[Table/Fig-1]:

Different stages of oocyte development and embryo development. A) Germinal vesicle oocyte enclosed cumulus cells. B) denuded germinal vesicle oocyte. C) MII oocyte with first polar body. D) Fertilized oocyte with 2 pronucleus bodies and 2 polar bodies. E) Two cells stage embryo .F) four cells stage embryo. G) Eight cells stage embryo. H) morula stage and I) blastocyst development. Bar = 40 mm.

Experiments were performed using 4-6 week old NMRI mice. Animals were handled according to the Guide for Care and Use of Laboratory Animals (Yasuj University of medical sciences) and maintained in a 12h light: 12h dark photoperiod under constant temperature and relative humidity. Adequate Food and water were provided. For ovarian stimulation 7.5 IU pregnant mare serum gonadotropin was injected intraperitoneally. After 48h the animals were sacrificed by cervical dislocation, and then ovaries were removed from animals and transferred to HEPES buffered human tubal fluid (HTF) medium supplemented with 5mg/ml bovine serum albumin (BSA). Cumulus oocyte complexes (COCs) [Table/Fig-1A] and Denuded oocytes (DOs) [Table/Fig-1B] were harvested. They were in germinal vesicle stage; and then randomly divided in 8 groups including: 1) DOs cultured in TCM199 without supplement; 2) DOs cultured in TCM199 supplemented with 5 ng/ml IGF-I; 3) DOs cultured in α-MEM without supplement; 4) DOs cultured in α-MEM supplemented with 100 ng/ml IGF-I; 5) COCs cultured in TCM199 without supplement; 6) COCs cultured in TCM199 supplemented with 100 ng/ml IGF-I; 7) COCs cultured in α-MEM without supplement; 8) COCs cultured in α-MEM supplemented with 5 ng/ml IGF-I. All media contain 100IU penicillin, 100ng/ml streptomycin, 75 mIU/ml FSH and 3mg/ml fetal bovine serum (FBS), 1% insulin transferring selenium (ITS). COCs and manually denuded oocytes were cultured in incubator with 5% CO₂ at 37°C. After 24 h mature (MII) oocytes were fertilized with sperm.

Male NMRI mice of 12 weeks age were killed by cervical dislocation and then spermatozoa was harvested from cauda epididymis into IVF medium T6 supplemented with 4 mg/ml BSA. The sperms were hanged in 200 μl droplet of the IVF medium covered with mineral oil and incubated at 37°C for 1- 2 hrs in humidified atmosphere of 5% CO₂ and 37°C for capacitation. Mature oocytes from each group (n=15-20) were separately placed in 200 μl droplet of IVF medium under mineral oil. Sperm mixture (10-20 μl) was added to each droplet to achieve a concentration of 1-2 × 10⁶ motile sperm/ml. After coincubation for 5h at 37°C, the oocytes were then taken away and rinsed in fresh T6 medium and put in a 5% CO₂ incubator at 37°C. After 6- 8 h, embryos were observed by using phase-contrast inverted microscope, and embryos were classified as pronucleus (PN) stage because two distinct pronuclei and a second polar body were seen. After 24h and 120h, embryos were observed for 2-cell stage and blastocyst development respectively. A total of 722 oocytes were obtained from 40 ovaries and they were used for in vitro maturation. The average number of collected oocytes was 18 per ovary.

Data were analyzed by SPSS version 17. We calculated the proportion of maturation rate of GV oocyte to MII and embryo development from MII to the blastocyst stage and then compared the proportion rate of each stage in different groups in a 2x2 tables and then summarized the results in [Table/Fig-2]. Chi-square test was used to compare the groups in terms of maturation, fertilization and developmental rate. The differences in the proportions were considered significant when p<0.05.

Statistical analysis has shown [Table/Fig-2] that higher maturation rate[Table/Fig-1C] (79.6%), fertilization rate [Table/Fig-1D] (87.2%), two cells development rate [Table/Fig-1E] (79.5%) and blastocyst rate [Table/Fig-1I] (43.2%) was seen in COC cultured in α-MEM with growth factor, while lower maturation rate (50.6%) fertilization rate (61%), two cells development rate (48.8%) and blastocyst rate (14.6%) were seen in cultured DOs in TCM199 without growth factor. As well as, maturation, fertilization, two cells development and blastocyst rates in COCs were higher than DOs. Meanwhile maturation, fertilization, two cells development and blastocyst rates was higher in α-MEM medium than TCM199 medium in all groups but they weren’t significant.

Comparison of maturation, fertilization rates, development of two cells and blastocyst stage with or without added IGF-1

Maturation, fertilization, two cells development and blastocyst rates were higher in medium with growth factor than medium without growth factor. However, significant differences were shown in maturation rate of COCs cultured in TCM199 (p<0.05) and blastocyst rate of COCs cultured in α-MEM (p<0.05) [Table/Fig-2].

Comparison of maturation, fertilization rates, development of two cell stage and blastocyst stage in COCs and DOs.

Maturation rate was significantly higher in COCs cultured in α-MEM with growth factor (p<0.05) and COCs cultured in TCM199 with growth factor groups (p<0.05) than other groups. As well as, fertilization rate was higher in COCs than DOs in same condition. Meanwhile these differences were significant in COCs cultured in α-MEM with growth factor group (p<0.05) and COCs cultured in TCM199 with growth factor group (p<0.05). Development to two cells and blastocyst stage was higher in COCs than DOs in same condition. Two cells development rate was significantly increased in COCs cultured in α-MEM with growth factor (p<0.05) and COCs cultured in TCM199 with growth factor groups (p<0.05) in comparison to other groups. Blastocyst rate was significantly higher in COCs cultured in α-MEM with growth factor (p<0.05) and COCs cultured in TCM199 with growth factor groups (p<0.05) than other groups [Table/Fig-2].

Comparison of maturation, fertilization rates, and development of two cells and blastocyst stage in MEM and TCM199.

Maturation, fertilization, two cells development and blastocyst rates were higher in α-MEM than TCM199, but they weren’t significant.

Comparison of effect of growth factor on maturation, fertilization rates, development of two cells and blastocyst stage in COCs and DOs.

Growth factor has significantly increased the maturation rate in COCs cultured in α-MEM medium (p<0.05) and TCM199 (p<0.05) compared to DOs. As well as fertilization rate was significantly higher in COCs cultured in α-MEM medium (p<0.05) and TCM199 (p<0.05) supplemented with growth factor.

Growth factor has significantly increased the two cells development rate in COCs cultured in both α-MEM medium (p<0.05) and TCM199 (p<0.05) compared to DOs. Meanwhile growth factor has significantly increased the blastocyst rate in COCs cultured in both α-MEM medium (p<0.05) and TCM199 (p<0.05) compared to Dos [Table/Fig-2].

Comparison of maturation, fertilization rates, and development of two cells and blastocyst stage in DOs in presence or absence of growth factor.

Maturation, fertilization, two cells development and blastocyst rates were higher in denuded oocyte cultured in presence of growth factor (60%, 66.7%, 60%, 21.7% respectively) than in denuded oocyte cultured in the absence of growth factor (54.4%, 64.9%, 54.02%, 13.5% respectively), but they were not significant [Table/Fig-2].

Comparison of maturation, fertilization rates, and development of two cells and blastocyst stage in COCs in presence or absence of growth factor.

Growth factor has increased the maturation rate of cultured COCs in both α-MEM (77.6% vs. 66.2%) and TCM199 (77% vs.62.5%) media supplemented with growth factor versus media without growth factor. This difference was significant in TCM199 medium. Fertilization rate of cultured COCs increased in both α-MEM (87.2% vs. 75.55%) and TCM199 (83% vs.70%) media supplemented with growth factor versus media without growth factor, however these changes weren’t significant. Meanwhile two cells development and blastocyst rate were higher in presence of growth factor than in absence of growth factor and this difference was significant in blastocyst rate in α-MEM medium (p<0.05) [Table/Fig-2].

Comparison of medium effect on maturation, fertilization rates, and development of two cells and blastocyst stage in COCs and denuded oocyte

Although the rates of maturation, fertilization, two cells and blastocyst development were higher in α-MEM groups than TCM199 groups, the differences were not significant [Table/Fig-2].

Our results have shown that presence of cumulus cells enhance the oocyte maturation from GV to MII, fertilization rate and embryo development; however DOs have low level in these events. This finding confirmed the fact that cumulus cells induce meiosis [14] and enhance cytoplasmic maturation [15]. Moreover, DOs are less able to survive because there is no harmony between nuclear and cytoplasmic maturation. In mammals, there is an important physical contact between oocytes and follicular somatic cells via granulosa cell extensions known as transzonal projections (TZPs). TZPs have important roles in oocyte development, including exchange of the metabolite by gap junction, heterocellular adhesion, regulation of cell cycle and bidirectional signaling [16,17].

On the other hand in oocyte maturation process meiosis-regulating molecules like cAMP is secreted by adjacent cumulus cells and through gap junction access to ooplasm and exert paracrine actions [18].

In addition, cumulus cells secrete progesterone that enhance sperm capacitation and penetration. As well as cumulus cells change the oocyte cytoplasm and zona pellucida to increase normal fertilization rate [19].

Moreover cumulus cells removal leads to precocious TZPs retraction and subsequently loss of cortical stability and loss of eccentric position of GV oocytes. The significance of eccentrically positioned GV is assisting in preservation of maternal transcript and vital organelles by separation of these components from developing spindle [20].

Our findings have shown that the maturation, fertilization rate and embryo development were increased in presence of IGF-1 and cumulus cells. These results are consistent with those of previous reports and studies asserting that IGF-I stimulates maturation in Xenopus oocytes [21], bovine oocyte and fertilization in vitro [22], and supports rabbit blastocyst development [23] and human oocyte and blastocyst development [24]. It is concluded that IGF-I have an important role in the nuclear maturation and cytoplasmic maturation of oocytes and exert its effect through cumulus cells.

In addition, IGF-I have many functions in reproductive system. It plays a potent mitogen role for granulosa cells and acts as a biological enhancer for FSH activity in the ovary [25] including; amplification of the luteinizing hormone (LH) receptor induction by FSH; improved FSH mediated granulosa cells differentiation, potentiating the action of FSH to raise adenylate cyclase which leads to cAMP production [26, 27]. Finally IGF-I affect the P-450 synthesis and then stimulates aromatase activity of human granulosa cells [28].

Our findings have shown that IGF-I is involved in the oocyte biology and improving the oocyte maturation, fertilization and embryo development to blastocyst competence in vitro. In addition, it has also shown that cumulus cells are vital for oocyte development when IGF-1 added to the medium.