Introduction
Hyaluronan (HA) is a major component of mammalian follicular, oviductal and uterine fluids, and plays an important role in optimal embryo development. Hyaluronan is a simple glycosaminoglycan with very complex biological functions. It is composed solely of 2000–25 000 carbohydrates, glucuronic acid and N-acetylglucosamine, and can reach a molecular mass of approximately 4 × 106 Da (Laurent, Reference Laurent1998). Previously known as hyaluronic acid, it is now more often referred as hyaluronan, reflecting the fact that in vivo it exists as a polyanion and not in the acid form. There is ample evidence indicating that, during embryogenesis, HA participates in several biological processes that utilize its unique biochemical and physical properties. Extracellular matrices rich in HA expand the extracellular space, facilitating cell migration and differentiation (Toole, Reference Toole1997). The viscoelastic properties of hyaluronan are essential for proper function of cartilage and joints (Gotoh et al., Reference Gotoh, Onaya, Abe, Miyazaki, Hamai, Horie and Tokuyasu1993). Hyaluronan also acts as an antioxidant, protecting body tissue from radical oxygen species caused by enzymatically produced hydroxyl (OH) radicals (Presti & Scott Reference Presti and Scott1994). Physiological importance of HA may be illustrated by the effects of hyaluronidase on certain biological processes, such as fertilization (Salustri et al. Reference Salustri, Yanagishita, Underhill, Laurent and Hascall1992), or from the correlation between HA accumulation and the presence of its receptors (CD44 and/or RHAMM), in mouse (Fenderson 1993), bovine (Furnus et al., Reference Furnus, Valcarcel, Dulout and Errecalde2003; Stojkovic et al., Reference Stojkovic, Krebs, Kolle, Prelle, Assmann, Zakhartchenko, Sinowatz and Wolf2003) and human (Campbell et al., Reference Campbell, Swann, Aplin, Seif, Kimber and Elstein1995) embryo development. Hyaluronan is especially abundant during embryogenesis (Fenderson et al., Reference Fenderson, Stamenkovic and Aruffo1993) and is considered to contribute to the optimal development of the gametes and the embryo (Gardner et al., Reference Gardner, Rodriguez-Martinez and Lane1999; Ulbrich et al., Reference Ulbrich, Schoenfelder, Thoene and Einspanier2004; Palasz et al., Reference Palasz, Rodriguez-Martinez, Beltran-Breña, Perez-Garnelo, Martinez, Gutierrez-Adan and De la Fuente2007). In contrast with other glycosaminoglycans that are synthesized in the Golgi apparatus and are attached to proteins, HA is synthesized at the inner face of the plasma membrane and is not associated with any protein (Weigel et al., Reference Weigel, Hascall and Tammi1997). Hyaluronan biosynthesis is determined by three related, yet distinct, hyaluronan synthase (Has) enzymes Has1, Has2 and Has3 (Itano et al., Reference Itano, Sawai, Yoshida, Lenas, Yamada, Imagawa, Shinomura, Hamaguchi, Yoshida, Ohnuki, Miyauchi, Spicer, McDonald and Kimata1999). The existence of three different Has isoforms with different characteristics implies that the wide range of biological and physiological functions are performed by HA (Spicer & McDonald, Reference Spicer and McDonald1998). Each of them functions independently. Has3 is intrinsically more catalytically active than Has2, which is in turn intrinsically more active than Has1 (Spicer & Nguyen, Reference Spicer and Nguyen1999). Furthermore, HA synthesized by each Has protein in vitro indicated that HA chain length/molecular weight (MW) is determined by the specific Has protein. Has1 and Has2 proteins polymerize hyaluronan chains of similar lengths (MW up to 2 × 106 Da), whereas Has3 polymerized much shorter hyaluronan chains, in the range of <2 × 105 Da to 3 × 105 Da (Tien & Spicer, Reference Tien and Spicer2005). During embryogenesis continuing changes of HA concentration and MW are prerequisite for embryo cell movement and proliferation. These functions require rapid polymerization and depolymerization of the HA chains, which in in vitro culture conditions is difficult to achieve or control. Presently, variable preparations of HA of different MW are used for embryo culture. Similarly, even concentrations as different as 6 mg/ml (Stojkovic et al., Reference Stojkovic, Krebs, Kolle, Prelle, Assmann, Zakhartchenko, Sinowatz and Wolf2003) and as low as 0.125 mg/ml of HA (Lane et al., Reference Lane, Maybach, Hooper, Hasler and Gardner2003) are used successfully for bovine embryo culture. Considering that almost all biological functions of HA depend on MW and on concentration, this finding may be the proof that full potential benefit of using HA in embryo culture has not yet been achieved. One important piece of information that may help to understand HA function, but was never tested in mammalian embryo culture, comes from the report by Kraemer & Barnhart (Reference Kraemer and Barnhart1978). They showed that cells cultured on solid surfaces, without surface active compound, produce larger amounts of HA than those grown in suspension. To verify this finding and to determine what specific MW of HA is synthesized during preimplantation bovine embryo development at different culture conditions, we quantified the gene expression patterns of HA synthases (Has 1, 2 and 3) and receptors CD44 and RHAMM in bovine day-7 blastocysts cultured up to day 4 (96 hpi) and without any protein or surface active compound. Quantification of mtDNA was used as an embryo quality assay. Considering that all biological functions of HA depend on MW and concentration we anticipate that results from this study will provide new scientific bases for the most effective use of HA for in vitro culture of bovine preimplantation embryos.
Materials and Methods
All chemicals for oocytes in vitro maturation, fertilization and embryo culture were purchased from Sigma Chemical Company unless otherwise stated.
In vitro maturation
Cumulus–oocyte complexes (COC) were aspirated from follicles of 2 to 7 mm in diameter from slaughterhouse-derived ovaries within 3 h of slaughter. After selection based on morphological evaluation (Shamsudin et al., 1994), COC were washed once in TALP medium (Bavister et al., Reference Bavister, Leibfried and Lieberman1983), buffered with 10 mM of MOPS (TALP–MOPS) and supplemented with 0.25 mM pyruvate and 0.5 mg/ml bovine serum albumin (BSA) fraction V, and then twice in the final maturation TCM-199 medium containing 10% fetal calf serum (FCS), 0.25 mM pyruvate and 100 ng/ml epidermal growth factor (EGF). Cumulus–oocyte complexes were matured in groups of 50 in 2 ml of medium (Avery et al. 1995) in Nunclon multi-well dishes (Nunc) without paraffin oil at 39 °C with 5% CO2, 5% O2 and 90% N2 in humidified air.
In vitro fertilization
After 24 h of incubation in the maturation medium, oocytes were washed once in TALP–MOPS and then twice in the final Fert–Talp medium supplemented with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/ml BSA–FAF and 10 μg/ml heparin (Parrish et al. 1988). Oocytes were inseminated in Nunclon dishes in groups of 50 in 250 μl Fert–Talp with 1 × 106 ml spermatozoa selected after swim-up trough in Fert–Talp medium supplemented with 4 mg/ml BSA–FAF.
In vitro culture
Twenty hours post insemination cumulus cells were removed by vortexing (150 s) and washed twice in TALP–MOPS and then twice in the final culture medium. All presumptive zygotes were randomly allocated to treatment groups. Zygotes were cultured in Falcon dishes (Becton Dickinson & Co.) in modified (Holm et al., 1999) Tervit's SOF culture medium (Tervit et al., 1972) under paraffin oil. SOF culture medium contains: 0.1% (v/v) of non-essential amino acids (modified Eagle medium; MEM) and 0.2% (v/v) essential amino acids (basal medium Eagle; BME). Cleavage rates were recorded 48 hpi. Zygotes that developed to ≤8 cells on day 4 (96 h pi) and number of embryos that developed to blastocysts on days 7, 8 and 9. Embryos were cultured under paraffin oil at 39 °C with 5% CO2, 5% O2 and 90% N2 in humidified air. All culture media used in this experiment were supplemented with 1 mM sodium pyruvate, 22 mM sodium lactate and 50 μg ml of gentamicin.
One to five day-7 blastocysts from each replicate and from each treatment were frozen for mRNA transcription and mtDNA copy analysis.
Assessment of embryo development
Cleavage rates were recorded at day 2 (48 hpi) as was the number of zygotes that developed to ≤8 cells on day 4 of culture (96 hpi). Developmental rates of embryos were based on the number of blastocysts that had developed at day 7, 8 and 9 of culture and were calculated based overall on the total number of cleaved oocytes, and separately on the number of zygotes that 96 hpi developed to ≤8 cells. Embryos with clearly visible blastocoel cavity up to two-thirds volume of the embryos were graded as blastocyst, as hatching blastocyst when the zona pellucida was open and part of embryo proper was out of the zona pellucida, and as hatched blastocyst when the entire embryo was out the zona pellucida.
Embryo's cell number counts
A total of 12 embryos from each treatment and from three replicates were taken randomly. Embryos were washed twice in 100 μl droplets of PBS containing 0.5 mg/ml ET–surfactant (Bioniche Inc.) and then fixed in a 100 μl droplet of 4% (w/v) paraformaldehyde solution in PBS, (pH 7.4) for 1 h at room temperature. Embryos were washed again three times in 100 μl droplets of PBS/ ET surfactant and transferred to a 50 μl microdrop of Hoechst 33342 dye. After 10 min of cell staining, embryos were transferred in a very small volume on previously coated glass with Sigmacoat (Sigma), embryos were allowed to settle and a coverslip was placed over the drop. Slides were stored in the dark at 4 °C until the nuclei count. The total number of nuclei was counted under a phase-contrast Nikon fluorescence microscope using a UV-2A/DH400 filter.
Experimental design
A total of 1966 presumptive zygotes in 11 replicates were cultured until 96 hpi in 30 μl (25 embryos per drop) of two culture media: Group 1, SOF supplemented with 5 mg/ml BSA; and Group 2, SOF without BSA supplement. On day 4 of culture, the number of zygotes that had developed to ≤8 cells was recorded and 20 μl of fresh culture medium were added as followed: Group 1a, SOF + BSA (no change); Group 1b, SOF + BSA + 1 mg/ml HA produced by bacteria fermentation (MAP-5; Bioniche Inc.); Group 2a SOF only (no change); Group 2b SOFaa + 1 mg/ml HA; and Group 3, SOF only (no change; negative control).
RNA Extraction and reverse transcription
Poly(A) RNA was prepared from groups of 10 day-7 in vitro blastocysts; 10 in vivo blastocysts exposed to PBS ~1 h were used as control. Blastocysts were washed in PBS supplemented with 0.1% (w/v) polyvinyl pyrrolidone and were stored at –80 °C until processed for real-time RT-PCR following the manufacturer's instructions using the Dynabeads mRNA Direct KIT (Dynal). Briefly, samples were lysed in 50 μl lysis/binding buffer (Dynal). After vortexing and a brief centrifugation, the samples were incubated at room temperature for 10 min. Ten microliters of pre-washed Dynabeads oligo (dT) 25 were added to each sample. After 5 min of hybridization, the beads were separated from the binding buffer using the Dynal magnetic separator. Then the beads were washed in buffer A and B (Dynal) and the poly(A) RNA was eluted from the beads by adding 11 μl of DEPC-treated water. The resultant poly(A) mRNA was used in the RT-PCR in a total volume of 20 μl using 2.5 μM random hexamer primer, 1× RT buffer, 20 IU RNase inhibitor, 50 IU MMLV reverse transcriptase enzyme (Promega, Madrid, Spain), 5 mM MgCl2 and 1 mM of each dNTP. Tubes were heated to 70 °C for 5 min to denature the secondary RNA structure before the addition of RT enzyme (Palasz et al. Reference Palasz, Beltrán-Breña, Martinez, Perez-Garnelo, Ramirez, Gutiérrez-Adán and De la Fuente2008). The RT reaction were then incubated at room temperature for 10 min and then at 42 °C for 60 min to allow the reverse transcription of RNA, followed by 93 °C for 1 min to denature the RT enzyme.
Quantitative RT-PCR
Quantitation of all gene transcripts was carried out by real-time quantitative RT-PCR in three replicates (Rizos et al. 2007). PCR was performed using a Rotorgene 2000 Real Time CyclerTM (Corbett Research) and SYBR Green (Molecular Probes) as a double-stranded DNA-specific fluorescent dye. The PCR reaction mixture (25 μl) contained 2.5 μl 10× buffer, 3 mM MgCl2, 2 U Taq Express (MWGAG Biotech), 100 μM of each dNTP, and 0.2 μM of each primer (Table 1). In addition, the double-stranded DNA dye, SYBR Green I, (1:3000 of 10 000× stock solution) was included in each reaction. The PCR protocol included an initial step of 94 °C (2 min), followed by 40 cycles of 94 °C (15 s), 56–59 °C (30 s) and 72 °C (30 s). Fluorescence data were acquired at 80–85 °C. The melting protocol consisted of holding at 40 °C for 60 s and then heating from 50 to 94 °C, holding at each temperature for 5 s while monitoring fluorescence. Product identity was confirmed by ethidium bromide staining and 2% agarose gel electrophoresis. As negative controls, tubes were always prepared in which RNA or reverse transcriptase was omitted during the RT reaction. The comparative CT method was used for quantitation of expression levels. The quantitation was normalized to the endogenous control histone H2a. Fluorescence data were acquired in each cycle in order to determine the threshold cycle or the cycle during the log–linear phase of the reaction at which fluorescence increased above background for each sample. Within this region of the amplification curve, each difference of one cycle is equivalent to a doubling of the amplified product of the PCR. According to the comparative CT method, the ΔCT value was determined by subtracting the H2a CT value for each sample from each gene CT value of the sample. Calculation of ΔΔCT involved using the highest sample ΔCT values (i.e. the sample with the lower target expression) as an arbitrary constant to subtract from all other ΔCT sample values. Fold changes in the relative gene expression of target were determined by using the formula, 2–ΔΔCT. The details of primers used for RT PCR are shown in Table 1.
Table 1 Details of primers used for RT-PCR.
Statistical analysis
Embryo development was analysed by chi-squared test with the Fisher's Exact test correction. Differences in embryo development between replicates were analysed by analysis of variance (ANOVA). Data on mRNA expression were analysed using the SigmaStat (Jandel Scientific) software package. One-way repeated-measures ANOVA (followed by multiple pair-wise comparisons using the Student–Newman–Kleus method) were used for the analysis of differences in mRNA expression assayed by quantitative RT-PCR.
Results
Embryo development
There was no effect of BSA on cleavage and embryo development until day 4 of culture. Zygotes that had been cultured in SOF supplemented with both non-essential (modified Eagle medium; MEM) and essential amino acids (basal medium Eagle; BME), sodium pyruvate, sodium lactate and l-glutamine, but without BSA or serum, all cleaved and developed to ≤8 cells 96 hpi at similar rates as in medium supplemented with BSA (Table 2). Although present results of embryo culture without any protein source until day 4 were not expected, the same patterns of cleavage and development were very consistent and did not differ among the 11 replicates. The majority of embryos developed to the blastocyst stage at day 7: Group 1, 23.1%; Group 1a, 19.2%; Group 2, 21.8%; Group 2a, 12.0% and Group 3, 3.5%. The total number of the blastocysts developed at day 7 and 8 is showed in Table 2. The relatively modest blastocyst production in our study may be related to the quality of the oocytes that we observed previously during summer time.
Table 2 Embryo development in SOF culture medium with or without BSA until day 4 and then supplemented with/without 0.5% BSA or 1 mg/ml HA.
a,b,cPercentages within a columns with values not in common differ (p < 0.05).
*Eight replicates.
**Based on the number of blastocysts remained in culture at day 8 (less blastocysts taken at day 7 for the PCR).
Total cell numbers
The total cell number of day-7 blastocysts from Group 1, cultured in SOF + BSA or SOF + BSA + HA and Group 2, SOF+BSA (added on day 4 of culture) or SOF + HA (added on day 4 of culture) were 99 ± 15.8, 108 ± 11.5, 77 ± 21.1 and 73 ± 26.2 respectively and were not different within the group but differed between the groups (p < 0.05). The total cell numbers of blastocysts from Group 3, cultured until day 7 in SOF only were 69 ± 26.1 and significantly (p < 0.05) lower than blastocysts from Group 1, but not different from blastocysts of Goup 2.
Hyaluronan receptors CD44 and RHAMM and synthases Has1, Has2 and Has3 gene expression patterns
Addition of BSA or HA, or both, 96 hpi to SOFaa medium with or without BSA have various effects on the levels of gene expression of HA receptors CD44 and RHAMM and synthases Has1, 2 and 3. Expression of RHAMM receptors was highly dependent on HA addition to media that contained BSA and were the highest (p < 0.05) in groups supplemented with BSA from day 1 of culture (24 hpi) and when HA was added at day 4, and lowest (p < 0.05) in the SOF-only group, as compared with both BSA-only supplemented groups that were not different. However, there was no effect of any treatment on the expression of CD44 genes except in the SOF-only group culture from days 1 to 9, without either BSA or HA (Fig. 1).
Figure 1 Relative abundance of hyaluronan receptors RHAMM and CD44 and hyaluronan synthases Has1, Has2 and Has3 in bovine blastocysts developed in SOF culture medium without BSA supplement until day 4 of culture.
Out of the three Has isoforms, only Has2 gene expression was increased (p < 0.05) in both BSA-only supplemented groups, but not in the remaining three groups in which expression did not differ.
Concentration of mitochondrial DNA
Mitochondrial DNA concentration was correlated to BSA presence in the culture medium from day 1 of embryo culture; addition of HA at day 4 to medium containing BSA further increased (p < 0.05) mtDNA copy number. However, addition of HA or BSA at day 4 to embryos that were culture in SOF-only medium had reduced (p < 0.05) expression of mtDNA copy number as compared with embryos cultured in SOF supplemented with BSA from day 1 (Fig. 2).
Figure 2 Mitochondrial copy number of bovine 16-cell and blastocysts embryo culture in SOF medium.
Discussion
The results of the present study demonstrate for the first time that preimplantation bovine embryos predominantly synthesise and utilize high- not low-molecular weight hyaluronan and are dependent on the composition of the culture medium.
Expression levels of all hyaluronan synthases Has1, 2 and 3 and corresponding HA receptors CD44 and RHAMM in day-8 blastocysts appear to be positive indicators and important prerequisites for the developing embryo. Of the three Has isoforms, only Has2 was expressed at an elevated level as compared with Has1 and 3; Has2 increased in both BSA-only groups but not in HA groups regardless of the time of BSA and/or HA addition. In contrast, the remaining synthases Has1 and 3 were at lower levels and were not affected by any changes in culture conditions. The lower expression levels of Has1 and Has3 syntheses in this study is most probably related to different catabolic activities of each HA synthase that differ at different stages of cell differentiation and development. The existence of three Has isoforms with distinctive characteristics suggests the broad variability of biological and physiological functions of each isoform are controlled in a different fashion (Itano & Kimata, Reference Itano and Kimata2002). Analysis of hyaluronan MW synthesized separately by each Has protein in vitro indicated that hyaluronan MW is determined by the specific Has protein. Has2 is the most expressed HA synthase throughout embryonic development in the mouse (McDonald & Camenisch, Reference McDonald and Camenish2003). It is significantly more active than Has1 and synthesizes high-molecular-weight HA (up to 2 × 106 Da; Camenisch et al., Reference Camenisch, Spicer, Brehm-Gibson, Biesterfeldt, Augustine, Calabro, Kubalak, Klewer and McDonald2000). Thus, the high expression of Has2 in our study indicates greater production of high-molecular-weight HA that could have significant effects on cell movement, differentiation (Scott & Heatley, Reference Scott and Heatley2002) and volume (development of blastocyst cavities). Mice deficient in Has2 activity have been found to have severe developmental defects, whereas mice deficient in Has1 or Has3 were viable (Camenisch et al., Reference Camenisch, Spicer, Brehm-Gibson, Biesterfeldt, Augustine, Calabro, Kubalak, Klewer and McDonald2000). These findings may suggest that either Has1 or Has3 is not required at early stages of embryonic development synthesis. However, Has3 is the most catabolically active of the three Has synthases, but is expressed late in embryonic development and synthesizes low-molecular-weight hyaluronan. Another possible cause that affects HA synthase activity is the alteration of molecular composition of the embryo membranes during in vitro culture, because HA is the only glycosaminoglycan that is synthesized at the inner layer of the cell membrane (Prehm, Reference Prehm1984). Any change in the phospholipid bilayer, which in in vitro conditions spontaneously loses phospholipids (Douard et al., Reference Douard, Hermier and Blesbois2000), may disrupt the HA synthesis process and can be reversed and missing lipids taken up from the culture medium if they are available. This scenario has been demonstrated with sperm cells (Spector & Yorek, Reference Spector and York1985; Graham & Foot, Reference Graham and Foote1987; He et al., Reference He, Bailey and Buhr2001) but not with oocytes or embryos.
Expression of the CD44 transmembrane receptor was not affected by any treatment but was significantly lower in blastocysts developed in SOF-only medium. Despite being widely distributed in the intracellular matrix, lack of expression of this receptor in CD44-null mice showed no obvious deleterious morphological defects (Schmits et al., Reference Schmits, Filmus, Gerwin, Senaldi, Kiefer, Kundig, Wakeham, Shahinian, Catzavelos, Rak, Furlonger, Zakarian, Simard, Ohashi, Paige, Gutierrez-Ramos and Mak1997). However, addition of HA at day 4 of culture dramatically increased expression of the RHAMM receptors and this effect was further magnified in presence of BSA. Such an outcome may partially explain the importance of exogenous HA and BSA in bovine embryo development that was previously demonstrated (Lane et al., Reference Lane, Maybach, Hooper, Hasler and Gardner2003). Specifically, addition of HA and BSA significantly improved the fine structure of embryo (Palasz et al., 2006) and is most probably one of the reasons for enhanced embryo cryo-survival (Lane et al., Reference Lane, Maybach, Hooper, Hasler and Gardner2003; Palasz et al., Reference Palasz, Rodriguez-Martinez, Beltran-Breña, Perez-Garnelo, Martinez, Gutierrez-Adan and De la Fuente2007). RHAMM intracellular receptors may play a part in embryo fine structure enhancement. It has been shown that RHAMM interacts with microtubules and actin filaments (Assmann et al., 1999), which may have further consequences regarding cytoskeleton organization. Increased expression of RHAMM proteins by HA in our study may indicate a more effective use of many of the growth factors (e.g. EGF, TGF-2α; Tzanakakis et al., 1995; Tirone et al., Reference Tirone, D'Alessandris, Hascall, Siracus and Salustri1997; Syrokou et al., Reference Syrokou, Tzanakakis, Hjerpe and Karamanos1999) and cytokines that have been shown to stimulates oocytes and embryo development in vitro (Salustri, 1992) and have HA affinity (Ruoslahti & Yamaguchi, Reference Ruoslahti and Yamaguchi1991). Increased synthesis of RHAMM receptors also means that, when needed, they bind to and immobilize large amounts of HA in specific locations.
We were not able to demonstrate that embryo culture in medium without surface active components, such as BSA, leads to more HA synthesis. Exclusion of exogenous protein from SOF containing essential and non essential amino acids, glutamine, sodium lactate and pyruvate did not affect cleavage and embryo development up to the ≤8-cell stage, but considerably reduced blastocysts development when compared with medium containing BSA. However, addition of BSA at day 4 of culture produced similar development and hatching rates to embryos cultured from day 1 with BSA supplement. Further improvement of embryo quality/hatching rates, but not blastocysts number, was observed when HA was added at day 4 of culture (≤8 cells stage) to media containing BSA. The positive effect of HA and BSA confirmed previously published reports (Lane et al., Reference Lane, Maybach, Hooper, Hasler and Gardner2003; Stojkovic et al., Reference Stojkovic, Krebs, Kolle, Prelle, Assmann, Zakhartchenko, Sinowatz and Wolf2003; Palasz et al., 2006). Normal embryo development in this study in medium without any proteins or surface active compound until day 4 of culture may come as a surprise, however we previously provided evidence that bovine follicular fluid and TCM-199 without protein supplementation showed similar values of surface tension (Palasz et al., Reference Palasz, Thundathil, Verrall and Mapletoft2000). For this reason this may be less of a surprise as TCM-199, as well as SOF culture medium, used in this study contains amino acids that possess some surface-active properties. This inclusion may facilitate the use of other components present in the culture medium and support embryo development until day 4 of culture. Also, the presence of glutamine in our media along with non-essential amino acids seems to be sufficient to support embryo development to ≤8 cell stage, which was previously shown with bovine (Steeves & Gardner, Reference Steeves and Gardner1999) and porcine (Suzuki & Yoshioka, Reference Suzuki and Yoshioka2006) embryos.
Visible changes in mitochondrial DNA expression were noticed in day-7 blastocysts cultured from day 1 in medium supplemented with BSA and these changes increased further after addition of HA at day 4 of culture. Whereas blastocysts that had been developed in media without BSA until day 4 showed lower and similar levels of mtDNA expression that were not affected by later addition of BSA or HA. This finding seems to be in agreement with the study by Hsien et al. (Reference Hsieh, Au, Yeh, Chang, Cheng and Tzeng2004) that showed that level of mitochondrial expression is closely related to fertilization/cleavage and, in general, the developmental potential of the embryo. The active transcription of mitochondrial DNA starts in bovines at the 8–16-cell stage (Thompson et al., Reference Thompson, McNaughton, Gasparrini, McGowan and Tervit2000), but mechanisms of activation are not very well established.
In summary, our data suggest that embryos cultured without BSA and/or HA up to 96 hpi increased gene expression of Has2 synthase and RHAMM receptors, but not Has1 and Has3 and CD44 receptors. Higher expression of the Has2 isoform compared with the Has1 and 3 isoforms indicates that only high-molecular-weight, not low-molecular-weight hyaluronan should be used for preimplantation embryo culture. Addition of both HA and BSA at day 4 increased mtDNA copy number at the blastocyst stage, which may indicate a higher developmental potential of embryos cultured under these conditions.
Acknowledgements
This study was supported by grant #OT 2006 002 and in part by grant AGL2003 05783 from the Ministry of Education and Science, Madrid, Spain.
