Effect of additives in medium on in-vitro maturation of goat oocytes

Efforts to increase productivity in goat has largely been limited to utilization of superior bucks in artificial insemination programme, rather than harvesting superior female germplasm from proven does using the technique of multiple ovulation and embryo transfer (MOET). In-vitro embryo production in goat provides an alternative to MOET. The environment in which the harvested oocytes are cultured during in-vitro maturation (IVM) plays an important role in in-vitro development of embryos. Maturation medium is critical in terms of providing proper environment for propagation of oocytes to nuclear and cytoplasmic maturation. The types and concentrations of energy substrates added to a culture medium might exert effect on the process of IVM of oocytes. Supplementations of protein, follicular fluid, hormone, growth factor, antioxidant etc. in maturation medium have been reported to enhance the process of IVM. However, there is paucity of literature on IVM of goat oocytes with regard to comparative efficacy of combining a growth factor with another as compared to other additives in a maturation media.

Goat ovaries were collected from local abattoirs of Guwahati (Assam) as soon as possible after the animals were slaughtered and transported to the laboratory in a thermosflask containing warm (37°C) Normal Saline Solution (N.S.S.) with antibiotic. The ovaries were than washed 3-4 times with N.S.S. containing antibiotic and washing medium prepared using TCM-199, Foetal Bovine Serum(4ml), L-glutamine (0.004g) and Gentamicin (200μl) per 40 ml of medium. The oocytes were recovered from the ovaries immediately after washing, examined under a stereo-zoom microscope and washed thoroughly. Only good quality oocytes surrounded by two or more complete layers of cumulus cells adhered to the zona pellucid were used for IVM.
        
For in-vitro maturation of oocytes three different combinations of additives were used separately in the control medium (Medium I) that consist of : Cysteamine (100μM/ml) + 17β-Oestradiol (1μg/ml) + pFSH (5μg/ml) + oLH (5μg/ml) + Follicular fluid (1ml) + Estrous goat serum (1ml) + Washing medium upto 10ml. The additives comprised: i) 10ng/ml Epidermal growth factor + 50ng/ml Insulin-like growth factor, ii) 10ng/ml Epidermal growth factor + 600ìM/ml Cysteine and iii) 10ng/ml Epidermal growth factor + 0.2mM/ml Sodium Pyruvate which were added to the control medium and constituted Medium II, III and IV respectively. After mixing of the ingredients, the media were filtered using 0.22μm syringe filter and then kept in a CO₂ incubator maintaining 5 per cent CO₂ at 38.5°C with 90-95 per cent relative humidity for overnight prior to use. Oocytes were then transferred into the maturation medium @ 4-5 oocytes/50ìl droplet in 35mm petri dish and covered with warmed (37°C-38°C) sterile mineral oil. The petri dish was then placed in a CO₂ incubator maintaining 5 per cent CO₂ and 90-95 per cent relative humidity for 24 hours at a temperature of 38.5°C.
       
After 24 hours of incubation in the maturation media, the degree of maturation of oocytes was determined microscopically by : (a) Visual assessment of expansion of cumulus cells layer of oocytes and (b) Assessment of nuclear maturation of oocytes. Confirmation of nuclear maturation of oocytes by extrution of polar body was done by nuclear-fluorescent staining (Hoechst 33342).       

The statistical analysis of the data was done using SAS Enterprise Guide 4.2 version.

The in-vitro maturation (IVM) performance of oocytes on the basis of cumulus cell expansion and nuclear maturation are presented in Table 1 and Table 2 respectively.
 

 

        
Data on rate of IVM of goat oocytes using 10 ng/ml EGF + 50 ng/ml IGF-I in TCM-based medium are apparently not available in the literature for comparison. Harper and Brackett (1993) reported 89.10 per cent cumulus cells expansion on IVM of bovine oocytes using 10 ng/ml EGF + LH in the maturation media. Lower percentage of cumulus cells expansion was observed than the present study by Nagar and Purohit (2005) in goat oocytes (42.25%) who used 10 ng EGF / ml in TCM-based medium. Lorenzo et al., (1995) reported lower cumulus cells expansion rate on IVM in medium with EGF (62.6%), IGF-I (40.6%) and EGF + IGF-I (74.2%) in bovine oocytes.
       
The percentage of oocytes with nuclear maturation using 10 ng/ml EGF in TCM-based media in the present study was similar to the findings of Yadav et al., (2013) in goat oocytes (60.00%). However, lower percentage was observed by Nagar and Purohit (2005) in goat (55.63%). In case of cysteine supplementation higher percentage (79.60 ± 1.70 %) of in-vitro nuclear maturation of goat oocytes was observed by Zhou et al., (2008) using 500µM cysteine in TCM-based media. The variations in the percentage of cumulus cell expansion and nuclear maturation during IVM might be due to differences in species, status of ovary, composition of the media, condition of maturation and concentrations of supplements used.
       
In the present study of IVM of oocytes based on both expansion of cumulus cells layer of oocytes and nuclear maturation of oocytes was significantly higher (P<0.05) in medium II than in media I, III and IV. The percentages of expansion of cumulus cell layer of oocytes and nuclear maturation on IVM of goat oocytes were found to be the highest in the TCM-based medium supplemented with EGF + IGF-1.
       
EGF has been demonstrated to have some positive effects on IVM of oocytes in cattle (Lonergan et al., 1996; Izadyar et al., 1998), pig (Abeydeera et al., 2000) and sheep (Guler et al., 2000). EGF was reported to be present in the ovary of cattle and performed cellular functions through EGF receptors (Yoshida et al., 1998). Stimulation of IVM by EGF could be due to improved cAMP production by the cumulus-oocyte complexes which induced breakdown of the germinal vesicle (Downs et al., 1991). EGF was found to be a mediator of the mitogenic activity of FSH in the granulosa cells (Roy and Greenwald, 1991). Increased cumulus expansion could be attributed to the differential mitogenic effect of  FSH on cumulus cells and granulosa cells and combined action with FSH and LH in the medium on cumulus cells could cause synthesis of pyruvate, thus stimulating the tetraacetic acid cycle leading to an increased availability of ATP for energy requirement of the oocytes (Nandi et al., 2002). EGF might act on the cumulus cells surrounding the oocyte and/or on the oocyte itself since mRNA for the EGF receptor was stated to be present in the bovine oocyte (Banwell and Thompson, 2008) which could be extrapolated to goat oocytes. Growth factors have been shown to bind to high-affinity receptors and promote the generation of signals and second messengers in the membrane and cytoplasm (Druker et al., 1989; Hill, 1989). Binding of EGF to its receptor could induce the activation of tyrosine kinase, an essential primary event in the EGF pathway. Tyrosine kinase activation initiates phosphorylation of several cellular proteins as well as the receptor itself (Rozengurt, 1983; Carpenter and Cohen, 1990). Several specific tyrosine kinase substrates were identified which might be involved in the signalling pathway leading to protein synthesis and phosphorylation. Such changes in protein synthesis and protein phosphorylation that were shown to be integral to bovine oocyte maturation could provide support for a physiological role of growth factors in goat oocytes maturation. EGF was demonstrated in follicular fluid of cattle (Parrish et al., 1986) and pig (Hsu et al., 1987) that exerted a beneficial influence on oocyte maturation in-vitro (Coskun et al., 1991; Harper and Brackett, 1991). The mechanism whereby growth factors regulate or modulate resumption of meiosis in oocytes might be mediated via the granulosa and/ or cumulus cells (Dekel   and Sherizly, 1985; Brucker et al., 1991). Study in pig oocytes (Coskun and Lin, 1992) also revealed that the action of EGF was mediated via the cumulus cells and gap junctions with the oocyte. By immunocytochemical methods, EGF was found to be localized in growing (small to medium preantral) follicles as well as in small antral follicles of hamster ovaries (Roy and Greenwald, 1990). An EGF-like substance was shown to be secreted by rat ovarian thecal/ interstitial cells (Skimmer et al., 1987). The goat cumulus cells were found to express EGF receptor (Gall et al., 2004) and EGF being a polypeptide with potent mitogenic activity was reported to trigger signalling through the MAPK (mitogen-activated protein kinase)  pathway during IVM in goat COCs (Gall et al., 2005). The intrinsic tyrosine kinase of EGF receptor (EGF-R) was activated by binding of EGF, resulting in EGF-R autophosphorylation and subsequent tyrosine phosphorylation of numerous substrates within the cell (Carpenter and Cohen, 1990). Goud et al., (1998) revealed that higher number of oocytes cultured in the absence of cumulus cells completed nuclear maturation in medium supplemented with EGF in comparison with their sibling oocytes cultured without EGF. This might be due to modulation of differentiation by EGF in ovarian folliculogenesis.
       
IGF-1 is known to stimulate protein synthesis when added to medium for mouse embryos in-vitro (Simmen et al., 1993) and known to increase oestradiol production by the theca granulosa cells in serum-free culture (Shores et al., 2000). The biological effects of IGF-1 are mediated by its interaction with the IGF type 1 receptor and modulated by IGF binding proteins (Jones and Clemmons, 1995). An increase in the level of IGF binding protein may alter the bioavailability of IGF, thus stimulating steroidogenesis and mitogenesis in developing follicles (Bridges et al., 2002). The growth factors acting in the presence of cumulus cells transfer a positive signal for oocyte maturation (Lorenzo et al., 1994) possibly by synthesis of new proteins. IGFs have an affinity for soluble binding proteins that can modulate receptor binding and hence influence their biological activity (Palma et al., 1997).
       
Lorenzo et al., (1993) observed that the combined effect of EGF and IGF-1 appeared to be mediated by surrounding cumulus cells. The factors secreted by cumulus cells that regulate the disruption of gap junction and cumulus expansion as shown for pig oocytes (Isobe and Terada, 2001) might also become stimulated by EGF and IGF-1in goat oocytes. EGF at concentration of 1-10 ng/ml (Kobayashi et al., 1994; Lonergan et al., 1996) and IGF-1 at concentration of 50-100 ng/ml (Herrler et al., 1992; Palma et al., 1997) were found to be effective in improving nuclear maturation of oocytes. Bovine oocyte activation is known to be associated with calcium dependent electrical events (Tosti et al., 2002). It is probable that growth factors alter these events by activating protein synthesis. EGF is known to decrease IGF-1 production by granulosa cells (Spicer and Chamberlain, 2000). Thus it was possible that a combination of EGF and IGF-1 under conditions in-vitro might have stimulated a cascade of events including protein synthesis which eventually generated positive signals for resumption of meiosis in oocytes. This might be ascribed for obtaining significantly higher percentage of IVM of goat oocyte in the present investigation with the supplementation of 10ng/ml EGF and 50 ng/ml IGF-I when used in combination.
       
Different workers observed the beneficial effects of sodium pyruvate on IVM of oocytes. The pattern of energy metabolism of zygote is determined in the oocyte before fertilization and pyruvate is the main energy substrate which can be used directly by the oocyte and zygote. Gamete metabolism is modified during the process of maturation, fertilization and development (Tosti and Boni., 2004). Isolated mouse cumulus cells formed pyruvate when incubated with glucose and lactate (Leese and Barton., 1985) and porcine oocyte-COCs were capable of producing pyruvate to meet metabolic needs (Eng et al., 1986). Pyruvate metabolism of bovine cumulus-free oocytes increased during IVM, while total glucose metabolism and the production of CO₂ from glucose were low and relatively constant throughout maturation (Rieger and Loskutoff., 1994). The higher IVM rate of goat oocytes in medium supplemented with EGF + sodium pyruvate in comparison with medium supplemented with EGF + cysteine and control medium in the present investigation could be attributed to the ability of the oocytes to utilize pyruvate during the process of IVM, in addition to the effect of EGF.
       
Availability of cysteine in the culture medium exerted influence on the synthesis of Glutathione (GSH) in-vitro required for cytoplasmic maturation (Meister and Tate, 1976; Chance et al., 1979) which could protect cells against the destructive effects of reactive oxygen species, regulated protein and DNA synthesis, and preserved meiotic spindle by altering redox status (Meister, 1983). GSH synthesis occurred during bovine oocyte maturation and it was stated occurred during bovine oocyte maturation and it was stated to be one of the indices of cytoplasmic maturation (Yi et al., 2003; Zhou et al., 2008). Addition of thiol containing precursors of GSH such as cysteine, cysteamine, 2-mercaptorthanol or use of a cysteine-rich medium (TCM 199 or Waymouth MB 75211) increased GSH content of oocytes after maturation (Bai et al., 2008; de Matos et al., 2002). The obtained significantly higher IVM rate in goat oocytes supplemented with cysteine as compared to control could be ascribed to increase in GSH concentration in the medium during maturation which is critical for protecting the oocytes from oxidative stress. Cysteine is a very unstable critical component amino acid of GSH which can be transferred from extracellular environment into the oocyte (Bai et al., 2008). Cysteine is known to be rapidly oxidised to cystine in the culture medium and its deficiency in the medium due to auto-oxidation to cystine might result in GSH synthesis failure in-vitro (Sagara et al., 1993). This might explain the significantly lower nuclear maturation rates of goat oocytes in medium supplemented with EGF + cysteine in comparison with that supplemented with EGF + sodium pyruvate.

Authors are thankful to the Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati- 781022 for the financial support and help during the research work.