Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes

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1 Indian Journal of Experimental Biology Vol. 50, December 2012, pp Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes Jolanta Opiela*, Ewa Latasiewicz & Zdzisław Smorąg Department of Biotechnology of Animal Reproduction; National Research Institute of Animal Production; Krakowska 1 st., Balice/Kraków, Poland Received 16 April 2012; revised 17 August 2012 With a view to search for optimal concentration of hyaluronan (HA) and plant protein (PP) in different culture systems for in vitro maturation of bovine oocytes, cumulus-oocyte complexes (COCs) were matured in vitro in 2 culture systems (first co-cultured with granulose cells and estrus calf serum (ECS) in 2 ml volume, second without co-culture where ECS was replaced by exogenous hormones and BSA or PP in 100 µl dose under mineral oil). Seven types of media were used; 3 in first system and 4 in second system. To evaluate HA and PP effect on oocytes after in vitro culture an estimation of meiosis stage and a level of DNA fragmentation was performed by TUNEL staining. The highest meiotic maturation (84%) was observed in oocytes cultured in medium enriched with ECS in co-culture with granulose cells (1 st system). The lowest meiotic maturation was noted in medium with addition of BSA (43%). The addition of HA in the medium enriched with BSA significantly increased the rate of matured oocytes (67%) and also didn t affect the chromatin quality of individual oocytes. The addition of HA to the culture medium supplemented with a PP decreased the rate of matured oocytes to 54% but no statistical differences were noted. The results of the present study showed that HA supplementation didn t have a detrimental impact on oocyte chromatin integrity and improved bovine oocytes meiotic maturation in medium supplemented only with BSA without co-culture of granulose cells. Keywords: Bovine oocytes, DNA fragmentation, Hyaluronan, IVM, TUNEL Maturation of oocytes in vitro is a critical step in the complex in vitro embryo production. Mature oocytes can be used in other biotechnological procedures such as cloning and transgenic animals production. During maturation in vitro more than 80% of oocytes reach metaphase II (MII), 70-80% of the fully mature oocyte are capable of fertilization and only half of them develop to the blastocyst stage during in vitro culture 1. The factors limiting a developmental potential of oocytes may be abnormal maturation due to suboptimal culture conditions 2,3 and two-dimensional nature of the in vitro cell culture, where many important biological functions are not properly regulated. HA, depending on its configuration and concentration in the aquatic environment creates intercellular 3D gelatinous network. This enhances an interaction between cell receptors and molecules of the external environment by better binding of growth factors important for early embryonic development 4. *Correspondent author Telephone: Fax: jolanta.opiela@izoo.krakow.pl Glycosaminoglycans, including HA are synthesized and secreted by cumulus cells under the stimulation of LH and FSH 5. Hyaluronan is a major component of the cumulus-oocyte complex (COCs) and for its expansion an interaction between HA and its receptor CD44 is required 6. An expanded cumulus facilitates a detachment of an oocyte from a follicle wall during ovulation. The expansion of the cumulus cells is accompanied by modification of transmembrane channels composed of hexameric proteins called connexins (CXS). A resumption of meiotic division in mares, pigs and rats is associated with decreased levels of connexin 43 (Cx43) 7. Yokoo et al. 8 revealed, that the interaction of hyaluronan with its receptor CD44 in cumulus cells during cumulus expansion lowers the levels of connexin 43 by tyrosine phosphorylation in Cx43. This blocks the transport of camp between cumulus cells and oocyte. As a result, the decrease in camp concentration in the oocyte activates MPF, which is responsible for the resumption of meiosis (GVBD) 8. Based on analysis of these results it was concluded that addition of exogenous HA to the culture of oocytes in vitro may increase the percentage of oocytes reaching metaphase II stage.

2 840 INDIAN J EXP BIOL, DECEMBER 2012 Attempts are being made to replace animal serum with safer and more standardized component for media used in animal reproduction biotechnology The reason is an ambiguous impact of serum on a quality of oocytes, embryos and cultured cells 10-13,16. Moreover, in vitro produced embryos during culture in medium supplemented with animal serum may transmit prions and viruses such as bovine viral diarrhea called virus (BVDV) 9 during the transfer of embryos. An alternative to the commonly used animal serum seems to be proteins of plant origin as they also provide nutrients, adhesion factors, growth factors and analogs 10. Homogenates of plant protein substituted for BSA in NCSU-23 medium for in vitro culture of pig embryos positively influenced the number of blastocysts produced 11. Plant protein can successfully replace BSA for in vitro culture of bovine fibroblasts as cells were characterized by better quality and faster rate of proliferation 12. The aim of this study is to search for optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes and to assess an impact of HA and PP supplementation on meiotic maturity as well as their quality measured by DNA fragmentation. As this was a completely new approach to the maturation medium composition, first of all the working concentrations of HA and plant protein were estimated which allow to achieve oocyte meiotic maturation and to check whether the new supplements do not have any detrimental effect on oocytes quality. As the IVF and in vitro embryo culture takes money, work and time such preliminary experiments were justified. Materials and Methods Unless otherwise indicated, entire plastic ware, i.e., culture vessels, dishes and tubes, used in the present experiments was obtained from Nunc, Germany, and all chemicals and media were purchased from Sigma, Poland. Immature oocytes were isolated from ovaries obtained from heifers and cows from a slaughterhouse, which were delivered to the laboratory in a thermo flask within 2-3 h after slaughter. Retrieval and selection of bovine cumulus-oocyte complexes (COCs) COCs were retrieved through syringe from ovaries from slaughtered cows by aspiration of follicular fluid of ovarian follicles. Follicular fluid was collected into tubes (Corning, 12 ml) and left for min at room temperature to sediment. The supernatant was collected and the remaining sediment was transferred into a holding medium (TCM Earle s salt with 25 mm HEPES containing 10% foetal calf serum). The COCs were recovered under stereomicroscope. Before placing COCs into in vitro maturation, they were washed twice, during which the selection was carried out. COCs with a homogenous cytoplasm and few compact layers of cumulus cells, without any features of apoptosis or necrosis (i.e. dark spots in oocyte cytoplasm and/or cumulus) were assigned to IVM. Degenerated oocytes and not fulfilling all above quality criteria were discarded. Maturation of oocytes in vitro Immature oocytes (COCs) were matured in TCM 199 Earle s salt buffered with sodium bicarbonate supplemented with L-glutamine. After adding the appropriate additions the medium was filtered and placed in petri dishes. (i) Culture System P1-TCM 199 supplemented with 20% bovine ECS and an additional 3 to granulose cells/ml. a) P1 + GC; control group. b) P1 + GC + HA (75 µl); medium 1 supplemented with 1% hyaluronan (HA; Croma Pharma GmbH, Austria) with a final concentration of 0.035%. c) P1 + GC + HA (150 µl); medium 2 supplemented with double concentration of 1% HA with a final concentration of 0.07%. 2 ml of medium co-cultured with granulose cells in groups of around 30 oocytes were taken in Petri dishes of 3 cm diameter (Nunc, Denmark). (ii) Culture system- P2-TCM 199 supplemented with FSH 10 µg/ml and estradiol 1µg/mL. d) P2 + BSA; medium supplemented with fatty acid free bovine serum albumin fraction at a concentration of 0.8%. e) P2 + BSA + HA; medium supplemented with fatty acid free bovine serum albumin fraction at a concentration of 0.8% and 1% HA with a final concentration of 0.025%. f) P2 + PP; medium supplemented with plant protein (PP) at a concentration of 0.6% (PP; Animal Pharma BV, Holland). g) P2 + PP + HA; medium supplemented with plant protein (PP) at a concentration of 0.6% and 1% HA with a final concentration of 0.025%.

3 OPIELA et al.: HYALURONAN, PLANT PROTEIN & BOVINE OOCYTES ml medium under mineral oil in 10 oocytes per well were taken in four-well dish (Nunc, Denmark). COCs in all tested media (a-g) were matured in vitro by h at 39 ºC in the presence of 5% CO 2 in the air at maximum humidity. Fixation of mature oocytes After in vitro maturation, oocytes were washed in PBS without calcium and magnesium ions. The oocytes were then deprived of cumulus cells surrounding the oocyte using 0.25% trypsin solution with EDTA by vigorous pipetting. In order to inactivate the enzyme, oocytes were washed in the manipulation medium supplemented with fetal serum, and then rinsed twice in 0.1% PVP in PBS. Denuded oocytes were fixed in 100 µl drop of 4% paraformaldehyde (PFA) in PBS (ph 7.4) for 1 h, at room temperature. Then the oocytes were transferred into 100 µl of 1% solution of PFA in PBS and kept at 4 C for no longer than 14 days. Evaluation of quality and maturity of oocytes TUNEL analysis: TUNEL analysis was performed using a kit of dead-end Fluorometric TUNEL System (Promega, Germany). To access the cell membrane permeability, oocytes were placed in a drop of 0.2% Triton X-100 solution for 5 min. After washing and equilibration, oocytes were transferred to the reaction mixture (equilibration buffer, the mixture of nucleotides and TdT enzyme) and incubated at 38 C in an atmosphere of 5% CO 2 in air for 1 h at maximum humidity13. The rinsing of oocytes in a solution of 2x SSC stopped the incorporation of fluorescein-labeled nucleotides. To stain DNA, oocytes were placed in a drop of VECTASHIELD + DAPI solution. Oocytes treated with UV light for 30 min immediately after maturation were used as a positive control. As a negative control oocytes placed in the reaction mixture lacking the rtdt enzyme were used Oocytes were analyzed under a fluorescent microscope. To visualize the stage of meiosis and DNA fragmentation of oocytes two filters were used filter with a length of > 460 nm, blue fluorescence of DAPI stained cells and filter with a wavelength of 520 ± 20 nm, the green fluorescence of apoptotic cells, which incorporate into the nucleus fluorescein-conjugated dutp. Assessment of meiotic maturity: On the basis of a blue fluorescent dye-dapi the classification of the stages of meiosis in oocytes was made as per Warzych et al 13. Stage of germinal vesicle (GV) was characterized by varying degrees of chromatin condensation, metaphase stage of first meiotic division (MI) was characterized by chromosomes arranged as a group of separated bivalents. The stage of metaphase second (MII) was characterized by the presence of two groups of chromosomes: (a) haploid set of chromosomes and (b) a compact group of chromosomes forming first polar body (PB1). Meiotic maturity was calculated as the ratio of MII stage oocytes to the total number of analyzed oocytes. Assessment of DNA fragmentation and apoptotic index: Oocytes with intense green fluorescence were evaluated as TUNEL positive and oocytes without green fluorescence were evaluated as TUNEL negative. Apoptotic index was expressed as a ratio of oocytes exhibiting intensive green fluorescence to the total number of analyzed oocytes from each group. Statistical analysis For statistical calculations χ 2 test was used. The differences were considered statistically significant at P <0.05, <0.01 and as highly significant at < Experimental design After COCs collection, their careful morphological selection was carried out. The COCs were classified on the basis of the compactness of cumulus cell layers and the homogeneity of oocyte cytoplasm. Due to the total lack of data regarding the HA supplementation for in vitro maturation of oocytes, different culture systems were used to test HA supplementation in presence of different proteins: ECS, BSA and PP. Considering big variety of media being tested, the routinely used IVM system served as control. Two culture systems were used: (i) 2 ml of medium co-cultured with granulose cells in groups of around 30 oocytes were taken in Petri dishes of 3 cm diameter (ii) 0.1 ml of medium under mineral oil in four-well dish where 10 oocytes per well were cultured. Oocytes of all groups after in vitro maturation were denuded of cumulus cells by gentle pipetting subsequently washed in PBS and fixed in PFA until TUNEL was performed. The oocyte meiotic maturity as well as DNA fragmentation rates and apoptotic index were recorded for each group. The statistical calculation by χ2 test was performed. Results Assessment of maturity and quality of bovine oocytes The TUNEL was analyzed in 471 oocytes maturing in two culture systems. There were 187 oocytes estimated from culture system I and

4 842 INDIAN J EXP BIOL, DECEMBER oocytes from culture system II. Altogether oocytes from 7 different culture media were subjected to TUNEL analysis. The supreme meiotic maturity (84%) was observed in oocytes cultured in medium with follicular granulose cells which served as control medium. The lowest meiotic maturity (43%) showed oocytes cultured in medium supplemented with bovine serum albumin (P2+BSA). High significant differences were observed (P <0.001; Table 1) between the meiotic maturity of oocytes cultured in medium P2+BSA and control, P1 + GC + HA (75 µl) and P2+PP media. As for oocytes cultured in medium P2+BSA also significant differences (P <0.05; Table 1) were observed between them and both oocytes cultured in P1 + GC + HA (150 µl) and P2+PP+HA media. The best medium P1 + GC also showed high significant differences (P <0.01; Table 1) with both media P1 + GC + HA (150 µl) and P2+PP+HA. Interestingly, addition of hyaluronan in medium supplemented with BSA (P2+BSA+HA) positively influenced the percentage of oocytes obtained in MII stage (67%). This medium turned to be significantly better (P<0.01, Table 1) than medium P2+BSA (43%). Figure 1A shows the example of chromatin configuration in metaphase II stage of the bovine oocyte after in vitro maturation. Out of 4 media supplemented with HA the highest meiotic maturity was found in oocytes cultured in P1+GC+HA (75 µl) (71%) (Table 1). Not much less oocytes (67%) matured in P2+BSA+HA medium (Table 1). Much less oocytes matured in medium P1+GC+HA (150 µl) supplemented with HA of final concentration 0.035% (62.3%). The lowest maturity exhibited in oocytes cultured in medium supplemented with plant protein and hyaluronian P2 + PP+HA (54%). The highest number of oocytes at metaphase I (Fig. 2A; Fig. 3A) and GV stage (Fig. 2C) was observed in medium supplemented with bovine albumin P2 + BSA medium. Assessment of DNA fragmentation of bovine oocytes Of the 73 oocytes cultured in medium supplemented with plant protein (P2 + PP), 4 oocytes showed DNA fragmentation and abnormal structure of chromatin in metaphase I (Fig. 3B). The oocytes apoptotic index (DCI) cultured in this medium was 5.5% (Table 2). Two oocytes presented abnormal DNA fragmentation and incorrect chromatin structure in germinal vesicle stage out of 65 oocytes matured in medium supplemented with plant protein and hyaluronan (P2 + PP + HA). The apoptotic index (DCI) of oocytes cultured in this medium was 3% (Table 2). In other media (P1 + GC +HA (75 µl), P1 + GC + HA (150 µl), P2 +BSA and P2 + BSA + HA), there were no DNA fragmentation (Table 2; Fig. 1B,D; Fig. 2B,D). Altogether 471 oocytes were subjected to TUNEL staining. Discussion In the present experiments, the impact of hyaluronan and plant protein supplementation on the meiotic maturation and DNA fragmentation of bovine Table 1 Effect of culture media used for oocyte meiotic maturity No Medium Oocytes (no.) Maturation Stage Maturity (%) Other Remark Culture system 1 MII MI GV 1 P1+GC* ** P1+GC+HA (75 µl) P1+GC+HA (150 µl) Culture system 2 4 P2+BSA *** P2+BSA+HA P2+PP nc 7 P2+PP+HA nc * - control medium control, used so far P1 = TCM199 medium supplemented with 20% serum estrus; GC = granulose cells of ovarian follicle; HA = 1% sodium hyaluronate; P2 = TCM199 medium supplemented with FSH and estradiol; PP = plant protein; nc = not correct chromatin structure Statistically significant differences (χ 2 test) at P < were shown between medium 4 and 1, 2 and 6; <0.01 between medium 1 and both 3 and 7; between medium 4 and 5; < 0.05 between medium 4 and both 3 and 7; between medium 1 and 5

5 OPIELA et al.: HYALURONAN, PLANT PROTEIN & BOVINE OOCYTES 843 Fig. 1 The example of chromatin configuration of bovine oocyte after in vitro maturation and subsequent TUNEL analysis (A) DAPI staining 1-chromatin of 1st polar body (200x), 2-oocyte chromatin in metaphase II (B) the same oocyte stained with fluorescein-conjugated dutp-the green fluorescence of apoptotic cells is not emitted therefore no DNA fragmentation is observed; (C) DAPI staining 1-chromatin of 1st polar body (200x), 2-oocyte chromatin in anaphase II (D) the same oocyte stained with fluorescein-conjugated dutp- the green fluorescence of apoptotic cells is not emitted therefore no DNA fragmentation is found. oocytes cultured in vitro at different concentrations and different media formulations were estimated. The experimental design used in culture system I showed interesting observation regarding the final concentration of exogenous hyaluronan, i.e., doubling the concentration of hyaluronan in the medium decreased the percentage of oocytes at metaphase II stage compared to the percentage of oocytes cultured in medium with a higher concentration of hyaluronan and the control medium. Surprisingly, low meiotic maturity (43%) was observed in oocytes cultured in medium supplemented with 0.8% fatty acid free bovine serum albumin. The results of the present study in relation to meiotic maturity are consistent with reports of Ali and Sirard 14. Culturing the oocytes in SOF medium supplemented with serum albumin (0.8% BSA-V) had a negative effect on the kinetics of oocytes maturation in vitro, as 71% of oocytes were observed in GV stage, and only 44% oocytes were in MII stage, after 6 and 18 h incubation culture respectively 14. This result is comparable with the percentage of oocytes at metaphase II stage (43%) in the experiment conducted, although the base culture medium was M199. The present result on meiotic maturity of oocytes cultured in medium supplemented fafbsa and hyaluronan is very interesting because more oocytes (67%) matured in this medium compared to medium supplemented with serum albumin (43%). These observations may suggest that hyaluronan influences nutrient supplementation to accelerate the meiotic maturity of oocytes cultured in the presence of fafbsa. So far nobody has studied the impact of adding exogenous hyaluronan to the in vitro oocyte culture. However, there is information regarding the impact of HA on the in vitro embryo culture. Hyaluronan added to the embryo culture medium supplemented with BSA positively influenced the embryo quality compared to embryos cultured only with BSA 18. Also, for the first time the use of plant protein in place of animal derived serum (e.g. BSA or estrus calf serum) for in vitro maturation

6 844 INDIAN J EXP BIOL, DECEMBER 2012 Fig. 2 The example of chromatin configuration of bovine oocyte after in vitro maturation and subsequent TUNEL analysis (A) DAPI staining 1-oocyte chromatin in metaphase I (200x) (B) the same oocyte stained with fluorescein-conjugated dutp- the green fluorescence of apoptotic cells is not emitted therefore no DNA fragmentation is observed; (C) DAPI staining 1-oocyte chromatin in germinal vesicle GV (D) the same oocyte stained with fluorescein-conjugated dutp- the green fluorescence of apoptotic cells is not emitted therefore no DNA fragmentation is found. Fig. 3 The example of chromatin configuration of bovine oocyte after in vitro maturation and subsequent TUNEL analysis (A) DAPI staining 1-oocyte chromatin in metaphase I (200x) (B) the same oocyte stained with fluorescein-conjugated dutp- the green fluorescence of apoptotic cells is emitted therefore DNA fragmentation is observed. of oocytes was tested. Research hypothesis assumed that the plant protein supplementation does not result in lowering the percentage of mature oocytes compared to oocytes matured in control medium and that will not affect the integrity of the oocytes chromatin after culture. There was observed significantly higher proportion of oocytes at metaphase II cultured in the presence of 0.6% of plant protein (70%) than in medium supplemented with BSA only. On the other hand, the plant protein supplementation along with hyaluronan resulted in even lower percentage of mature oocytes compared to oocytes cultured only

7 OPIELA et al.: HYALURONAN, PLANT PROTEIN & BOVINE OOCYTES 845 Table 2 Effect of culture media used for DNA fragmentation of oocytes after 24 h in vitro culture Medium Culture system 1 Oocytes (no.) DNA Fragmentation Index DCI rate of Apoptotic Oocytes P1+GC* 56-0 P1+GC+HA 62-0 (75µL) P1+GC+HA (150µL) 69-0 Culture system 2 P2+BSA 79-0 P2+BSA+HA 67-0 P2+PP 73 4 (MI) 5.5 P2+PP+HA 65 2 nc (GV) 3.0 * - control medium, used so far P1 = TCM199 medium supplemented with 20% serum estrus; GC = granulose cells of ovarian follicle; HA = 1% sodium hyaluronate; nc = not correct chromatin structure with plant protein. This may suggest an unfavorable interaction between hyaluronan and plant protein. In our opinion, it is possible that plant protein blocks receptors for HA such as CD44 which prevents the resumption of meiosis or block of camp flow is insufficient and leads to the arrest of meiosis at metaphase stage of first meiotic division. Zhuo et al. 19 showed that inter-alpha-trypsin inhibitors (ITIs) contained in serum, bind to hyaluronan and stabilize the extracellular matrix of cumulus cells during its expansion 19. This finding perfectly explains the present results regarding the positive impact of hyaluronan in presence of ECS and BSA and negative in presence of plant protein. Plant protein, contrary to serum, does not contain inter-alpha-trypsin inhibitors, which facilitate the interaction of hyaluronan with its receptor or receptors. Studies concerning the use of plant protein in media focused their attention on the embryo culture 11,18. In one of the first experiments done, the plant protein was used instead of serum albumin in medium for freezing embryos. The studies showed that replacement of serum albumin by 0.18% plant peptides does not affect the viability and quality of embryos after thawing 15. Recent studies of Gajda et al. 16 showed a higher average number of cells in blastocysts cultured in NCSU-23 supplemented with 0.4% plant protein compared to blastocysts cultured in the same medium supplemented with BSA 16. Considering the trend, this observation is consistent with the result obtained in the present experiment, where the proportion of mature oocytes in medium supplemented with PP is higher than in medium with BSA, although different concentrations of plant protein were applied. Assessment of DNA fragmentation in oocytes cultured in all experimental media was a vital part of the experiment because it was assumed that TUNEL positive reaction would be a marker of detrimental effect of media on the oocytes. DNA fragmentation was found only in two media tested, both were supplemented with plant protein while one of them additionally with hyaluronan demonstrated apoptotic index (3 and 5.5%) is very low, but implies the proapoptotic action of plant protein and/or plant protein hyaluronan interaction in the oocytes cultured in vitro. Furthermore, this can also mean that the P2+PP and P2+PP+HA media are not sufficiently balanced therefore the culture environment is sub-optimal. The present results regarding proapoptotic effect of plant protein correlate with study where level of glutathione was analyzed 17. Glutathione protects embryos against oxidative stress and therefore can serve as a marker of embryo quality and viability. In embryos cultured in presence of wheat and cotton peptones the lower level of glutathione was observed 17. This indicate on increased oxidative stress during embryo culture in medium supplemented with peptons 17. Also in other studies on the effects of plant protein on pigs embryo quality the increased apoptotic index in the medium supplemented with vegetable protein (13%) was observed comparing to control medium (BSA, 6.5%) 11. It is interesting that oocytes cultured in medium P2 + PP + HA had lower apoptotic index (3%) compared with oocytes cultured in medium P2 + PP (5.5%), as regarding meiotic maturity medium P2 + PP proved to be more optimal compared to P2 + PP + HA. This observation may be explained by the study of Scott et al. 20, which proved that the glycol group present in the structure of hyaluronan plays the protective function during lipid peroxidation 20. Therefore it can be assumed, that HA added to the medium supplemented with plant protein may have contributed to a reduction in oocytes apoptotic index by protecting them against oxidative stress caused by the action of reactive oxygen species. In conclusion, present results provide evidence that hyaluronan may be used as a supplement for maturation of bovine oocytes in vitro, preferably in conjunction with BSA or estrus serum and follicular

8 846 INDIAN J EXP BIOL, DECEMBER 2012 granular cells. However, plant protein supplementation along with HA seems to be responsible for suboptimal culture conditions. It is possible that optimization of IVM medium for PP requires an innovative approach in terms of ingredients and concentrations used. Importantly, the developmental competence of the oocytes cultured in the experimental media must be estimated by in vitro fertilization and embryo culture to the blastocyst stage to definitely prove the utility of the proposed maturation systems. Considering that HA and PP were not tested before as supplements for oocyte in vitro maturation and due to high costs of IVF/IVP procedure the preliminary experiments on oocytes were justified. Acknowledgement This research was supported by the Statutory Activity of National Research Institute of Animal Production Thanks are due to Bożenna Ryńska for technical help in retrieval and preparation of follicular granulose cells for oocytes maturation in I culture system. 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