are thought to be responsible for the poor survival rate of parthenogenetic embryos. In this study, HPRT1 expression was upregulated (P < 0.05) at the 2–4-cell stage of haploid embryos. Latham (Reference Latham2005) reported that the developmental arrest in early androgenones may be, in part, due to reduced expression of essential X-linked genes, particularly those near the X inactivation centre, whereas the developmental defects of gynogenones and parthenogenones, by contrast, may be partially due to overexpression of X-linked genes in extraembryonic tissues, possibly those farthest away from the X-inactivation center. Most of the X-linked genes upregulated in female embryos exhibited a fold-change lower than 2, a finding that suggested that X-chromosome inactivation was only partial. Unfortunately, very little information is known about X-chromosome inactivation during preimplantation development in other species than mouse (Davies et al., Reference Davies, Isles and Wilkinson2005).
Cx43 expression was significantly (P < 0.05) downregulated in all stages of haploid embryos compared with the control, whereas it was upregulated (P < 0.05) in 2–4-cell and morula stages of diploid embryos. Cx43 or Gap junctions have been reported to play a pivotal role in co-ordinating embryonic development. Cx43 and Cx31 mRNA has been found to be expressed from the compacted 8-cell stage onward in bovine (Reuss et al., Reference Reuss, Hellmann, Traub, Butterweck and Winterhager1997). Cell coupling is governed by several mechanisms, including connexin expression (Kihara et al., Reference Kihara, de Castro, Moriscot and Hamassaki2006). Improper cell communication is thought to be one of the reasons for the poor development of parthenogenetic embryos compared with IVF embryos. To our knowledge, no comparable study for caprine parthenogenetic embryos is available in the literature but Amarnath et al. (Reference Amarnath, Li, Kato and Tsunoda2007) observed that Cx43 expression was lower in cumulus cell nuclear-transferred (CNT) embryos compared with IVF-derived embryos in bovine, a finding that is fundamentally supportive to our study. In the case of Sox2, expression was significantly (P < 0.05) downregulated at the morula-cell stage of haploid embryos compared with the control, whereas it was upregulated (P < 0.05) at the 8–16-cell stage of diploid embryos. Sox2 is associated with pluripotency (Chen et al., Reference Chen, Rebecca, Rebecca, Rachel, Ernst and Nicholas2012) and overexpression of a dominant-negative form of Sox2 leads to differentiation of ES cells into cells with a trophectoderm phenotype (Li et al., Reference Li, Pan, Cui, Liu, Xu and Pei2007; Masui et al., Reference Masui, Nakatake, Toyooka, Shimosato, Yagi, Takahashi, Okochi, Okuda, Matoba, Sharov, Ko and Niwa2007). Because of the imbalance expression of the Sox2 gene, parthenogenetic embryos may not survive long term. One similar study revealed that the relative abundance of Sox2 was statistically different among the three sources of embryos at the 2-cell stage. Embryos produced by IVF, showed a higher level of Sox2 expression compared with in vivo derived and parthenogenetic embryos (IVF versus in vivo derived and parthenogenetic, P < 0.05). Sox2 transcript abundance was decreased at the 4-cell stage in in vivo-derived embryos (in vivo versus IVF and parthenogenetic embryos, P < 0.05). At the blastocyst stage, embryos produced by IVF expressed a lower amount of Sox2 transcript than the in vivo derived or parthenogenetic embryos (IVF versus parthenogenetic and in vivo, P < 0.05) (Magnani & Cabot, Reference Magnani and Cabot2008).
Genomic imprinting, a specific genetic mechanism in mammals, plays important roles in the regulation of fetal growth, development, placental function, and postnatal behavior (Li et al., Reference Li, Keverne, Aparicio, Ishino, Barton and Surani1999; Ono et al., Reference Ono, Nakamura, Inoue, Naruse, Usami, Wakisaka-Saito, Hino, Suzuki-Migishima, Ogonuki, Miki, Kohda, Ogura, Yokoyama, Kaneko-Ishino and Ishino2006; Liu et al., Reference Liu, Zhu, Liang, Yin, Ola, Hou, Chen, Schatten and Sun2008). Genomic imprinting is controlled by DNA methylation, histone modifications, non-coding RNA and specialized chromatin structure (Pauler & Barlow, Reference Pauler and Barlow2006). Unipaternal fetuses, including parthenotes and androgenotes, show disrupted expression of several imprinted genes (Ogawa et al., Reference Ogawa, Wu, Komiyama, Obata and Kono2006). In the present study, Mest gene expression, which is imprinted in several species, was upregulated (P < 0.05) at the 2–4-cell stage of both haploid and diploid embryos, whereas it was downregulated in 8–16-cell stage diploid embryos compared with control. Morula stage parthenogenetic mouse embryos showed 100% (11/11) hyper-methylation strands, a finding that indicated that diploid parthenogenetic embryos have the capacity to restore the methylation imprints of Mest on two sets of maternal chromosomes (Liu et al., Reference Liu, Zhu, Liang, Yin, Ola, Hou, Chen, Schatten and Sun2008). Heavy methylation imprinted genes may account for the low expression of Mest in parthenogenetic fetuses (Ogawa et al., Reference Ogawa, Wu, Komiyama, Obata and Kono2006; Liu et al., Reference Liu, Zhu, Liang, Yin, Ola, Hou, Chen, Schatten and Sun2008) that support to our present study.
In the case of IGF2R gene, parthenogenetic embryos showed downregulated expression (P < 0.05) in both morula stages of haploid and diploid embryos as compared with control. In contrast, Bebbere et al. (Reference Bebbere, Bogliolo, Ariu, Fois, Leoni, Succu, Berlinguer and Ledda2010) observed no difference in IGF2R expression up to the morula stage between parthenogenetic and IVF embryos in ovine. The higher expression of IGF2R at the morula stage of parthenogenetic embryos in the present study may be due to early expression of this gene as compared with IVF embryos. In another study, it has been shown that the expression of imprinted genes like IGF2, IGF2R and H19 was found to be aberrant in deceased newborn cloned calves and relatively normal in surviving adult clones (Yang et al., Reference Yang, Chavatte-Palmer, Kubota, O'Neill, Hoagland, Renard, Taneja, Yang and Tian2005). Deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs such as brain, bladder, heart, kidney, liver, lung, spleen, and thymus. This disruption of expression of imprinting genes in bovine clones could possibly be due to incomplete reprogramming of donor cell nuclei during nuclear transfer (Yang et al., Reference Yang, Chavatte-Palmer, Kubota, O'Neill, Hoagland, Renard, Taneja, Yang and Tian2005). Such abnormal genomic modification, especially for imprinted genes, cannot be changed into normal patterns when translated into oocytes (Kato et al., Reference Kato, Rideout, Hilton, Barton, Tsunoda and Surani1999). The mechanisms are not clear, but abnormal epigenetic modification of imprinted genes might lead to abnormal early embryonic development-related gene expression (Li et al., Reference Li, Kato and Tsunoda2005).
In conclusion, parthenogenetic embryos showed aberrant gene expression levels for developmentally important genes such as HPRT1, Cx43, Sox2, Mest and IGF2R in comparison with IVF embryos. This factor may be one of the major reasons for poor developmental competence of caprine parthenogenetic embryos. It is suggested that the expression of development-related genes present in embryonic cells must be tightly controlled to allow proper parthenogenetic embryo development.
Acknowledgements
The work was supported by National Agricultural Innovative Project (NAIP), ICAR, Govt. of India.
