| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ovary |
Institute of Physiology, Technical University of Munich, D-85354 Freising, Germany
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
cumulus cells, gene regulation, granulosa cells, oocyte development
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
During COC expansion in mammalian species, HAS2 [3, 17] was suggested to synthesize the huge amount of HA found in ovarian follicles [18]. Recently, receptors for HA have been identified and include the transmembrane receptors CD44 (named by clusters of differentiation) and RHAMM (receptor for HA-mediated motility). The glycoprotein CD44, probably the most common HA receptor, is characterized by a variety of isoforms caused by alternative splicing and posttranslational modifications [19]. This receptor is responsible for cell-to-cell and cell-to-extracellular matrix (ECM) interactions [20], inhibition of apoptosis [21], endocytosis of HA [22], augmentation of tumor cell motility and metastasis [23], and stimulation of lymphocytes [24]. In contrast, little information is available concerning the regulation of CD44 gene expression, though recent studies indicate that CD44 may influence fertility and quality of human oocytes [25].
The other known HA receptor, RHAMM, possesses two potential HA-binding sites [26]. Interactions between HA and RHAMM could activate protein kinases that modulate cellular behavior. Known to promote cell movement, RHAMM is located both in the cell as well as at the cell surface [27]. Furthermore, the HA receptor may be involved in wound healing [28], migration of smooth muscle cells [29], and modulation of ciliary beating of the airway epithelial cells [30].
The aim of the present study was to characterize the expression profiles of HAS1, HAS2, and HAS3 as well as the corresponding receptors, CD44 and RHAMM, during in vitro maturation (IVM) of bovine COCs. By use of real-time reverse transcriptase-polymerase chain reaction (RT-PCR), the mRNA transcripts of HAS1, HAS2, HAS3, CD44, RHAMM, ubiquitin, and 18S rRNA were relatively quantified. Additionally, immunohistochemistry of CD44 and localization of HA by biotinylated HA-binding protein (bHABP) were used to describe the HA system in ovarian cumulus and GCs when under the influence of gonadotropin.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Noncystic bovine ovaries were collected, irrespective of stage of the estrous cycle, at a local abattoir and transported at 37°C in sterile Ringer solution to the laboratory within 1 h. Immediately, COCs were recovered by aspiration and sorted out for grade 1 COCs with a compact and multilayered cumulus oophorus as well as homogeneous ooplasm [31]. To reduce estrous cycle-dependent effects, groups of 10 COCs underwent IVM [32, 33]. The COCs were cultured in 400 µl of bicarbonate-buffered modified Parker medium (MPM, pH 7.2) supplemented with 10% (w/w) fetal calf serum (Seromed; Biochrom KG, Berlin, Germany). The MPM was composed of 15.0 g/L of Medium 199 (M-2520; Sigma, Taufkirchen, Germany), 2.2 mg/ml of sodium bicarbonate (S-5761; Sigma), 50 µg/ml of gentamicin (Selectevet; Germany), 0.23 mg/ml of pyruvate (15220; Serva, Heidelberg, Germany), 50 µg/ml of ascorbic acid (A-4034; Sigma), 0.55 mg/ml of calcium lactate (Merck, Darmstadt, Germany), and 0.01 IU/ml of bovine FSH (NIH B1 activity) as well as 0.01 IU/ml of LH (NIH LH B10; Sioux Biochemicals, Sioux Center, IA).
Granulosa cells were recovered by rinsing antral follicles with PBS (0.24 g/L of KH2PO4, 8.0 g/L of NaCl, 0.2 g/L of KCl, and 1.44 g/L of Na2HPO·2H2O; pH 7.4) using a 10-ml, single-use syringe (Braun, Melsungen, Germany) connected to a 20-gauge needle (Sterican; Braun); washing three times in culture medium; and seeding into 12-well uncoated tissue-culture plates (4 x 105 cells per 2-ml well). To reduce contamination of atretic GCs, only small follicles up to 8 mm were punctured. Potential influence of simultaneously extracted COCs was prevented by sorting out the GC cultures for oocytes. Gonadotropin stimulation with FSH, LH, or both in combination started 1.5 days after seeding (onset), when all GCs were attached to the bottom and proliferating. The FSH and LH were each used at final concentrations of 0.01 IU, as indicated above. Sampling was carried out at the onset, after 4 h, and after 24 h by removing culture medium and cell lysis for total RNA extraction directly in culture dishes.
RNA Extraction and RT-PCR
For total RNA extraction, all culture media were removed. Before cell lysis, all cultured COCs and GCs were washed twice in sterile PBS and put through RNA extraction performed by spin columns (NucleoSpin RNA II; Macherey & Nagel, Düren, Germany) including DNase1 digestion (Macherey & Nagel) to reduce contaminating DNA. The RNA integrity was verified by optical density (OD) 260/OD280-nm absorption (Biophotometer; Eppendorff, Hamburg, Germany). Total RNA (COC, 200 ng; GC, 500 ng) was reverse transcribed with 200 IU of MMLV-Reverse Transcriptase (Promega, Madison, WI) using 2.5 µM random hexamer primers (Gibco BRL, Carlsbad, CA) and 0.5 mM dNTPs (Roche Diagnostics, Heidelberg, Germany) as described previously [33].
The following primer sets were selected based on published data as indicated. Optimal annealing temperatures (AT) were established in a gradient cycler (Mastercycler; Eppendorff) and tested for quantification temperature (QT) by a melting-point analysis in the LightCycler (Roche): 18S rRNA, forward 5'-AAG TCT TTG GGT TCC GGG-3', reverse 5'-GGA CAT CTA AGG GCA TCA CA-3' (EMBL, AF176811; product, 365 base pairs (bp); AT, 60°C; QT, 82°C); ubiquitin, forward 5'-AGA TCC AGG ATA AGG AAG GCA T-3', reverse 5prime;-GCT CCA CCT CCA GGG TGA T-3' (EMBL, Z18245; product, 198 bp; AT, 60°C; QT, 85°C), HAS1, forward 5'-GGT ACA ACC AGA AGT TCC TGG G-3', reverse 5'-CGG AAG TAC GAC TTG GAC CAG-3' (EMBL, AB017803; product, 184 bp; AT, 55°C; QT, 79°C); HAS2, forward 5'-GGM TGT GTC CAG TGC ATT AGC GGA C-3', reverse 5'-CAG CAC TCG GTT CGT TAG RTG CCT G-3' (EMBL, U54804; product, 144 bp; AT, 68°C; QT, 80°C); HAS3, forward 5'-ACA GGT TTC TTC CCC TTC TTC C-3', reverse 5'-GCG ACA TGA AGA TCA TCT CTG C-3' (EMBL, AJ293889; product, 166 bp; AT, 60°C; QT, 84°C); HA-receptor CD44, forward 5'-TAT AAC CTG CCG ATA TGC AGG-3', reverse 5'-CAG CAC AGA TGG AAT TGG G-3' (EMBL, X62881; product, 221 bp; AT, 64°C; QT, 83°C); and HA-receptor RHAMM, forward 5'-TGT TGA ATG AAC ATG GTG CAG CTC-3', reverse 5'-CCT TAG AAG GGT CAA AGT GTT TGA T-3' (EMBL, AF310973; product, 249 bp; AT, 61°C; QT, 77°C). To verify specificity of each gene amplification, PCR products were isolated and sequenced (TopLab, Martinsried, Germany).
Quantitative Real-Time PCR
For each sample, a standard 10-µl real-time PCR reaction mix (Roche) was prepared containing the following components: 1 µl of LightCycler mix, 4 mM MgCl2, 0.4 mM primers, and sample cDNA (COC, 0.33 ng/µl; GC, 0.83 ng/µl). The QTs were set below the individual melting peak of each PCR product. For specific gene amplification, a standard protocol of 50 cycles was used in the LightCycler: After initial polymerase activation at 95°C for 10 min, primer-specific amplification and quantification cycles were run at AT and QT as indicated above. To evaluate specific amplification, a final melting curve was created (60–99°C) under continuous fluorescent measurement.
Relative quantification was determined using sample crossing points analyzed with LightCycler software 3.5 (Roche) by the second derivative maximum method.
PCR Efficiency and Relative Quantification
PCR efficiency
The PCR efficiency (E) of each primer set was determined within distinct detection ranges (2.7 pg to 8.33 ng of cDNA) and calculated according to the following equation [34]:
 | (1) |
Relative quantification
A mathematical model adopted from the LightCycler software package [35] served to calculate the relative differences between groups. Relative expression ratios were calculated by the following equation:
 | (2) |
To evaluate the efficiencies of the preceding RT reaction, unregulated reference genes are suggested as control [36]. Therefore, all cDNA samples were compared to endogenous standards. Because of its highly balanced expression (P = 0.092), ubiquitin and 18S rRNA served as the main housekeeping genes in our system. To ensure reproducibility and accuracy of real-time PCR, assay precision was confirmed using ubiquitin PCR within one LightCycler run (n = 3) or at three different days using different batches of LightCycler premixes showing an intraassay variation of 6.05% and an interassay variation of 8.40%.
Full-Length cDNA Sequencing
Full-length cDNA sequencing of the bovine HAS2 was performed using the SMART-RACE cDNA-Kit (Clontech, Palo Alto, CA) introducing total RNA of bovine COCs. One microgram of RNA and 200 IU of MMLV-Reverse Transcriptase were used for first-strand synthesis in the 5'- as well as 3'-direction. Resulting cDNA libraries were subsequently used for amplification of specific PCR products by introducing the described HAS2 primers in a thermal cycler (Master Gradient Cycler; Eppendorff) performing 25 cycles of denaturation at 94°C for 5 sec, annealing at 68°C for 10 sec, and elongation at 72°C for 2 min and 30 sec. The PCR products were sequenced commercially (TopLab).
Statistical Analysis
One-way ANOVA was used to test the effects of LH, FSH, and FSH+LH application. Differences between groups (n = 6) were identified by least significant difference Student t-test (P < 0.05). Analyses were performed with SAS release 8.01 (SAS, Inc., Cary, NC). Raw data are presented as the crossing point (mean ± SD) and the variation quotient (in %). Results were depicted by the calculated expression ratios according to the control group.
Histological Localization of HA and Immunodetection of CD44
Cultured bovine COCs and ovary were embedded in TissueTek (Miles, Inc., Torrance, CA) and shock-frozen in liquid nitrogen. Serial cross-sections (COC: thickness, 14 µm; ovary: thickness, 7 µm) were performed using a cryotome (HM 505E; Microm, Walldorf, Germany) and mounted on preheated (37°C) HistoBound glass slides (Marienfeld). The GCs were grown and treated directly on sterilized glass slides provided with flexiPERM chambers (Vivascience AG, Germany). Before specific staining, all cryosections as well as cultured GCs were air-dried for 30 min at 37°C and fixed with ethanol for 5 min at room temperature at -30°C for HA localization or with 3.7% formaldehyde in PBS for 15 min at room temperature for CD44 immunodetection.
For HA localization, all tissues were incubated for 30 min with 5 ng/ml of bHABP (catalog no. 385911; Calbiochem, San Diego, CA) diluted in PBS. Unbound bHABP was removed by washing twice in PBS. To detect bound bHABP, slides were treated for 30 min with a streptavidin fluorescein conjugate (SAF; 1 µg/ml, catalog no. 189734; Calbiochem), washed twice in PBS, counterstained for 5 min with propidium iodide in PBS (2.5 µg/ml), and mounted in antifading solution Citiflour AF1 (Agar Scientific, Essex, U.K.). Negative controls were performed either without bHABP or without SAF.
Immunolocalization of bovine HA-receptor CD44 was accomplished by using rat-anti-porcine CD44 immunoglobulin (Ig) G (1:100 dilution; SM488, lot 130900; DPC Biermann, Bad Nauheim, Germany) as first antibody and horseradish peroxidase (HRP)-labeled goat-anti-rat IgG (1:200 dilution; R1378HRP, lot 6563; DPC Biermann) for visualization by diaminobenzidine (DAB; D-5905; Sigma). After fixation in formaldehyde, samples were washed twice in PBS and incubated for 30 min in 1% H2O2 to block endogenous peroxidases. Background was reduced by treatment with 10% normal goat serum for 30 min. First-antibody incubation was done overnight, followed by three washing steps in PBS and incubation with HRP-labeled goat-anti-rat IgG for 1 h. Specific staining of CD44 were visualized by 0.05% DAB in 0.01% H2O2. Additionally, samples were counterstained by Mayers Haemalaune, dehydrated with graded alcohols, and embedded in Eukitt (Kindler, Freiburg, Germany). Specificity of the immunodetection was proved by several negative controls: 1) replacement of the first antibody, 2) replacement of secondary antibody, and 3) incubation with DAB alone to show efficiency of blocking endogenous peroxidases. All samples were visualized with a Axioscope microscope (Zeiss, Jena, Germany) and documented by digital imaging (Zeiss AxioCam MR with AxioVision software) or color slides (Kodak Elite 100; Eastman Kodak, Rochester, NY).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
Sequencing of the bovine HAS2 mRNA by use of rapid amplification of cDNA ends technology lead to an mRNA of 2946 bases possessing the 5' untranslated area and the entire 3' untranslated region containing a putative polyadenylation signal (AATAAA) at base 2888, leading further into the poly-A-tail. An open reading frame was identified between bases 545 and 2201 encoding a 552-amino acid protein (63 kDa). This HAS2 cDNA sequence has been submitted to a nucleic acid database to permit public access (EMBL, BTA004951). To our knowledge, it is the first description of the complete bovine HAS2 mRNA sequence derived from bovine COC RNA, enabling further transcript characterization in cattle. The deduced open reading frame showed high homologies to known HAS2 cDNA as well as to deduced protein sequences of other species (cDNA homology, 93.6% human, 90.7% rat, and 88.9% mouse; protein homology, 97%–98%).
Furthermore, a functional PCR setup amplifying bovine RHAMM transcripts was introduced (Fig. 1). Sequencing of this cDNA provided a new partial sequence of this bovine HA-receptor type, including two potential HA-binding sites (EMBL, AJ439694) with high homologies to other RHAMM sequences (cDNA homology, 98% sheep, 89% human, and 82% rat).
HA Synthases
In both cumulus as well as GCs, HAS2 showed the most prominent increase in mRNA expression after gonadotropin treatment (FSH+LH in COCs and LH in GCs). In COCs, HAS2 transcripts reached the highest levels (120-fold) within 4 h (Fig. 2A) and decreased thereafter, reaching a plateau at 60-fold higher when compared to the experimental onset (P < 0.001). In GCs, the HAS2 elevation (Fig. 3A) was highest under FSH (5.5-fold, P = 0.04), followed by FSH+LH in combination (3.5-fold, P = 0.003) and then LH (2.6-fold, P = 0.05). After 24 h, HAS2 transcripts returned to or dropped below the baseline level.
|
|
Although HAS3 increased, if not as much as HAS2, significant alteration of HAS3 transcripts was detected after 12- and 24-h IVM in COCs (Fig. 2B). The relative concentration at the onset of IVM and 4 h later (indicated by low crossing points) as well as the increase after 12 h in HAS3-specific mRNA (2-fold, P = 0.003) was always below the height found for HAS2. In contrast, cultured GCs always showed a significant decline of HAS3 transcripts within the first 4 h compared to the onset (Fig. 3B). Regardless of gonadotropin treatment, the HAS3 expression was adjusted to the lowest levels at the end of each experiment after 24 h.
In bovine COCs as well as in GCs, HAS1 mRNA was not detectable with the real-time RT-PCR system (2 pg of cDNA). The validity of these primers was demonstrated using whole ovarian follicle mRNA (Fig. 1).
HA-Receptors CD44 and RHAMM
Within the first 4 h of COC maturation, very low mRNA quantities of the HA-receptor CD44 were present (Fig. 2C). After 12 h of IVM, CD44 expression increased more than 100-fold (P = 0.001) and stayed high until the end of maturation (24 h, P = 0.047). In GCs, response to both gonadotropins was detected, but at a lower level (Fig. 3C). In these cells, LH induced the highest increase (2.2-fold, P = 0.019) within the first hours, followed by FSH (1.9-fold, P = 0.047). In addition, FSH showed a more prolonged stimulus on CD44 expression after 24 h (P = 0.028). However, no effect was observed when both gonadotropins were supplemented simultaneously (4 h, P = 0.546; 24 h, P = 0.496).
The PCR results for RHAMM indicated that its mRNA is detectable at all stages of IVM in COCs (Fig. 2D) as well as during GC culture (Fig. 3D). The initial expression of RHAMM decreased until the end of COC maturation and reached significant differences compared to the control after 4 and 24 h of IVM. In contrast, during the first hours, the RHAMM mRNA levels in GCs stayed unchanged, but it reached significantly higher levels in all groups after 24 h. Neither FSH, LH, or their combination provoked significant alterations compared to the untreated control.
Localization of HA-Receptor CD44 in the Ovary, COCs, and Cultured GCs
Serial cross-sections of the whole bovine ovary showed a distinct staining for immunoreactive CD44 in small antral follicles (Fig. 4A). Specific staining could be detected for the theca interna as well as for the theca externa. However, no specific localization for CD44 was observed in mural GCs or in COCs of immature follicles (Fig. 4A).
|
In sections obtained from matured bovine COCs with an expanded cumulus, specific staining for CD44 was observed only for several cumulus cells (Fig. 4B). In contrast, cells of both corpora lutea (Fig. 4C) and cultured mural GCs (Fig. 4D) were positive for the HA-receptor CD44. Furthermore, in GC cultures, an irregular staining was observed (Fig. 4D, arrows).
Localization of HA in COCs and GCs
In histological cross-sections of COCs, it could be shown that the cumulus forms a compact and multilayered envelope around the oocyte (Fig. 5A). At the onset of IVM, all cumulus cells were tightly attached to each other, and only a faint HA-specific staining could be detected in the extracellular space. After 24 h of IVM (Fig. 5B), an increase of HA, indicated by the green fluorescence, could be observed. Most of the cumulus cells were dispersed from the oocyte but were anchored in a large coat of HA. Only a single-cell layer, the corona, was still attached to the oocyte. All the controls remained free of unspecific staining (data not shown). In cultured GCs, HA was hardly detectable at the beginning of the experiment (Fig. 5C), but after 4 h, sporadic signals specific for HA became visible (Fig. 5D). In GC cultures grown in medium supplemented with both gonadotropins, increased and cloud-like, HA-specific fluorescence was observed (Fig. 5E). However, 24 h after gonadotropin stimulation, all cultured GCs appeared to be nearly depleted of cell-bound HA molecules (Fig. 5F).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
These findings were supported by the excessive local accumulation of HA, shown using histological techniques, that matched the increased enzyme transcription. These rapid changes indicate extensive alterations of the microenvironment during bovine IVM, leading to cumulus expansion, probably under peripheral hormonal control. Ubiquitous HA is known to influence major cellular events, such as cell migration and proliferation [37], and might be an important polymer during final oocyte development and fertilization [13]. Compared to HAS3, HAS2 is the most abundantly expressed HA-synthesizing enzyme during bovine COC expansion, as has been reported in mouse cumulus cells as well [38]. However, in contrast to the rapid mRNA expression of HAS2 in the cow, recent reports by Kimura et al. [3] concerning porcine IVM showed a significant increase of HAS2, but not until 24 h. Additionally, HAS3 mRNA was located exclusively in porcine oocytes and was lowered by substitution with gonadotropins. These lower amounts of porcine HAS3 expression are comparable to the present bovine system when LH and FSH were added to the maturation medium. However, the lower transcript concentration does not exclude HAS3 from also being important for HA production during cumulus expansion [16] when considering the diverse processing characteristics of these enzymes, as shown earlier [39]. The HAS3 possesses intrinsically higher catalytic activity than other HAS enzymes, and the chain length of HA molecules synthesized by HAS3 is shorter when compared to HAS2-generated polymers. Low-molecular-weight HA will directly influence the expression of cytokines and cell shape in eosinophils [40], but the proportion as well as the effect of the different HA length classes in the bovine cumulus is unknown. The immediate response of HAS2 to gonadotropins indicates its local importance, but the influence of the later, much-less-expressed HAS3 has to be established in the bovine COC relative to different functions of the resulting polymer. Independent of biomechanical effects during ovulation, HA could transmit signals through binding to receptor proteins and, potentially, influence cell functions and properties in a paracrine manner [41].
Although little is known about regulation of the HA-receptors CD44 and RHAMM in reproductive tissue, the few experiments that have been conducted suggest that CD44 plays an important role during human oocyte maturation [25] and prevents apoptosis in human GCs [21]. The CD44 protein has been found by immohistochemistry in a variety of mammalian epithelial cells, including oviduct, uterus, and vagina [42]. Such observations suggest a local communication between the cumulus-derived HA and the HA receptors on the reproductive epithelium (e.g., the oviduct). Cycle-dependent regulations of CD44 in the human tissues have been described with increased levels during the uterine secretory phase, indicating a possible steroid influence [43]. Conversely, our results suggest an endocrine influence on the mRNA expression of the HA-receptor CD44. Although GCs produced steroid hormones under FSH or LH application, the production of progesterone and estradiol lagged behind the initial increase of CD44 mRNA at 4 h [44]. Therefore, the major influence on CD44 induction in GCs is likely based on gonadotropins rather than on steroid hormones. In contrast to the lack of CD44 modulation in GCs, with simultaneous treatment of LH and FSH the same conditions led to a significant increase of CD44 mRNA transcripts during COC maturation. Hence, our results suggest both an endocrine as well as a paracrine regulation of CD44 through gonadotropins and, probably, oocyte factors, respectively. A few so-called oocyte-derived factors (e.g., growth and differentiation factor 9) may be potential modulators for the essential interplay between the oocyte and the surrounding somatic cells [45]. Additionally, the altered expression pattern of HAS3 in oocyte-surrounding cumulus cells when compared to the GCs indicates especially the oocyte's feasible influence. Although RHAMM was unregulated under gonadotropins, possible functions of this HA receptor in the bovine reproductive tract could be important, because basic expression levels during IVM were found to be compared to CD44 expression. The RHAMM may mediate movement and attachment of human cumulus cells [46], or it could modulate ciliary beating of oviduct epithelia, as described for the respiratory system [30]. Recent work discovered CD44 as the essential HA receptor throughout development of the preimplantation human embryo [47] and during trophoblast implantation [48]. Further investigations will be necessary to elucidate the detailed functions of both receptor types in the bovine reproductive system.
Our in vitro results indicate that a balance of FSH and LH is capable of modulating the main components of the HA system in bovine follicular cells. These data support an influence of the endogenous gonadotropin surge before ovulation and, subsequently, stimulating HAS2 expression within 24 h in native follicular GCs [49]. The common bovine IVM system appears to be comparable to normal physiological situations leading to fully mature COCs. An exogenous application of HA during bovine IVM showed beneficial effects on both IVM and in vitro production of embryos [50]. Additionally, Saito et al. [18] postulate that the concentration of HA in the follicular fluid could be used as an indicator to estimate oocyte fertilization capacity.
Previously described effects of HA surrounding the oocyte have indicated that it could serve as a protective shield as well as a reservoir for different growth factors. Glycosaminoglycans are negatively charged compounds and, therefore, bind unspecifically to many other substances, including growth factors. The interactions between GAGs and cytokines are one of the important mechanisms underlying communication processes between cells that are mediated by secreted and locally acting factors. Various studies have shown that quite a few cytokines are associated with the ECM by polysaccharide-binding motives [51]. For example, vascular endothelial growth factor protein has been found attached in increasing amounts at the bovine COC during final maturation [32], and such HA-rich oocyte microspheres may serve as a pool for such potent cytokines. Rapid remodeling of the ECM of COCs may trigger cell function through a possible cleavage of cytokines from that matrix. Still another mechanism of HA action together with bound cytokines could alter cell dynamics through intracellular uptake of HA via CD44-mediated incorporation [22]. A mucosal host defense has been proposed based on HA protecting cells from ciliary clearance in the lung [52]. This may draw future attention to interesting interactions between the cumulus and the oviduct after ovulation of the COCs.
In summary, our findings assign HAS2 and the corresponding receptor, CD44, a major role during final bovine oocyte maturation facilitating cumulus expansion. Interestingly, CD44 appeared to be differentially expressed in bovine cumulus in contrast to GCs: LH and FSH in combination induced an altered reaction in GCs. Considering such a diverse response, oocyte-derived local factors likely play a role in regulating the CD44 expression during IVM independent of endocrine signals. The results of investigations with HA components indicate a complex, local, and fine-tuned system during final oocyte maturation in the cow. These findings may further complete our understanding of the complex network of circulating hormones, local growth factors, and ECM components enabling optimal development of the oocyte that leads to reproductive success. Furthermore, the growing knowledge of such essential physiological processes supporting oocyte maturation will enable an improved understanding of assisted reproduction.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Ralf Einspanier, Institute of Physiology, Weihenstephaner Berg 3, D-85354 Freising, Germany. FAX: 49 8161 71 4204; einspanier{at}wzw.tum.de
Received: 20 September 2002.
First decision: 9 October 2002.
Accepted: 19 February 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-trypsin inhibitor family. J Biol Chem 1992 267:12380-12386This article has been cited by other articles:
|
|
 |
|
  H M Picton, S E Harris, W Muruvi, and E L Chambers The in vitro growth and maturation of follicles Reproduction, December 1, 2008; 136(6): 703 - 715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Assidi, I. Dufort, A. Ali, M. Hamel, O. Algriany, S. Dielemann, and M.-A. Sirard Identification of Potential Markers of Oocyte Competence Expressed in Bovine Cumulus Cells Matured with Follicle-Stimulating Hormone and/or Phorbol Myristate Acetate In Vitro Biol Reprod, August 1, 2008; 79(2): 209 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Borg and M. Holland The effect of glycosaminoglycans on rat gametes in vitro and the associated signal pathway Reproduction, March 1, 2008; 135(3): 311 - 319. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P.A. van Montfoort, J. P.M. Geraedts, J. C.M. Dumoulin, A. P.M. Stassen, J. L.H. Evers, and T. A.Y. Ayoubi Differential gene expression in cumulus cells as a prognostic indicator of embryo viability: a microarray analysis Mol. Hum. Reprod., March 1, 2008; 14(3): 157 - 168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. G Clarke, S. A Hope, S. Byers, and R. J Rodgers Formation of ovarian follicular fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans Reproduction, July 1, 2006; 132(1): 119 - 131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Stuhlmeier and C. Pollaschek Adenovirus-mediated Gene Transfer of Mutated I{kappa}B Kinase and I{kappa}B{alpha} Reveal NF-{kappa}B-dependent as Well as NF-{kappa}B-independent Pathways of HAS1 Activation J. Biol. Chem., December 30, 2005; 280(52): 42766 - 42773. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ohno-Nakahara, K. Honda, K. Tanimoto, N. Tanaka, T. Doi, A. Suzuki, K. Yoneno, Y. Nakatani, M. Ueki, S. Ohno, et al. Induction of CD44 and MMP Expression by Hyaluronidase Treatment of Articular Chondrocytes J. Biochem., May 1, 2004; 135(5): 567 - 575. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
