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RESEARCH |
1 AgResearch, Wallaceville Animal Research Centre, Ward Street, PO Box 40063, Upper Hutt, New Zealand, 2 AgResearch Molecular Biology Unit, Department of Biochemistry, University of Otago, Dunedin, New Zealand
Correspondence should be addressed to J Juengel; Email: jenny.juengel{at}agresearch.co.nz
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The BMP subfamily members, BMP2, 4, 6 and 7 have been shown to be expressed by follicular cells in several species such as mice, rats, chickens and cows (Shimasaki et al. 1999, Erickson & Shimasaki 2003, Onagbesan et al. 2003, Glister et al. 2004) and furthermore, to regulate follicular cell function in vitro (Shimasaki et al. 1999, Dooley et al. 2000, Lee et al. 2001, Mulsant et al. 2001, Otsuka et al. 2001, Souza et al. 2002, Fabre et al. 2003, Onagbesan et al. 2003, Nilsson & Skinner 2003, Glister et al. 2004, 2005, Lee et al. 2004, Pierre et al. 2004). While it is known that follicular cells in sheep express receptors capable of responding to these BMPs (Wilson et al. 2001, Souza et al. 2002) and that BMP2 and 4 can regulate granulosa cell function (Mulsant et al. 2001, Souza et al. 2002, Fabre et al. 2003, Pierre et al. 2004) the ovarian cell-types that express BMP2, 4, 6 and 7, as well as the effects of BMP6 and BMP7 on granulosa cell function are presently unknown in sheep. Given the importance of BMP15 and GDF9 in regulation of follicular growth and ovulation rate as well as the known effect of mutations in the BMPRIB gene on ovarian activity in sheep (Shimasaki et al. 2004, McNatty et al. 2005a), the characterization of the ovarian cell-types that produce other BMP family members as well as the effects of these proteins on granulosa cell function is central to further elucidation of their roles in regulating ovarian activity. The objectives of this study were to determine the patterns of expression of BMP2, 4, 6 and 7 in the ovine ovary and to determine the effects of these ligands on ovine granulosa cell proliferation and progesterone production. In addition, we determined the role of these BMPs in rat granulosa cells under identical culture conditions to be able to compare more directly the effects of BMPs on granulosa cells of sheep and the more extensively studied species, the rat.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Cloning of BMP2, 4, 6 and 7 and in situ hybridization
Except where indicated, laboratory reagents were obtained from BDH Chemicals New Zealand Ltd (Palmerston North, New Zealand), Invitrogen (Auckland, New Zealand) or Roche Diagnostics N.Z. Ltd (Auckland, New Zealand). Complimentary cDNAs encoding a portion of ovine BMP2, 4, 6 and 7 were generated using standard reverse transcription(RT)-PCR techniques. Primers and conditions for PCR are listed in Table 1
. Sequences of resulting plasmids were confirmed prior to use for in situ hybridization. In situ hybridization was performed as previously described (Tisdall et al. 1999) with minor modifications. Briefly, 4–6 µm tissue sections were incubated overnight at 50–55 ° C with 45 000 c.p.m./µl (approximately 48 000 d.p.m./µl) of 33P-labelled antisense RNA. Non-specific hybridization of RNA was removed by RNase A digestion followed by stringent washes (2 x SSC, 50% formamide, 65 ° C and 0.2 x SSC at 37 ° C). Following washing, sections were dehydrated, air dried and coated with autoradiographic emulsion (LM-1 emulsion; Amersham Pharmacia Biotech New Zealand). Emulsion-coated slides were exposed at 4 ° C for 3–4 weeks, developed for 3.5 min in D19 developer (Eastman Kodak, Rochester, NY, USA). Development was stopped using a 1 min incubation in 1% acetic acid and slides were fixed with a 10 min incubation in Ilfofix II (Ilford Limited, Cheshire, England). Sections were stained with hematoxylin and then viewed and photographed using both light and dark field illumination on an Olympus BX-50 microscope (Olympus New Zealand Ltd). At least 8 animals were examined for expression of each of the BMP genes. These included both lambs and adult ewes and no differences were noted in the pattern of gene expression related to the age of the animal. In addition, as the expression of BMP4 and 7 was not commonly observed in the ovarian sections, a positive control tissue of fetal kidney (n = 2) was included with each in situ hydridization. This tissue had been collected from female ovine fetuses collected on day 40 of gestation, which were part of another approved study. Follicles at each defined stage of development were observed in at least 3 animals for all genes examined. Classification of follicles at each developmental stage was based on the system outlined (Lundy et al. 1999). Briefly, type 1/1a follicles consist of an oocyte surrounded by a single layer of flattened or mixed flattened and cuboidal cells. Type 2 follicles contain 1 < 2 layers of cuboidal granulosa cells, whereas type 3 follicles contain 2 < 4 layers of cuboidal granulosa cells. Type 4 follicles have > 4 layers of granulosa cells and a well defined theca, but have not yet formed an antrum. Type 5 follicles have multiple layers of granulosa cells, a well defined theca and a defined antrum. All follicles with signs of degeneration (i.e. pyknotic granulosa cells, lack of a distinct basement membrane or degenerate oocytes) were considered atretic. Non-specific hybridization was monitored by hybridizing the sense RNA for each receptor to tissue collected from at least one animal per age group. Hybridization was considered specific when the intensity of silver grains, as measured by visual assessment, over a cellular type was greater than that observed in the area of the slide not containing tissue. For all genes, hybridization of the sense RNA over the tissue section was similar or lower in intensity to that observed on the areas of the slide not containing tissue of both the sense and antisense hybridized slides and thus was considered non-specific.
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Granulosa cell culture
Collection of granulosa cells
Granulosa cells were isolated from 1–2 mm follicles from ovine ovaries collected from the local abattoir and from Sprague–Dawley rats (23–26 day old; University of Otago, Dunedin, New Zealand). Granulosa cells were also collected from sheep and rats approximately 96 h after subcutaneous administration of implants containing diethylstilbestrol (DES). Rats received a single implant of approximately 1.8 cm with an internal diameter of 3.35 mm (Chen et al. 2001). Ewes received 2 implants based on previous results with implants containing oestradiol (McNatty et al. 1989). Oocytes and follicular debris were removed from the cells using a micro-glass pipette. Remaining cells were collected by centrifugation at 300 g for 5 min at room temperature, washed once in 5 ml Leibovitz media, twice in 5 ml McCoys media (Sigma, Auckland, New Zealand) and resuspended using a syringe and needle. Cell viability was determined using trypan blue exclusion, 100 000 viable cells per well (250 µl total volume) were added in McCoys media containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM Gluta-MAX-1 and 0.1% BSA. Media for cells used for progesterone and DNA determination also contained 5 ng/ml selenium (Sigma), 10 ng/ml insulin (Sigma), 5 µg/ml apotransferrin (Sigma), 30 ng/ml androstenedione (Sigma), 3 ng/ml ovine FSH (purified in our laboratory; 1.4 X USDA-oFSH-19-SIAFP RP2), and 1 ng/ml IGF-1 (Long-R3, GroPep, Adelaide, SA, Australia).
Determination of 3H-thymidine incorporation
Cells (100 000 viable cells per well) were incubated in media described above with the addition of 6-3H-thymidine (Perkin Elmer, Boston, MA, 20 Ci/mmol, 1.0 µCi per well) with or without human (h) BMP2, hBMP4, hBMP6 or hBMP7 (3 µg/ml; R&D Systems Inc, MN USA) for 48 h at 37 ° C in a 5% CO2 incubator. This standardized dose of BMP added to the cultures was chosen after analysis of dose-response assays with both sheep and rat granulosa cells (see Fig. 1 for representative data from sheep). At the termination of culture, cells were harvested with a cell harvester onto a thick filter mat. Incorporation of 3H-thymidine was determined using a Wallac Trilux MicroBeta 1450 liquid scintillation counter (Biolab, Auckland, New Zealand). Average values for incorporation of 3H-thymidine were determined for each treatment, outlier replicates (outside of 30% of the mean) were identified and discarded.
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for representative data from sheep). Every 48 h, 200 µl of media was removed from each well and replaced with 200 µl of warmed media that had been prepared at the start of the culture and stored at 4 ° C. Media samples from the first (rat only) and last 48 h of treatment were collected on the appropriate day of treatment and frozen at –20 ° C for later determination of progesterone concentrations by RIA. Unattached cells were removed by 2 washes with McCoys media at 37 ° C. Attached cells were lysed by incubating cells at 37 ° C in 100 µl distilled water for 1 – 2 h followed by freezing at –70 ° C.
Measurement of progesterone
Concentrations of progesterone in media were determined by RIA as described (Lun et al. 1998). The sensitivity of the assay (90% maximum binding) was 16 pg/ml and the intra- and interassay co-efficients of variation (CV), averaged for a standard pool sample at approximately 20, 50 and 80% binding, was 11 and 16%, respectively. No samples were below the sensitivity of the assay. Average values for progesterone concentrations were determined for each treatment within each independent granulosa cell bioassay, outlier replicates (outside of 20% of the mean) were identified and discarded.
Measurement of DNA
The amount of DNA present in each well was determined by comparing binding of Hoechst 33258 dye (Sigma, final concentration of 10 µg/ml in well) in samples to calf thymus DNA standard measured with a Wallac 1420 plate reader at 350 nm for excitation and 460 nm for emission. Sensitivity of the assay (+ two S.D. of control buffer value) was 20 ng per well and the intra- and interassay CV, based on variability of the 100, 250, 1000 and 2500 ng standard curve points were 6 and 6%, respectively. No samples were below the sensitivity of the assay.
Statistical analysis
Amount of progesterone produced per µg DNA was calculated individually for each well. Replicates that were not within 30% of the mean were discarded. For each variable, effects of specified treatment were determined by comparison to the untreated control sample using the 2-tailed paired t-test function in Microsoft Excel 2003. As the BMPs were not always run in the same assay, no statistical comparisons were made between the effects of the various BMPs. For ease of presentation, all values have been converted to a ratio of the appropriate controls which were assigned a value of 1.00.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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). Atretic type 5 follicles expressed BMP2 mRNA in the degenerating granulosa cells and this was most pronounced in follicles in the late stages of atresia (Fig. 2).
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). Expression of BMP4 mRNA was often observed in the surface epithelium and around blood vessels. In addition, expression was observed in a concentric circle around some antral follicles. This expression did not appear to be in the theca interna or externa of these follicles although the boundary between the theca externa and stromal cells of the ovary can be difficult to discern. Often, these follicles were atretic. The positive controls (i.e. sections of kidney tissue collected on day 40 of fetal life) expressed mRNA encoding BMP4 (Fig. 3).
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). BMP6 mRNA was not evident in either the granulosa or thecal cells of follicles of any size (Fig. 4).
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). When rete tubules were present on the slide (n = 3), positive expression of BMP7 mRNA was noted. The positive controls (i.e. sections of kidney tissue collected on day 40 of fetal life) expressed mRNA encoding BMP7 (Fig. 5).
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). However, in all cases, the strength of the signal was weak compared with the positive control sample.
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) as assessed either over the first 48 h of culture by thymidine incorporation or during the longer term (6 days) through direct measurement of DNA. In contrast, to the lack of effect observed on cellular proliferation, concentrations of progesterone in media were decreased when compared with control cultures following treatment of granulosa cells with either BMP2, 4, 6 or 7. When the effects of the BMPs were assessed on a per cell basis, concentrations of progesterone/ng DNA were decreased in granulosa cells treated with BMP4, 6 or 7 when compared with control cultures (Fig. 8).
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). However, the concentrations of progesterone in media were decreased by all BMPs and a similar trend (P < 0.10) was observed when progesterone was expressed on a per cell basis (Fig. 8).
Rat
Without DES
Treatment of rat granulosa cells with either BMP4, 6 or 7, but not BMP2, resulted in an increased thymidine uptake over the first 48 h of culture (Fig. 9). The amount of DNA at the end of culture was not significantly higher than observed for control cultures, although the absolute values were increased between 40–60% (Fig. 9). All BMPs reduced progesterone concentrations after 2 days in culture and progesterone produced per cell at 6 days in culture (Fig. 10).
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). BMP2 had no effect on thymidine uptake or DNA concentrations. Concentrations of progesterone in media were decreased in all BMP treated cultures when compared with control cultures following 2 days of culture but only BMP6 showed a decreased concentration of progesterone compared with controls at 6 days of culture (Fig. 10). However, all BMPs tested decreased progesterone synthesis at 6 days of culture when the concentrations of progesterone were expressed relative to DNA concentration (Fig. 10). |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The expression patterns for BMP2, 4 and 7 were more restricted in sheep follicles when compared with other species that have been examined to date. For instance, while expression of BMP2 mRNA was not observed in granulosa cells, theca cells, cumulus cells or oocytes of healthy follicles and was restricted to atretic follicles in the sheep ovary in situ, both the granulosa and thecal cells of non-atretic rat follicles as well as atretic follices expressed BMP2 as detected by in situ hybridization (Erickson & Shimasaki 2003). In chickens, expression of BMP2 mRNA was detected in both the theca and granulosa of non-atretic antral follicles using RT-PCR (Onagbesan et al. 2003) while in cattle BMP2 protein was predominately observed in thecal cells of antral follicles with some oocytes also staining positive using immunocytochemistry (Fatehi et al. 2005). In the present study, BMP4 and 7 mRNA was not observed in granulosa cells, theca cells, cumulus cells or oocytes of non-atretic ovine follicles in situ. In contrast, both BMP4 and 7 mRNA was detected in rat thecal cells using in situ hybridization (Erickson & Shimasaki 2003) and in both granulosa and thecal cells in chickens as detected by RT-PCR (Onagbesan et al. 2003). Moreover, both BMP4 and 7 immunoreactive proteins were detected in bovine thecal cells by immunocytochemistry (Glister et al. 2004, Fatehi et al. 2005). In the present study, BMP6 mRNA was observed only in oocytes of sheep follicles and was not observed in granulosa cells, theca cells or cumulus cells as determined by in situ hybridization, whereas in cows (Glister et al. 2004) and rats (Erickson & Shimasaki 2003) BMP6 was detected in both the oocytes and granulosa cells.
In some instances, these findings may represent true species differences. However, there is also a possibility that the differences are due to different methodologies. One key difference between the present study and that of Glister et al.(2004) is that the bovine granulosa and thecal cells had been cultured for 6 days before examination compared with in situ examination of ovine granulosa and theca cells at tissue recovery. Given the low level of expression detected by RT-PCR for these genes in isolated granulosa cells, it is possible that the culture conditions may have induced expression of the BMPs in bovine granulosa and theca cells.
In ovine granulosa cells, neither BMP2, 4, 6 nor 7 stimulate granulosa cell proliferation/survival. In contrast, while some differences were noted among the rat assays according to animal treatment and end measurement overall, BMP4, 6 and 7 all stimulated granulosa cell proliferation/survival in rat granulosa cells and the effects of BMP2 on DNA content at the end of culture approached significance for the DES treated rat cells (P < 0.06). Previously, BMP4, 6 and 7 have been shown to increase cell numbers in cultures of bovine granulosa cells (Glister et al. 2004) and BMP 7 stimulated thymidine uptake and numbers of cultured granulosa cells in rats (Lee et al. 2001) and chickens (Onagbesan et al. 2003) whereas BMP4 or 6 had no effect on cell numbers in the chicken (Onagbesan et al. 2003) or thymidine incorporation of rat granulosa cells (Otsuka et al. 2001), respectively. The reasons for the differences between these findings, including the differences between the DES-treated and non-treated rats in the present study, may relate to differences in methodologies. Multiple factors, including the stage of maturation of the granulosa cells, differences in media composition such as addition of factors such as FSH and IGF-1, or differences in length of the culture period may influence the responsiveness to BMPs. In addition, determination of cell numbers/DNA content will take into account both cell mitosis and cell death, whereas measuring thymidine uptake only measures the intent for cell division. However, it is important to note that in the present study, rat and ovine granulosa cells were cultured under identical conditions suggesting that true differences between species in their responses to BMPs exist. Interestingly, the effects of BMP15 and GDF9, other TGFß superfamily members, have also been shown to differ subtly between even the closely related species sheep and cow when tested under identical conditions in the same laboratory suggesting the roles of the BMPs may vary even between closely related species (McNatty et al. 2005b).
BMP2, 4, 6 and 7 all suppressed progesterone production. This is in agreement with the suppressive effects of the BMPs in a number of mammalian species with respect to progesterone production by granulosa cells (Shimasaki et al. 1999, Otsuka et al. 2001, Fabre et al. 2003, Glister et al. 2004, Pierre et al. 2004). In chickens, BMP4 and 7 stimulate progesterone production by granulosa cells but this difference may be related to the differing roles of progesterone in regulation of gonadotrophins between mammalian and avian species (Onagbesan et al. 2003).
Sheep with a mutation in activin receptor-like kinase 6 (Alk6, also known as BMPRIB) have increased ovulation rates (Mulsant et al. 2001, Souza et al. 2001, Wilson et al. 2001) and the Alk6 receptor is found in both oocytes and granulosa cells (Wilson et al. 2001, Souza et al. 2002). The mechanisms by which this mutation results in increased ovulation rate, including the preferred ligand for this receptor in sheep is not known. Based on the expression patterns of BMP2, 4 and 7, these proteins do not appear to be strong candidates as the preferred physiological ligands for Alk6 in sheep granulosa cells. However, BMP6, which has been shown to be able to signal through Alk6 (Juengel & McNatty 2005), might be one of the preferred ligands regulating follicular maturation and ovulation rate through this receptor (Shimasaki et al. 2004). However, it is important to note that BMP15 also has been shown to act through Alk6 (Moore et al. 2003). Futhermore, the BMP15 and Alk6 mutations in sheep have been shown to have synergistic effects on ovulation rate (Davis et al. 1999). Since mice lacking an active BMP6 gene show no apparent fertility phenotype (Solloway et al. 1998), the role that BMP6 plays in regulating ovarian follicular development and ovulation rate in sheep needs further clarification.
In conclusion, several members of the BMP subfamily of the TGFß superfamily, namely BMP2, 4, 6 and 7 were shown to have similar actions in regulating ovine granulosa cells. Specifically, these proteins suppressed concentrations of progesterone secreted into the media without affecting cell number. This is consistent with a luteinization inhibition factor. However, based on their localization patterns in developing ovarian follicles, BMP6 would be the most likely candidate to play a physiological role in the local regulation of follicular growth in sheep, as mRNA encoding BMP2, 4 and 7 could not be detected by in situ hybridization in non-atretic follicles.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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