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Reproductive Endocrinology Center, University of California, San Francisco, California 94143-0556; and the Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99164-4231
Address all correspondence and requests for reprints to: Dr. Michael K. Skinner, Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99164-4231. E-mail: skinner{at}mail.wsu.edu
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Abstract |
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Introduction |
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Cell-cell interactions between thecal and granulosa cells are essential for follicular development in the ovary. These mesenchymal-epithelial cell interactions are in part mediated by keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and Kit ligand (KL). Mesenchymal-derived thecal cells express the KGF and HGF genes and secrete the proteins (1). KGF and HGF can regulate granulosa cell granulosa cell function and growth (1) Epithelial-derived granulosa cells express the KL gene and secrete the protein (2). KL can regulate thecal cell function and growth (2). The expression of all of these factors increases to the highest levels in large antral follicles (2, 3, 4). Therefore, the actions of KGF, HGF, and KL are postulated to be involved in folliculogenesis, particularly during later stages of follicular development. The potential interactions among these growth factors has not been investigated and is the focus of the current study.
KGF and HGF are mesenchymal-derived growth factors that act on adjacent epithelial cells in a number of tissues (1, 5, 6, 7, 8). KGF is a member of the fibroblast growth factor family (FGF7), and HGF is the ligand for the c-met receptor. KL is a multipotent growth factor that is important for ovarian follicular development, germ cell migration, melanocytes, and hematopoietic cells (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Although the expression and actions of KGF, HGF, and KL have been examined in many tissues, no information is available on the potential paracrine interactions of these factors among each other. In the ovary, KGF and HGF are produced by mesenchyme-derived thecal cells and regulate the growth and steroidogenesis of epithelial-derived granulosa cells (1, 3, 4). KL is expressed by granulosa cells and has effects on oocytes, thecal cells, and ovarian stromal cells (2, 15, 23, 24, 25). The hypothesis has been developed that thecal cell-derived KGF and HGF and granulosa cell-derived KL mediate a positive feedback loop between thecal cells and granulosa cells. This novel feedback loop is proposed to be important for ovarian follicular development and hormone (i.e. gonadotropin) action.
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Materials and Methods |
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Preparation of RNA
Thecal and granulosa cells were cultured as confluent monolayers
in six-well plates for 72 h. Medium was removed, and total RNA was
prepared by either of two methods. First, cultured thecal and granulosa
cell total RNA was extracted using guanidium isothiocyanate, followed
by centrifugation through a cesium chloride gradient (27).
Alternatively, total RNA was prepared using the RNA-Stat 60 kit
(Tel-Test, Friendswood, TX). In either procedure, denaturing extraction
buffer was added directly to the culture plate to prevent RNA
degradation. RNA was stored at -70 C until use.
Quantitative RT-PCR assays
Steady state levels of KGF, HGF, KL, and cyclophilin
(i.e. IB15) messenger RNAs (mRNAs) were analyzed using a
specific quantitative RT-PCR assay for each gene. These quantitative
RT-PCR assays have previously been described in detail (2, 3, 4). The
primers used in this quantitative analysis of KGF, HGF, KL, and 1B15
were: KGF, 5'-ATA CTG ACA TGG ATC CTG CCA AGT TTG CTC TAC AGA TCA TGC
TTC-3' (5'-primer; 45-mer) and 5'-TCC AAC TGC CAC GGT CCT GAT-3'
(3'-primer; 21-mer), which generated a specific 306-bp KGF PCR product;
HGF, 5'-ACA GCT TTT TGC CTT CGA GCT ATC GGG GTA AAG ACC TAC AGG-3'
(5'-primer; 42-mer) and 5'-CAT CAA AGC CCT TGT CGG GAT A-3' (3'-primer;
22-mer), which generated a specific 292-bp HGF PCR product; KL, 5'-GGA
CAA GTT TTC GAA TAT TTC TGA AGG CTT GAG TAA TTA TTG-3' (5'-primer;
42-mer) and 5'-AGG CCC CAA AAG CAA ACC CGA TCA CAA GAG-3' (3'-primer;
30-mer), which generated a specific 452-bp KL PCR product [this
soluble form of KL (i.e. KL1) is the primary form expressed
by bovine granulosa cells (2) ]; and 1B15, 5'-ACA CGC CAT AAT GGC ACT
GGT GGC AAG TCC ATC-3' (5'-primer; 33-mer) and 5'-ATT TGC CAT GGA CAA
GAT GCC AGG ACC TGT ATG-3' (3'-primer; 33-mer), which generated a
specific 105-bp product from all cell types, demonstrating the
integrity of the RNA samples. Before RT, tubes containing total RNA and
specific 3'-primers were heated to 65 C for 10 min to facilitate
denaturing and were cooled to room temperature to facilitate annealing.
Total RNA (1 µg) was reverse transcribed for 1 h at 37 C using
the following conditions: 1 µg total RNA, 1 µM specific
3'-primers of interest (up to four different primers, including 1B15),
0.1 mM deoxy (d)-NTPs, 10 mM dithiothreitol, 40
U ribonuclease inhibitor (Promega, Madison, WI), and 200 U Moloney
murine leukemia virus reverse transcriptase (Life Technologies,
Gaithersburg, MD) in 40 µl RT buffer (50 mM Tris-HCl, pH
8.3; 75 mM KCl; and 3 mM MgCl2).
After 1 h, samples were heated to 95 C for 5 min to inactivate the
reverse transcriptase enzyme. Samples were immediately diluted
2.5-fold, and carrier DNA (Bluescript plasmid, Stratagene) was added to
a final concentration of 10 ng/µl. This concentration of Bluescript
carrier DNA (10 ng/µl) was included in all subsequent dilutions of
samples and standards. Immediately before amplification, each unknown
sample was further diluted 1:10 to improve the fidelity of the PCR
reaction. Plasmid DNAs (i.e. Bluescript) containing bovine
KGF, HGF, KL, or 1B15 subclones were used to generate standard curves
from 1 attogram/µl (10-15 g/µl) to 10 pg/µl (10
x 10-9 g/µl), each containing 10 ng/µl Bluescript
carrier DNA. Identical 10-µl aliquots of each sample and standard
were pipetted in duplicate into a 96-well reaction plate (Marsh
Biomedical Products, Rochester, NY) and sealed with adhesive film
(Marsh Biomedical Products) for PCR amplification. By this design it
was possible to simultaneously assay 5 known standard concentrations
and 40 unknown samples for each gene. Amplification was performed in a
Perkin-Elmer 9600 (Perkin-Elmer, Norwalk, CT) equipped with a heated
lid using the following conditions: 0.4 µM of each
primer, 16 µM dNTPs, and 1.25 U AmpliTaq polymerase in 50
µl GeneAmp PCR buffer (containing 1.5 mM
MgCl2; Perkin-Elmer). Each PCR amplification consisted of
an initial denaturing reaction (5 min, 95 C); 25–31 cycles of
denaturing (30 sec, 95 C), annealing (1 min, 60 C), and elongation (2
min, 72 C) reactions; and a final elongation reaction (10 min, 72 C).
At least 0.25 µCi 32P-labeled dCTP (Redivue, Amersham
Life Sciences, Arlington Heights, IL) was included in each sample
during amplification for detection purposes. Specific PCR products were
quantitated by electrophoresing all samples on 4–5% polyacrylamide
gels, simultaneously exposing the gels to a phosphor screen for 8–24
h, followed by quantitating the specific bands on a Storm
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Each gene was
assayed in separate PCR reactions from the same RT samples. Equivalent
steady state mRNA levels for each gene were determined by comparing
each sample to the appropriate standard curve. All KGF, HGF, and KL
data were normalized for 1B15.
The optimal cycle number for amplification was determined for each assay to achieve maximum sensitivity while maintaining linearity (i.e. logarithmic phase of PCR reactions). KGF quantitative PCR products were amplified for 28 cycles, HGF and KL PCR products were amplified for 31 cycles, and 1B15 PCR products were amplified for 25 cycles. The sensitivity of each quantitative PCR assay was below 1 fg, which corresponds to less than 125 fg target mRNA/µg total RNA. For each assay, all samples were simultaneously measured in duplicate, resulting in intraassay variabilities of 11.3% (KGF), 13.6% (HGF), 8.9% (KL), and 6.5% (1B15).
Statistical analysis
All data were analyzed by a JMP 3.1 statistical analysis program
(SAS Institute, Cary, NC). Effects of growth factors or gonadotropins
on KGF, HGF, and KL mRNA levels were analyzed by one-way ANOVA.
Observed significance probabilities of 0.05 (P > F) or
less were considered evidence that an ANOVA model fits the data.
Significant differences between treated cells and control (untreated)
cells were determined using Dunnett’s test, which guards against the
high 
(type I) error rate across the hypothesis tests (28). This
multiple comparison test is recommended for multiple comparisons with
controls (Dunnett’s) (29, 30).
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Results |
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Steady state levels of KL mRNA in granulosa cells were stimulated by
treatment with KGF (Fig. 1
). Similarly,
levels of granulosa cell KL mRNA were also stimulated by HGF (Fig. 1).
Similar observations were made with KL concentrations of 25 and 50
ng/ml (data not shown). These results demonstrate that KGF and HGF
regulate the gene expression of granulosa-derived growth factors such
as KL. Therefore, KGF and HGF can influence granulosa cell growth (1)
and growth factor (i.e. KL) expression.
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). The combined effects of FSH and hCG
on KL gene expression were not synergistic, suggesting that KL mRNA
levels were maximally stimulated in granulosa cells. The level of
stimulation obtained with KGF and HGF was similar to that obtained with
gonadotropins (Figs. 1
and 2). The effects of gonadotropins on bovine
granulosa cell KL expression are consistent with the effects of
(Bu)2cAMP on mouse granulosa cells (15). These
results suggest that granulosa cell-derived KL in part mediates the
stimulatory actions of gonadotropins on folliculogenesis.
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). Similar
results were obtained with 25 and 50 ng/ml concentrations of these
growth factors (data not shown). The ability of KL to stimulate KGF and
HGF expression in thecal cells suggests that granulosa cells feed back
on thecal cells to promote follicular growth. The ability of
gonadotropin (i.e. hCG) to stimulate KGF and HGF mRNA levels
is shown in Fig. 4. The LH agonist hCG
stimulated both KGF and HGF mRNA levels in a manner similar to that of
KL (Figs. 3 and 4). The ability of these growth factors to act in a
paracrine manner to stimulate thecal and granulosa cell gene expression
establishes a novel feedback loop that may be important for
folliculogenesis (Fig. 5). The ability of
gonadotropins to regulate the expression of these growth factors also
suggests that the hormonal regulation of follicular development may in
part be mediated by the feedback loop involving these growth
factors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During follicular development, mesenchymal-derived thecal cells produce a number of growth factors that include KGF and HGF. Both KGF and HGF are mesenchymal-derived growth factors that act on adjacent epithelial cells in a number of tissues. Gene expression of KGF and HGF is developmentally and hormonally regulated in thecal cells during follicular development (3, 4). Thecal cells have been shown to produce and secrete these growth factors (1). Granulosa cells have been shown to proliferate in response to KGF and HGF in vitro (1). These observations suggest that KGF and HGF may be important mediators of ovarian mesenchymal-epithelial cell interactions that promote folliculogenesis. The current study demonstrates that thecal cell-derived KGF and HGF also stimulate KL expression in granulosa cells. This is the first report of the regulation of KL by these mesenchymal growth factors. Although these results show that KGF and HGF regulate KL mRNA levels, the effects of KGF and HGF on KL protein production remain to be elucidated. The actions of KGF and HGF on granulosa cell KL expression indicate that these growth factors also alter cellular parameters other than cell growth (1). As granulosa cell-derived KL is important for oocyte maturation, thecal cells may indirectly regulate oocyte function by influencing granulosa cell production of KL.
KL is expressed in granulosa cells of healthy developing follicles. Granulosa cell-derived KL promotes follicular development and is important for oocytes, thecal cells and stromal-interstitial cells (2, 15, 23, 24, 25). As gonadotropins directly stimulate granulosa cell functions and promote folliculogenesis, the regulation of KL expression in granulosa cells by gonadotropins was investigated. Two previous studies addressed the direct regulation of KL in granulosa cells by gonadotropins and produced conflicting results. KL expression was stimulated by (Bu)2cAMP in mouse granulosa cells, suggesting that gonadotropins may stimulate KL expression (15). In human granulosa-luteal cells, KL expression was decreased in response to FSH and hCG, suggesting that gonadotropins inhibit KL expression (31). In addition, in vivo experiments suggest that both mouse and rat granulosa cell KL expression can be increased by LH, hCG, and PMSG (32, 33). In the current study, both FSH and hCG stimulated gene expression of KL in purified bovine granulosa cells. These results suggest that gonadotropins (i.e. FSH and LH) may in part promote follicular development by directly stimulating KL expression in granulosa cells. The explanation of why gonadotropins decreased KL expression in human granulosa cells is not known. Potential differences in luteinized granulosa cells need to be investigated. Analysis of the potential regulation of KL by gonadotropins in other species also remains to be elucidated.
Epithelium-derived granulosa cells are proposed to feedback on mesenchyme-derived thecal cells to regulate thecal cell functions. Previous work has shown that granulosa cell-derived KL acts on thecal cells to promote cell proliferation and differentiation (2). The possibility that KL feedback on thecal cells may also regulate KGF and HGF expression was investigated in the current study. Treatment of thecal cells with KL stimulated both KGF and HGF gene expression. The ability of KL to stimulate thecal cell KGF and HGF expression is significant because it establishes a positive feedback loop between granulosa cells and thecal cells. Thecal cell-derived KGF and HGF can stimulate granulosa cell KL expression, and granulosa cell-derived KL can stimulate thecal cell KGF and HGF expression. As the expression of KGF, HGF, and KL is greatest in large antral follicles (2, 3, 4), the positive feedback among these factors may be particularly important during later stages of follicular development. Communication between thecal cells and granulosa cells through the production and actions of KGF, HGF, and KL appears to be a mesenchymal-epithelial cell interaction that may in part promote folliculogenesis.
Hormones such as FSH and LH stimulate follicular growth in vivo (34, 35, 36, 37, 38). The actions of such hormones in the ovary are necessary for follicular development and reproductive viability. Although these hormones increase follicular growth in vivo, no proliferative effect is apparent on purified cells in vitro. As a result, the hypothesis has developed that gonadotropins and steroids indirectly stimulate follicular growth by influencing local mesenchymal-epithelial cell interactions in the ovary. The current study supports previous work suggesting that hormones can directly regulate KGF and HGF expression in thecal cells. The current study also demonstrates that FSH and hCG directly stimulate KL expression in granulosa cells. The ability of gonadotropins to influence KGF, HGF, and KL gene expression provides an indirect mechanism for gonadotropins to regulate folliculogenesis. Gonadotropins may also indirectly influence oocyte functions by stimulating granulosa cell functions such as KL expression. These observations provide an expanded mechanism to explain hormone-induced follicular growth in the ovary.
The current hypothesis involving mesenchymal-epithelial cell
interactions and these growth factors suggested by this study is
summarized in Fig. 5. This schematic of thecal and granulosa cells
depicts both the endocrine regulation and the paracrine regulation of
thecal and granulosa cell functions. It is well established that
gonadotropins regulate thecal and granulosa cell differentiated
functions (see FSH and LH, Fig. 5). Steroid-mediated thecal
cell-granulosa cell interactions via androgen and estrogen are critical
and are influenced by gonadotropins. Locally produced growth factors,
such as KGF, HGF, and KL, may also be important for regulating thecal
and granulosa cell functions. The combined endocrine and paracrine
regulation of KGF, HGF, and KL expression in the ovary is postulated to
be important for follicular development. The autocrine actions of these
growth factors may also be a factor (not shown in Fig. 5). For example,
previous observations have suggested that HGF may act on thecal cells
in an autocrine manner in the rat (39). It is anticipated that a
complex network of autocrine, paracrine, and endocrine cell-cell
interactions involving these and other growth factors will be needed
for optimal follicular development. The current study used purified
cell populations to demonstrate a potentially important cell-cell
interaction in the follicle. Future studies involving coculture and
in vivo studies will be required to assess the physiological
importance of this interaction to follicle development and hormone
actions. The novel positive feedback loop between thecal cells and
granulosa cells suggested in the current study provides insight into
potential mesenchymal-epithelial cell interactions required for
follicular development.
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Acknowledgments |
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Footnotes |
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Received December 1, 1997.
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