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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.007716
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BIOLOGY OF REPRODUCTION 68, 60–66 (2003)
DOI: 10.1095/biolreprod.102.007716
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

Developmental Regulation of Hyaluronan-Binding Protein (RHAMM/IHABP) Expression in Early Bovine Embryos1

Miodrag Stojkovic2,a, Ottheinz Krebsa, Sabine Kölleb, Katja Prellea, Volker Assmannc, Valeri Zakhartchenkoa, Fred Sinowatzb, and Eckhard Wolfa

a Department of Molecular Animal Breeding and Biotechnology, LMU, D-85764 Oberschleissheim, Germany b Department of Veterinary Anatomy II, LMU, D-80539 Munich, Germany c Institute of Tumor Biology, D-20246 Hamburg, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS and METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyaluronan or hyaluronic acid (HA) is a normal component of mammalian follicular, oviduct, and uterine fluids. Granulosa and expanding cumulus cells secrete large amounts of HA, and when HA is added in maturation and culture media, it improves the developmental potential of oocytes and embryos. HA regulates gene expression, signaling, proliferation, motility, adhesion, and morphogenesis. Many of these biological activities of HA are mediated through binding to the receptor for HA-mediated motility/intracellular HA-binding protein (RHAMM/IHABP). We evaluated the presence and dynamics of RHAMM/IHABP mRNA and protein expression in different stages of in vitro-produced bovine embryos using quantitative reverse transcriptase-real time-polymerase chain reaction and immunohistochemistry. We also analyzed the effects of different culture systems on the relative abundance of RHAMM/IHABP transcripts. RHAMM/IHABP mRNA levels decreased from the 2-cell to the 16-cell stage, increased again at the morula stage, and reached their highest level at the expanded blastocyst stage. RHAMM/IHABP mRNA abundance was significantly (P < 0.05) lower in embryos recovered in serum-containing medium than in embryos from serum-free media. Immunohistochemistry revealed the presence of RHAMM/IHABP first in 8-cell stages. Whereas RHAMM staining in 8-cell and morula stages was intense, it was weaker in blastocysts. Embryonic secretion of HA increased from the 2-cell stage until the 8-cell stage and then decreased in 16-cell embryos. After this, HA secretion increased in expanded and hatched blastocyst stages. These data suggest that the positive effects of HA on in vitro-produced bovine embryos may be mediated at least in part by RHAMM/IHABP.

developmental biology, early development, embryo, gene regulation, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS and METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The extracellular matrix (ECM) polysaccharide hyaluronan or hyaluronic acid (HA) is a nonsulfated linear polymer of (1-ß-4) D-glucuronic acid and (1-ß-3) N-acetyl-D-glucosamine [1]. This large glucosaminoglycan (average molecular mass ~106) is ubiquitously distributed [2] and is a normal component of mammalian follicular, oviductal, and uterine fluids [3]. Granulosa and expanding cumulus cells secrete large amounts of HA [4–6], and added in maturation and culture media HA improves the developmental potential of bovine [7] and porcine [8] oocytes and embryos [8–10]. Furthermore, greater rates of implantation and fetal development after transfer of mouse blastocysts to recipients were observed when HA was added to the culture medium [11].

HA regulates cellular events such as gene expression, signaling, proliferation, motility, adhesion, metastasis, and morphogenesis [1, 12]. Many of the biological activities of HA are mediated through binding to known cellular HA receptors, namely the cell surface glycoprotein CD44 [13] and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) [14], or to a receptor for HA-mediated motility/intracellular HA-binding protein (RHAMM/IHABP) [15, 16]. CD44 is a mediator for HA-induced cell proliferation [17] and is present in human [18], porcine [19, 20], and bovine cumulus cells, oocytes, and preimplantation embryos ([21] and our unpublished data). LYVE-1 receptor is expressed on human lymph vessels, placenta, and uterus and supports HA-mediated adhesion to CD44 [22].

Although it is widely accepted that CD44 represents the principal cell surface receptor for HA, the role of RHAMM/IHABP is still controversial [23–25]. RHAMM/IHABP is encoded by a single gene [26] and is an intracellular protein that interacts with microtubules and actin filaments, and might thereby play an important role in the organization of the cytoskeletal network [23, 24]. RHAMM/IHABP is expressed in a variety of mammalian cell types, including smooth muscle cells, endothelial cells, nerve cells, macrophages, several tumors, and sperm [27–29]. Other studies have suggested that RHAMM/IHABP may be a cell surface receptor for HA [30–32]. Binding of HA to RHAMM/IHABP has been shown to activate a signal transduction cascade leading to tyrosine phosphorylation of several intracellular proteins, one of which has been identified as the focal adhesion kinase pp125FAK [30]. The phosphorylation/dephosphorylation of pp125FAK as well as the activation of proto-oncogene pp60c-src, another important modulator of cytoskeletal organization, are believed to induce rearrangements in actin filament and cell-matrix adhesion structures that are required for cell locomotion [16, 24, 30, 31]. By activating p42/44 extracellular-regulated kinases (ERKs), which are invoked during receptor tyrosine kinase signaling [23, 24, 27], RHAMM/IHABP is involved in cell signaling and migration via interactions with HA, microtubules, and calmodulin [24, 30–32]. Inhibition of RHAMM/IHABP receptor signaling in H-ras-transformed fibrosarcoma cells results in suppression of cdc2/cyclin B1 expression and cell cycle arrest in the G2/M phase of the cell cycle, implicating a role for RHAMM/IHABP in regulating cell growth and metastasis [33].

The aim of this study was to evaluate whether RHAMM/IHABP mRNA and protein are present in different stages of early bovine embryos using quantitative reverse transcriptase (RT)-real time-polymerase chain reaction (PCR) and immunohistochemistry. In addition, we analyzed the effects of different culture systems on the relative expression of RHAMM/IHABP mRNA and whether in vitro-cultured bovine embryos are able to synthesize and secrete HA.


   MATERIALS and METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS and METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Production of Bovine Embryos

Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Company (St. Louis, MO).

In vitro maturation of oocytes Bovine oocytes were obtained from slaughtered cows as previously described [34] with some minor modifications. Briefly, only follicles with a diameter of 2–8 mm were aspirated from the ovaries using a 20-gauge needle and a vacuum pressure of approximately 100 mm Hg. The cumulus-oocyte complexes (COCs) were collected in a 50-ml centrifuge tube and washed twice with preincubated (39°C; 5% CO2) tissue culture medium 199 (TCM199) supplemented with 10% (v/v) estrous cow serum (ECS). Only COCs with a complete dense cumulus and a dark, evenly granulated cytoplasm were selected for in vitro maturation. The COCs were washed in TCM199 supplemented with 10% ECS and 0.01 U of bovine FSH and bovine LH (Sioux Biochem., Sioux Center, IA) and matured in this medium for 22 to 24 h at 39°C in an atmosphere of 5% CO2 in air and maximum humidity.

In vitro fertilization After maturation, the COCs were maintained in a Tyrode albumin-lactate-pyruvate medium containing 6 mg/ml BSA, 10 µg/ml heparin, and frozen/thawed semen (106 spermatozoa/ml) that had been subjected to a swim-up procedure. COCs were maintained in this medium under the same conditions as those used for in vitro maturation.

In vitro culture Eighteen hours after insemination (hpi) cumulus cells were removed from presumptive zygotes by vortexing (120 sec) and by gentle pipetting. Then, groups of 30 presumptive zygotes were washed three times and cultured in 1) 400 µl of synthetic oviduct fluid medium (SOF) supplemented with 0.1% nonessential amino acids (modified Eagle medium; MEM), 0.2% basal medium Eagle (BME) amino acids (SOF), and 6 mg/ml BSA (SOF-BSA); 2) SOF + 10% (v/v) ECS (SOF-ECS); or 3) SOF + 1 mg/ml BSA + 6 mg/ml HA (SOF-HA) [10].

Experimental Design

In the first experiment, we identified bovine RHAMM/IHABP sequences using RT-PCR.

In the second experiment, we analyzed the dynamics of RHAMM/IHABP mRNA within different embryonic stages recovered in SOF-BSA by RT-real time-PCR. Embryos were collected as previously described by Laurincik et al. [35] with some minor modifications. Briefly, 2-cell, 4-cell, 8-cell, and 16-cell stages as well as morulae, blastocysts, expanded blastocysts, and hatched blastocysts were collected at 20–22, 36–38, 46–54, 62–70, 108–120, 144–152, 168–176, and 192–200 hpi, respectively. In addition, we investigated the effect of different culture systems (SOF-ECS, SOF-BSA, and SOF-HA) on the relative expression of RHAMM/IHABP mRNA in morula, blastocyst, expanded blastocyst, and hatched blastocyst stages.

In the third experiment, the presence and localization of RHAMM/IHABP protein was evaluated in different embryonic stages (as described above) recovered in SOF-BSA medium using immunohistochemistry.

In the fourth experiment, we measured the amount of newly synthesized HA by 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, expanded blastocyst, and hatched blastocyst stages cultured in SOF supplemented with 1 mg/ml polyvinyl alcohol (PVA) using a radiometric HA assay.

Reverse Transcriptase-Polymerase Chain Reaction

Total RNA was extracted from freshly dissected bovine ovary cortex using TriPure Isolation Reagent (Roche Diagnostics, Mannheim, Germany). DNase I (Roche Diagnostics)-treated total RNA was used as a template for first-strand cDNA synthesis using random hexamer primers (Gibco Invitrogen, Karlsruhe, Germany). Moloney murine leukemia virus (MMLV) RT was used for the extension, according to the manufacturer's protocol (Gibco Invitrogen). Second-strand cDNA was synthesized during a single PCR cycle with a thermostable polymerase (Qiagen, Hilden, Germany) using RH37 forward and RH37 reverse oligonucleotide primers specific for bovine RHAMM/IHABP (Table 1). The partial bovine RHAMM/IHABP cDNA was amplified by 35 cycles of denaturation (60 sec at 94°C), annealing (30 sec at 60°C), and elongation (30 sec at 72°C), followed by a final step at 72°C for 5 min.


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  		TABLE 1. Primers used for PCR

Primers for RT-real time-PCR analysis of RHAMM/IHABP (RH427 forward and RH427 reverse, Table 1) were designed from the partial cDNA sequence shown in Figure 1.



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  		FIG. 1. Comparison of partial bovine and human RHAMM/IHABP cDNA sequences. The sequence amplified by RT-real time-PCR analysis is bold and underlined

RT-Real Time-PCR Analysis of Embryonic RHAMM/IHABP Messenger RNA Expression

Embryos cultured in SOF-BSA medium were washed in PBS supplemented with 0.1% (w/v) polyvinyl pyrrolidone (Sigma) and pools of four embryos of the same developmental stage removed from the same culture medium were stored at -80°C until processed for RT-real time-PCR. Total embryonic RNA was isolated as described previously [36, 37]. Briefly, 100 µl of extraction buffer (0.2 M NaCl, 0.025 M Tris pH 7.2, 1 mM EDTA) and 4 ng of carrier synthetic A-oligonucleotides (Amersham Bioscience, Freiburg, Germany) were added. RNA was precipitated with 75 µl of chloroform:isoamyl alcohol (24:1) and 75 µl of phenol. Then the aqueous phase was mixed again with 100 µl of chloroform:isoamyl alcohol (24:1) and after centrifugation, 2 µl of the coprecipitant, seeDNA (Amersham Bioscience), and 0.1x volume of the aqueous phase of 3 M sodium acetate were added. The RNA pellet was washed once in 70% ethanol and dissolved in 20 µl of RNase-free water. For the RT reaction, 10 µl of total embryonic RNA were mixed with 10 µl of RT master mix containing 0.66 µg of random hexamer primers (Roche Diagnostics), 1 mM dNTPs (MBI Fermentas, St. Leon-Rot, Germany), 0.01 M dithiothreitol, 20 IU MMLV RT and 5x RT buffer (Invitrogen).

The real time-PCR reaction mix (25 µl) contained 2 µl of embryonic cDNA, 2x SYBR Green Master Mix, 0.25 IU AmpErase (Applied Biosystems, Weiterstadt, Germany), and 0.4 µM of the bovine RH427 forward and RH427 reverse primers (Table 1) or 0.3 µM of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers (bGAPDH, Table 1) in separate PCR tubes. To create standards for calculating amplification efficiency (E) during real time-PCR five dilutions of embryonic cDNA (0.8, 0.4, 0.2, 0.1, and 0.05 blastocyst RNA units) were primed separately (in different PCR tubes) for RHAMM/IHABP and GAPDH proved to be a nonregulated housekeeping gene. PCR runs began with the activation of AmpErase for 2 min at 50°C, followed by 10 min of denaturation at 95°C and 40 cycles of denaturation (95°C, 15 sec) and annealing/extension (60°C, 1 min). Quantification of mRNA abundance was performed by real time PCR detection using an ABI PRISM 7700 Sequence Detector (PE Biosystems, Weiterstadt, Germany). Amplification and quantification were performed following the standard curve method described previously [36, 37].

The relative expression of RHAMM/IHABP mRNA (expressed in arbitrary units [AUs]) obtained for different embryonic stages cultured in SOF-BSA were referenced to the corresponding values of the 2-cell stage (set to 1). The expression levels obtained for embryos (morula, blastocyst, expanded blastocyst, and hatched blastocyst) recovered from different culture systems (SOF-ECS, SOF-BSA, and SOF-HA) were referenced to the corresponding values obtained for the same stages recovered in SOF-BSA medium.

Immunohistochemistry

Ten to 15 embryos from each stage were removed from SOF-BSA medium, washed in PBS, and transferred to 3-aminopropylene-ethoxysilane-coated slides. After drying, fixation in Bouin fluid, and dehydration in a graded series of ethanol (30%–100%), the embryos were frozen at -70° C for at least 24 h. After thawing, specimens were hydrated in a graded series of ethanol (100%–30%) and were washed in PBS. Slides were blocked with normal swine serum (dilution 1:20 in PBS) for 130 min at room temperature. Specimens were incubated with the RHAMM antibody [23] at a dilution of 1:300 at 4°C overnight. The secondary antibody was biotinylated rabbit anti-swine immunoglobulin G (IgG; DAKO, Hamburg, Germany; dilution 1:300). After incubating the slides with avidin-streptavidin-biotin horseradish peroxidase complex (DAKO) for 1 h at 20°C (dilution 1:150 in PBS-1% BSA), proteins were detected by 0.05% diaminobenzidine in PBS supplemented with 1% H2O2. Controls were performed by 1) omitting the RHAMM antibody, and 2) omitting of the secondary antibody.

Determination of Newly Synthesized HA in Embryo-Conditioned Media

Fifteen embryos at each stage (2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, expanded blastocyst, and hatched blastocyst) were cultured in 150 µl of SOF-PVA for 6 h. Then, 100 µl of embryo-conditioned media were collected and frozen at -20°C until further use. The concentration of HA in media was determined using the radiometric assay HA-Test (Pharmacia Upjohn Diagnostics AB, Uppsala, Sweden). The test is based on the reaction between HA in sample and labeled 125HABP (hyaluronic acid binding proteins) in a test solution. The unbound 125HABP was quantified by incubation with HA covalently coupled to Sepharose (Pharmacia Upjohn) particles. The radioactivity bound to the particles was measured in a gamma counter (CB 2111; Berthold, Munich, Germany). All measurements were repeated at least three times. Culture medium without embryos was always used as a negative control.

Statistical Analysis

All data were analyzed using the SPSS statistical program (version 10.0; SPSS Inc., Chicago, IL). Data are presented as the mean percentage ± SEM. When a significant (P < 0.05) F statistic was found, ANOVA followed by the least significant differences (LSD) test was used. Differences were considered significant when P < 0.05. All experiments were repeated at least three times.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS and METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Bovine RHAMM/IHABP Complementary DNA Sequence

The homology between the human (GenBank accession number NM_012484.1) and partial bovine RHAMM/IHABP cDNA sequences, corresponding to the nucleotide positions 359–1018, was 88% (Fig. 1). The partial bovine RHAMM/IHABP sequence was published under GenBank accession number AF499008.

Experiment 2: RT-Real Time-PCR

The cleavage rates for 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, expanded blastocyst, and hatched blastocyst stages cultured in SOF-BSA were 81.2 ± 1.4%, 72.9 ± 1.6%, 60.8 ± 0.9%, 51.1 ± 1.4%, 44 ± 1.3%, 37.2 ± 1.9%, 32.8 ± 1.1%, and 25.4 ± 1.3%, respectively.

The abundance of mRNA encoding RHAMM/IHABP was calculated using the standard curve method with determination of PCR amplification efficiency and normalization for GAPDH mRNA as the internal reference. The expression of this housekeeping gene was assumed not to vary between the different stages.

RHAMM/IHABP mRNA expression decreased constantly from the 2-cell to the 16-cell stage; it increased again at the morula stage, reached the highest level at the expanded blastocyst stage, and then decreased again at the hatching blastocyst stage (Fig. 2). Statistical analysis of normalized RHAMM/IHABP mRNA abundance revealed significant (P < 0.05) differences when the 2-cell and 4-cell stages were compared with the 8-cell stage. The 16-cell stage contained significantly (P < 0.01) less RHAMM/IHABP mRNA than other evaluated stages. The RHAMM/IHABP mRNA abundance in expanded blastocysts was significantly (P < 0.001) higher than in other embryonic stages.



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  		FIG. 2. Quantification of RHAMM/IHABP mRNA during early bovine embryogenesis by RT-real time-PCR. The relative levels of RHAMM/IHABP mRNA (expressed in arbitrary units) obtained for different embryonic stages recovered in SOF-BSA were referenced to the corresponding values of the 2-cell stage (set to 1). Expression of RHAMM/IHABP mRNA was normalized for GAPDH expression. Normalized expression data for embryos were analyzed by one-way ANOVA followed by the LSD post hoc test. Significant differences between means (P < 0.05) are indicated by different superscripts. m, Morula; bl, blastocyst; ebl, expanded blastocyst; hbl, hatched blastocyst

There was a highly significant (P < 0.001) effect of culture conditions on the relative abundance of RHAMM/IHABP mRNA in embryos (ANOVA, F = 7.65). At morula and expanded blastocyst stages, RHAMM/IHABP mRNA abundance was significantly (P < 0.05) lower for embryos recovered in SOF-ECS medium than in embryos from the SOF-BSA and SOF-HA media (Fig. 3, a and c). Blastocysts, expanded blastocysts, and hatched blastocysts cultured in SOF-HA contained significantly (P < 0.01) more RHAMM/IHABP mRNA than the same stages recovered in SOF-BSA or SOF-ECS media (Fig. 3, b–d).



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  		FIG. 3. Effect of different culture conditions on the expression of RHAMM/IHABP mRNA. The expression levels obtained for embryos (morula, blastocyst, expanded blastocyst, and hatched blastocyst stages) recovered from different culture systems (SOF-ECS or SOF-BSA or SOF-HA) were referenced to the corresponding values obtained for the same stages recovered in SOF-BSA medium (set to 1). Expression of RHAMM/IHABP mRNA was normalized for GAPDH expression. Normalized expression data for embryos were analyzed by one-way ANOVA followed by the LSD post hoc test. Significant differences between means (P < 0.05) are indicated by different superscripts

Experiment 3: RHAMM/IHABP Immunohistochemistry

Early embryonic stages revealed the presence of RHAMM/IHABP first in 8-cell (Fig. 4a) stages. Whereas RHAMM/IHABP staining in 8-cell (Fig. 4a) and morula stages (Fig. 4b) was very intense, it was weaker in blastocysts (Fig. 4c). However, staining was visible in both in inner cell mass (ICM) cells and in trophectoderm (TE) cells (Fig. 4c). Omission of the RHAMM/IHABP antibody or of the secondary rabbit anti-swine IgG resulted in no staining (Fig. 4d).



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  		FIG. 4. Localization of RHAMM/IHABP protein in early bovine embryos. Immunohistochemistry of RHAMM/IHABP in early bovine embryos: 8-cell (a), morula (b), blastocyst (c), and blastocyst used as negative control (d); omission of the RHAMM/IHABP antibody. Note very intense staining (arrows) for RHAMM/IHABP in 8-cell (a) and morula (b) stages and lower expression of RHAMM/IHABP in both the inner cell mass (*) and trophectoderm cells (arrowhead) at the blastocyst stage (c). Magnification x250

Experiment 4: Radiometric HA Assay

From the 2-cell stage, synthesis of HA increased until the 8-cell stage and then decreased in 16-cell embryos (Table 2). After this, HA synthesis increased again at the morula stage, decreased in blastocyst and expanded blastocyst stages, and then increased considerably in hatched blastocysts. Morula stages secreted significantly (P < 0.05) more HA than all other stages except the hatching blastocyst stage. The latter stage secreted significantly (P < 0.001) more HA than all embryonic stages (Table 2). HA was not detected in culture media without embryos (data not shown).


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  		TABLE 2. Newly synthesized HA by different stages of in vitro-produced bovine embryos


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS and METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our study we report for the first time the presence and developmental regulation of RHAMM/IHABP mRNA and protein within different bovine embryonic stages.

Using quantitative analysis of RHAMM/IHABP mRNA expression we found that the relative abundance of RHAMM mRNA decreased from the 2-cell stage to the 16-cell stage; it increased again at morula, blastocyst, and expanded blastocyst stages; and decreased again at the hatching blastocyst stage. Until now, no studies have established the role of the HA-RHAMM/IHABP system in development of mammalian embryos. It is known that supplementation of media with HA preserves motility and increases human sperm velocity [38, 39] through RHAMM [29]. We have previously demonstrated that sequential addition of HA in culture medium increases cleavage, cell number, and the survival rate of frozen/thawed in vitro-produced bovine embryos [10, 40]. Now we have demonstrated that embryos produced in the presence of HA express higher levels of RHAMM/IHABP mRNA compared with embryos produced in the presence of BSA or ECS. This confirms findings that different culture conditions affect gene expression in mammalian embryos [36, 37] and that information concerning mRNA dynamics is helpful in attempts to optimize in vitro culture conditions.

Immunohistochemistry showed that RHAMM/IHABP was more abundant in early embryonic stages than in blastocysts. In the latter stage, the signal was still visible in both ICM and TE cells. In mice, the strongest HE staining was visible in ICM cells and its associated endoderm derivates [41]. RHAMM/IHABP has been found in association with tubulin and actin cytoskeletal elements [23, 24], suggesting that RHAMM/IHABP might play an important role in movement and reorganization of blastomeres within early embryonic development. In addition, RHAMM/IHABP is associated with mitochondria, and in conjunction with its cytoskeletal interaction it also regulates mitochondrial motility and positioning [42], which is important for cytoplasmic maturation and the further developmental potential of mammalian oocytes [43, 44].

Using biotin-labeled HA-binding complexes, Brown and Papaioannou [41] demonstrated that in mice HA was first secreted by visceral endoderm cells of the early egg cylinder on Day 5.5 postcoitum. Contrary to this, using a very sensitive radiometric HA assay, we found that newly synthesized HA was already detectable at the 2-cell stage. HA secretion increased until the 8-cell stage, decreased at the 16-cell stage, and increased again at the morula and hatched blastocyst stages. Greater RHAMM/IHABP mRNA expression and HA secretion at the morula stage could be related to the polarization/differentiation of ICM/TE cells. In mice, TE fragments revealed no HA secretion during outgrowth during in vitro conditions [41]. On the other hand, endodermal derivatives of differentiating ICM cells showed significant synthetic activity [41].

HA synthesis is greater in actively dividing cells and peaks during mitosis [45–48]. Because hatched blastocysts produced considerably more HA, the role of HA in elongation and later implantation processes of bovine embryos deserves further investigation. In mice, implantation of the blastocyst into the uterine wall triggers a rapid loss of HA from the ECM of decidual cells on the antimesometrial side of the uterus [49], and the presence of HA within angiogenic regions of the decidua basalis implies a functional role for this molecule in placental vascularization in this species [50]. Greater levels of RHAMM/IHABP mRNA in expanded blastocysts and high levels of newly synthesized HA in hatched blastocysts is similar to the presence of HA in cavities of Xenopus embryos [51, 52] and can be considered as a protective against damage by environmental compressive forces due to the viscoelastic properties of HA [10, 40, 41, 51, 52].

We emphasize the differences in the appearance of RHAMM/IHABP mRNA and protein. RHAMM/IHABP transcripts were present from the 2-cell stage onward, but RHAMM/IHABP immunoreactivity was first visible at the 8-cell stage. Whereas RHAMM/IHABP mRNA synthesis was considerably high during morula and blastocyst stages, RHAMM/IHABP staining decreased in the latter stage. Such a time delay between mRNA and protein synthesis has already been reported for several receptor proteins in bovine embryos (e.g., for the growth hormone receptor) [37]. In addition, in interpreting these results, the semiquantitative nature of immunohistochemistry should be not overlooked.

In conclusion, the present study demonstrated for the first time the developmental changes in the abundance of RHAMM/IHABP mRNA and protein during bovine preimplantation development. Thus, in addition to CD44, RHAMM/IHABP may be involved in the mediation of the profound effects of HA on in vitro-produced embryos. Future studies will aim at the dissection of these pathways by using selective gene inactivation techniques, such as RNA interference [53].


   ACKNOWLEDGMENTS
 
We are grateful to Petra Stojkovic and Gudrun Boie for their excellent technical support.


   FOOTNOTES
 
1 This study was supported by grants from the Bayerische Forschungsstiftung and from the Deutsche Forschungsgemeinschaft. Back

2 Correspondence. FAX: 49 89 315 2799; m.stojkovic{at}gen.vetmed.uni-muenchen.de Back

Received: 29 May 2002.

First decision: 19 June 2002.

Accepted: 8 July 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS and METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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