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Reproduction (2006) 131 449-460
DOI: 10.1530/rep.1.00581
Copyright © 2006 Society for Reproduction and Fertility
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RESEARCH

Dissociation of oocyte nuclear and cytoplasmic maturation by the addition of insulin in cultured bovine antral follicles

A A Fouladi-Nashta and K H S Campbell1

Roslin Institute, Roslin, Midlothian, Edinburgh EH13 9HN, UK and 1 School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, UK

Correspondence should be addressed to A A Fouladi-Nashta; Email: ali.fouladi-nashta{at}nottingham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Follicles of 4–8 mm in diameter were dissected from ovaries and cultured in Waymouth culture medium in the presence or absence of insulin (5 µg/ml) at 39 °C in a humidified atmosphere of 45% O2, 5% CO2 and 50% N2 for 24 h. Following follicle culture, the oocytes were collected and examined for developmental potential, total protein profile and ultrastructural aspects. Oocytes aspirated directly from follicles of the same size were used as controls. Addition of insulin to the follicle culture medium significantly reduced expression of the low molecular weight insulin-like growth factor-binding proteins (IGFBPs) in the follicular fluid, and significantly reduced the cleavage rate of subsequently matured and fertilised oocytes (0.52 vs 0.61). However, there were no differences in the proportion of cleaved embryos which developed to the blastocyst stage (0.30 vs 0.28), nor embryo quality as assessed by total cell number (137 ± 8.53 vs124.6 ± 6.95). The total protein profiles of immature oocytes recovered after 24 h of follicle culture were compared by PAGE. There were marked differences between the two groups, unmatured oocytes recovered from the insulin-positive follicle group showed a protein pattern similar to that of matured oocytes. In addition, examination of ultrastructural features by transmission electron microscopy indicated that oocytes from follicles cultured in the presence of insulin undergo many of the cytoplasmic changes associated with oocyte maturation. In conclusion, follicle culture in the presence of insulin is beneficial for follicular survival and significantly reduces cleavage but has no detrimental effects on the development of cultured embryos. However, many of the cytoplasmic changes associated with oocyte maturation occur prior to the induction of nuclear maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The growth and development of ovarian follicles, oocytes and embryos depend upon the spatial and temporal expression of specific genes and on a continuous supply of energy, hormones and growth factors. Insulin, which is primarily involved in the regulation of glucose concentration in the circulation, also regulates cell growth and development in a wide variety of cell types (Starus 1981, Hsueh et al. 1984). The importance of insulin as a regulator of ovarian function was revealed in patients with altered insulin concentrations. Insulin-dependent diabetes mellitus in women is commonly accompanied by ovarian hypo-function and amenorrhea, whilst hyper-insulinaemia is associated with ovarian hyperstimulation and hyperan-drogenism (Poretsky & Kalin 1987, Poretsky 1991, Fulghesu et al. 1997). Mouse models of maternal hypoin-sulinaemia and hyperglycaemia produce oocytes and developing ovarian follicles of smaller sizes, reduced percentage of germinal vesicle breakdown and an increased occurrence of apoptosis (Chang et al. 2005). In cattle, high insulin concentrations in non-lactating heifers were associated with increased numbers of small follicles (<4 mm) (Gong et al. 1993a) and an increased response to superovulatory treatment (Gong et al. 1993b).

In vitro, insulin has repeatedly been reported to stimulate proliferation and steroidogenesis of granulosa and theca cells (Campbell et al. 1995, Duleba et al. 1997) and is routinely included in tissue and cell culture media. In porcine (May et al. 1980) and human (Hill & Osteen 1992) ovarian tissue, insulin stimulates granulosa cell progesterone secretion and granulosa cell luteinisation (Channing et al. 1976) and induces granulosa cell luteinising hormone (LH)/human chorionic gonadotrophin (hCG) receptors (May et al. 1980). It also increases rat granulosa cell oestrogen and progestin production in response to follicle-stimulating hormone (FSH) (Davoren & Hsueh 1984).

A role for insulin in oocyte maturation has been described. Insulin induced meiotic maturation in Xenopus laevis oocytes. Tsafriri & Channing (1975) demonstrated that insulin stimulates maturation of pig oocytes beyond first metaphase and extrusion of the first polar body. Subsequently, Lessman & Schuetz (1981) reported that insulin facilitates germinal vesicle breakdown in leopard frogs (Rana pipiens). In cattle, mRNA encoding receptor for insulin has been detected at all embryonic stages from the one-cell zygote to the blastocyst (Schultz et al. 1992). However, insulin does not improve bovine oocyte maturation (Betteridge et al. 1989) and has no effect on the fertilisation rate or subsequent development (Stubbings et al. 1990). In the mouse, protein synthesis increases in the presence of insulin at the compacted morulae stage of development (Rao et al. 1990, Kaye et al. 1992) when the insulin receptor appears (Harvey & Kaye 1988).

The insulin-like growth factors (IGFs) are small proteins, structurally related to proinsulin, that stimulate growth and differentiation of a wide variety of cell types (Jones & Clemmons 1995). In the circulation and body fluids, IGFs are complexed with specific binding proteins (IGFBPs) and association of IGFs with their binding proteins in the circulation prolongs their half-life by decreasing their clearance rate (Jones & Clemmons 1995).

The IGFBP content of ovarian follicular fluid from a number of species has been examined using Western ligand blots and immunoblots. For example, in the cow, binding proteins of molecular mass 40–44 kDa (IGFBP-3), 34 kDa (IGFBP-2), 29–27 kDa (IGFBP-5) and 22 kDa (IGFBP-4) have been identified (Echternkamp et al. 1994). The actions of IGFs in ovarian cell cultures have been shown to be inhibited by IGFBPs (Mason et al. 1992, Monget et al. 1993). A decrease in follicular IGFBP production would therefore be expected to increase the biological activity of locally produced IGF and thus increase the sensitivity of the follicle to gonadotrophins. The observed changes in the concentration of IGFBPs in follicular fluid during follicle development agree with this hypothesis and indicate that IGFBPs are key autocrine and paracrine regulators of ovarian function.

We have previously reported a system for the culture of bovine intact antral follicles in vitro (Fouladi-Nashta et al. 1998). Oocytes recovered following 24 h of follicle culture remained at the germinal vesicle (GV) stage and gave a significantly greater frequency of development compared with oocytes aspirated from fresh follicles, following subsequent maturation, fertilisation and culture. Insulin is generally added to tissue culture including ovarian follicles as a survival/mitogenic factor in high concentrations. Use of supraphysiological concentrations of insulin may be beneficial for the survival of ovarian follicular somatic cells, but may have a detrimental effect on oocyte quality and development. The aims of the experiments reported here were to elucidate the effects of addition of insulin to the follicle culture medium on follicular survival, via the evaluation of embryonic development and the quality of oocytes following 24 h of follicle culture. The effect of insulin on the ultrastructure of immature oocytes derived following follicle culture in the presence of insulin was examined. In addition, the effects of insulin on IGFBP and total protein profiles in follicles and oocytes were investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
All chemicals and reagents were purchased from Sigma (Poole, Dorset, UK) unless otherwise stated.

Isolation and culture of antral follicles
Ovaries were obtained from local abattoirs and maintained at 28–35 °C during transport to the laboratory (1–2 h). Using a pair of scissors and forceps, follicles of 4–8 mm diameter were dissected from the surrounding connective tissues and cultured as previously described (Fouladi-Nashta et al. 1998). Briefly, non-atretic follicles were selected on the basis of morphological criteria, including translucency, lack of free particles in the antrum and the presence of blood vessels in the follicular wall (Kruip & Dieleman 1982).

Selected follicles were cultured in six-well culture dishes fitted with netwell inserts of 500 µm pore diameter containing Waymouth medium MB752/1 (Invitrogen, Paisley, Strathclyde, UK) supplemented with 0.23 mM pyruvic acid, 50 mg/l streptomycin sulphate, 75 mg/l penicillin G and 3 mg/ml BSA (pH 7.4, 280 mOsmol/kg H2O) in the presence or absence (control) of 5 µg/ml insulin. A gaseous atmosphere of 45% O2, 50% N2 and 5% CO2 was used according to Moor et al.(1973). Before use, the culture medium was placed in an anaerobic jar which was gassed for 5 min, sealed and warmed to 39 °C in an incubator. After transferring the follicles into the culture dishes, they were replaced in the anaerobic jar which was gassed for 5 min before being sealed and returned to the incubator.

Oocyte collection
Cumulus–oocyte complexes (COCs) were aspirated from follicles 4–8 mm in diameter using a 10 ml syringe fitted with a hypodermic needle (1.2 mm internal diameter). The aspirated follicular fluid was placed into sterile universal containers (Nunc, Rosklide, Denmark), in a warm air chamber (39 °C) and allowed to settle for 10–15 min. The majority of the follicular fluid was then removed by surface aspiration and the remaining follicular material diluted with an equal volume of dissection medium (TCM199 with Earl’s salts (Invitrogen, Paisley, UK) 75.0 mg/l kanamycin, 7.08 g/l Hepes (pH 7.8, osmolarity 279 mOsmol/kg H2O] supplemented with 10% fetal calf serum (FCS). The diluted follicular fluid was transferred into an 85 mm Petri dish (Bibby Sterilin, Stone, UK) and examined for COCs under a dissecting microscope.

Oocytes from follicle culture treatments were collected after opening the follicles with forceps. After 24 h of follicle culture, COCs from both groups (n = 106 in insulin-positive and n = 98 in insulin-negative groups) were collected and washed in dissection medium as previously described.

Oocyte maturation
COCs isolated following 24 h of follicle culture were washed twice in dissection medium and transferred into 500 µl pre-gassed maturation medium (TCM199 with Earl’s salts, 75 mg/l kanamycin, 4.75 g/l Hepes, 2.29 g/l NaHCO3 (pH 7.8, osmolarity 280 mOsmol/kg H2O) supplemented with 0.006 IU/ml FSH/LH (Pergonal; Serono Laboratories, Herts, UK) and 10% FCS in 35 mm tissue culture dishes. COCs were then incubated in a humidified atmosphere of 5% CO2 in air at 39 °C for 24 h.

In vitro fertilisation
In vitro matured oocytes were fertilised as previously described (Fouladi-Nashta et al. 1998). Briefly, COCs were gently pipetted in order to remove adhering granulosa cells and break up aggregated COCs. Disaggregated COCs were then washed once in oocyte wash medium containing 6.8 g/l NaCl, 0.23 g/l KCl, 168 mg/l NaHCO3, 47 mg/l Na2HPO4, 4.8 g/l Hepes, 75 mg/l kanamycin monosulphate, 11 mg/l pyruvic acid, 6 g/l BSA, 1.86 ml/l 60% syrup lactic acid, 100 mg/l MgCl2.6H2O, 0.84 g/l CaCl2.2H2O (pH 7.4, osmolarity 282 mOsmol/kg H2O) and transferred into 45 µl microdrops of fertilisation medium (five to ten oocytes/drop) containing sperm (1.5 x 106/ml) and maintained for 24 h at 39 °C in a humidified incubator in 5% CO2 in air.

In vitro embryo culture
At 46–48 h after co-incubation of the spermatozoa and oocytes, cleaved embryos with at least four cells were selected, washed twice in a Hepes-buffered synthetic oviductal fluid (SOF) medium and transferred to 20 µl droplets (5 µl/embryo) of SOF medium (Thompson et al. 1991) supplemented with 4 mg/ml Pentex crystalline BSA (Bayer, Elkhart, Germany) overlaid with mineral oil. Embryo culture was carried out in 35 mm cell culture dishes at 39 °C, in a humidified incubator with a gaseous atmosphere of 5% CO2 and 5% O2.

Total cell counting
Blastocyst stage embryos were incubated for 15 min in dissection medium containing 5 µg/ml bisbenzimide (Hoechst 33258; Sigma). The embryos were then placed onto clean glass slides in 3 µl drops of diazabicyclo (2.2.2) octane (DABCO; Sigma) and secured with 22 mm2 coverslips. Counting took place using an inverted, differential interference contrast microscope fitted with epifluorescence (Nikon, Garden City, NY, USA).

Transmission electron microscopy of oocyte ultrastructure
COCs were collected following 24 h of follicle culture in the presence (n = 18) or absence (n = 18) of insulin. A proportion of oocytes from each group were matured in vitro (n = 6 in each treatment). Immature and in vitro matured oocytes were processed for electron microscopy using a modification of the protocol described by Hyttel & Madsen (1987). Briefly COCs were fixed in 3% glutaradehyde in 0.1 M Millonigs buffer (Sigma; pH 7.4), for 1 h at 4 °C, then washed three times each for 20 min in 0.2 M buffer and post-fixed in 1% OsO4 in 0.1 M buffer for 1 h at 4 °C. The oocytes were then washed in distilled water for 30 min, placed in 0.5% uranyl acetate (TAAB Laboratories, Reading, UK) for 2 h, and dehydrated in ascending concentrations of ethanol, cleared in inhibisol (1.1.1.tri-chloroethane), and impregnated and embeded in Araldite. Ultrathin sections (300 Å) of oocytes were cut perpendicular to the plating surface. Oocyte sections were stained for 3 min with uranyl acetate (25% uranyl acetate in 50% ethanol; Watson 1958) and 7 min with lead citrate (Reynolds 1963). Ultra-structural observations were made using a Philips EM300 electron microscope (Eindhoven, The Netherlands).

Total protein profile of oocytes
Oocyte preparation
Non-mature oocytes (total of 54 oocytes in three replicate cultures) were collected following 24 h of follicle culture in the presence (n = 27) or absence (n = 27) of insulin, transferred into 500 µl drops of dissection medium containing 300 units/ml hyaluronidase (Sigma) and incubated at 39 °C for 15 min. After incubation, the oocytes were transferred into dissection medium and the cumulus cells removed by repeated pipetting. Denuded oocytes were placed into 1.5 ml Eppendorf tubes containing 15 µl sample buffer. The oocytes were then lysed by multiple rapid freeze–thaw cycles in liquid nitrogen and methanol respectively. The prepared samples were stored at –20 °C until PAGE analysis.

One dimensional SDS-PAGE
A 6% acrylamide gel for protein separation and 4% stacking gel were freshly prepared in a mini-protean II (BioRad, Hemel Hempstead, UK). Prepared samples were thawed on ice, boiled for 3 min to denature the proteins and then stored on ice until loading onto the gel. Molecular weight markers (29 000–116 000) were run on each gel to ascertain the molecular weight in kDa of the separated proteins. A total of five oocytes were loaded/sample well. The gel was run for 6 h at a constant voltage of 200 V. After running, gels were fixed in a solution of 10% glacial acetic acid and 40% methanol in distilled water for at least 1 h.

Silver staining
After fixation, the gels were silver stained using a modification of the method described by Blum et al.(1987). Briefly, the gels were washed twice for 20 min in double-distilled water (DDW) followed by a 20-min dehydration in 50% ethanol and pretreated in a 0.2 mg/ml solution of Na2S2O3.5H2O (BDH, Poole, Dorset, UK) for 1 min. They were then passed through three quick washes in DDW and placed into a solution containing 2 µg/ml silver nitrate (BDH) and 0.75 µl/ml 36% formaldehyde (TAAB Laboratories) for 30 min to allow silver impregnation. After staining, the gels were given two quick washes in DDW and the protein bands were visualised in a development solution containing 60 mg/ml Na2CO3 (BDH), 0.5 µl/ml 36% HCHO and 0.5 µg/ml Na2S2O3.5H2O for 3–4 min. Development was monitored visually and stopped by placing the gel into a solution containing 18.6 mg/ml EDTA (Sigma) and 0.2 mg/ml thiomersol for 30 min. The gels were then washed in DDW for 30 min and equilibrated in a solution containing 33% ethanol and 3% glycerol at least for 1 h.

Analysis of IGFBP profiles
IGFBP profiles in both follicular fluid from individual follicles cultured in the presence (n = 14 in 2–4 mm and n = 8 in 4–8 mm) and absence of insulin (n = 12 in 2–4 mm and n = 8 in 4–8 mm) and culture media were analysed by Western ligand blot. Samples of follicular fluid were diluted (2 µl follicular fluid + 23 µl distilled H2O) and the 25 µl follicular sample or 25 µl culture media were mixed with 25 µl Laemmli sample buffer (10 ml 0.5 M Tris–HCl, pH 6.8, 10 ml 10% SDS and 10 ml glycerol, containing bromophenol blue in 100 ml dH2O).

Samples were solubilised and loaded onto 12% (w/v) non-reducing SDS-PAGE gels and electroblotted onto PVDF membrane. Non-specific binding sites on the membrane were blocked using 3% (w/v) BSA in PBS–0.1% (v/v) Tween 20 (blocking solution). The blot was then left overnight in 1% BSA and 0.1% Tween 20 in TS buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.4) containing 3 x 106 c.p.m. 125I-labelled ligand (in 20 ml). After labelling, the blots were washed four times, each for 30 min, in 0.2% Nonidet P40 in TS buffer. All washing steps were performed at 4 °C. The nitrocellulose was then dried and exposed to a Cronez 4 film (Sterling Diagnostic Imaging Inc, Newark, DE, USA) for 3 days. The films were developed using an autoradiograph developer.

Statistical analysis
The data from the proportion of oocytes which cleaved after fertilisation and the proportion of cleaved embryos which became blastocysts were analysed by fitting replicates as a source of random variation in a general linear mixed model (Breslow & Clayton 1993).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
The developmental competence of oocytes and embryo quality
The effects of addition of insulin to follicle culture media are summarised in Table 1Go. There was a significant reduction in the proportion of cleaved embryos in the insulin-treated group as compared with the control (means ± S.E.M.; 0.52 ± 0.029 vs 0.61 ± 0.032) (P < 0.05). However, there was no significant difference in the proportion of cleaved embryos that became blastocysts (0.30 ± 0.038 in the insulin-positive group vs 0.28 ± 0.047 in the control group; 0.15 ± 0.089 vs 0.18 ± 0.075 of oocytes) nor in the quality of embryos between the two groups as assessed by total cell number (137 ± 8. 53 vs 124.6 ± 6.95).


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  		Table 1 Effect of addition of insulin during follicle culture on the developmental competence of the recovered oocytes. Insulin adversely reduced the cleavage (P < 0.05) rate but there were no differences in either the number of quality of blastocysts between the two groups. The values are presented as means ± S.E.M.
 
Total protein profile in the oocytes
Total protein profiles of immature oocytes (groups of five oocytes in each category) collected either directly from follicular aspiration (non-mature oocytes aspirated from fresh follicles; NM) or following follicle culture in the presence of insulin (insulin-treated follicles; FNMI) as well as the in vitro matured oocytes from these categories (24 h of oocyte maturation (M) and 24 h of follicle culture + 24 h of oocyte maturation (FM)) were compared. A representative profile of total proteins in the oocytes is shown in Fig. 1Go. Non-matured oocytes recovered from follicle culture in the presence of insulin (immature oocytes from insulin-treated follicles; FNMI) had a remarkably different profile from both immature NM and non-mature oocytes collected after follicle culture (FNM) (insulin-negative) oocytes. This group of oocytes (insulin-positive group) showed a profile more similar to that of matured oocytes. The differences were observed either as reduction in the concentration or disappearance of protein bands such as the 34, 39, 42, 50 and 55 kDa proteins that were also observed following maturation in vitro. There were no detectable differences in the total protein profile between the two groups following subsequent in vitro maturation (FM vs M). However, there was a significant reduction in the 50 kDa protein in follicles cultured for 24 h in the presence of insulin + 24 h of oocyte maturation (FMI) oocytes.


Figure 1
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  		Figure 1 Effects of addition of insulin during the culture of bovine large antral follicles on total protein profile of oocytes. Immature oocyte from insulin-treated follicles (FNMI) displayed a protein profile identical to matured oocytes. There was no difference in the protein profile of oocytes after maturation. NM, non-mature oocyte aspirated from fresh follicle; FNM, non-mature oocyte collected after follicle culture; FM, 24 h of follicle culture + 24 h of oocyte maturation; M, 24 h of oocyte maturation; FMI, 24 h of follicle culture in the presence of insulin + 24 h of oocyte maturation.

 
Ultrastructure of oocytes
A total of 36 oocytes were sectioned and analysed (n = 24 immature and 12 matured). Representative images of the oocytes from both treatments are shown in Figs 2Go–7GoGoGoGoGo. The ultrastructure of immature oocytes recovered following follicle culture in the absence of insulin and in vitro matured oocyte are shown in Figs 2Go and 3Go respectively. Immature oocytes were characterised by peripherally located clusters of mitochondria and cortical granules (Fig. 2Go). In matured oocytes, the mitochondria become evenly distributed throughout the cytoplasm and the cortical granules became individually located in the marginal area under the oocyte membrane (Fig. 3Go). The ultrastructure of immature oocytes recovered following 24 h of follicle culture in the presence of insulin is shown in Figs 4Go and 5Go. In these oocytes, the cortical granules changed their position from being in clusters to becoming individually located in the marginal area. The mitochondria became dispersed throughout the cytoplasm. The number of vesicles reduced and these moved towards the central area. The majority of these oocytes exhibited an organelle-free area under the zona pellucida. This feature was recorded in 75% of the oocytes examined (n = 9). The majority of the oocyte microvilli remained attached to the zona pellucida. However, some of them became terminated in the previtelline space. Similar features were observed in in vitro matured oocytes following follicle culture in the absence (Fig. 6Go) and presence of insulin (Fig. 7Go).


Figure 2
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  		Figure 2 Electron micrograph showing part of an immature oocyte following 24 h of follicle culture (FNM) in the control group. Note the clusters of mitochondria (M) and cortical granules (CG) located peripherally and distribution of fat vesicles (V) throughout the cytoplasm. ZP, zona pellucida.

 

Figure 3
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  		Figure 3 Electron micrograph showing part of an in vitro matured control oocyte. Note relocation of the mitochondria (M) throughout the cytoplasm and location of the cortical granules (CG) in the marginal area. The number of fat vesicles (V) has reduced. ZP, zona pellucida; Gr, granulosa cell.

 

Figure 4
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  		Figure 4 Electron micrograph showing part of an immature oocyte following culture of large antral follicles in the presence of insulin. The oocyte gained the features of a matured oocyte. The mitochondria (M) became evenly distributed throughout the cytoplasm (see inset). Some of the small vesicles fused together and formed giant vesicles (V). Note the organelle-free area in the marginal area. The cortical granules (CG) became located individually in the margin of oocyte. The inset shows high magnification of a group of mitochondria. ZP, zona pellucida.

 

Figure 5
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  		Figure 5 Electron micrograph showing part of an immature oocyte following culture of large antral follicles in the presence of insulin. Cortical granules (CG) are located individually in the margin of oocyte. Many of the microvilli are disconnected and terminate in the perivitelline space (see inset) but some of them still penetrated into the zona pellucida (ZP). MV, microvilli.

 

Figure 6
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  		Figure 6 Electron micrograph showing part of an in vitro matured oocyte following follicle culture in the absence of insulin. Note the relocation of mitochondria (M) throughout the cytoplasm and relocation of the cortical granules (CG) in the marginal area (see inset). The number of fat vesicles (V) has reduced. ZP, zona pellucida; G, Golgi apparatus.

 

Figure 7
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  		Figure 7 Electron micrograph showing part of an in vitro matured oocyte following follicle culture in the presence of insulin. Cortical granules (CG) are located individually in the margin of oocyte. Microvilli are disconnected and terminate in the perivitelline space (see inset). V, fat vesicle; M, mitochondria; ZP, zona pellucida.

 
IGFBPs
The IGFBPs present in follicular fluid and the culture media were compared following culture of small (2–4 mm)- and medium (4–8 mm)-sized follicles in the presence or absence of insulin. The control contained samples from the pool of follicular fluid collected from the aspiration of oocytes that were used for subsequent maturation. Follicular fluid and culture media collected from the culture of individual follicles cultured in the presence (n = 22 follicles) and absence (n = 20 follicles) of insulin were analysed by Western blotting (n = 5 Western blots repeats). A representative profile of IGFBPs is shown in Fig. 8Go. Whilst the control group showed moderate amounts of IGFBPs, there were major qualitative differences in both culture medium and follicular fluid between the insulin-positive and control groups. In insulin-treated follicles two strong bands corresponding to IGFBP-4 (19–22 kDa) and -2 (29–34 kDa) disappeared in both the follicular fluid and the culture media of 4–8 mm diameter follicles. In contrast, smaller follicles retained the same pattern of IGFBPs.


Figure 8
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  		Figure 8 Effects of addition of insulin during culture of bovine antral follicle on the IGFBP profile. Follicles from two size ranges of small (2–4 mm) and medium (4–8 mm) were cultured in the presence (I+) or absence of insulin (I–). Follicular fluid (FF) and culture medium (CM) were collected from two size groups of follicle following 24 h of follicle culture. Controls, follicular fluid from directly aspirated follicles. a vs b, P < 0.001; b vs c, P < 0.01; a vs c, P < 0.01; d vs e, P < 0.05; f vs g, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
The results of the present studies have demonstrated that the presence of insulin has a number of effects on the immature or GV stage oocyte, including changes in protein profile and ultrastructure. In addition, following subsequent maturation and fertilisation, a decrease in cleavage rates was observed in insulin-treated oocytes. Transcripts for the receptors of insulin have been detected at all developmental stages of preimplantation bovine embryos (Watson et al. 1994). Insulin stimulates both amino acid uptake and protein synthesis (Harvey & Kaye 1988, Kaye et al. 1992) in mouse preimplantation embryos. We observed significant quantitative changes in the total protein profile of the immature oocytes following follicle culture in the presence of insulin. For example, proteins with molecular weights of 34, 39, 42 and 50 kDa were decreased or became undetectable. The involvement of proteins of these molecular weights has previously been documented in a number of different species (Cicirelli et al. 1988, Mutter et al. 1988, Naz et al. 2001). The 50 kDa protein that was reduced in FMI oocytes may be the oocyte membrane antigen that is important for binding to sperm during fertilisation (Naz et al. 2001). Reduction in the expression of this protein may have contributed to the lower cleavage rate of the FMI oocytes.

There is a limited number of studies on the effects of insulin on oocyte maturation and preimplantation bovine embryo development. Over-stimulation of IGF-I (Armstrong et al. 2001) and probably insulin (Armstrong et al. 2003) is detrimental to follicle and oocyte development. In mice, high concentrations of IGF-I and insulin induced a down-regulation of the IGF-I receptor in blastocysts, with a subsequent decrease in signalling of IGF-I receptor-associated pathways (Chi et al. 2000). This decrease in IGF-I receptor reduced glucose uptake and triggered apoptosis. In addition, women with polycystic ovary syndrome exhibit elevated concentrations of insulin and IGF-I and experience increased pregnancy losses (Tulppala et al. 1993, Zephne et al. 2004, Dumesic et al. 2005). A threshold may exist at which the level of growth hormone, IGF-I or insulin goes from being beneficial to detrimental on oocyte and embryo development. Matsui et al.(1997) reported that insulin at concentrations of 0.5–10 µg/ml had a beneficial effect on the rate of development to the morula stage on day 5 of embryo culture and that this stimulation of embryonic development was mediated through the IGF-I receptor. However, these authors did not report the frequency of development to the blastocyst stage. In the studies reported here, insulin at a concentration of 5 µg/ml was added to the follicle culture medium. This level of insulin, which is routinely used for the culture of preantral follicles, is greater than the physiological concentrations of insulin reported in biological fluids (0.5–10 ng/ml) (Diamond et al. 1985). However, although this high concentration of insulin resulted in a decrease in oocyte cleavage, the frequency of blastocyst production was not affected. In addition, the quality of blastocysts produced (as assessed by total cell number) was not significantly different between the two groups. In the present study, we have used Waymouth medium to culture follicles. Waymouth medium is extensively used for culture of follicles in different species. However, it contains a high concentration of glucose that may affect oocyte maturation. In oocytes, glucose is essential for optimal completion of nuclear maturation to metaphase II stage, but supraphysiological levels may be detrimental and compromise subsequent developmental capacity (Hashimoto et al. 2000). Exposure of mouse oocytes to diabetic conditions during folliculogenesis has a negative effect on meiotic maturation and subsequent development, due to decreased communication between the somatic and germ cell compartments (Colton et al. 2003). In the mouse, alterations in energy substrate supplementation profoundly influence meiotic regulation in vitro. For example, high glucose induces germinal vesicle breakdown in COCs in the presence of meiotic inhibitors (Downs & Mastropolo 1994). In the present study, a lower cleavage rate was observed in oocytes derived from the insulin-treated group. It seems that insulin supplied a surplus amount of glucose for oocytes that resulted in an alteration of oocyte cytoplasmic maturation which adversely affected cleavage rate. However, no differences were observed in the developmental competence of oocytes as assessed by frequency of development to blastocyst stage and total cell numbers. Indeed, although not significant, slightly higher numbers of blastocysts were produced in oocytes derived from the insulin-treated follicle culture group. In addition, these blastocysts contained slightly increased total cell numbers than the oocytes from the insulin-negative group.

Any effects of the insulin family of growth factors on the oocyte in vivo are possibly regulated by IGFBPs (Funston et al. 1996). IGFBPs have a regulatory role in follicle development (Echternkamp et al. 1994) and are developmentally regulated in bovine granulosa and theca cells (Spicer & Echternkamp 1995). IGFBP-2 and possibly IGFBP-4 and -5 are high in small- and medium-sized follicles, but reduce or become undetectable in large or dominant follicles, resulting in increased IGF bioactivity and enhancing the amplification of gonadotrophin action on follicular cells (Spicer & Echternkamp 1995, Funston et al. 1996, de la Sota et al. 1996). In contrast, atresia is characterised by an increase in concentrations of the low molecular weight IGFBPs in the follicular fluid (Besnard et al. 1997).

The present study on the effects of insulin on IGFBPs in both culture media and follicular fluid indicate that in insulin-treated follicles of >4 mm in diameter, two protein bands with estimated molecular weights of 19–22 and 29–34 kDa corresponding to the low molecular weight IGFBP-4 and 2 were significantly reduced to undetectable levels. It is possible that this reduction of low molecular weight IGFBPs resulted in protection of larger follicles against atresia. However, in small follicles, insulin did not protect follicles against atresia, so the concentrations of IGFBPs increased. During follicular development in cattle, the IGFBP-3 content of follicular fluid increases (Armstrong et al. 1996). In addition, growth of follicles larger than 4 mm in diameter is FSH dependent (Gong et al. 1995). The presence of insulin may have a compensatory role in this group of follicles, resulting in healthier follicles. On the other hand, the disappearance of low molecular weight IGFBPs in larger follicles may be explained by intrinsic changes in the follicle and any effect of insulin on the follicle are ignored.

Analysis of the ultrastructure of the recovered oocytes showed that insulin-treated follicles yield oocytes which have features normally associated with matured oocytes. This may explain the reduced cleavage rate after fertilisation. As these oocytes were cultured in maturation medium for a further 24 h, to allow nuclear maturation, the early occurrence of cytoplasmic changes may suggest that the oocytes became hypermature or aged, thus resulting in a lower fertilisation rate than the control group. In porcine (May & Schomberg 1981), human (Hill & Osteen 1992) and bovine (Spicer et al. 1993) ovarian tissue, insulin stimulates granulosa cell progesterone secretion, granulosa cell luteinisation (Channing et al. 1967) and induces granulosa cell LH/hCG receptors (Osteen et al. 1985, May & Schomberg 1981). In the experiments presented here, the levels of progesterone were not compared between the two groups. If insulin supplementation increases progesterone level in the follicular fluid, then it may mimic the phenomenon at the preovulatory surge of LH that leads to resumption of meiosis in the oocytes.

In conclusion, follicle culture in the presence of insulin has no detrimental effect on oocyte structure and does not improve bovine oocyte quality or preimplantation embryo development in vitro, but appears to promote cytoplasmic maturation. In addition, the presence of insulin during follicle culture may prevent the process of atresia in medium-sized antral follicles.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors would like to thank Dr David Armstrong and Mrs C Hogg for IGFBP analysis. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 24 November 2004
First decision 3 February 2005
Revised manuscript received 19 August 2005
Accepted 22 November 2005

A A Fouladi-Nashta is now at School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, UK


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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