Synergistic Effect of Insulin on in vitro Development of Immature Bovine Oocytes

Problem statement: Development of efficient culture system to support embryonic development would be valuable when quality of produ ced embryos was important. However, the rate of bovine embryo production in vitro was still lower than expected. Present study, incl uding of three experiments, was carried out to investigate the eff ct of insulin on nuclear maturation and subsequent development of immature bovine oocytes and i vitro fertilized embryos. Approach: Grade one cumulus-oocyte-complexes harvested from slaughterho use ovaries were selected and randomly allocated in each treatment groups. In experiment 1 , in vitro maturation medium (Hepes-buffered medium 199 + fetal calf serum + gonadotrophins + an tibiotics) supplemented with 0 (control), 1, 10, 20 and 100 μg mL −1 of insulin. In experiment 2, to eliminate the effe ct of serum and hormones, Hepesbuffered medium 199 was supplemented with 1 mg mL −1 polyvinyl alcohols (PVA) and same levels of insulin. In experiment 3, the effect of insulin on bovine in vitro embryo development was assessed. Presumptive zygotes were randomly cultured in synth etic oviductal fluid added with 0 (control), 1, 10, 20 and 100 μg mL −1 of insulin. Results: In experiment 1, nuclear maturation and embryo deve lopment rates were significantly higher in 1 and 10 μg mL −1 compared with other groups (P<0.05). In experiment 2, both maturation and cleavage rate sig nificantly increased in 1 and 10 μg mL −1 insulin. The only treatment resulted in higher hatchability was 10 μg mL −1 insulin (17.1±2.34%) compared with control (11.34±3.94). In experiment 3, cleavag e and morula rates were significantly greater in 1 and 10 μg mL −1 insulin compared with other groups; although the h ighest rates resulted by using 10 μg mL. Conclusion: Obtained results show that inclusion of 10 μg mL −1 insulin in maturation and culture medium exerted beneficial effects on nuclea r m turation of bovine oocytes and in vitro embryo development till morula stage.


INTRODUCTION
In recent years, in vitro embryo production (IVEP) systems have been acknowledged to increase the rate of transferable embryos in bovine (Galli et al., 2004;Spicer et al., 2007). Commercial and research purposes have been enhancing the demand for in vitro production of bovine embryos (Smiljakovic, 2009). In vitro embryo production system includes three consequent steps: in vitro oocyte maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). The final target for IVEP is the production of transferrable embryos and birth of healthy offspring. To achieve these objectives, each step has to be efficient. Although preimplantation embryos may develop during IVC period, obvious differences exist between their developmental rates compared to those developed in vivo. For instance, approximately 60-80% of in vivo matured bovine oocytes are competent to reach to the metaphase II stage while only 25-40% of in vitro matured oocytes reach to this stage (Blondin et al., 2002;Dieleman et al., 2002;Avery et al., 2003). Failure to fertilize and develop to the blastocyst stage may show the lack of vital factors in the culture media which normally are available in vivo. Therefore, it is necessary to modify the culture medium and conditions to support higher percentage of embryo development in vitro. Several factors such as, hormones, proteins and growth factors supplemented to the culture medium may have crucial roles on the outcome of IVEP. It has been shown that insulin plays a crucial role for growth of variety of cells in IVC. Insulin bind to cell surface receptor and its action is receptor-mediated. It has been demonstrated that insulin stimulates glucose and amino acid uptake and protein synthesis (Harvey and Kaye, 1988;Kane et al., 1997) of mouse embryos (Velazquez et al., 2009;Kaye and Harveyt, 1995). Several studies have indicated that insulin increases the in vitro oocyte maturation and development of human (Dashtizad et al., 2003), mice (Demeestere et al., 2004) and porcine embryos . Insulin receptor has also been detected in all stages of bovine embryos (Makarevich and Markkula, 2002). However, the results of using insulin are controversial for in vitro bovine embryo production and need more investigation. Therefore, this experiment was designed to study the effect of different concentration of insulin on bovine IVEP.

Chemicals:
All chemicals and reagents used in the current study were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA) unless otherwise stated.

Evaluation of nuclear maturation:
After completion of maturation period, in order to assess the nuclear maturation status, oocytes were denuded using 0.1% (w/v) hyaluronidase (Type 1-S) in Hepes-buffered medium 199. Denuded oocytes were washed twice with working solution and fixed in aceto-ethanol (1:3 v/v) solution at 4°C for 24 h. Five to ten fixed oocytes were mounted on a clean glass slide in a microdroplet (10-20 µL) between two parallel lines of wax-vaseline mixture (1:20). A cover slip was slowly placed on the lines and gently pressed down until it touched the microdroplet and secured the oocytes between the slide and the cover slip. Staining solution, 1% aceto-orcein was passed once via introduction from one side of the cover slip and blot-dried from the opposite side by a piece of filter paper. After 3-5 min, when the staining was completed, decolorizing solution (acetic acid: distilled water: glycerol: 1:3:1) was passed through to remove the stain residuals. The cover slip was sealed with a colorless nail varnish to provide a permanent storage for future examination. The stained oocytes were evaluated under a phase contrast microscope at 400X magnification to assess the status of nuclear maturation.

In Vitro Fertilization (IVF):
In vitro matured COCs with expanded cumulus cells were used for in vitro fertilization. The fertilization method was previously described by Parrish et al. (1988) with some modifications. Briefly, following maturation period for 22-24 h matured COCs were washed 2 times in working solution and in two droplets of tyrode's albumin lactate pyruvate buffered with HEPES (IVF-TALP solution). Subsequently 6-8 COCs were placed in a 48 µL of pre-equilibrated IVF-TALP droplets covered with sterile mineral oil (M-5310). Frozen semen straws (250 µL) were thawed in water bath at 37°C for 45-60 second and content was poured into a 1.5 ml centrifuge tube containing 1 ml pre-warmed BoviExtend (Nidacon Laboratories AB, Gothenburg, Sweden) buffer solution and smoothly mixed. The amount of 1 ml of the diluted semen was gently loaded on the top of the adjusted BoviPure density gradient in the conical tube and centrifuged for 20 min at 300×g at room temperature. After centrifugation, the supernatant was carefully discarded. The sperm pellet was resuspended with 5 mL of pre-warmed sperm-TALP medium (Parrish et al., 1988) supplemented by 6 mg mL −1 bovine serum albumin fatty acid free (BSA-FAF) (A-8806, Sigma) and centrifuged again for 10 min at 300×g. The final pellet was resuspended in 150-200 µL of pre-equilibrated IVF-TALP. Spermatozoa were checked for motility and counted by a haemocytometer to give the final concentration of 1×10 6 spermatozoa/ml. Based on concentration, spermatozoa were added gently to each IVF droplet containing 6-8 mature oocytes. Thereafter, 2 µL of PHE mixture consisting of 20 µM D-penicillamine (P-4875) 10 µM Hypotaurine (H-1384) and 1 µM Epinephrine (E-4250) were added to each IVF droplet. In vitro fertilization was accomplished by co-incubation of sperm-oocytes at humid environment with 5% CO 2 and 38.5°C for 18-20 h.
In Vitro Culture (IVC): At the end of fertilization period, oocytes were freed of cumulus cells by gentle mechanical pipetting. After denuding, the presumptive zygotes and embryos were washed 2 times in fresh preequilibrated working solution. Then, denuded zygotes were washed through embryo culture droplets. Approximately 15-20 presumptive zygotes and embryos were transferred into each well of 4-well dish containing 400 µL of synthetic oviductal fluid (SOFaaci;Holm et al., 1999) supplemented with 5% adult bovine serum (B-9433) and 1 µg mL −1 gentamycin under sterile mineral oil. During 10 days, at every two day interval, 200 µL of SOF solution was replenished by 200 µL warm and CO 2 equilibrated IVC medium. Cleavage, morula, blastocyst and hatched blastocyst rates were recorded at days 2, 4, 7 and 9 post inseminations, respectively.

Experimental design:
In the first experiment, supplementation of insulin in IVM medium and its effects on nuclear maturation of bovine oocytes and subsequent embryo development in vitro were evaluated. Hepes-buffered medium 199 supplemented with gonadotrophins, fetal bovine serum and antibiotics added with 0 (control), 1, 10, 20 and 50 µg mL −1 insulin were used as maturation medium.
In the second experiment, influence of insulin in serum and hormone free IVM medium on in vitro maturation of bovine immature oocytes and consequent embryo development was evaluated. To avoid the interaction effect of insulin with IVM ingredients, Hepes-buffered medium 199 supplemented with 1 mg mL −1 PVA and the same levels of insulin were used as maturation medium. In both experiments 1 and 2, after 22-24 h of maturation, representative oocytes were randomly selected to examine their nuclear maturation by using aceto-orcein staining method. The rest of in vitro matured bovine oocytes were subsequently fertilized and cultured in SOF solution for 9 days. The cleavage, blastocyst and hatched blastocyst rates were assessed at day 2, 7 and, 9 respectively.
In the third experiment, effect of insulin on development of bovine in vitro fertilized embryos was investigated. Complete IVM medium without insulin was used as maturation medium. Following IVM and IVF, presumptive denuded zygotes were randomly transferred to SOF solution supplemented with 0 (control), 1, 10, 20 and 50 µg mL −1 insulin for 9 days. The cleavage, morula, blastocyst and hatch blastocyst rates were assessed at day 2, 4, 7 and 9, respectively.

Statistical analysis:
All experiments were repeated six times. Significant differences among treatments were revealed by one-way analysis of variance followed by Duncan's multiple range test for mean comparisons (p<0.05) using SAS software ver. 9.1.
In the second experiment, a total of 1079 immature bovine oocytes were used to evaluate the effect of supplementing insulin in serum and hormone free IVM medium on in vitro maturation of bovine immature oocytes and consequent embryo development. Data are summarized in Table 2. The percentage of immature bovine oocytes that reached metaphase II was noticeably higher in 1 and 10 µg mL −1 (80.51±2.02% and 82.15±2.35%, respectively) compared with other groups, but there was no difference between them. All of the treatment groups had higher cleavage rate (p<0.05) with the highest in 1 µg mL −1 (71.92±3.82%) and 10 µg mL −1 (73.30±2.14%) compared with 20 and 50 µg mL −1 (60.14±3.53% and 70.35±2.57%, respectively). Addition of 1 and 10 µg mL −1 insulin in serum and hormone free IVM medium significantly increased the proportion of inseminated oocytes developed to the blastocyst stage than the control group. To evaluate the further viability of the blastocysts produced after addition of different insulin levels, hatchability of embryos were assessed. The hatching rates of blastocysts, remarkably improved in 1 and 10 µg mL −1 insulin (10.93±3.48% and 10.85±2.41%, respectively) compared with the control (6.45±2.68%).

DISCUSSION
In the present study, the potential role of insulin on IVM of immature bovine oocytes and in vitro embryo development were investigated. The results demonstrated that presence of 1 or 10 µg mL −1 insulin in the maturation media showed a positive effect on maturation and cleavage rates of bovine immature oocytes in vitro. In subsequent bovine embryo development in vitro, insulin at 10 µg mL −1 enhanced viability of blastocyst embryos and improved rate of hatchability. However, findings of previous studies regarding using insulin are controversial for in vitro bovine oocyte development. Our results are in agreement with previous reports in which addition of insulin to the IVM medium showed a positive effect on in vitro development of bovine oocytes, when oocytes were cultured in TCM-199 medium supplemented with FCS (Dieleman et al., 2002). On the other hand, Zhang et al. (1991) reported that although inclusion of insulin in maturation medium enhanced cumulus expansion scores and the level of maturation, the yield of in vitro embryo production were not affected. Furthermore, Matsui (1995) showed that addition of insulin at 10 µg mL −1 to the oocyte maturation medium had no effect on the nuclear maturity, fertilization and development of bovine embryos toward the blastocyst stage. It has been shown that insulin (0.1-10 µg mL −1 ) enhanced the mitosis of bovine granulosa cells and accelerated progression of meiosis in oocytes enclosed with cumulus cells. Both granulosa cells and oocyte normally express insulin receptor. Percentage of apoptotic cells in the group of oocytes evaluated immediately after recovery was very low but it noticeably increased during in vitro maturation without any additional supplementation. This incidence of apoptotic DNA degeneration was reduced by addition of insulin, insulin like growth factor I (IGF I) and other growth factors to the maturation medium (Wasielak and Bogacki, 2007). Addition of insulin to IVM and subsequent embryo development media reduced not only the incidence of spontaneous apoptosis in bovine embryos (Augustin et al., 2003) but also blocked apoptosis induced by exogenous factors, such as heat shock (Jousan and Hansen, 2004).
The obtained data from this study probably shows an interaction between insulin and FSH/or E 2 on the bovine COCs. Suzuki et al., (2006) reported that stimulating effect of insulin on the proliferation of bovine granulosa cells is synergistic with gonadotrophins. Therefore, the existence of FSH and E 2 in the IVM medium might interfere the real effects of insulin on the maturation of bovine cumulus-intact oocytes. For this reason we have designed the second experiment to find out the pure effect of insulin on oocyte maturation. Therefore, a serum and hormones free IVM medium was used (Table 2) in which PVA was added instead of serum. The results of the current study undoubtedly confirmed that insulin by itself had a positive effect on in vitro bovine embryo production system. Our finding propose that supplementation of maturation medium with insulin (1-50 µg mL −1 ) improves the fertilization rate. Cumulus cells surrounding bovine oocytes synthesize glycosaminoglycans (GAGs) such as hyaluronic acid. GAGs are able to induce the acrosome reaction of bovine sperm (Handrow et al., 1982). It has been reported that granulosa cells are able to secrete heparinlike GAGs which are recognized as in vitro sperm capacitating agent (Bellin et al., 1986). It is presumed that the addition of insulin to the maturation medium stimulates GAGs secretion from cumulus cells and improves the fertilization rate as a result of the promotion of cumulus-induced sperm capacitation (Matsui, 1995).
It has been shown that insulin has broad effects on preimplantation embryos (Kim et al., 2005). The third experiment was carried out to determine the effect of different concentrations of insulin in IVC media on in vitro bovine embryo development. The results clearly demonstrated that supplementation of insulin (1 and 10 µg mL −1 ) to the culture medium improved cleavage rate and development of morula but did not show positive effect on blastocyst production rate (Table 3). Addition of higher concentration of insulin (20 and 50 µg mL −1 ) did not show positive effect on in vitro embryo development. This could be due to differences in glucose utilization of the early embryo (morula), compared to the late embryos (blastocyst) (Harvey and Kaye, 1990). The post-compacted embryos are more sensitive to insulin (Zhandi et al., 2009). Scientists demonstrated a stage-specific binding by insulin which could be related to embryonic metabolism and switch from lactate to glucose uptake. They also showed that insulin stimulates glucose uptake and amino acid transport by embryonic cells which can improve embryo development (Tareq et al., 2007). Furthermore, the proportion of embryos reaching to the 8-cell stage was considerably enhanced by addition of 10 µg mL −1 insulin to the culture medium. However, development to the morula and blastocyst stages was not significantly increased. Insulin stimulates embryo development via insulin receptors which are expressed at the 8-cell stage. Our findings are in agreement with previous results of Harvey and Kaye (1990). In contrast, Quetglas and co-researchers (2001) added 5 µg mL −1 insulin to the culture medium and the percentage of embryos reaching to the morula stage was not affected by addition of insulin alone, but was significantly increased when amino acids were included with insulin. Therefore, it seems that insulin has not a strong stimulatory effect on the growth of late stage embryos (Navarrete et al., 2004). Based on our results, it seems that addition of insulin (10 µg mL −1 ) to the culture medium would slightly improve blastocyst hatchability in the bovine IVC system. The proportion of hatched blastocysts can be considered as an indicator to evaluate quality and viability of the transferable bovine embryos. Insulin increases the cell number of the inner cell mass and stimulates morphological development of mouse blastocysts in vitro (Liu et al., 2009).

CONCLUSION
Obtained results demonstrated that insulin promotes in vitro bovine embryos production. Supplementation of 10 µg mL −1 insulin in maturation medium, exerted beneficial effects on nuclear maturation, cleavage and subsequent bovine embryo development in vitro. Furthermore, addition of insulin (10 µg mL −1 ) to the culture medium showed positive effect on bovine embryo development till morula stage and slightly enhanced hatchability of produced blastocysts.