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Research Article |
Department of Theriogenology and Biotechnology,2 College of Veterinary Medicine, Seoul National University, Seoul 151–742, Korea
The Xenotransplantation Research Center,3 Seoul National University Hospital, Seoul 110–744, Korea
School of Agricultural Biotechnology,4 Seoul National University, Seoul 151–742, Korea
ABSTRACT
To investigate whether insulin and/or metformin improve the developmental competence of porcine oocytes and embryos, insulin (100 ng/ml) and/or metformin (10–5 M) were supplemented during in vitro maturation (IVM) and/or in vitro culture (IVC) periods. Insulin (33 to 34% vs. 21%) or insulin plus metformin (27 to 39% vs. 21%) significantly (P < 0.01) increased the rates of blastocyst formation, whereas metformin alone had no effect when added during the first half (0–22 h), the latter half (22–44 h), or the entire (0–44 h) maturation period. No additional effect of insulin plus metformin on increasing blastocyst formation was observed compared to insulin alone. When supplemented during IVC, insulin (34% vs. 23%) or insulin plus metformin (35% vs. 23%) significantly (P < 0.05) increased the rates of blastocyst formation, whereas metformin alone had no effect. Compared to insulin alone, no additional effect of insulin plus metformin on increasing blastocyst formation was observed. When added during the entire IVM and IVC, insulin (40% vs. 24%) or insulin plus metformin (52% vs. 21%) significantly increased the rates of blastocyst formation, whereas metformin alone had no effect. In addition, the effect of insulin was enhanced when supplemented with metformin compared to insulin alone (52% vs. 40%). After IVM, oocyte glutathione (GSH) content and tyrosine kinase activity were measured. Insulin significantly (P < 0.01) increased oocyte GSH content (6.2 pmol vs. 4.3 pmol) and metformin significantly (P < 0.01) enhanced the action of insulin on GSH content (7.3 pmol vs. 6.2 pmol) and tyrosine kinase activity (1.9 arbitrary units [AU] vs. 1.5 AU) when compared to insulin alone. In conclusion, insulin increased the developmental potential of porcine oocytes and embryos, and metformin enhanced the action of insulin when supplemented during the entire IVM and IVC. The effects of insulin and metformin were associated with oocyte GSH content and tyrosine kinase activity.
embryo, gamete biology, insulin, in vitro fertilization, metformin, oocytes, ovum, pigs
INTRODUCTION
The establishment of a reliable in vitro production (IVP) system for porcine embryos is important not only for use in basic research but also for use in embryo manipulation procedures including gene manipulation and somatic cell nuclear transfer. The IVP system for porcine embryos comprises the sequential procedures of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). Although the IVP system has been successful in producing porcine embryos [1–5], these embryos' developmental potential is still lower than that of oocytes matured and fertilized in vivo. Major problems accounting for the lower efficiency of IVP include improper nuclear and cytoplasmic maturation of oocytes and polyspermic fertilization. Despite the progress in IVM research, the quality of oocytes matured in vitro, defined as their potential to develop into viable offspring, is still not satisfactory, limiting the improvement of other reproductive techniques in pigs [6]. Several studies performed to improve the developmental competence of IVP porcine embryos have demonstrated that many factors are involved in IVM, embryo development, and embryo viability after transfer. These factors include specific physical conditions (osmolarity, ionic composition, temperature, pH, oxygen tension, and culture volume) and less well-defined factors (e.g., serum, glutathione [GSH] content of oocytes, and cumulus cells) [7]. Because the earliest stages of embryonic development are largely dependent on events that occur during oocyte maturation, provision of an adequate supply of appropriate metabolic substrates to the cumulus-oocyte complexes (COCs) during IVM is likely to be important for subsequent embryo developmental potential [8].
In most mammals, insulin stimulates granulosa cell proliferation and production of progesterone and enhances luteal cell steroidogenesis [9]. In women, granulosa and theca cells from dominant follicles localized more insulin receptor (INSR) mRNA than cells from small antral follicles [10, 11]. Similarly, porcine granulosa cells from small follicles have fewer INSRs compared to medium or large follicles [12]. Metformin (N, N'-dimethylbiguanide) is an oral antihyperglycaemic drug that is extensively used in treating diabetes mellitus [13]. Metformin acts as an insulin-sensitizing agent (ISA) that increases insulin-stimulated glucose uptake in rat adipocytes [14] and elevates receptor tyrosine kinase activity and inositol 1, 4, 5-Tris-phosphate (IP3) production in Xenopus oocytes [15]. Other reports have demonstrated that metformin has effects on ovarian steroidogenesis in humans [16]. Taken together, these data suggest that insulin and metformin might have beneficial effects on porcine oocyte maturation and/or embryo development, but no data have been reported on the effect of insulin and metformin on porcine IVM and IVC.
Accordingly, to improve IVP of porcine embryos, we investigated the effects of insulin and metformin on maturation of porcine oocytes and subsequent embryo development after IVF. Further, we investigated possible mechanisms of the action of insulin and metformin.
MATERIALS AND METHODS
All chemicals were purchased from Sigma-Aldrich Corp. unless otherwise stated.
Recovery and Culture of Cumulus-Oocyte Complexes for IVM
Pig ovaries were collected from gilts at a local slaughterhouse and transported to the laboratory in physiological saline supplemented with penicillin G (100 U/ml) and streptomycin sulfate (100 mg/ml) at 30–35°C within 2 h. Follicular fluid and COCs from follicles 3–6 mm in diameter were aspirated using an 18-gauge needle attached to a 5-ml disposable syringe. Compact COCs were selected and cultured in tissue culture medium (TCM)-199 (Life Technologies), supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml eCG (Intervet) and hCG (Intervet), and 10% porcine follicular fluid (pFF). The pFF was aspirated from 3- to 7-mm follicles of prepubertal gilt ovaries. After centrifugation at 300 x g for 30 min, supernatants were collected and filtered sequentially through 1.2- and 0.45-µm syringe filters (Gelman Sciences). Prepared pFF was stored at –20°C until use. Each group of 50 COCs was cultured in 500 µl of TCM-199 incubated at 39°C in a humidified atmosphere of 5% CO2 and 95% air. After culturing for 22 h (the first half of IVM culture), COCs were washed three times and cultured in eCG- and hCG-free TCM-199 medium for another 22 h (Table 1).
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In Vitro Fertilization and Embryo Culture
Fresh boar semen were purchased from Davi Bred Co., frozen and stored in the liquid nitrogen at –196°C. A straw of frozen semen was thawed at 39°C for 1 min in a water bath, diluted in 10 ml Dulbecco PBS (Life Technologies) supplemented with 0.1% BSA (Cat. No. A-9418), 75 µg/ml potassium penicillin G, and 50 µg/ml streptomycin sulfate, and centrifuged twice at 350 x g for 3 min. The sperm pellet was resuspended in modified Tris-buffered medium (mTBM) to give a concentration of 2 x 107 spermatozoa/ml. The mTBM consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2, 20 mM Tris (Cat. No. T-1410), 11 mM glucose, 5 mM sodium pyruvate and 8 mg/ml BSA (Cat. No. A-6003). At 44 h of IVM culture, oocytes were freed from cumulus cells by repeated pipetting in 0.1% hyaluronidase in TCM-199 medium and washed three times with pre-equilibrated mTBM. The matured oocytes were selected and used for IVF and IVC. After washing, 20 to 25 oocytes were placed in 45-µl drops of mTBM covered with prewarmed paraffin oil, and 5 µl sperm suspension was added to each fertilization drop to give a final sperm concentration of 2 x 106 spermatozoa/ml. The basic IVC medium was North Carolina State University (NCSU)-23 medium supplemented with 4 mg/ml fatty acid-free BSA (Cat. No. A-6003). After coincubation of gametes for 6 h, the oocytes were freed from excess spermatozoa by repeated pipetting, washed three times in NCSU-23 medium, and cultured in 25 µl drops of the modified NCSU-23 medium supplemented with 0.5 mM pyruvate:5.0 mM lactate instead of 5.5 mM glucose (without glucose) in 5% O2, 5% CO2, and 90% N2 at 39°C for 56 h after insemination. At 56 h after insemination, the embryos were transferred to the IVC medium and cultured for 40 h. At 96 h after insemination, the embryos were transferred to fresh IVC medium containing 10% FBS (Cat. No. 16000–044; Life Technologies) and cultured for 48 h (Table 1). Blastocyst formation was evaluated under a stereomicroscope at 144 h after insemination and confirmed by the total cell number in blastocysts. Any blastocysts with a total cell number less than 20 were not considered normal and were excluded from study. The total cell number in blastocysts was determined after nuclear staining with 20 µg/ml bisbenzimide (Hoechst 33342) in 100% ethanol for fixation and staining at 4°C. Fixed and stained whole blastocysts were mounted and assessed for cell number using epifluorescence microscopy (Nikon Corp.) [17, 18].
Treatment with Insulin and Metformin
Porcine insulin (Cat. No. I5523) and metformin (Cat. No. D5035) were purchased from Sigma-Aldrich Corp. Insulin and metformin were prepared as 100x stocks. In order to determine effective concentrations of insulin and metformin for improving developmental potential of the oocyte during IVM, various concentrations of insulin (100 ng/ml or 10 µg/ml) or metformin (10–6, 10–5, or 10–4 M) were added to each IVM medium during the entire maturation period of 44 h (the first half, eCG- and hCG-containing TCM-199 medium for 22 h; and the latter half, eCG- and hCG-free TCM-199 medium for 22 h). The experiment was replicated nine times. To investigate whether effects of insulin and metformin on developmental potential of oocytes were dependent on the timing of maturation, the effective concentrations of insulin (100 ng/ml), metformin (10–5 M) or insulin plus metformin were added to the IVM medium during either the first half, the second half, or the entire maturation period. The experiment was replicated nine times. To investigate the effects of insulin and metformin on developmental potential of embryos during IVC, embryos cultured in the modified NCSU-23 medium were transferred to basic NCSU-23 medium supplemented with insulin (100 ng/ml), metformin (10–5 M), or insulin plus metformin at 56 and 96 h after insemination, respectively. The experiment was replicated six times. To investigate the effects of insulin and metformin during IVM and IVC, COCs were matured in IVM medium containing insulin (100 ng/ml), metformin (10–5 M), or insulin plus metformin for the entire maturation period, and subsequently the embryos were cultured in IVC medium supplemented with insulin (100 ng/ml), metformin (10–5 M), or insulin plus metformin at 56 h and 96 h after insemination, respectively. The experiment was replicated six times. The percentage of blastocysts formed at 144 h after insemination was evaluated as a parameter of the developmental potential of porcine oocytes and embryos.
Assay of Glutathione Content in Porcine Oocytes
After IVM during the entire maturation period of 44 h with insulin (100 ng/ml), metformin (10–5 M) or insulin plus metformin, the amount of GSH in the oocytes was assayed using a microglutathione assay with slight modifications [19]. Briefly, cumulus cells were removed from the oocytes by treating them with 0.1% hyaluronidase and by aspirating in and out of a narrow-bore pipette. Denuded oocytes were washed three times in pre-equilibrated mTBM, and 30–40 oocytes were placed in 50 µl Cytobuster protein extraction reagent (Merck & Co.). The reagent containing oocytes was transferred to a 96-well microtiter plate and incubated in a shaker at room temperature for 15 min. The samples were frozen at –70°C for 10 min and thawed at room temperature for 5 min. This process was repeated two times. The samples were then transferred to 1.5 ml Eppendorf tubes and centrifuged at 12 000 x g for 10 min. After centrifugation, the supernatants were collected and transferred to a 96-well microtiter plate (45 µl/well) and 5 µl phosphoric acid (1.25 M) was added. The 100 µl reaction mixture consisted of 5 ml 5,5'-Dithio-bis(2)-nitrobenzoic acid (Cat. No. D8130; 1 mM), 5 ml NADPH (Cat. No. N7505; 1 mM), 5.75 ml NaPO4 buffer (100 mM) and 0.1 ml GSH reductase (Cat. No. G3664: 200 U/ml) was added and the plate was immediately placed in a microtiter plate reader (Biorad). The formation of 5-thio-2-nitrobenzoic acid was monitored every 30 sec for 3 min. Standards were prepared for each assay, and GSH content per sample was determined from a standard curve (Sigma Plot/Enzyme Kinetics). The GSH concentrations (pmol/oocyte) were calculated by dividing the total concentration per sample by the number of oocytes present in the sample. The number of oocytes per replicate was 30 (control), 40 (insulin), 30 (metformin), and 40 (insulin plus metformin), respectively. The experiment was replicated five times.
Measurement of Tyrosine Kinase Activity in Porcine Oocytes
After IVM for 44 h with insulin (100 ng/ml), metformin (10–5 M), or insulin plus metformin, the tyrosine kinase activity in porcine oocytes was examined using Western blot assay. Briefly, cumulus cells were removed from the oocytes by treating them with 0.1% hyaluronidase and by aspirating in and out of a narrow-bore pipette. Denuded oocytes were washed three times in pre-equilibrated mTBM. Groups of 100 oocytes were homogenized with a lysis buffer (Promega, Madison, WI) and protein was measured using the Bradford kit (Bio-Rad, Hercules, CA). Quantification of Western blot analyses was performed using the Multi Gauge Program, version 2.02 (Korea Fujifilm Co.). Equal amounts of protein were separated on SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Pharmacia). Subsequently, the membranes were blocked for 1 h and incubated for 2 h at room temperature with antiphosphotyrosine antibody (CalBiochem). After washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibody and visualized using the Westzol enhanced chemiluminescence detection kit (Intron). The bands were detected with LAS-3000 (Fujifilm) as insulin receptor substrate 1 (IRS1) and anti-IRS1 (Upstate) identified a band at the same location. Relative expression level of tyrosine kinase was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. The experiment was replicated three times.
Statistical Analysis
All data were subjected to one-way ANOVA followed by a Tukey test using Sigmastat (Systat Software Inc.) to determine statistical differences among experimental groups. Statistical significance was determined when P < 0.05.
RESULTS
Effect of Insulin and Metformin Supplementation During In Vitro Maturation on Developmental Potential of Porcine Oocytes after In Vitro Fertilization
As shown in Table 2, insulin significantly (P < 0.01) increased the rate of blastocyst formation at both 100 ng/ml (34.2 ± 1.0%) and 10 µg/ml (33.1 ± 2.9%) when included in the IVM medium during the entire maturation period compared to the control (21.2 ± 1.0%). No significant difference between the two concentrations of insulin was observed. Metformin had no effect on the rate of blastocyst formation at any of the concentrations. As shown in Table 3, insulin significantly (P < 0.01) increased the rate of blastocyst formation when included during the first half (31.3 ± 2.3%), the latter half (31.9 ± 1.4%), and the entirety (37.5 ± 1.7%; P < 0.01) of the IVM period compared to the control (21.3 ± 1.4%), whereas metformin had no effect. Similarly, compared to the control (21.3 ± 1.4%), insulin plus metformin significantly (P < 0.01) increased the rate of blastocyst formation when present during the first half (27.3 ± 2.1%), the latter (30.2 ± 1.9%) and the entirety (39.4 ± 2.2%) of the maturation period. No additional effect of insulin plus metformin on increasing blastocyst formation was observed compared to insulin alone.
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Effect of Insulin and Metformin During In Vitro Culture (IVC) or In Vitro Maturation and IVC Periods on Developmental Potential of Porcine Oocytes after In Vitro Fertilization
As shown in Table 4, insulin (34.1 ± 2.7%) and insulin plus metformin (34.6 ± 3.0%) significantly (P < 0.05) increased the rate of blastocyst formation compared to the control (23.1 ± 1.4%), whereas metformin alone had no effect when added during the IVC period. Compared to insulin alone, no additional effect of insulin plus metformin on increasing blastocyst formation was observed. When present during both IVM and IVC periods (IVP period), compared to the control (21.2 ± 1.4%), insulin alone (40.4 ± 2.3%) and insulin plus metformin (52.4 ± 2.7%) significantly (P < 0.01) increased the incidence of blastocyst formation, whereas metformin alone had no effect. In addition, insulin and metformin together significantly (P < 0.01) increased the rate of blastocyst formation compared to insulin alone (52.4 ± 2.7% vs. 40.4 ± 2.3%).
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Effect of Insulin and Metformin on Glutathione Content and Tyrosine Kinase Activity in Porcine Oocytes Matured In Vitro
As shown in Figure 1, compared to the control (4.3 ± 0.1 pmol/oocyte), insulin (6.2 ± 0.3 pmol/oocyte) significantly (P < 0.01) increased the GSH content in oocytes after IVM during the entire maturation period of 44 h, whereas metformin did not. The GSH content of oocytes was increased significantly (P < 0.01) more with insulin plus metformin (7.3 ± 0.1 pmol/oocyte) than with insulin alone (6.2 ± 0.3 pmol/oocyte). As shown in Figure 2, compared to the control (1.0 ± 0 arbitrary units [AU]), insulin and metformin increased the tyrosine kinase activity. Supplementation with insulin and metformin together (1.9 ± 0.2 AU) further increased (P < 0.05) tyrosine kinase activity compared insulin alone (1.5 ± 0.1 AU).
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DISCUSSION
Insulin has direct stimulatory effects on ovarian steroidogenesis and granulosa cell stimulation, which are beneficial effects on in vivo oocyte maturation. Metformin acts as an insulin-sensitizing agent. To improve the developmental competence of IVP porcine embryos, insulin and/or metformin were supplemented during the IVM and/or the IVC period, and we examined the frequency of blastocyst formation, oocyte GSH content, and tyrosine kinase activity. Insulin increased the developmental potential of porcine oocytes and embryos, and metformin enhanced the action of insulin, when given during the entire IVM and IVC. Metformin alone had no beneficial effect. The effects of insulin and metformin were associated with oocyte GSH content and tyrosine kinase activity.
The effects of insulin on cultured ovarian cells, including stimulation of granulosa cell mitogenesis, granulosa and luteal cell progesterone production, and theca cell androgen production appear to be similar among different species. In pigs, the ED50 for insulin on granulosa cell mitogenesis and estradiol production is 100 ng/ml. Studies indicate that the ED50 for insulin on LH-induced theca cell androgen biosynthesis is 200 ng/ml. According to previous reports on insulin's action in the ovarian cells (for reviews, see [9]), the experimental concentrations of insulin and metformin used here were selected and evaluated for their effects on porcine oocyte maturation and embryo development. We demonstrated that insulin had a significant (P < 0.01) effect at 100 ng/ml or 10 µg/ml on the developmental potential of porcine oocytes during IVM, with no significant difference observed between the two concentrations, whereas metformin alone had no effect at any concentration (Table 2).
Maturation of oocytes includes two aspects: nuclear and cytoplasmic maturation. Generally, an oocyte is considered to be morphologically mature when the first polar body is extruded (nuclear maturation) and the oocyte is arrested at metaphase of the second meiotic division (MII) stage. Although oocytes exhibiting nuclear maturation can be fertilized, they may be developmentally incompetent because of a deficiency in the cytoplasmic factors needed for full development (cytoplasmic maturation; for review, see [20]). In the present study, insulin had a significant (P < 0.01) effect on developmental potential of porcine oocytes independently of the maturation period, and no significant difference between the first half and the latter half of the maturation period was observed (Table 3). Related to its weak efficacy, metformin may not improve blastocyst formation when added alone or with insulin, although it can act independently of insulin to stimulate receptor kinase activity and IP3 production in oocytes [15]. Our results suggest that the effect of insulin on developmental potential of porcine oocytes is not associated with the meiotic stage of oocytes during IVM.
Insulin stimulates granulosa cell mitogenesis and has a positive effect on ovarian cell proliferation [9]. In this study, insulin increased the rates of blastocyst formation (P < 0.05) when IVF oocytes were cultured with them during IVC (Table 4). Furthermore, when oocytes were matured with insulin and/or metformin during the entire IVM period, fertilized, and cultured with insulin and/or metformin during IVC, the rates of blastocyst formation (insulin, 2-fold; insulin plus metformin, 2.5-fold) were dramatically increased. Metformin with insulin has a significant (P < 0.01) effect on the rate of blastocyst formation compared with insulin alone only when added during both IVM and IVC periods. These results suggest that insulin affects both oocyte maturation and embryo mitosis in vitro and the effects of insulin and metformin depend on the time length of supplementation.
Oocytes matured in vitro seem to be deficient in some, as yet unidentified, cytoplasmic factors, and therefore are less developmentally competent compared to in vivo matured oocytes. For IVP of porcine embryos, GSH is a crucial intraoocyte factor for supporting events after maturation. Synthesis of GSH during oocyte maturation occurs and promotes male pronuclear formation after sperm penetration [21, 22]. It has an important role in providing cells with a reducing environment and in protecting against oxidative damage [23]. GSH also acts as a substrate of glutathione peroxidase, which is a scavenger of free radicals in oocytes, enhancing their competence as a whole [24]. Porcine oocytes and embryos differ from oocytes and embryos of most species in having a large quantity of endogenous lipids. The role of these lipids in oocyte maturation and embryo development is unclear, although they may have a potential role as reserve fuels [25]. Insulin increases glucose uptake, glycogenesis, glycolysis, and lipogenesis in fat cells, which are also lipid-abundant cells. Metformin increases insulin-stimulated glucose uptake and enhances glycogen synthase activity when induced by insulin [14, 26]. Another action of metformin is to reduce fatty acid oxidation in an apparently insulin-independent manner, which serves to redress the imbalance in the glucose-fatty acid cycle [27]. In this study, insulin increased the intracellular GSH level and the tyrosine kinase activity in oocytes during IVM (Figs. 1 and 2) and improved embryo development during the IVC and IVP periods (Table 4). Furthermore, metformin enhanced the action of insulin on oocyte GSH content, tyrosine kinase activity during IVM, and the rate of blastocyst formation during IVP. These results suggest that insulin and metformin may increase cytoplasmic maturity of oocytes during IVM and save the embryo from the harmful effects of lipid peroxidation during IVC. Because cumulus cells support IVM of porcine oocytes to the MII stage and are involved in cytoplasmic maturation [28, 29], the stimulatory findings may be attributable in part to effects of insulin or insulin plus metformin on the accompanying cumulus cells that may have led to intraoocyte changes, as well as to the direct effects of insulin or insulin plus metformin on the oocytes.
In conclusion, insulin increased the developmental potential of porcine oocytes and embryos during IVM and IVC. The beneficial effect of insulin was independent of the maturation period and was directly associated with increased oocyte GSH content and oocyte tyrosine kinase activity. Furthermore, metformin enhanced the actions of insulin as an ISA on oocyte maturation and preimplantaion embryo development in vitro during IVP. Taken together, our results demonstrated that insulin and metformin can be beneficial for the production of transferable embryos and offspring in pigs.
FOOTNOTES
1 Correspondence. FAX: 822 884 1902; hwangws{at}snu.ac.kr 
Received: 21 February 2005.
First decision: 15 March 2005.
Accepted: 16 August 2005.
REFERENCES
-glutamyl compounds: biosynthesis and utilization. Ann Rev Biochem 1976 45:559-604[CrossRef][Medline]This article has been cited by other articles:
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  L. Tosca, C. Chabrolle, S. Uzbekova, and J. Dupont Effects of Metformin on Bovine Granulosa Cells Steroidogenesis: Possible Involvement of Adenosine 5' Monophosphate-Activated Protein Kinase (AMPK) Biol Reprod, March 1, 2007; 76(3): 368 - 378. [Abstract] [Full Text] [PDF]
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 E. Elia, V. Sander, C.G. Luchetti, M.E. Solano, G. Di Girolamo, C. Gonzalez, and A.B. Motta The mechanisms involved in the action of metformin in regulating ovarian function in hyperandrogenized mice Mol. Hum. Reprod., August 1, 2006; 12(8): 475 - 481. [Abstract] [Full Text] [PDF]
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