Lysyl Oxidase Gene Expression and Enzyme Activity in the Rat Ovary: Regulation by Follicle-Stimulating Hormone, Androgen, and Transforming Growth Fact
Abstract3r, http://www.100md.com
Lysyl oxidase (LOX) catalyzes the final enzymatic reaction required for cross-linking of collagen and elastin fibers and therefore has a crucial role in regulating the formation and maintenance of extracellular matrix in the ovary. LOX mRNA is abundantly expressed in rat granulosa cells. To examine how regulation of LOX in the ovary might influence follicular development, we studied LOX mRNA expression and enzyme activity in rat granulosa cells from late preantral/early antral follicles in vitro. FSH dose dependently inhibited LOX mRNA and enzyme activity (50% reduction at 10 ng/ml) in vitro, and FSH action was mimicked by 8-bromo-cAMP, suggesting FSH action via elevation of cAMP. Dihydrotestosterone alone enhanced LOX mRNA and enzyme activity, but potentiated the effect of FSH, causing a further reduction. TGFß1 alone dose dependently enhanced LOX mRNA (5-fold increase at 10 ng/ml) and activity (1.5-fold increase). FSH dose dependently inhibited the increase in LOX mRNA and activity caused by TGFß1 (by up to 84% and 80%, respectively). Growth differentiation factor-9 (GDF-9) and activin A, at the same concentration as TGFß1 (10 ng/ml), stimulated LOX mRNA and activity within 6 h, although overall expression was higher at 48 h. All three factors when combined with FSH further reduced both mRNA and enzyme activity (by up to 60%) compared with FSH alone. These findings indicate control of LOX at endocrine, paracrine, and autocrine levels within the ovary and suggest coordinated regulation of ovarian extracellular matrix during follicular development, with FSH determining whether local factors act as stimulators or inhibitors of LOX.
Introductionxdd, 百拇医药
LYSYL OXIDASE (LOX; EC 1.4.3.13) activity catalyzes the final enzymatic reaction required in the biosynthesis of cross-linked mature collagens and elastin and, as such, is critical in the formation and deposition of extracellular matrix (ECM) (1). LOX is synthesized and secreted as a 50-kDa inactive, N-glycosylated proenzyme (2). It is enzymatically cleaved to a 32-kDa mature, active enzyme; this cleavage has been shown to be effected by procollagen C proteinases, identified as the expressed products of the bone morphogenetic protein-1 gene and the closely related Tll1 and Tll2 genes (3, 4, 5).xdd, 百拇医药
We have recently described expression of the LOX gene in rat granulosa cells using differential display RT-PCR and in situ hybridization techniques and found this gene to be negatively regulated by FSH in pre-/early antral follicles (6). These studies also highlighted another gene, connective tissue growth factor (CTGF), also negatively regulated by FSH. In gingival fibroblasts the expression of LOX and CTGF mRNA and LOX enzyme activity are up-regulated by TGFß (7), but in these cells and in osteoblastic cells (8) LOX activity does not always correlate with mRNA levels. However, to date the only description of LOX activity in the ovary is the observation of peak LOX activity in rabbit follicles just after ovulation (9). The expression of the LOX gene in the ovary has likewise received little attention. There is enhanced expression during progestin-induced ovulation in the perch ovary (10), and also in dehydroepiandrosterone-treated rats, suggesting a potential role for LOX in the development of polycystic ovary syndrome, as LOX gene expression was shown to be up-regulated by androgen (11). Two genes encoding the related proteins, lysyl oxidase-like (LOXL2) protein and LOXL3 protein, are highly expressed in the ovary (12, 13), but their functions remain unknown.
Development of ovarian follicles from primordial to early antral stages and beyond requires a tightly coordinated set of paracrine interactions between both follicular cells (granulosa and theca) and the oocyte. In addition, endocrine factors such as gonadotropins impinge upon and modulate these paracrine events. However, central to the actions of a number of growth factors is the ECM, which comprises the backbone of the follicular basement membrane and is also present in subendothelial basement membrane and surrounding smooth muscle cells of arterioles of the theca (14) as well as thecal stroma (15). ECM binds and sequesters pools of active growth factors such as TGFß (16) and basic fibroblast growth factor (17), and ECM components, including type I collagen, laminin, and fibronectin, can modulate sheep granulosa cell steroidogenesis in vitro (18). Thus, ECM not only provides a structural component to tissues, but is an active modulator of paracrine signals within a given tissue. Given the importance of both endocrine (FSH) and paracrine [growth differentiation factor-9 (GDF-9), activin, TGFß, and androgens] signals in the process of folliculogenesis and the dependence of such follicular growth and development on basement membrane and ECM remodeling/growth, we now report the regulation of gene expression and enzymatic activity of lysyl oxidase in the rat preantral/early antral ovarian follicle.
Materials and Methods;}, 百拇医药
Hormones and tissue culture reagents;}, 百拇医药
Diethylstilbestrol (DES), 5{alpha} -dihydrotestosterone (5{alpha} -DHT), testosterone, 8-bromo-cAMP (8-br-cAMP), recombinant human TGFß1, and BSA (fraction V) were obtained from Sigma (Poole, UK). Recombinant human FSH (3860 IU/mg) was donated by Dr. C. Howles (Serono Laboratories, Inc., Welwyn Garden City, UK). Recombinant mouse GDF-9 protein (a gift from Dr. M. Matzuk, Baylor College of Medicine, Houston, TX) was produced as previously described (19), using CHO cells carrying the full-length mouse GDF-9 cDNA in the pHTop expression vector (a gift from Genetics Institute, Cambridge, MA). Recombinant human activin A was donated by Dr. T. Woodruff (Northwestern University, Evanston, IL). Culture medium was medium 199 with 25 mM HEPES supplemented with 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin (all supplied by Life Technologies, Inc., Paisley, UK), and 0.1% (wt/vol) BSA. Donor calf serum and Dulbecco’s PBS were obtained from Life Technologies, Inc.
Animals3p, 百拇医药
Twenty-one-day-old female Wistar rats (Charles River Laboratories, Inc., Margate, UK) were housed under temperature-controlled conditions on a 12-h light, 12-h dark cycle and fed rat chow ad libitum. Handling and treatment of animals were according to the Animals (Scientific Procedures) Act, 1986. Proliferating, but essentially undifferentiated, granulosa cells were induced by giving two daily sc injections of DES (2 mg/d) in ethanol/propylene glycol (5:95) to stimulate preantral/early antral follicular development.3p, 百拇医药
Granulosa cell isolation and culture3p, 百拇医药
Rats were killed by CO2-induced asphyxiation, and ovaries were recovered immediately. Granulosa cells were isolated by puncturing follicles with a 25-gauge hypodermic needle and gently expelling the cells into the culture medium. Pooled cells were centrifuged, resuspended in fresh medium, and their viability was assessed by trypan blue exclusion in a hemocytometer. Cell viability was 25–30%. Tissue culture grade 24-well plastic dishes (Corning, Inc., Corning, NY) were precoated with 0.25 ml donor calf serum, incubated overnight at 37 C, and washed twice with Dulbecco’s PBS (0.5 ml) before inoculation with 0.25 ml culture medium containing 1 x 105 viable cells. After overnight preincubation at 37 C in a humidified atmosphere containing 5% CO2 in air, 0.25 ml prewarmed medium containing hormone treatments was added (24–36 wells/treatment), and the incubation was continued for 6 or 48 h. All cultures were repeated a minimum of three times using cells from separate sets of rats to permit analyses of statistically significant treatment effects.
RNA isolation and Northern blot analysis^|, 百拇医药
Total RNA was isolated from rat granulosa cell monolayers using RNAzol B (Tel-Test, Friendswood, TX), as previously described (6). Total RNA (5 µg) was size fractionated by electrophoresis on a 1% agarose-formaldehyde denaturing gel for 3 h at 80 V and visualized with ethidium bromide. The RNA was transferred overnight to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Little Chalfont, UK), which was then baked for 2 h at 80 C. Rat LOX transcripts were detected by probing with a 560-bp cDNA clone corresponding to nucleotides 1868–2428 of the full-length rat LOX cDNA (20) (GenBank accession no. U11038), as described previously (6). Northern blot hybridization was performed using standard methods (21). Hybridization was quantified by electronic autoradiography using an Instant Imager (Packard, Downers Grove, IL), and exposed to autoradiographic film (XAR-5, Eastman Kodak Co., Rochester, NY) for 2–24 h at -70 C. After stripping, blots were reprobed with a rat 18S rRNA cDNA probe (donated by Dr. G. Scobie, Medical Research Council Human Reproductive Sciences Unit, Edinburgh, UK) to allow correction for gel loading and transfer. Northern analysis data are presented relative to unstimulated control values.
LOX activity assayi, 百拇医药
LOX enzyme activity secreted into culture medium was assessed using tritiated recombinant human tropoelastin, expressed in and purified from Escherichia coli as previously described (22, 23). The plasmid was provided by Dr. Joel Rosenbloom (University of Philadelphia School of Dental Medicine, Philadelphia, PA). Assays were performed as described previously (7), with minor modifications. Briefly, 0.5- to 0.75-ml samples were assayed in triplicate in the presence or absence of the specific LOX inhibitor ß-aminopropionitrile (BAPN; 0.5 mM) in a final volume of 1.5 ml assay buffer (0.1 M borate and 0.15 M NaCl, pH 8.0) and 140,000 cpm tritiated recombinant human tropoelastin at 55 C for 3 h. After collection of released tritiated water by vacuum distillation and quantification by liquid scintillation spectrometry, values for BAPN-containing samples were subtracted from samples assayed in the absence of BAPN to give the specific counts per minute released by LOX. Separate experiments were normalized on the basis of cell number per well. Data from experimental treatments are presented as fold/percent changes compared with untreated control cultures.
Data analysis60y;e@], 百拇医药
Data were analyzed by one-way ANOVA, using Student’s t test to identify significant differences between treatments. In cases where the data were not normally distributed, log transformation was performed before analysis.60y;e@], 百拇医药
Results60y;e@], 百拇医药
Inhibition of LOX mRNA expression and enzyme activity by FSH60y;e@], 百拇医药
Northern analysis revealed an approximately 5.2-kb LOX mRNA transcript, with an associated, less abundant, approximately 3.8-kb transcript. LOX mRNA was clearly inhibited after 48-h exposure to FSH, with 10 ng/ml FSH reducing expression to less than 60% of the control value (Fig. 1, A and B). LOX activity was dose dependently inhibited by FSH, being reduced 50% by 10 ng/ml FSH (Fig. 1C).60y;e@], 百拇医药
fig.ommitteed60y;e@], 百拇医药
Figure 1. Dose-dependent effects of FSH on rat granulosa cell LOX mRNA (A and B) and LOX activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing dose-dependent inhibition of the approximately 5.2-kb LOX transcript and the less abundant, approximately 3.8-kb transcript compared with the signal for 18S rRNA (18S). B, Composite data from five separate experiments. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 4.74; P = 0.045). a, P < 0.05 compared with control. C, LOX activity after 48 h culture. Bars indicate the mean ± SEM levels in culture medium in four separate experiments, after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. a, P < 0.05 compared with control.
Intermediary role of cAMP in suppression of LOX mRNA expression and enzyme activity, and the role of androgenssr, 百拇医药
The cAMP analog 8-br-cAMP (1.0 mM) had an inhibitory effect on LOX mRNA, similar to that observed with FSH (Fig. 2, A and B). The nonaromatizable androgen DHT (1 µM) further enhanced the inhibition of LOX by FSH, and this effect was near the level of statistical significance (P = 0.08; see FSH + DHT in Fig. 2, A and B). DHT had no significant additive effect on 8-br-cAMP inhibition of LOX mRNA (Fig. 2, A and B). DHT alone slightly increased LOX mRNA (P = 0.07; Fig. 2, A and B). LOX activity was inhibited 40–50% by both FSH and 8-br-cAMP (P < 0.001), whereas DHT alone increased activity by 20% (P < 0.05; Fig. 2C). DHT further reduced the inhibition of activity by FSH (P < 0.01), but did not affect the inhibition by 8-br-cAMP (Fig. 2C).ssr, 百拇医药
fig.ommitteedssr, 百拇医药
Figure 2. Actions of FSH (10 ng/ml), 8-br-cAMP (cAMP; 1 mM), and 5{alpha} -DHT (1 µM) on rat granulosa cell LOX mRNA (A and B) and LOX activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing LOX mRNA compared with the signal for 18S rRNA (18S). B, Composite data from three separate experiments. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography. All data were normalized to the control value. a, P < 0.05 compared with controls; b, P = 0.07 compared with controls; c, P = 0.08 compared with FSH. C, LOX activity after 48-h culture. Bars indicate the mean ± SEM levels in culture medium in three separate experiments after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 182.45; P < 0.001). a and b, P < 0.001 compared with control; c, P < 0.05 compared with control; d, P < 0.01 compared with FSH; e, P = NS compared with cAMP.
TGFß1 dose dependently enhances LOX mRNA expression and enzyme activityg, 百拇医药
TGFß1 dramatically enhanced LOX mRNA expression, with 10 ng/ml TGFß1 producing a 5-fold increase (P < 0.05), and lower doses of TGFß-1 causing a modest stimulation indicative of a dose-dependent response (Fig. 3, A and B). LOX enzyme activity was also dose dependently increased by TGFß1, although to a lesser extent, with 10 ng/ml increasing levels by 50% (P < 0.01; Fig. 3C).g, 百拇医药
fig.ommitteedg, 百拇医药
Figure 3. Dose-dependent effects of TGFß1 on rat granulosa cell LOX mRNA (A and B) and LOX activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing LOX mRNA compared with the signal for 18S rRNA (18S). B, Composite data from five separate experiments. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography. All data were normalized to the control value. ANOVA showed a trend, although not significant, due to effect of treatment (F = 2.17; P = 0.17). a, P < 0.05 compared with controls. C, LOX activity after 48-h culture. Bars indicate the mean ± SEM levels in culture medium in five separate experiments after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 20.54; P < 0.001). a, P < 0.05 compared with controls.
Interaction between FSH and TGFß1 action on LOX mRNA expression and enzyme activityn, 百拇医药
The increase in LOX mRNA expression produced by TGFß1 was dose dependently inhibited by FSH, with 10 ng/ml FSH reducing the expression by over 80% (P < 0.05; Fig. 4, A and B). TGFß1-stimulated LOX enzyme activity was also dose dependently suppressed by FSH, with 10 ng/ml FSH again reducing activity by 80% (P < 0.01; Fig. 4C).n, 百拇医药
fig.ommitteedn, 百拇医药
Figure 4. Dose-dependent effects of FSH on TGFß1-stimulated rat granulosa cell LOX mRNA (A and B) and LOX activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing LOX mRNA compared with the signal for 18S rRNA (18S). B, Composite data from three separate experiments. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F =100.38; P < 0.001). a, P < 0.05 compared with controls; b, P < 0.05 compared with TGFß1 alone. C, LOX activity after 48-h culture. Bars indicate the mean ± SEM levels in culture medium in three separate experiments after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 277.14; P < 0.001.). a, P < 0.01 compared with controls; b, P < 0.01 compared with TGFß1.
Time-dependent effects of TGFß1 and other TGFß superfamily members, GDF-9 and activin A, on LOX mRNA expression and enzyme activity/7[, 百拇医药
At 6 h, GDF-9 and activin A (both at 10 ng/ml) stimulated LOX mRNA expression in a similar fashion as TGFß1 (P < 0.05; Figs. 5 and 6A). FSH inhibited both basal and TGFß1-, GDF-9-, and activin A-stimulated LOX mRNA expression (P < 0.05; Figs. 5 and 6A), although there was no additional effect of the growth factors on inhibition by FSH at this time point (compare with Fig. 6B). At 48 h, TGFß1, GDF-9, and activin A all stimulated LOX mRNA expression (P < 0.05; Figs. 5 and 6B). FSH significantly inhibited all treatments (P < 0.05), and importantly, when any of the growth factors was combined with FSH, LOX mRNA was significantly suppressed compared with FSH treatment alone (P < 0.05; Figs. 5and 6B). LOX enzyme activity was measurable only at very low levels at 6 h (results not shown). At 48 h the pattern of LOX enzyme activity followed that of mRNA expression very closely, although GDF-9 did not stimulate (Fig. 6C). Once again, all of the growth factors when combined with FSH reduced LOX enzyme activity to below the level with FSH alone (P < 0.05; Fig. 6C).
fig.ommitteed-jo, 百拇医药
Figure 5. Comparison of the effects of TGFß1, GDF-9, and activin (Act) (all at 10 ng/ml) in the presence or absence of FSH (10 ng/ml) on rat granulosa cell LOX mRNA after 6 and 48 h of treatment. Shown is an autoradiograph from a typical Northern analysis showing LOX mRNA compared with the signal for 18S rRNA (18S).-jo, 百拇医药
fig.ommitteed-jo, 百拇医药
Figure 6. A, Composite data from three separate experiments comparing the effects of TGFß1, GDF-9, and activin (Act; all at 10 ng/ml) in the presence or absence of FSH (10 ng/ml) on rat granulosa cell LOX mRNA after 6 h of treatment. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography in three experiments. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 33.0; P < 0.0001). a, P < 0.01 compared with controls; b, P < 0.05 compared with controls; c, P < 0.05 compared with FSH plus TGFß1; d, P < 0.001 compared with FSH plus GDF-9; e, P < 0.01 compared with FSH plus activin. B, As in A, but continued for 48 h, with data normalized to the control value at 48 h. ANOVA showed a significant effect of treatment (F = 31.8; P < 0.0001). a, P < 0.05 compared with controls; b, P < 0.01 compared with FSH and TGF treatments alone; c, P < 0.05 compared with FSH and GDF-9 treatments alone; d, P < 0.05 compared with FSH and activin treatments alone. C, LOX activity after 48-h culture. Bars indicate the mean ± SEM levels in culture medium in three separate experiments after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 2.82; P < 0.05). a, P < 0.05 compared with controls; b, P < 0.05 compared with FSH; c, P = NS compared with controls.
Discussionwh, http://www.100md.com
Although considerable attention has focused on the factors regulating breakdown of the ECM in tissue remodeling of the ovary (24), there is scant evidence for the regulation of ECM deposition.wh, http://www.100md.com
In this study we demonstrate for the first time the detailed analysis of LOX in the mammalian ovary and show that both mRNA and enzyme activity are regulated by FSH, androgen, and TGFß superfamily members. LOX is a key enzyme in ECM formation in a wide range of tissues and cell types, being responsible for the final catalytic cross-linking of collagen and elastin monomers (1, 25). Although the cellular origins of the type IV collagen-rich basement membrane of the follicle are a matter of controversy (26), there is evidence that granulosa cells have the capacity to secrete many of its components (14). We have previously demonstrated LOX mRNA expression restricted to the granulosa cell compartment of the ovary, which points to a local mechanism for assembly of the basement membrane ECM collagen component (6). However, expression of type VI collagen has been demonstrated in the theca of developing human follicles (27), and the newly identified type XXVI collagen is expressed in prethecal cells of the mouse neonate (28). We have also observed high levels of LOX enzyme activity in human theca cells (Rae, M., and C. R. Harlow, unpublished observation), suggesting that transfer of LOX into the theca may occur.
We previously demonstrated that LOX mRNA is expressed in granulosa cells from late antral/early preantral rat follicles and is down-regulated by FSH both in vitro and in vivo (6). We now confirm that expression of LOX mRNA is dose dependently regulated by FSH in vitro, and that this is reflected in reduced activity of the mature LOX enzyme. Thus, LOX, a key enzyme in the assembly of mature collagen and elastin, can now be added to the repertoire of granulosa cell FSH-regulated genes that includes steroidogenic enzymes (29, 30) and LH receptors (31). However, like CTGF, which we recently showed was also under FSH regulation in granulosa cells (21), LOX is down-regulated by FSH through elevation of cAMP. Interestingly, cAMP also suppresses both basal and TGFß-induced collagen and LOX mRNA in human lung fibroblasts (32), although it has the opposite effect on LOX mRNA and enzyme activity in vascular smooth muscle cells (33). FSH receptor mRNA is developmentally regulated in the human follicle, being absent at the primordial stage, found intermittently in primary and two-layered preantral follicles, and expressed ubiquitously in multilayered follicles (34), raising the possibility of developmental regulation of LOX by FSH in the ovary.
By interacting with FSH, androgens are known to directly enhance many differentiation processes, such as estrogen and progestogen biosynthesis (35, 36), induction of LH receptors (37), and carbohydrate metabolism (38). The mechanism of action of androgens in this process appears to be through alterations in cAMP metabolism (39). By contrast, in the presence of FSH, further down-regulation of LOX mRNA and enzyme activity by DHT was observed in the same fashion that we described previously for CTGF (21). FSH receptor expression is up-regulated in the primate ovary by receptor-mediated androgen action (40). We await direct evidence of androgen receptor involvement in LOX expression, but it seems reasonable to assume that the mechanisms are similar.(%6, 百拇医药
Interestingly, DHT alone caused a modest increase in both LOX mRNA and enzyme activity, which parallels our observation of DHT action on CTGF mRNA described previously (21) and is in keeping with the finding that dehydroepiandrosterone treatment of rats increases ovarian LOX expression (11). Androgens also act in bovine aorta smooth muscle cells in vitro (41) and mouse cervix in vivo (42) to enhance LOX enzyme activity.
TGFß has been identified as a key regulator of LOX in diverse cell types, up-regulating both mRNA and enzyme activity in lung fibroblasts (43), osteoblasts (8), aortic smooth muscle cells (44), and human gingival fibroblasts (7). In the DES-primed immature rat ovary, TGFß1 mRNA expression is principally located in thecal/interstitial cells of preantral follicles (45). Immunolocalization studies showed intense TGFß1 staining in follicles around the time of antrum formation (46). Therefore, our observation of a dose-dependent stimulation of LOX by TGFß1 in rat granulosa cells points to a local paracrine regulation in the ovary.-0k@1+, 百拇医药
In embryonic lung fibroblasts, prostaglandin E2 reverses TGFß up-regulation of LOX mRNA via a cAMP-mediated mechanism (32), and cAMP is able to block TGFß-stimulated collagen synthesis (47). As cAMP mimicked the effects of FSH on LOX mRNA and enzyme activity, we believe that the dose-dependent inhibition by FSH of TGFß1-stimulated LOX is probably cAMP mediated. Paradoxically, TGFß1 combined with FSH consistently reduced LOX mRNA and enzyme activity to below the level achieved with FSH alone, although this could be explained by the actions of TGFß1 in enhancing FSH receptor expression (48, 49). Overall, we have demonstrated that the stimulatory action of TGFß1 on LOX appears insufficient to overcome the more potent inhibitory effect of FSH on rat granulosa cells from preantral/early antral rat follicles in vitro.
FSH action on granulosa cells is subject to paracrine modulation, not only by theca-derived TGFß1 (50), but also by GDF-9 originating from the oocyte (51, 52). Activin is another structurally related member of the TGFß superfamily of granulosa cell origin. It has autocrine effects on mitogenic activity (53), 17ß-hydroxysteroid dehydrogenase type I mRNA expression and enzyme activity (54), FSH and LH receptor mRNA expression (55), and P450 aromatase activity (56). Activin also modulates aromatase activity indirectly, by paracrine inhibition of LH-induced thecal androgen production (57). Moreover, activin A enhances the expression of FSH receptor mRNA in immature rat granulosa cells (58), and there is preferential synthesis of activin over inhibin at the earliest stages of follicular development (59). We recently showed that all three of these TGFß superfamily members enhance CTGF mRNA expression, but are inhibitory in the presence of FSH (21). We can now add that LOX, at the levels of mRNA and active enzyme, is under a similar control mechanism, and that the direction of action (stimulatory or inhibitory) of these factors is dependent on the degree of exposure of the granulosa cells to FSH. Furthermore, although inhibition of LOX by FSH alone was observed at 6 h, the additional presence of TGFß superfamily members did not have an inhibitory effect until 48 h. This delay may reflect the time necessary for these factors to induce FSH receptor expression (60, 61). Given the links established between TGFß1, CTGF, LOX, and insoluble collagen accumulation in human gingiva (7), we speculate that these factors will have a crucial role in coordinating the accumulation of ECM in the ovary and may be involved in basement membrane formation, thecal cell recruitment, follicular expansion, ovulation and the repair of the ovarian surface, and corpus luteum formation. LOX may also have a role in laying down the collagen type IV component of Call-Exner bodies, whose walls are lined with basal lamina and whose cavities contain large aggregates of convoluted basal lamina (14). The number of positively staining collagen type IV Call-Exner bodies is reduced in antral follicles (14) at a time when LOX is also decreasing (6), which is further evidence for a possible role of LOX in the development of these structures.
We have previously developed a working hypothesis for CTGF as a transducer of FSH and intrafollicular factors in follicle development (21). We now extend this to include the actions of LOX as a key factor in the synthesis of ECM. The presence of LOX at the early stages of follicular development (6) and the regulation of expression and enzyme activity by GDF-9 point to a possible role of LOX, in addition to CTGF (6), in transducing oocyte-derived GDF-9 signaling of basement membrane and thecal development (Fig. 7A). The additional stimulation of LOX by TGFß1 and activin at a time of low levels of FSH and low FSH receptor availability suggests communication between thecal and granulosa cell layers in multilayered preantral follicles, which may facilitate expansion of the ECM components in the basement membrane and theca during antrum formation (Fig. 7B). Our in vitro findings are substantiated by the observation that granulosa cells freshly isolated from preantral follicles of unstimulated or DES-treated 23-d-old rats have high endogenous LOX mRNA expression (6). However, in the antral follicle, thecal androgens and TGFß have up-regulated FSH receptors, and there is greater availability of FSH, resulting in these TGFß superfamily members enhancing the inhibitory effects of FSH on LOX (Fig. 7C), although GDF-9 may not be impinging on mural granulosa cells due to gradient effects resulting from spatial removal of the oocyte from the mural granulosa cells (Fig. 7C) (62). At this time, we observed a massive increase in aromatase mRNA expression at the precise point when LOX mRNA expression is declining in vivo (6). In other words, conditions that promote differentiation of granulosa cells toward maximal estradiol secretion switch off LOX activity. However, the factors that regulate LOX activity in more fully differentiated granulosa cells remain to be determined.
fig.ommitteed:iqy9o, 百拇医药
Figure 7. A working hypothesis of FSH and TGFß superfamily actions in the developing follicle. A, In the primary follicle, in the absence of FSH stimulation, GDF-9 and activin stimulate LOX mRNA expression in granulosa cells. We suggest the secreted LOX enzyme initiates mature collagen synthesis in the emerging thecal cell layer and orchestrates ECM deposition in the developing basement membrane. B, The multilayered granulosa cells of the preantral follicle have relatively few FSH receptors and are exposed to low levels of FSH. In this environment, GDF-9, activin, and TGFß stimulate high expression of LOX mRNA and the secreted enzyme, which could coordinate theca and basement membrane expansion as well as formation of the antral cavity. LOX could also synthesize the basal lamina components of Call-Exner bodies. C, In the antral follicle, greater FSH receptor availability and higher FSH levels would serve to switch the effects of activin and TGFß from stimulatory to inhibitory, resulting in reduced LOX mRNA expression and enzyme activity concomitant with the rapid mobilization of aromatase activity. GDF-9 may have less of an effect on mural granulosa cells due to gradient effects, as the oocyte is now spatially removed from the mural granulosa cells.
In summary, granulosa cells from late preantral/early antral rat follicles express LOX mRNA and mature LOX enzyme, which are regulated at endocrine, paracrine, and autocrine levels by FSH, androgen, and TGFß superfamily members coming from the oocyte, thecal cells, and the granulosa compartment itself. TGFß1, GDF-9, and activin are stimulatory, whereas FSH, by raising cAMP levels, is inhibitory. In the presence of FSH, TGFß superfamily members lose their stimulatory effect and enhance the inhibitory effect of FSH. This coordinated control of a key enzyme in ECM deposition predicts that the regulation of LOX will have a crucial role in tissue remodeling and ECM formation during all phases of follicular development./%8qi], 百拇医药
Received June 25, 2002./%8qi], 百拇医药
Accepted for publication September 13, 2002./%8qi], 百拇医药
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Erickson GF, Shimasaki S 2000 The role of the oocyte in folliculogenesis. Trends Endocrinol Metab 11:193–198(Christopher R. Harlow Mick Rae Lindsay Davidson Philip C. Trackman and Stephen G. Hillier)
Lysyl oxidase (LOX) catalyzes the final enzymatic reaction required for cross-linking of collagen and elastin fibers and therefore has a crucial role in regulating the formation and maintenance of extracellular matrix in the ovary. LOX mRNA is abundantly expressed in rat granulosa cells. To examine how regulation of LOX in the ovary might influence follicular development, we studied LOX mRNA expression and enzyme activity in rat granulosa cells from late preantral/early antral follicles in vitro. FSH dose dependently inhibited LOX mRNA and enzyme activity (50% reduction at 10 ng/ml) in vitro, and FSH action was mimicked by 8-bromo-cAMP, suggesting FSH action via elevation of cAMP. Dihydrotestosterone alone enhanced LOX mRNA and enzyme activity, but potentiated the effect of FSH, causing a further reduction. TGFß1 alone dose dependently enhanced LOX mRNA (5-fold increase at 10 ng/ml) and activity (1.5-fold increase). FSH dose dependently inhibited the increase in LOX mRNA and activity caused by TGFß1 (by up to 84% and 80%, respectively). Growth differentiation factor-9 (GDF-9) and activin A, at the same concentration as TGFß1 (10 ng/ml), stimulated LOX mRNA and activity within 6 h, although overall expression was higher at 48 h. All three factors when combined with FSH further reduced both mRNA and enzyme activity (by up to 60%) compared with FSH alone. These findings indicate control of LOX at endocrine, paracrine, and autocrine levels within the ovary and suggest coordinated regulation of ovarian extracellular matrix during follicular development, with FSH determining whether local factors act as stimulators or inhibitors of LOX.
Introductionxdd, 百拇医药
LYSYL OXIDASE (LOX; EC 1.4.3.13) activity catalyzes the final enzymatic reaction required in the biosynthesis of cross-linked mature collagens and elastin and, as such, is critical in the formation and deposition of extracellular matrix (ECM) (1). LOX is synthesized and secreted as a 50-kDa inactive, N-glycosylated proenzyme (2). It is enzymatically cleaved to a 32-kDa mature, active enzyme; this cleavage has been shown to be effected by procollagen C proteinases, identified as the expressed products of the bone morphogenetic protein-1 gene and the closely related Tll1 and Tll2 genes (3, 4, 5).xdd, 百拇医药
We have recently described expression of the LOX gene in rat granulosa cells using differential display RT-PCR and in situ hybridization techniques and found this gene to be negatively regulated by FSH in pre-/early antral follicles (6). These studies also highlighted another gene, connective tissue growth factor (CTGF), also negatively regulated by FSH. In gingival fibroblasts the expression of LOX and CTGF mRNA and LOX enzyme activity are up-regulated by TGFß (7), but in these cells and in osteoblastic cells (8) LOX activity does not always correlate with mRNA levels. However, to date the only description of LOX activity in the ovary is the observation of peak LOX activity in rabbit follicles just after ovulation (9). The expression of the LOX gene in the ovary has likewise received little attention. There is enhanced expression during progestin-induced ovulation in the perch ovary (10), and also in dehydroepiandrosterone-treated rats, suggesting a potential role for LOX in the development of polycystic ovary syndrome, as LOX gene expression was shown to be up-regulated by androgen (11). Two genes encoding the related proteins, lysyl oxidase-like (LOXL2) protein and LOXL3 protein, are highly expressed in the ovary (12, 13), but their functions remain unknown.
Development of ovarian follicles from primordial to early antral stages and beyond requires a tightly coordinated set of paracrine interactions between both follicular cells (granulosa and theca) and the oocyte. In addition, endocrine factors such as gonadotropins impinge upon and modulate these paracrine events. However, central to the actions of a number of growth factors is the ECM, which comprises the backbone of the follicular basement membrane and is also present in subendothelial basement membrane and surrounding smooth muscle cells of arterioles of the theca (14) as well as thecal stroma (15). ECM binds and sequesters pools of active growth factors such as TGFß (16) and basic fibroblast growth factor (17), and ECM components, including type I collagen, laminin, and fibronectin, can modulate sheep granulosa cell steroidogenesis in vitro (18). Thus, ECM not only provides a structural component to tissues, but is an active modulator of paracrine signals within a given tissue. Given the importance of both endocrine (FSH) and paracrine [growth differentiation factor-9 (GDF-9), activin, TGFß, and androgens] signals in the process of folliculogenesis and the dependence of such follicular growth and development on basement membrane and ECM remodeling/growth, we now report the regulation of gene expression and enzymatic activity of lysyl oxidase in the rat preantral/early antral ovarian follicle.
Materials and Methods;}, 百拇医药
Hormones and tissue culture reagents;}, 百拇医药
Diethylstilbestrol (DES), 5{alpha} -dihydrotestosterone (5{alpha} -DHT), testosterone, 8-bromo-cAMP (8-br-cAMP), recombinant human TGFß1, and BSA (fraction V) were obtained from Sigma (Poole, UK). Recombinant human FSH (3860 IU/mg) was donated by Dr. C. Howles (Serono Laboratories, Inc., Welwyn Garden City, UK). Recombinant mouse GDF-9 protein (a gift from Dr. M. Matzuk, Baylor College of Medicine, Houston, TX) was produced as previously described (19), using CHO cells carrying the full-length mouse GDF-9 cDNA in the pHTop expression vector (a gift from Genetics Institute, Cambridge, MA). Recombinant human activin A was donated by Dr. T. Woodruff (Northwestern University, Evanston, IL). Culture medium was medium 199 with 25 mM HEPES supplemented with 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin (all supplied by Life Technologies, Inc., Paisley, UK), and 0.1% (wt/vol) BSA. Donor calf serum and Dulbecco’s PBS were obtained from Life Technologies, Inc.
Animals3p, 百拇医药
Twenty-one-day-old female Wistar rats (Charles River Laboratories, Inc., Margate, UK) were housed under temperature-controlled conditions on a 12-h light, 12-h dark cycle and fed rat chow ad libitum. Handling and treatment of animals were according to the Animals (Scientific Procedures) Act, 1986. Proliferating, but essentially undifferentiated, granulosa cells were induced by giving two daily sc injections of DES (2 mg/d) in ethanol/propylene glycol (5:95) to stimulate preantral/early antral follicular development.3p, 百拇医药
Granulosa cell isolation and culture3p, 百拇医药
Rats were killed by CO2-induced asphyxiation, and ovaries were recovered immediately. Granulosa cells were isolated by puncturing follicles with a 25-gauge hypodermic needle and gently expelling the cells into the culture medium. Pooled cells were centrifuged, resuspended in fresh medium, and their viability was assessed by trypan blue exclusion in a hemocytometer. Cell viability was 25–30%. Tissue culture grade 24-well plastic dishes (Corning, Inc., Corning, NY) were precoated with 0.25 ml donor calf serum, incubated overnight at 37 C, and washed twice with Dulbecco’s PBS (0.5 ml) before inoculation with 0.25 ml culture medium containing 1 x 105 viable cells. After overnight preincubation at 37 C in a humidified atmosphere containing 5% CO2 in air, 0.25 ml prewarmed medium containing hormone treatments was added (24–36 wells/treatment), and the incubation was continued for 6 or 48 h. All cultures were repeated a minimum of three times using cells from separate sets of rats to permit analyses of statistically significant treatment effects.
RNA isolation and Northern blot analysis^|, 百拇医药
Total RNA was isolated from rat granulosa cell monolayers using RNAzol B (Tel-Test, Friendswood, TX), as previously described (6). Total RNA (5 µg) was size fractionated by electrophoresis on a 1% agarose-formaldehyde denaturing gel for 3 h at 80 V and visualized with ethidium bromide. The RNA was transferred overnight to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Little Chalfont, UK), which was then baked for 2 h at 80 C. Rat LOX transcripts were detected by probing with a 560-bp cDNA clone corresponding to nucleotides 1868–2428 of the full-length rat LOX cDNA (20) (GenBank accession no. U11038), as described previously (6). Northern blot hybridization was performed using standard methods (21). Hybridization was quantified by electronic autoradiography using an Instant Imager (Packard, Downers Grove, IL), and exposed to autoradiographic film (XAR-5, Eastman Kodak Co., Rochester, NY) for 2–24 h at -70 C. After stripping, blots were reprobed with a rat 18S rRNA cDNA probe (donated by Dr. G. Scobie, Medical Research Council Human Reproductive Sciences Unit, Edinburgh, UK) to allow correction for gel loading and transfer. Northern analysis data are presented relative to unstimulated control values.
LOX activity assayi, 百拇医药
LOX enzyme activity secreted into culture medium was assessed using tritiated recombinant human tropoelastin, expressed in and purified from Escherichia coli as previously described (22, 23). The plasmid was provided by Dr. Joel Rosenbloom (University of Philadelphia School of Dental Medicine, Philadelphia, PA). Assays were performed as described previously (7), with minor modifications. Briefly, 0.5- to 0.75-ml samples were assayed in triplicate in the presence or absence of the specific LOX inhibitor ß-aminopropionitrile (BAPN; 0.5 mM) in a final volume of 1.5 ml assay buffer (0.1 M borate and 0.15 M NaCl, pH 8.0) and 140,000 cpm tritiated recombinant human tropoelastin at 55 C for 3 h. After collection of released tritiated water by vacuum distillation and quantification by liquid scintillation spectrometry, values for BAPN-containing samples were subtracted from samples assayed in the absence of BAPN to give the specific counts per minute released by LOX. Separate experiments were normalized on the basis of cell number per well. Data from experimental treatments are presented as fold/percent changes compared with untreated control cultures.
Data analysis60y;e@], 百拇医药
Data were analyzed by one-way ANOVA, using Student’s t test to identify significant differences between treatments. In cases where the data were not normally distributed, log transformation was performed before analysis.60y;e@], 百拇医药
Results60y;e@], 百拇医药
Inhibition of LOX mRNA expression and enzyme activity by FSH60y;e@], 百拇医药
Northern analysis revealed an approximately 5.2-kb LOX mRNA transcript, with an associated, less abundant, approximately 3.8-kb transcript. LOX mRNA was clearly inhibited after 48-h exposure to FSH, with 10 ng/ml FSH reducing expression to less than 60% of the control value (Fig. 1, A and B). LOX activity was dose dependently inhibited by FSH, being reduced 50% by 10 ng/ml FSH (Fig. 1C).60y;e@], 百拇医药
fig.ommitteed60y;e@], 百拇医药
Figure 1. Dose-dependent effects of FSH on rat granulosa cell LOX mRNA (A and B) and LOX activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing dose-dependent inhibition of the approximately 5.2-kb LOX transcript and the less abundant, approximately 3.8-kb transcript compared with the signal for 18S rRNA (18S). B, Composite data from five separate experiments. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 4.74; P = 0.045). a, P < 0.05 compared with control. C, LOX activity after 48 h culture. Bars indicate the mean ± SEM levels in culture medium in four separate experiments, after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. a, P < 0.05 compared with control.
Intermediary role of cAMP in suppression of LOX mRNA expression and enzyme activity, and the role of androgenssr, 百拇医药
The cAMP analog 8-br-cAMP (1.0 mM) had an inhibitory effect on LOX mRNA, similar to that observed with FSH (Fig. 2, A and B). The nonaromatizable androgen DHT (1 µM) further enhanced the inhibition of LOX by FSH, and this effect was near the level of statistical significance (P = 0.08; see FSH + DHT in Fig. 2, A and B). DHT had no significant additive effect on 8-br-cAMP inhibition of LOX mRNA (Fig. 2, A and B). DHT alone slightly increased LOX mRNA (P = 0.07; Fig. 2, A and B). LOX activity was inhibited 40–50% by both FSH and 8-br-cAMP (P < 0.001), whereas DHT alone increased activity by 20% (P < 0.05; Fig. 2C). DHT further reduced the inhibition of activity by FSH (P < 0.01), but did not affect the inhibition by 8-br-cAMP (Fig. 2C).ssr, 百拇医药
fig.ommitteedssr, 百拇医药
Figure 2. Actions of FSH (10 ng/ml), 8-br-cAMP (cAMP; 1 mM), and 5{alpha} -DHT (1 µM) on rat granulosa cell LOX mRNA (A and B) and LOX activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing LOX mRNA compared with the signal for 18S rRNA (18S). B, Composite data from three separate experiments. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography. All data were normalized to the control value. a, P < 0.05 compared with controls; b, P = 0.07 compared with controls; c, P = 0.08 compared with FSH. C, LOX activity after 48-h culture. Bars indicate the mean ± SEM levels in culture medium in three separate experiments after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 182.45; P < 0.001). a and b, P < 0.001 compared with control; c, P < 0.05 compared with control; d, P < 0.01 compared with FSH; e, P = NS compared with cAMP.
TGFß1 dose dependently enhances LOX mRNA expression and enzyme activityg, 百拇医药
TGFß1 dramatically enhanced LOX mRNA expression, with 10 ng/ml TGFß1 producing a 5-fold increase (P < 0.05), and lower doses of TGFß-1 causing a modest stimulation indicative of a dose-dependent response (Fig. 3, A and B). LOX enzyme activity was also dose dependently increased by TGFß1, although to a lesser extent, with 10 ng/ml increasing levels by 50% (P < 0.01; Fig. 3C).g, 百拇医药
fig.ommitteedg, 百拇医药
Figure 3. Dose-dependent effects of TGFß1 on rat granulosa cell LOX mRNA (A and B) and LOX activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing LOX mRNA compared with the signal for 18S rRNA (18S). B, Composite data from five separate experiments. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography. All data were normalized to the control value. ANOVA showed a trend, although not significant, due to effect of treatment (F = 2.17; P = 0.17). a, P < 0.05 compared with controls. C, LOX activity after 48-h culture. Bars indicate the mean ± SEM levels in culture medium in five separate experiments after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 20.54; P < 0.001). a, P < 0.05 compared with controls.
Interaction between FSH and TGFß1 action on LOX mRNA expression and enzyme activityn, 百拇医药
The increase in LOX mRNA expression produced by TGFß1 was dose dependently inhibited by FSH, with 10 ng/ml FSH reducing the expression by over 80% (P < 0.05; Fig. 4, A and B). TGFß1-stimulated LOX enzyme activity was also dose dependently suppressed by FSH, with 10 ng/ml FSH again reducing activity by 80% (P < 0.01; Fig. 4C).n, 百拇医药
fig.ommitteedn, 百拇医药
Figure 4. Dose-dependent effects of FSH on TGFß1-stimulated rat granulosa cell LOX mRNA (A and B) and LOX activity (C) after 48-h culture. Controls (Con) received no treatment. A, Autoradiograph from a typical Northern analysis showing LOX mRNA compared with the signal for 18S rRNA (18S). B, Composite data from three separate experiments. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F =100.38; P < 0.001). a, P < 0.05 compared with controls; b, P < 0.05 compared with TGFß1 alone. C, LOX activity after 48-h culture. Bars indicate the mean ± SEM levels in culture medium in three separate experiments after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 277.14; P < 0.001.). a, P < 0.01 compared with controls; b, P < 0.01 compared with TGFß1.
Time-dependent effects of TGFß1 and other TGFß superfamily members, GDF-9 and activin A, on LOX mRNA expression and enzyme activity/7[, 百拇医药
At 6 h, GDF-9 and activin A (both at 10 ng/ml) stimulated LOX mRNA expression in a similar fashion as TGFß1 (P < 0.05; Figs. 5 and 6A). FSH inhibited both basal and TGFß1-, GDF-9-, and activin A-stimulated LOX mRNA expression (P < 0.05; Figs. 5 and 6A), although there was no additional effect of the growth factors on inhibition by FSH at this time point (compare with Fig. 6B). At 48 h, TGFß1, GDF-9, and activin A all stimulated LOX mRNA expression (P < 0.05; Figs. 5 and 6B). FSH significantly inhibited all treatments (P < 0.05), and importantly, when any of the growth factors was combined with FSH, LOX mRNA was significantly suppressed compared with FSH treatment alone (P < 0.05; Figs. 5and 6B). LOX enzyme activity was measurable only at very low levels at 6 h (results not shown). At 48 h the pattern of LOX enzyme activity followed that of mRNA expression very closely, although GDF-9 did not stimulate (Fig. 6C). Once again, all of the growth factors when combined with FSH reduced LOX enzyme activity to below the level with FSH alone (P < 0.05; Fig. 6C).
fig.ommitteed-jo, 百拇医药
Figure 5. Comparison of the effects of TGFß1, GDF-9, and activin (Act) (all at 10 ng/ml) in the presence or absence of FSH (10 ng/ml) on rat granulosa cell LOX mRNA after 6 and 48 h of treatment. Shown is an autoradiograph from a typical Northern analysis showing LOX mRNA compared with the signal for 18S rRNA (18S).-jo, 百拇医药
fig.ommitteed-jo, 百拇医药
Figure 6. A, Composite data from three separate experiments comparing the effects of TGFß1, GDF-9, and activin (Act; all at 10 ng/ml) in the presence or absence of FSH (10 ng/ml) on rat granulosa cell LOX mRNA after 6 h of treatment. Bars indicate the mean ± SEM LOX mRNA intensity relative to 18S rRNA measured by electronic autoradiography in three experiments. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 33.0; P < 0.0001). a, P < 0.01 compared with controls; b, P < 0.05 compared with controls; c, P < 0.05 compared with FSH plus TGFß1; d, P < 0.001 compared with FSH plus GDF-9; e, P < 0.01 compared with FSH plus activin. B, As in A, but continued for 48 h, with data normalized to the control value at 48 h. ANOVA showed a significant effect of treatment (F = 31.8; P < 0.0001). a, P < 0.05 compared with controls; b, P < 0.01 compared with FSH and TGF treatments alone; c, P < 0.05 compared with FSH and GDF-9 treatments alone; d, P < 0.05 compared with FSH and activin treatments alone. C, LOX activity after 48-h culture. Bars indicate the mean ± SEM levels in culture medium in three separate experiments after subtraction of values for samples assayed in the presence of the LOX inhibitor BAPN. All data were normalized to the control value. ANOVA showed a significant effect of treatment (F = 2.82; P < 0.05). a, P < 0.05 compared with controls; b, P < 0.05 compared with FSH; c, P = NS compared with controls.
Discussionwh, http://www.100md.com
Although considerable attention has focused on the factors regulating breakdown of the ECM in tissue remodeling of the ovary (24), there is scant evidence for the regulation of ECM deposition.wh, http://www.100md.com
In this study we demonstrate for the first time the detailed analysis of LOX in the mammalian ovary and show that both mRNA and enzyme activity are regulated by FSH, androgen, and TGFß superfamily members. LOX is a key enzyme in ECM formation in a wide range of tissues and cell types, being responsible for the final catalytic cross-linking of collagen and elastin monomers (1, 25). Although the cellular origins of the type IV collagen-rich basement membrane of the follicle are a matter of controversy (26), there is evidence that granulosa cells have the capacity to secrete many of its components (14). We have previously demonstrated LOX mRNA expression restricted to the granulosa cell compartment of the ovary, which points to a local mechanism for assembly of the basement membrane ECM collagen component (6). However, expression of type VI collagen has been demonstrated in the theca of developing human follicles (27), and the newly identified type XXVI collagen is expressed in prethecal cells of the mouse neonate (28). We have also observed high levels of LOX enzyme activity in human theca cells (Rae, M., and C. R. Harlow, unpublished observation), suggesting that transfer of LOX into the theca may occur.
We previously demonstrated that LOX mRNA is expressed in granulosa cells from late antral/early preantral rat follicles and is down-regulated by FSH both in vitro and in vivo (6). We now confirm that expression of LOX mRNA is dose dependently regulated by FSH in vitro, and that this is reflected in reduced activity of the mature LOX enzyme. Thus, LOX, a key enzyme in the assembly of mature collagen and elastin, can now be added to the repertoire of granulosa cell FSH-regulated genes that includes steroidogenic enzymes (29, 30) and LH receptors (31). However, like CTGF, which we recently showed was also under FSH regulation in granulosa cells (21), LOX is down-regulated by FSH through elevation of cAMP. Interestingly, cAMP also suppresses both basal and TGFß-induced collagen and LOX mRNA in human lung fibroblasts (32), although it has the opposite effect on LOX mRNA and enzyme activity in vascular smooth muscle cells (33). FSH receptor mRNA is developmentally regulated in the human follicle, being absent at the primordial stage, found intermittently in primary and two-layered preantral follicles, and expressed ubiquitously in multilayered follicles (34), raising the possibility of developmental regulation of LOX by FSH in the ovary.
By interacting with FSH, androgens are known to directly enhance many differentiation processes, such as estrogen and progestogen biosynthesis (35, 36), induction of LH receptors (37), and carbohydrate metabolism (38). The mechanism of action of androgens in this process appears to be through alterations in cAMP metabolism (39). By contrast, in the presence of FSH, further down-regulation of LOX mRNA and enzyme activity by DHT was observed in the same fashion that we described previously for CTGF (21). FSH receptor expression is up-regulated in the primate ovary by receptor-mediated androgen action (40). We await direct evidence of androgen receptor involvement in LOX expression, but it seems reasonable to assume that the mechanisms are similar.(%6, 百拇医药
Interestingly, DHT alone caused a modest increase in both LOX mRNA and enzyme activity, which parallels our observation of DHT action on CTGF mRNA described previously (21) and is in keeping with the finding that dehydroepiandrosterone treatment of rats increases ovarian LOX expression (11). Androgens also act in bovine aorta smooth muscle cells in vitro (41) and mouse cervix in vivo (42) to enhance LOX enzyme activity.
TGFß has been identified as a key regulator of LOX in diverse cell types, up-regulating both mRNA and enzyme activity in lung fibroblasts (43), osteoblasts (8), aortic smooth muscle cells (44), and human gingival fibroblasts (7). In the DES-primed immature rat ovary, TGFß1 mRNA expression is principally located in thecal/interstitial cells of preantral follicles (45). Immunolocalization studies showed intense TGFß1 staining in follicles around the time of antrum formation (46). Therefore, our observation of a dose-dependent stimulation of LOX by TGFß1 in rat granulosa cells points to a local paracrine regulation in the ovary.-0k@1+, 百拇医药
In embryonic lung fibroblasts, prostaglandin E2 reverses TGFß up-regulation of LOX mRNA via a cAMP-mediated mechanism (32), and cAMP is able to block TGFß-stimulated collagen synthesis (47). As cAMP mimicked the effects of FSH on LOX mRNA and enzyme activity, we believe that the dose-dependent inhibition by FSH of TGFß1-stimulated LOX is probably cAMP mediated. Paradoxically, TGFß1 combined with FSH consistently reduced LOX mRNA and enzyme activity to below the level achieved with FSH alone, although this could be explained by the actions of TGFß1 in enhancing FSH receptor expression (48, 49). Overall, we have demonstrated that the stimulatory action of TGFß1 on LOX appears insufficient to overcome the more potent inhibitory effect of FSH on rat granulosa cells from preantral/early antral rat follicles in vitro.
FSH action on granulosa cells is subject to paracrine modulation, not only by theca-derived TGFß1 (50), but also by GDF-9 originating from the oocyte (51, 52). Activin is another structurally related member of the TGFß superfamily of granulosa cell origin. It has autocrine effects on mitogenic activity (53), 17ß-hydroxysteroid dehydrogenase type I mRNA expression and enzyme activity (54), FSH and LH receptor mRNA expression (55), and P450 aromatase activity (56). Activin also modulates aromatase activity indirectly, by paracrine inhibition of LH-induced thecal androgen production (57). Moreover, activin A enhances the expression of FSH receptor mRNA in immature rat granulosa cells (58), and there is preferential synthesis of activin over inhibin at the earliest stages of follicular development (59). We recently showed that all three of these TGFß superfamily members enhance CTGF mRNA expression, but are inhibitory in the presence of FSH (21). We can now add that LOX, at the levels of mRNA and active enzyme, is under a similar control mechanism, and that the direction of action (stimulatory or inhibitory) of these factors is dependent on the degree of exposure of the granulosa cells to FSH. Furthermore, although inhibition of LOX by FSH alone was observed at 6 h, the additional presence of TGFß superfamily members did not have an inhibitory effect until 48 h. This delay may reflect the time necessary for these factors to induce FSH receptor expression (60, 61). Given the links established between TGFß1, CTGF, LOX, and insoluble collagen accumulation in human gingiva (7), we speculate that these factors will have a crucial role in coordinating the accumulation of ECM in the ovary and may be involved in basement membrane formation, thecal cell recruitment, follicular expansion, ovulation and the repair of the ovarian surface, and corpus luteum formation. LOX may also have a role in laying down the collagen type IV component of Call-Exner bodies, whose walls are lined with basal lamina and whose cavities contain large aggregates of convoluted basal lamina (14). The number of positively staining collagen type IV Call-Exner bodies is reduced in antral follicles (14) at a time when LOX is also decreasing (6), which is further evidence for a possible role of LOX in the development of these structures.
We have previously developed a working hypothesis for CTGF as a transducer of FSH and intrafollicular factors in follicle development (21). We now extend this to include the actions of LOX as a key factor in the synthesis of ECM. The presence of LOX at the early stages of follicular development (6) and the regulation of expression and enzyme activity by GDF-9 point to a possible role of LOX, in addition to CTGF (6), in transducing oocyte-derived GDF-9 signaling of basement membrane and thecal development (Fig. 7A). The additional stimulation of LOX by TGFß1 and activin at a time of low levels of FSH and low FSH receptor availability suggests communication between thecal and granulosa cell layers in multilayered preantral follicles, which may facilitate expansion of the ECM components in the basement membrane and theca during antrum formation (Fig. 7B). Our in vitro findings are substantiated by the observation that granulosa cells freshly isolated from preantral follicles of unstimulated or DES-treated 23-d-old rats have high endogenous LOX mRNA expression (6). However, in the antral follicle, thecal androgens and TGFß have up-regulated FSH receptors, and there is greater availability of FSH, resulting in these TGFß superfamily members enhancing the inhibitory effects of FSH on LOX (Fig. 7C), although GDF-9 may not be impinging on mural granulosa cells due to gradient effects resulting from spatial removal of the oocyte from the mural granulosa cells (Fig. 7C) (62). At this time, we observed a massive increase in aromatase mRNA expression at the precise point when LOX mRNA expression is declining in vivo (6). In other words, conditions that promote differentiation of granulosa cells toward maximal estradiol secretion switch off LOX activity. However, the factors that regulate LOX activity in more fully differentiated granulosa cells remain to be determined.
fig.ommitteed:iqy9o, 百拇医药
Figure 7. A working hypothesis of FSH and TGFß superfamily actions in the developing follicle. A, In the primary follicle, in the absence of FSH stimulation, GDF-9 and activin stimulate LOX mRNA expression in granulosa cells. We suggest the secreted LOX enzyme initiates mature collagen synthesis in the emerging thecal cell layer and orchestrates ECM deposition in the developing basement membrane. B, The multilayered granulosa cells of the preantral follicle have relatively few FSH receptors and are exposed to low levels of FSH. In this environment, GDF-9, activin, and TGFß stimulate high expression of LOX mRNA and the secreted enzyme, which could coordinate theca and basement membrane expansion as well as formation of the antral cavity. LOX could also synthesize the basal lamina components of Call-Exner bodies. C, In the antral follicle, greater FSH receptor availability and higher FSH levels would serve to switch the effects of activin and TGFß from stimulatory to inhibitory, resulting in reduced LOX mRNA expression and enzyme activity concomitant with the rapid mobilization of aromatase activity. GDF-9 may have less of an effect on mural granulosa cells due to gradient effects, as the oocyte is now spatially removed from the mural granulosa cells.
In summary, granulosa cells from late preantral/early antral rat follicles express LOX mRNA and mature LOX enzyme, which are regulated at endocrine, paracrine, and autocrine levels by FSH, androgen, and TGFß superfamily members coming from the oocyte, thecal cells, and the granulosa compartment itself. TGFß1, GDF-9, and activin are stimulatory, whereas FSH, by raising cAMP levels, is inhibitory. In the presence of FSH, TGFß superfamily members lose their stimulatory effect and enhance the inhibitory effect of FSH. This coordinated control of a key enzyme in ECM deposition predicts that the regulation of LOX will have a crucial role in tissue remodeling and ECM formation during all phases of follicular development./%8qi], 百拇医药
Received June 25, 2002./%8qi], 百拇医药
Accepted for publication September 13, 2002./%8qi], 百拇医药
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