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Ovary |
1
a School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, United Kingdom
b School of Biological and Molecular Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Intense staining with TGF antibody (but not with EGF antibody) was detected in oocytes both before and after coculture. Experiments involving addition of TGF to granulosa cells confirmed that the peptide mimicked the effects of oocytes on cell proliferation and on FSH- and IGF-induced hormone secretion. These experiments indicate that bovine oocytes secrete a factor(s) capable of modulating granulosa cell proliferation and responsiveness to FSH and IGF in terms of steroidogenesis and production of inhibin-related peptides, bovine oocytes express TGF but not EGF, and TGF is a prime candidate for mediating the actions of oocytes on bovine granulosa cells.
activin, follicle, follistatin, granulosa cells, inhibin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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More recent in vitro studies in rodents involving oocyte-granulosa cell coculture and/or comparison of intact and oocytectomized cumulus-oocyte complexes (COCs) have demonstrated that the oocyte secretes a factor(s) capable of influencing granulosa cell proliferation and differentiation. Oocyte-derived factors have been reported to promote granulosa cell proliferation [5, 6], inhibit FSH-stimulated steroidogenesis [7, 8], and suppress LH receptor formation on granulosa cells [9] in mice. Moreover, comparison of mural and cumulus granulosa cell subpopulations revealed that the more distally located mural cells have a greater steroidogenic capacity and LH receptor expression than the cumulus cells proximal to the oocyte [10, 11]. In comparison with studies on mice, relatively few researchers have examined potential effects of oocytes on granulosa cell function in large domesticated species. However, the observation that an oocyte factor(s) can suppress FSH-induced steroid production has been extended to pigs [12] and cattle [5]. Candidate factors that are known to be expressed by oocytes and that may mediate these actions on granulosa cell function include several members of the transforming growth factor (TGF) ß superfamily (GDF-9, BMP-15/GDF-9B, BMP-6 [13–18], epidermal growth factor [EGF], and TGF [19, 20]).
In addition to producing steroids, mainly estradiol (E2) and progesterone (P4), granulosa cells are a major site of inhibin, activin, and follistatin (FS) expression [21, 22]. Like steroids, inhibin-related proteins exert both local intragonadal actions and peripheral endocrine actions. In a reciprocal manner, regulation of the synthesis and secretion of steroids and inhibin-related peptides by granulosa cells involves interactions between systemic factors (principally pituitary gonadotropins) and intraovarian autocrine/paracrine factors secreted by theca cells, granulosa cells, and the oocyte. Recent evidence suggests that inhibin-related peptides of granulosa cell origin (mural and/or cumulus) can modulate oocyte maturation and developmental competence [23–26]. To our knowledge, however, there have been no studies to investigate whether the oocyte in turn might contribute to the regulation of inhibin-related peptide production by granulosa cells.
Because of the paucity of information available on oocyte-granulosa cell interactions in ruminants, the aim of this study was to determine whether denuded bovine oocytes cocultured with mural granulosa cells are able to modify cell proliferation or basal, FSH-induced, and IGF-induced secretion of inhibin-related peptides and steroids. The pronounced effects of oocytes on granulosa cell hormone secretion and cell proliferation were remarkably similar to those elicited by EGF and TGF in the same culture system. We therefore used immunocytochemistry to determine whether bovine oocytes express TGF or its related peptide EGF.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mural granulosa cells were harvested from cattle ovaries obtained from an abattoir as described by Glister et al. [27]. Follicles 4–6 mm in diameter and lacking obvious signs of atresia (i.e., follicles with a translucent appearance, well-vascularized theca, and clear follicular fluid with no visible debris or blood) were dissected, aspirated, and hemisected, and the granulosa cell layer was gently disrupted with the aid of a plastic inoculation loop. Cells were pelleted by centrifugation (800 x g for 10 min) and subjected to an osmotic shock treatment to lyse any red blood cells present (double-distilled water for 10 sec before restoring isotonicity with 3x concentrated PBS). Cells were pelleted and resuspended in a small volume of culture medium consisting of McCoy 5A modified medium supplemented with 1% (v/v) antibiotic-antimycotic solution, 10 ng/ml bovine insulin, 2 mM L-glutamine, 10 mM Hepes, 5 µg/ml apotransferrin, 5 ng/ml sodium selenite, 0.1% BSA, and 10-7 M androstenedione (all from Sigma, St. Louis, MO). Cells were then counted using a hemocytometer. Cell viability, assessed using trypan blue staining, was routinely 20–30%.
The COCs were aspirated from follicles 3–8 mm in diameter using a 19-gauge needle connected via a 20-cm length of polyethylene tubing to a 25-ml universal container maintained at negative pressure with a suction pump. The aspirated follicular fluid was passed through a mesh screen filter (100-µm mesh; Sigma) to remove free cells and cell clumps, and the retained COC-enriched suspension was searched for intact COCs with the aid of a zoom stereomicroscope (M8; Wild, Heerbrugg, Switzerland). COCs with at least one layer of compact cumulus cells were retrieved, treated with hyaluronidase (0.25 mg/ml) for 1 min, and gently agitated to remove attached cumulus cells. Denuded oocytes were washed in culture medium before transfer to experimental plates.
Granulosa cells were seeded at a density of 75 x 103 viable cells/50 µl culture medium into wells of a 96-well tissue culture plate (Nunclon; Life Technologies Ltd., Paisley, U.K.) containing 200 µl preequilibrated culture medium with and without treatments. Plates were incubated at 38.5°C in 5% CO2 and 95% air for 6 days. Conditioned medium was removed and replaced with fresh media with and without treatments every 48h. Conditioned media samples were stored at -20°C for immunoassay. At the end of the culture period, the number of viable granulosa cells was determined by uptake of neutral red dye as described elsewhere [27, 28]. Visual observation of neutral red dye uptake by oocytes at the end of the culture period indicated that >80% of these cells remained viable throughout. The majority of oocytes had become reassociated with granulosa cells by the end of the culture period.
Preparation and Addition of Treatment Factors
Ovine FSH (0.037, 0.33, and 3.0 ng/ml, oFSH-19SIAPP; NIADDK), recombinant IGF-I analogue (2, 10, and 50 ng/ml, Long R3 IGF-I; Sigma), and human recombinant TGF (0.1, 1.0, and 10 ng/ml; Autogen Bioclear Ltd., Wiltshire, U.K.) were dissolved in Hanks balanced salt solution (HBSS) containing 0.3% (w/v) BSA, and the stock solution was sterilized by passing it through a 0.2-µm membrane filter (Minisart; Sartorius AG, Gottingen, Germany) before further dilution in sterile culture medium. Test substances were added to replicate wells in a volume of 25 µl, and an equal volume of culture medium alone was added to control wells.
Immunoassays
Concentrations of inhibin A (inh A) were determined using the two-site immunoradiometric assay described by Knight and Muttukrishna [29]. Purified 32-kDa bovine inhibin [30] was used as the standard. The detection limit of the assay was 250 pg/ml with mean intra- and interassay coefficients of variation (CVs) of 5.2% and 6.2%, respectively. Total activin A (act A) (i.e., free and FS bound) levels were measured using a two-site ELISA [31]. Human recombinant act A (NIBSC, Potters Bar, Herts, UK) was used as a standard, and the assay sensitivity was 100 pg/ml. Intra- and interassay CVs were 6.2% and 10.1%, respectively. Total FS levels were determined using the ELISA described by Tannetta et al. [32]. Values are expressed in terms of hr-FS-288 preparation provided by NIDDK. Assay sensitivity was 100 pg/ml, and intra- and interassay CVs were 6.3% and 8.8%, respectively.
Concentrations of P4 were determined by direct RIA as described elsewhere [33]. The detection limit was 0.1 ng/ml, and intra- and interassay CVs were 10.5% and 11.7%, respectively. Concentrations of E2 were determined by direct RIA using a commercial kit (Biostat Diagnostic Systems, Woking, Surrey, U.K.) that was modified and validated for bovine granulosa cell-conditioned culture medium as described previously [27]. The detection limit of the assay was 1.5 pg/ml, and intra- and interassay CVs were 2.8% and 9%, respectively.
Immunofluorescent Staining of Oocytes
Retrieved denuded oocytes were submitted for immunostaining either before or after 6 days of coculture with granulosa cells. After washing (twice in PBS for 10 min each time), oocytes were fixed overnight in 4% paraformaldehyde in PBS (pH 7.4), washed (twice in PBS for 10 min each time), and then permeabilized using 0.1% Triton X-100 in PBS for 10 min. After washing again (as above), oocytes were blocked for 1 h in PBS containing 0.1% NaN3, 2% BSA, and 3% normal goat, donkey, or horse serum (matched to the species in which secondary antibody was raised). Oocytes were incubated (overnight at room temperature) with primary antibody (or control IgG) diluted in PBS containing 0.5% BSA and 0.1% NaN3, washed (three times in PBS for 10 min each time), and incubated for 1 h with the secondary antibody diluted in PBS containing 0.5% BSA, 0.1% NaN3, and 2% normal serum. Oocytes were washed in 0.1% Triton X-100 in PBS for 1 h and then in PBS (three times for 10 min each time). Oocytes were mounted under raised coverslips in an antifading mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and imaged on a Leica TCS-NT scanning laser confocal microscope (Leica Lasertechnik GmBH, Heidelberg, Germany).
Primary antibodies used were affinity-purified goat anti-human TGF (T-0563; Sigma), rabbit anti-human EGF (ABC503; Autogen Bioclear, Wiltshire, U.K.), goat anti-human GDF-9 (SC-7407; Santa Cruz Biotechnology, Santa Cruz, CA), and a mixture of two mouse monoclonal antibodies (clones 22 and 53) raised in one of our laboratories (N.P.G.) against a 28-mer carboxy terminal sequence of human GDF-9; all primary antibodies were used at a concentration of 5 µg/ml. For controls, IgG preparations obtained by protein G-agarose fractionation of normal goat, rabbit, and mouse serum (Sigma) were used at the same concentration (5 µg/ml). The secondary antibodies were fluorescein-conjugated goat anti-rabbit IgG (FI-1200; Vector), Alexa Fluor 488-conjugated donkey anti-goat IgG (Molecular Probes, Eugene, OR), and fluorescein-conjugated horse anti-mouse IgG (FI-2000; Vector), all used at 10 µg/ml.
Statistical Analysis
Two-way ANOVA was used to evaluate the effect of oocytes alone and in combination with FSH or IGF and of TGF alone and in combination with FSH or IGF on hormone secretion during different periods of culture and on cell number at the end of culture. Data were log-transformed before analysis to reduce heterogeneity of variance. One-way ANOVA and post hoc Fisher protected least squares difference tests were subsequently used to make individual comparisons within a given treatment group when the initial two-way ANOVA indicated a significant (P < 0.05) effect of that treatment. Unless stated otherwise, results presented are arithmetic means ± SEM based on combined data from four independent cultures. Results are only presented for the final 96–144 h of culture during which responsiveness to the various test substances was greatest.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Consistent with previous studies [27], both FSH (Fig. 1) and IGF (Fig. 2) caused dose-dependent stimulation of E2 (20-fold and 23-fold increases for FSH and IGF, respectively), inh A (9- and 8.7-fold), act A (10- and 18-fold), FS (6.9- and 6-fold), and P4 (3- and 4.6-fold) secretion with maximal responses observed during the final (96–144 h) culture period with doses of 0.33 ng/ml FSH and 50 ng/ml IGF. As observed previously [27], responses to FSH and IGF were minimal during the first period (0–48 h) and intermediate during the second period (48–96 h) (data not shown).
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When denuded oocytes (0, 2, 8, or 20 oocytes/well) were cocultured with granulosa cells in the absence of FSH or IGF, no effect on any of the five measured hormones was observed (Figs. 1, a–e, and 2, a–e). However, in the presence of FSH or IGF, oocytes had a marked dose-dependent inhibitory effect on the secretion of all five hormones. Addition of oocytes to FSH-stimulated cells (Fig. 1) dose-dependently suppressed (P < 0.0001) E2 (4.6-fold maximum suppression during the final culture period), inh A (6-fold), act A (5.5-fold), FS (3.6-fold), and P4 (2.4-fold), with the degree of suppression increasing with FSH dose. Likewise, oocytes dose-dependently suppressed (P < 0.05) IGF-induced secretion of E2 (1.6-fold), inh A (1.6-fold), act A (1.4-fold), and FS (1.8-fold). Conversely, IGF-induced P4 secretion was moderately enhanced (P < 0.05) in the presence of oocytes (1.7-fold; Fig. 2e).
Treatment with FSH alone promoted a 1.8-fold increase (P < 0.005) in cell number determined at the end of the 144-h culture period (Fig. 1f). Coculture with oocytes in the absence of FSH also increased cell number. Addition of 8 oocytes/well increased cell number 1.8-fold (P < 0.001); addition of 2 and 20 oocytes/well also increased cell number (1.3-fold and 1.4-fold, respectively; P < 0.01), but responses were suboptimal relative to the 8 oocytes/well. Treatment with IGF alone promoted a substantial dose-dependent increase in cell number (3-fold; P < 0.001) (Fig. 2f). Coculture with oocytes in the presence of high IGF doses caused no further increase in cell number.
Effects of TGF on Basal, FSH-Induced, and IGF-Induced Hormone Secretion and Cell Number
Addition of TGF in the absence of FSH or IGF caused a significant reduction (P < 0.001) in the secretion of E2 (5-fold), inh A (2.5-fold), act A (1.6-fold), and FS (2.3-fold) (Figs. 3 and 4). TGF alone also increased cell number 1.3-fold (P < 0.0001). When tested in combination, TGF had a marked dose-dependent suppressive effect (P < 0.0001) on FSH-induced (Fig. 3) and IGF-induced (Fig. 4) secretion of E2 (53- and 32-fold maximum suppression for FSH- and IGF-induced secretion, respectively), inh A (14- and 8-fold), act A (12- and 17-fold), and FS (17- and 18-fold) but did not modify P4 secretion. Conversely, TGF in combination with either FSH or IGF had an additive stimulatory effect on cell number (maximal 1.6-fold increase with TGF + FSH, 2.6-fold increase with TGF + IGF, compared with 1.4-fold increase with TGF alone; P < 0.001).
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Immunostaining of TGF, EGF, and GDF-9 in Oocytes Before and after Coculture with Granulosa Cells
Immunostained freshly isolated denuded bovine oocytes and those retrieved after 6 days of coculture with granulosa cells are shown in Figure 5. Intense immunofluorescence staining was observed with TGF antibody for both freshly isolated (Fig. 5A) and cocultured (Fig. 5A*) oocytes. Staining was weak with the EGF antibody and not significantly different from the control for both sets of oocytes (Fig. 5, B and B*). Both sets of controls (incubated with normal rabbit/goat IgG) stained very weakly, with immunofluorescence barely detectable in both freshly isolated (Fig. 5, C and D) and cocultured (Fig. 5, C* and D*) oocytes.
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In addition, both goat and mouse GDF-9 antibodies produced intense staining of oocytes before (Fig. 5, E and F) and after (Fig. 5, E* and F*) culture, with their respective controls (normal goat/normal mouse IgG) exhibiting only weak staining (Fig. 5, D, D*, G, and G*).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because FSH-stimulated E2 and P4 were both inhibited by oocytes in this and other studies (excluding those on mice), the oocyte-secreted factor(s) appears to be generally suppressing gonadotropin-induced steroidogenesis. However, although IGF-stimulated E2 secretion in the present study was also suppressed by the oocyte-secreted factor(s), IGF-induced P4 secretion was actually enhanced. This finding alludes to a mechanism whereby the oocyte-secreted factor(s) interacts with other regulatory signals impinging on the follicle to differentially modulate steroidogenesis. During bovine follicular development, FSH may act as an early stimulator whereas IGF may act later in follicle development as an amplifier of FSH action [35]. Thus, by stimulating IGF-induced P4 secretion while suppressing IGF-induced E2 secretion, the oocyte in the later stages of folliculogenesis may promote luteinization and corpus luteum formation, an event essential for the successful continuance of the embryo.
The ability of oocytes to promote follicular cell proliferation was first described in the mouse [6], where an oocyte-secreted factor(s) stimulated proliferation of undifferentiated granulosa cells from preantral follicles and more differentiated mural and cumulus granulosa cells from antral follicles. Recently, bovine COCs were shown to have a greater rate of incorporation of [3H]thymidine than mural granulosa cells. However, this incorporation was reduced when COCs were oocytectomized such that levels in OOX and mural cells were similar [5]. In the present study, we also observed a positive effect of oocytes on cell proliferation. This proliferative effect of an oocyte-secreted factor(s) occurred independently of FSH or IGF stimulation. When cell proliferation was stimulated by higher doses of FSH or IGF, the effect of the oocyte was no longer apparent, indicating that the mitogenic action acts via a mechanism different from the FSH/IGF-linked system that modifies steroidogenesis. This mitogenic effect of the oocyte could be important in stimulating somatic cell proliferation throughout follicle development. However, the growth-promoting activity of the oocyte appears to be specific to granulosa cells, with other somatic cell lines unaffected by coculture with oocytes [36].
We are not aware of any previous studies in any species reporting the effects of oocytes or an oocyte-secreted factor(s) on inh A, act A, or FS peptide secretion by granulosa cells. In the present study, the presence of oocytes dose-dependently suppressed FSH- and IGF-stimulated secretion of inh A, act A, and FS but had no effect on basal secretion of these peptides. Inhibin- mRNA levels increased in rat ovary cultures upon the addition of high doses of GDF-9, one of several recently identified oocyte-specific factors [15]; however, GDF-9-deficient ovaries expressed comparable or increased inhibin- mRNA levels [37]. These observations indicate that GDF-9 signaling is not necessary for inhibin- expression in vivo. Addition of another oocyte-specific factor, BMP-15 (=GDF-9B), to rat granulosa cell cultures did, however, suppress FSH-induced inhibin/activin subunit mRNA expression [38], consistent with our finding of an oocyte-mediated reduction in inh A and act A peptide secretion in the bovine model.
The effects of oocytes on FSH- and IGF-induced hormone secretion (steroids and inhibin-related peptides) and granulosa cell proliferation were very similar to those we observed previously when bovine granulosa cells were treated with EGF [27], i.e., enhanced cell proliferation and attenuation of FSH- and IGF-induced hormone secretion. Evidence from several species other than cattle indicates that TGF and/or EGF are expressed by the oocyte [19, 20] and by theca cells [39, 40]. In the present study, immunofluorescent staining of both freshly isolated and cocultured oocytes revealed the clear presence of immunoreactive TGF in bovine oocytes; however, EGF immunoreactivity was apparently absent. This finding prompted us to test whether addition of TGF to cultured granulosa cells could mimic the effects of denuded oocytes. Our experiments confirmed that the modulatory actions of TGF on bovine granulosa cell hormone secretion and proliferation were very similar to those elicited by denuded oocytes. The only difference noted was that oocytes, at the dose levels tested, had no effect on basal hormone secretion (in the absence of FSH or IGF), whereas TGF did. However, at the lowest dose tested (0.1 ng/ml), TGF also had no effect on basal hormone secretion but clearly suppressed FSH- and IGF-induced secretion of E2, inh A, act A, and FS in a manner comparable to that observed for oocytes (at 20 oocytes/well). Given the expression of multiple peptides by oocytes, including GDF-9, BMP-15 (GDF-9B), BMP-6, and EGF/TGF [13–18], it seems unlikely that the effects of oocytes are mediated by a single secreted factor. In this study, we also confirmed the presence of GDF-9 immunoreactivity in bovine oocytes. Unfortunately, we currently do not have at our disposal any GDF-9 to directly test in this bovine culture system. In a recent study [41], we showed that BMP-6, another oocyte-secreted factor, can enhance both basal and IGF-induced secretion of E2, inh A, act A, and FS from bovine granulosa cells but had no effect on FSH-induced hormone secretion. These actions are quite distinct from those elicited by oocytes or TGF in the present study. However, BMP-6 suppressed basal and IGF-induced P4 secretion and enhanced cell number in a manner similar to that of oocytes and TGF reported here. The observation that different oocyte-derived factors exert different effects on basal and FSH- and IGF-induced hormone secretion highlights the value of monitoring several different markers of granulosa cell function (i.e., E2, P4, and inhibin-related peptides).
To our knowledge, effects of EGF-related peptides on granulosa cell production of inhibin, activin, and FS proteins have been examined in only one previous study [27]. TGF of thecal origin is probably the main intrafollicular ligand for granulosa cell EGF receptors in the cow [39, 40]. Our observations that both EGF [27] and TGF (present study) can enhance cell proliferation and uniformly suppress FSH- and IGF-induced secretion of four distinct granulosa cell markers (E2, inh A, act A, and FS) further supports the view that paracrine growth factors secreted by theca cells and/or the oocyte interact with granulosa cells to modulate both their proliferation and their hormone-dependent function. BMP-6 [41] of oocyte origin, other BMPs, and GDF-9 most likely also interact with granulosa cells in a similar manner.
Several precautionary points must be considered in relation to these observations. We and others have investigated the effects of oocytes on mural granulosa cells but not on cumulus granulosa cells, which would presumably experience greatest exposure to oocyte-secreted factors in vivo. Given the proposal that an oocyte-secreted factor(s) determines whether granulosa cells differentiate into mural or cumulus granulosa cells [9] and that this differentiation is dependent upon the dose of the factor, observations made on already differentiated cells (i.e., mural or cumulus) may be misleading. Because the oocyte expresses a range of factors (including GDF-9, BMP-15, BMP-6, and TGF), each of which may act in a paracrine fashion on surrounding granulosa cells, until receptors for each factor are identified (possibly stage-specific receptor expression?) and the associated post-receptor events are analyzed, it is difficult to conclude whether one or multiple factors are responsible for the observed effects.
These findings confirm and extend knowledge obtained from previous studies in other species and clearly show that the bovine oocyte secretes a factor(s) that regulates granulosa cell function with regard to cell proliferation, FSH- and IGF-induced steroidogenesis, and FSH- and IGF-stimulated production of inhibin-related peptides. This study also provides evidence pointing to TGF as at least one of several oocyte-derived peptides that mediate this action. The potential involvement of TGF may have been overlooked in other recent studies that have focused on more recently discovered TGFß superfamily members, including GDF-9 and BMP-15. Although the precise mechanisms by which the oocyte influences granulosa cell activity remain to be defined, as does the definitive identification of the oocyte-secreted factor or combination of factors, further investigations should ultimately reveal an important facet of follicular development and control.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 44 118 931 0180; e-mail: p.g.knight{at}reading.ac.uk
Received: 25 June 2002.
First decision: 24 July 2002.
Accepted: 5 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit on the developmental competence of in vitro matured bovine oocytes. J Reprod Fertil 1999 115:381-388C subunit on intrafollicular levels of activin A, inhibin A and follistatin. J Endocrinol 1998 157:157-168[Abstract] present in bovine follicular fluid prevents the suppression of follicle-stimulating hormone-induced production of estradiol by bovine granulosa cells cultured in vitro. Biol Reprod 1997 57:341-346[Abstract]This article has been cited by other articles:
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C. Glister, S. L. Richards, and P. G. Knight Bone Morphogenetic Proteins (BMP) -4, -6, and -7 Potently Suppress Basal and Luteinizing Hormone-Induced Androgen Production by Bovine Theca Interna Cells in Primary Culture: Could Ovarian Hyperandrogenic Dysfunction Be Caused by a Defect in Thecal BMP Signaling? Endocrinology, April 1, 2005; 146(4): 1883 - 1892. [Abstract] [Full Text] [PDF]
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