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Regular Article |
a Department of Animal Science, Cornell University, Ithaca, New York 14853
ABSTRACT
Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin
subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F1 and F3 + F4 follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F3 + F4 granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F3 + F4 cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F1 and F3 + F4 granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F3 + F4 granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.
activin, follicle, follicular development, granulosa cells, inhibin
INTRODUCTION
Activin is a dimeric protein hormone that stimulates pituitary FSH secretion. It is synthesized as a homo- or heterodimer of the distinct but quite similar ß subunits (ßA or ßB), which combine to form either activin A (ßA + ßA), activin B (ßB + ßB), or activin AB (ßA + ßB). Interestingly, inhibin, which has an endocrine function to suppress pituitary FSH secretion, is composed of an inhibin-specific subunit combined with one of the activin ß subunits. As reviewed by many authors [1–5], activin has diverse functions in a wide array of tissues, including many paracrine/autocrine functions in the ovary.
The ovary of the domestic laying hen provides a unique model for studying follicular development. The ovary contains five to eight large, yolk-filled, preovulatory follicles that have advanced from a pool of smaller follicles (<12 mm in diameter). The theca and granulosa layers surrounding the developing follicles are distinct and can be separated easily from one another. The large follicles are arranged in a hierarchy according to size, with the largest follicle designated as the F1 follicle, the second largest as the F2 follicle, and so on. The development of these hierarchical follicles is tightly regulated, with an interval of 24–26 h between each consecutive ovulation. After ovulation, succeeding follicles each advance one place in the hierarchy, and an additional follicle is recruited from the pool of small, nonhierarchical follicles.
Using a specific two-site enzyme-linked immunosorbent assay, Lovell et al. [6]) examined the content of activin A and inhibin A in the theca and granulosa layers of the four largest follicles. They reported that activin A was present in both the granulosa and theca cells, but that the amount of activin A was approximately 35-fold greater in the theca layer compared to the granulosa layer. Inhibin A production was largely confined to the granulosa layer. The content of inhibin A was reported to increase approximately 40-fold from the F4 granulosa layer to the F1 granulosa layer.
The mRNA expression pattern of the inhibin/activin subunits in the granulosa layer from hen preovulatory follicles [7–9] is consistent with the findings reported by Lovell et al. [6]. The mRNA for the inhibin subunit is abundant in the large hierarchical follicles [7–9] and undetectable by Northern blot analysis in the small (<5 mm) nonhierarchical follicles [8]. Expression of the inhibin/activin ßA subunit is much greater in the F1 follicle compared to the other large preovulatory follicles [7–9].
Addition of LH or FSH to granulosa cell cultures from the large preovulatory follicles of domestic hens has been reported to stimulate immunoreactive inhibin subunit production [10, 11] and to support higher levels of mRNA for the inhibin subunit [11]. The role of activin in the ovary of the hen has not been determined. In granulosa cell cultures from mammalian species, activin A has been reported to stimulate secretion of inhibin A, to increase the expression of mRNA for the inhibin and ßA subunits [12], and to enhance the FSH-stimulated expression of mRNA for the LH [13] and the FSH [14, 15] receptors. Additionally, activin has been reported to influence progesterone production [14, 16–27] and in vitro granulosa cell growth [21, 22, 28]. To determine possible roles of activin A in the hen ovary, we examined the effect of activin A on the expression of mRNA for the LH and FSH receptors and on the mRNA expression and protein secretion of the inhibin subunit. In addition, the influence of activin A on progesterone secretion and granulosa cell growth was also examined. These experiments were conducted in the presence of FSH or LH and used cultured granulosa cells isolated from the F1 and F3 + F4 follicles.
MATERIALS AND METHODS
Animals
Single-comb White Leghorn hens of the Babcock B-300 strain were individually caged, with egg laying monitored at 2-h intervals during daylight. The hens were maintained on a lighting schedule of 14L:10D (lights-on at 0600 h), with free access to water and food. Hens between 7 and 13 mo of age and laying regular sequences were selected and killed at midsequence 2–3 h after an oviposition for collection of follicles. All animal procedures were approved by the Institutional Animal Care and Use Committee of Cornell University.
Cell Culture
The F1, F3, and F4 follicles were removed from five birds for each replicate experiment. The granulosa layer was isolated from each follicle, and the pooled granulosa layers from the F1 follicle and the combined F3 and F4 follicles were then dispersed as previously described [11]. Cells were plated in six-well tissue culture plates (Corning Costar, Cambridge, MA) at a density of 2.5 x 106 cells/well. The granulosa cell culture media and incubation conditions have been previously described [11]. Cell number and viability were estimated using a hemocytometer with trypan blue exclusion. Cell viability was always greater than 95% at the start and finish of all experiments.
Experiment 1
Granulosa cells were cultured with or without highly purified ovine (o) FSH (NIH oFSH-19-SIAFP, generously provided by the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Child Health and Human Development, and U.S. Department of Agriculture [USDA]) at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A (a generous gift from Dr. J.N. Coulombe, Uniformed Services University of the Health Sciences, Bethesda, MD). Bioactivity of the recombinant chicken activin A was tested in a somatostatin expression bioassay by Dr. Coulombe (personal communication). The dose of FSH was previously found to be maximally effective in stimulating subunit expression in cultured granulosa cells from hierarchical follicles [11]. Cell cultures were terminated 48 h after plating. Total RNA was extracted from the cells, and the cell culture medium was saved for RIA. Each treatment consisted of three wells, and the cells were combined before RNA extraction. This experiment was replicated two more times (n = 3).
Experiment 2
Experiment 2 followed the same protocol as experiment 1, except that oLH (NIH oLH-26, a generous gift provided by the National Hormone and Pituitary Program of the NIDDK, NICHHD, and USDA) at 50 ng/ml was used instead of FSH. Similar to FSH, this dose of LH was previously found to be maximally effective in stimulating subunit expression in cultured granulosa cells [11]. This experiment was performed four times (n = 4).
Experiment 3
To examine the effects of activin A on growth, granulosa cells from each follicle type were cultured with either 0, 10, or 50 ng/ml of recombinant chicken activin A; 50 ng/ml of either oLH or oFSH; or an individual gonadotropin combined with 10 ng/ml of recombinant chicken activin A. After 48 h of culture, the cells were washed from the wells, resuspended, and counted using a hemocytometer as previously described [29]. Three replications of each experiment with separate pools of cells were performed (n = 3). Because no RNA isolation was involved in this experiment, each treatment consisted of one well rather than three.
RNA Extraction and Northern Blot Analysis
Total RNA was extracted from the cells using the guanidine isothiocyanate/phenol-chloroform method [30]. Ten micrograms of total RNA for each sample were used in Northern blot analysis for chicken inhibin subunit, LH receptor, FSH receptor, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as previously described [8]. The chicken LH [31] and FSH [32] receptor cDNA clones were both generously provided by Dr. A.L. Johnson (University of Notre Dame, Notre Dame, IN). The hybridization and densitometry procedures were also the same as those described previously [8]. The GAPDH mRNA expression was used to verify and to correct for equality of RNA loading and transfer. After correction of loading differences, the intensity of each sample within a blot was then expressed relative to the strongest signal, which was given a value of one.
Radioimmunoassay
Media progesterone and inhibin subunit concentrations were determined by RIA procedures that have been described previously [11]. The antibody used for the inhibin subunit RIA detects both free subunit and intact inhibin [11]. Intraassay coefficients of variations for a high- and a low-granulosa cell culture medium pool were less than 10% for both the inhibin subunit and progesterone RIA.
Statistics
Analysis of variance was performed for each follicle type using the General Linear Model procedure of SAS (Cary, NC) or Minitab (State College, PA). Protected least-significant difference test was used to determine differences between sample treatments. Differences were considered to be significant when P values were less than 0.05.
RESULTS
Experiment 1
Addition of FSH alone or in combination with activin to the granulosa cell cultures from the F1 and F3 + F4 follicles significantly enhanced the expression of mRNA for the inhibin subunit (Fig. 1, top). The addition of activin A alone to the granulosa cell cultures from each follicle type had no effect on inhibin subunit mRNA expression. The accumulation of immunoreactive inhibin subunit paralleled the results obtained for the expression of mRNA for the inhibin subunit (Fig. 1, middle). Addition of FSH to the F1 and the F3 + F4 granulosa cell cultures significantly increased the production of progesterone from these cells compared to untreated cells (Fig. 1, bottom). Accumulation of progesterone in the media of the F1 and F3 + F4 cell cultures was unaffected by adding activin A to the culture medium.
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Addition of FSH and/or activin to the F1 granulosa cell cultures had no effect on the expression of mRNA for the FSH receptor (Fig. 2, top). In the F3 + F4 granulosa cell cultures, however, the addition of the combination of FSH with the highest dose of activin A resulted in an increased expression of mRNA for the FSH receptor compared to the untreated controls or the cultures treated with activin alone. The mRNA expression of the LH receptor was unaffected by the addition of FSH, activin, or the combination of FSH and activin to the granulosa cell culture medium (Fig. 2, bottom).
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Experiment 2
The relative densities of mRNA for the subunit are shown in the top panel of Figure 3. The addition of activin A alone to the granulosa cell cultures from both follicle types had no effect on mRNA expression of the inhibin subunit. When LH was added alone or in combination with either dose of activin A to the granulosa cell cultures from both the F1 and F3 + F4 follicles, the expression of mRNA for the inhibin subunit was significantly enhanced.
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Compared to the untreated control cell cultures, addition of LH to the F1 and F3 + F4 granulosa cell cultures significantly increased the accumulation of the inhibin subunit protein in the media from these cultures (Fig. 3, middle). Both doses of activin A were equally effective in stimulating immunoreactive inhibin subunit production over control levels. Addition of the combination of LH and activin A to the F3 + F4 granulosa cell cultures only stimulated the accumulation of inhibin subunit protein to an amount equal to that obtained with LH alone. In the F1 granulosa cell cultures, the combination of the lower dose of activin A with LH stimulated greater inhibin accumulation than LH alone.
As seen in experiment 1, the addition of activin A to the culture media of either follicle type had no effect on progesterone production (Fig. 3, bottom). The addition of LH to the culture media of both the F1 and F3 + F4 granulosa cells significantly increased the secretion of progesterone from these cells compared to untreated control granulosa cells, and the addition of activin A along with LH did not alter the positive response to LH.
In the F1 granulosa cell cultures, the addition of LH or of activin alone did not significantly change expression of FSH receptor mRNA (Fig. 4, top). In the presence of LH and the highest dose of activin, FSH receptor mRNA was increased. In the granulosa cultures from the F3 + F4 follicles, only LH plus the low dose of activin A significantly increased FSH receptor expression.
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In the F3 + F4 cultures, the addition of LH significantly increased the expression of its own receptor compared to untreated controls, but this was not significant for the F1 granulosa cultures (Fig. 4, bottom). In the F3 + F4 cultures, addition of the highest dose of activin inhibited expression of mRNA for the LH receptor. Addition of the highest dose of activin with LH significantly increased the expression of mRNA for the LH receptor in the F1 granulosa cell cultures compared to the untreated control cultures or the cultures treated with activin alone. Adding either dose of activin with LH to the F3 + F4 cultures prevented LH from stimulating expression of its own receptor.
Experiment 3
The mean ± SEM (n = 3) number of cells per well for each follicle size and for each treatment is presented in Table 1. Viability of the granulosa cells from both follicle types and for all treatments was greater than 95%. In both the F1 and F3 + F4 granulosa cell cultures at 48 h of culture, a significant increase was observed in the number of cells (from the original 2.5 x 106 cells plated per well) in all treatments except for the cultures treated with the highest dose of activin by itself, in which the number of cells after 48 h was still equivalent to the amount that was initially plated. The rate of cell proliferation in the F1 granulosa cells treated with gonadotropins was significantly greater compared to the untreated controls, and the addition of 10 ng/ml of activin A with the gonadotropins did not alter this proliferation. In the F3 + F4 granulosa cell cultures, the addition of LH and FSH both significantly increased the rate of cell proliferation compared to untreated controls, but the addition of 10 ng/ml activin A with LH prevented LH from significantly enhancing cell proliferation beyond the growth rate obtained in the untreated controls.
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DISCUSSION
The present study produced several novel findings. First, activin A suppressed the expression of mRNA for the LH receptor in the F3 + F4 granulosa cells. Second, activin A suppressed the LH-stimulated proliferation of F3 + F4 granulosa cells. Third, gonadotropin-stimulated progesterone secretion was not altered by the addition of activin A to cultured F1 and F3 + F4 granulosa cells.
LaPolt et al. [12] reported that inhibin subunit mRNA expression and immunoreactive inhibin secretion were both increased in cultured rat granulosa cells treated with activin A, and that the addition of FSH with activin enhanced these effects. Subsequently, Yokota et al. [33] reported that activin A treatment of cultured follicles significantly increased inhibin secretion in follicles obtained from immature mice but not from adult mice. In the present experiments, addition of activin A alone or in combination with FSH or LH had a tendency to increase the expression of mRNA of the inhibin subunit. This tendency for increased expression of mRNA of the inhibin subunit was also reflected in the secretion of immunoreactive inhibin subunit. Immunoreactive inhibin subunit secretion was increased by both doses of activin in experiment 2. In experiment 1, a clear and consistent trend of activin increasing secretion of the inhibin subunit was observed; however, the increase was not statistically significant (as it was in experiment 2).
The addition of LH or FSH to the chicken granulosa cell cultures from both follicle sizes also increased progesterone secretion. Addition of activin A to the granulosa cultures failed to increase progesterone secretion or to alter the increase in production stimulated by FSH or LH. The lack of any alteration of progesterone production by activin A in the cultured chicken granulosa cells was somewhat unexpected, given its well-documented effects in mammalian systems. Activin enhances gonadotropin-stimulated progesterone production by nondifferentiated granulosa cells obtained from immature follicles, but it suppresses progesterone production in differentiated granulosa cells from mammalian species [14, 16–27]. Specifically, in nondifferentiated granulosa cells, activin increases FSH-induced progesterone production by stimulating cholesterol side-chain cleavage cytochrome P450 mRNA, but in differentiated granulosa cells, activin actually inhibits cholesterol side-chain cleavage cytochrome P450 transcription [22]. Therefore, in mammalian species, it appears that locally produced activin stimulates steroidogenesis in small follicles but inhibits the luteinization of large antral follicles. Granulosa cells from hierarchical follicles of the hen would be considered well-differentiated cells compared to granulosa cells from smaller follicles with respect to LH receptor abundance and progesterone production [31, 34, 35]. Additionally, the expression of cholesterol side-chain cleavage cytochrome P450 mRNA increases substantially from the F3 to F1 follicle, which has the highest expression [36], and this F1 follicle also produces the most progesterone [35]. Based on the results obtained with mammalian species, progesterone production could have been expected to decrease in these well-differentiated cells exposed to activin. That this did not occur suggests activin A may not be a potent negative regulator of the transcription of cholesterol side-chain cleavage cytochrome P450 in differentiated chicken granulosa cells (as it is in mammalian cells).
The production of immunoreactive inhibin and progesterone could be influenced by cell number in the present experiments. Although we did not correct the production of immunoreactive inhibin subunit or progesterone by cell numbers, the values obtained for the gonadotropin treatments would decrease, and those for the activin treatments would increase, if cell number was used to adjust the secretion values. In fact, if media progesterone accumulation was corrected for cell number in experiments 1 and 2, the progesterone production by the cells treated with 50 ng/ml of activin alone would be significantly greater than that produced by the untreated controls. Our decision not to correct the progesterone and inhibin subunit secretion values for cell number stemmed from our experience with previous experiments [11, 29], in which changes in the production of these two products were disconnected, in part, from the total number of granulosa cells.
Interestingly, the granulosa cells from both the F1 and F3 + F4 follicles treated with the highest dose of activin proliferated significantly less than the untreated control cultures. This result contrasts with findings in mammalian species. Activin has been reported to stimulate the proliferation of cultured rat granulosa cells [21, 22, 28] and human granulosa luteal cells from preovulatory follicles [37]. The most interesting inhibition of the cell proliferation rate in the present study was the prevention of LH-stimulated proliferation by the addition of activin to the F3 + F4 granulosa cell cultures.
It seems very likely that LH was unable to increase cell proliferation in the F3 + F4 cultures when it was added with activin because activin prevented LH from stimulating expression of its own receptor. Expression of mRNA for the LH receptor is greatest in the largest hierarchical follicles [31], and only the combination of the highest dose of activin with LH stimulated expression of the LH receptor in the F1 granulosa cell cultures. In granulosa cell cultures derived from the less-mature F3 + F4 follicles, however, the addition of LH alone induced the expression of mRNA for its own receptor. Activin did not augment this response, however, as the results from the F1 follicle might have suggested. Instead, the lowest dose of activin added with LH to the F3 + F4 granulosa cell cultures prevented LH from inducing expression of its own receptor. Furthermore, addition of the highest dose of activin, either alone or in combination with LH, resulted in a drastic reduction of the expression of mRNA for the LH receptor.
Interestingly, although addition of the highest dose of activin to the F3 + F4 cell cultures reduced the expression of mRNA for the LH receptor in the presence of added LH, progesterone production was still stimulated by the presence of LH in these cultures. These seemingly contradictory results need further investigation. It is possible that the effect of LH on progesterone production was mediated quickly during the initial phase of the 48-h culture period, whereas the decrease in mRNA expression of the LH receptor and in granulosa cell proliferation occurred later. During previous research [11], we noted that the increased accumulation of progesterone detected at 48 h in F3 + F4 granulosa cells cultured with LH compared to untreated controls was actually produced during the first 24 h of culture, with 50% of it actually being produced during the first 4 h of culture.
The actions of activin A in the F3 + F4 granulosa cell cultures suggest that activin A derived from the theca layer could modulate the maturation of smaller hierarchical follicles by negatively regulating expression of the LH receptor. This activin A is likely to be theca-derived activin, because in the granulosa layers of the F3 through F6 follicles, a very high level of expression for mRNA of the inhibin subunit is found [8], which would favor the formation of inhibin over activin if both species of mRNA were translated. In fact, Lovell et al. [6] detected very little activin A in the granulosa layer of the F1 through F4 follicles, but in the theca layers of these follicles, activin A was much more abundant.
Addition of activin A to the F1 and F3 + F4 granulosa cell cultures had negligible effects on the expression of mRNA for the FSH receptor. Although activin induces the expression of FSH receptors in cultures of mammalian granulosa cells [14, 15], it may not be surprising that this effect was not observed in the present study. The number of FSH receptors [38] and the expression of mRNA for the FSH receptor [32] decrease with follicular development, and as stated previously, the F1 and F3 + F4 granulosa cells are highly differentiated cells. Future experiments should focus on determining if activin is responsible for the induction of FSH receptor mRNA that is seen as the large, white follicles mature into small, yellow follicles [32]. Using activin B rather than activin A might be a more logical choice in such future experiments, because the ßB subunit is the only inhibin/activin subunit that is expressed abundantly in these small follicles [8].
In summary, we discovered very dramatic effects of activin A in cultured chicken granulosa cells, although these effects may differ from some reported in mammalian granulosa cells. The observed differences between our findings and the previously reported effects of activin A in mammalian granulosa cell cultures may be due to differences in the maturation of the follicle from which the granulosa cells were obtained. Our most significant finding was that activin A could inhibit the expression of mRNA for the LH receptor in F3 + F4 granulosa cells. This was likely responsible for the inhibitory effect of activin A on LH-stimulated proliferation in F3 + F4 cells. Local activin A (possibly theca derived) may modulate the growth rate of avian hierarchical follicles.
FOOTNOTES
1 Supported by U.S. Department of Agriculture grant 97-35203-4979. 
2 Correspondence: Patricia Johnson, 202 Morrison Hall, Department of Animal Science, Cornell University, Ithaca, NY 14853. FAX: 607 255 9829; paj1{at}cornell.edu
3 Current address: Department of Poultry Science, University of Georgia, Athens, GA 30602.
Accepted: June 13, 2001.
Received: April 4, 2001.
REFERENCES
subunit levels in granulosa and thecal layers of developing preovulatory follicles in the chicken. Endocrinology 1998; 139:1164-1171 and inhibin/activin ßA subunits in the granulosa layer of the large preovulatory follicles of the hen. Biol Reprod 1996; 55:450-454[Abstract] and ßA subunit messenger ribonucleic acid by competitive reverse transcription-polymerase chain reaction in chicken granulosa cells during follicular development. Biol Reprod 1998; 59:1047-1054 subunit mRNA and immunoreactive protein in cultured chicken granulosa cells. Gen Comp Endocrinol 1999; 116:90-103[CrossRef][Medline]- and ßB-subunit mRNA in avian granulosa cells. Gen Comp Endocrinol 2000; 119:308-316[CrossRef][Medline]This article has been cited by other articles:
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  T. M Lovell, S. L Al-Musawi, R. T Gladwell, and P. G Knight Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles Reproduction, June 1, 2007; 133(6): 1159 - 1168. [Abstract] [Full Text] [PDF]
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 T M Lovell, P G Knight, and R T Gladwell Differential expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and activin type-I and type-II receptors in preovulatory and prehierarchical follicles of the laying hen ovary J. Endocrinol., February 1, 2006; 188(2): 241 - 249. [Abstract] [Full Text] [PDF]
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 A.L. Johnson, J.T. Bridgham, and D.C. Woods Cellular Mechanisms and Modulation of Activin A- and Transforming Growth Factor {beta}-Mediated Differentiation in Cultured Hen Granulosa Cells Biol Reprod, December 1, 2004; 71(6): 1844 - 1851. [Abstract] [Full Text] [PDF]
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