| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Reproductive Endocrinology Center, University of California, San Francisco, California 94143-0556; and 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: 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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Keratinocyte growth factor (KGF) is produced by mesenchymal-theca cells and stimulates epithelial granulosa cell proliferation during follicular development in the ovary (8). In situ hybridization has confirmed the localization of KGF gene expression in theca cells in the ovary (6). The ovarian follicle is one of the most rapidly proliferating normal tissues known in vivo, and granulosa cell growth accounts for the majority of this follicle expansion (9). Factors, such as KGF, that promote the growth of the granulosa cell population (8) help establish the microenvironment required for oocyte maturation and fertility in the female. Endocrine hormones, such as estrogen and LH, have been shown to stimulate follicle development and granulosa cell growth in vivo, but neither of these hormones acts as a granulosa cell mitogen in vitro (10, 11, 12, 13, 14). Therefore, the effects of estrogen and LH on follicular growth in vivo are proposed to be indirectly mediated by factors such as KGF.
KGF is a 28-kDa protein that is a member of the fibroblast growth factor family (FGF-7) (15, 16). KGF is primarily produced by mesenchymal-derived cells in many tissues and acts as an epithelial-cell-specific mitogen (17, 18, 19). The receptor to KGF (KGFR) is a splice variant of the FGF receptor family (FGFR-2) that is primarily localized on epithelial cells (17, 20, 21, 22). KGF mediates mesenchymal-epithelial cell interactions in many tissues, including the ovary (8), placenta (23), endometrium (24), bladder (25, 26), hair follicle (27), stomach (28), lung (29, 30), ventral prostate (31), and seminal vesicle (32). Expression of KGF can be regulated by endocrine hormones such as androgen, progesterone, and glucocorticoids (24, 33, 34). These and other studies have established that KGF can mediate the actions of endocrine hormones and act as a paracrine mediator of mesenchymal-epithelial cell interactions in many tissues.
The current study was designed to investigate the role of KGF as a paracrine mediator of mesenchymal-epithelial cell interactions during ovarian follicular development. Experiments address the developmental and hormonal regulation of KGF in theca cells and the KGFR in granulosa cells. Estrogen and LH are proposed to stimulate granulosa cell proliferation in vivo, in part, by stimulating theca cell KGF production. Effects of KGF on granulosa cell steroid production are also examined.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Preparation of RNA and PCR
Follicles were dissected from the bovine ovaries and separated
into pools of small (<5 mm), medium (5–10 mm), and large (>10 mm)
follicles. Granulosa and theca cell total RNA was extracted from each
pool of samples using a guanidium thiocyanate procedure, followed by
centrifugation through a cesium chloride gradient (36). Alternatively,
total RNA was prepared using the RNA-Stat 60 kit (Tel-Test,
Friends-wood, TX). For qualitative analysis of gene expression, 10
ug total RNA was reverse transcribed with Moloney’s murine leukemia
virus RT (Gibco BRL, Gaithersburg, MD) at 37 C for 1 h using
oligodeoxythymidine12–18 primers (Gibco BRL). This
complementary DNA (cDNA) template was amplified by PCR using specific
primers for KGF, KGFR, or the constitutively expressed gene cyclophilin
(1B15). The KGF primers were 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 from bovine theca cells. The KGFR primers were 5'-GAA GTG
CTG GCT CTG TTC AAT GTG AC-3' (5' primer, 26-mer) and 5'-TTG GAG TTC
ATG GAG GAG CTG GAC TC-3' (3' primer, 26-mer), which generated a
specific 359-bp KGFR PCR product from bovine granulosa cells. The
primers for 1B15 were: 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. Amplification was performed with AmpliTaq DNA polymerase
(Perkin Elmer, Foster City, CA) for 35 cycles using the following
conditions: 0.8 µM of each primer, 100 µM
deoxynucleotide triphosphates (dNTPs), 1.5 mM
Mg++, 1.25 U Taq polymerase in 50 µl total
vol. Each PCR amplification consisted of an initial denaturing reaction
(5 min, 95 C); 35 cycles of denaturing (30 sec, 95 C), annealing (2
min, 60 C), and elongation (3 min, 72 C) reactions; and a final
elongation reaction (10 min, 72 C). PCR products were visualized by UV
illumination (312 nm) of 2% agarose gels stained with ethidium
bromide.
The KGF, KGFR, and 1B15 PCR products were subcloned into the Bluescript plasmid (Stratagene, La Jolla, CA) at the SmaI site. Each subclone was sequenced in both directions and confirmed to be bovine KGF, KGFR, and 1B15. These subclones were used as standard templates in the quantitative PCR procedure below.
Quantitative RT-PCR assays
Steady-state levels of KGF, KGFR, and 1B15 messenger RNAs
(mRNAs) were measured using a specific quantitative RT-PCR assay
for each gene. The primers used in this quantitative analysis of KGF,
KGFR, and 1B15 were the same as described above. These were 3' primers,
which significantly increased the reproducibility of the reverse
transcription and eliminated the variability normally found with
oligodeoxythymidine. This eliminated the need for internal standards in
the PCR. Before reverse transcription, tubes containing total RNA and
specific 3'-primers were heated to 65 C for 10 min to facilitate
denaturing and 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 4 different primers, including 1B15), 0.1
mM dNTPs, 10 mM dithiothreitol, 40 U
ribonuclease inhibitor (Promega, Madison, WI), and 200 U M-MLV RT
(Gibco BRL) in 40 µl RT buffer (50 mM Tris-HCl (ph 8.3),
75 mM KCI, 3 mM MgCl2). After
1 h, samples were heated to 95 C for 5 min to inactivate the RT
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 (37). Plasmid
DNAs containing bovine KGF, KGFR, 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, equipped with a
heated lid, using the following conditions: 0.4 µM 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 uCi of 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 with the appropriate standard curve. All KGF and KGFR data
were normalized for 1B15. The IB15 PCR was performed on the same sample
and reverse transcribed together with the appropriate 3' primers. The
expression of IB15 did not vary on a per-cell basis between cells and
was similar, independent of source. The IB15 corrected for cell number
and for amount and integrity of the RNA sample used.
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, KGFR 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), 10.2% (KGFR), and 6.5% (1B15).
Assay for aromatase activity in granulosa cells
Aromatase activity was assessed by the release of
3H2O from aromatization of
[1b-3H]androstenedione, as previously described (38),
with the following modifications: Approximately 1.5 million granulosa
cells/cm2 were plated in 24-well plates and immediately
treated with no-factor (control), KGF (50 ng/ml) or FSH (100 ng/ml) in
the presence or absence of KGF. After 20 h, 0.5 ml medium containing
0.5 mCi [1b-3H]androstenedione (New England Nuclear,
Boston, MA) was added and incubated for 6 h at 37 C in a
CO2 atmosphere. The culture was then sonicated, and an
aliquot was removed for DNA analysis. The remaining sample was
transferred to a tube and incubated with dextran-coated charcoal
[0.2% (wt/vol) 70K dextran (Sigma), activated 2% (wt/vol) charcoal,
10 mM Tris, and 1 mM EDTA, pH 7.5] for 2
h at 4 C, then centrifuged at 13,000 x g for 15 min.
Aliquots of the supernatant, containing 3H2O,
were then combined with 5 ml scintillation cocktail (ICN, Costa Mesa,
CA) and counted for 5 min in a scintillation counter. Blank values were
established from identical incubations, in the absence of cells, and
contained less than 100 cpm. Aromatase activity, measured in counts per
min of 3H2O released, was normalized, per
microgram DNA, using an ethidium bromide procedure described previously
(35).
Assay for progesterone production
Progesterone production by granulosa cells was determined by
quantitating progesterone accumulation in the culture medium.
Approximately 1.5 million granulosa cells/cm2 were plated
in 24-well plates and immediately treated with no-factor (control), KGF
(50 ng/ml), or hCG (100 ng/ml) in the presence or absence of KGF. After
72 h, the medium was collected and assayed for progesterone using the
ImmunoChem 125I progesterone kit (ICN). The sensitivity of
the progesterone assay was 0.01 ng/ml. Progesterone data were
normalized, per total DNA per well, as previously described (35).
Statistical analysis
All data were analyzed by a JMP 3.1 statistical analysis program
(SAS Institute Inc., Cary, NC). Effects of hormones on KGF or KGFR mRNA
levels and on granulosa cell aromatase activity and progesterone
production were analyzed by a one-way ANOVA. Significant differences
between treated cells and control (untreated) cells were determined
using the Dunnett’s test, which guards against the high 
-size (Type
I) error rate across the hypothesis tests (39). Effects of follicle
size on steady-state KGF or KGFR mRNA levels were analyzed by a one-way
ANOVA, as described above. Significant differences between small,
medium, and large follicles were determined using the Tukey-Kramer HSD
(honestly significant difference) test which protects the significance
tests of all combinations of pairs (40, 41, 42). These multiple comparisons
tests are recommended for multiple comparisons with control
(Dunnett’s) or multiple comparisons of all pairs (Tukey-Kramer HSD)
(43, 44).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
The 359-bp KGFR PCR product was observed in granulosa cell RNA from
small, medium, and large follicles (Fig. 1B). These PCR products were
blunt-end subcloned into the Bluescript plasmid, sequenced, and
confirmed to be bovine KGF and KGFR cDNA (data not shown). KGF and KGFR
subclones were used below as standards for quantitative RT-PCR.
Although KGF and KGFR are generally expressed in mesenchymal-derived
cells and epithelial cells, respectively, their expression patterns
have not been well characterized in the ovary. KGF mRNA expression has
been shown in bovine theca cells (8), but KGFR expression has not been
directly examined in the ovary. The results in Fig. 1 indicate that
KGFR is expressed by epithelial granulosa cells in ovarian follicles.
These results also demonstrate that KGF and KGFR are expressed
throughout follicular development, because KGF and KGFR mRNAs were
observed from small, medium, and large follicles.
|
as template, to generate standard curves.
Samples, consisting of total RNA from freshly isolated theca cells or
granulosa cells, were reverse transcribed using the specific 3' primers
of the gene(s) of interest. These unknown samples were simultaneously
amplified by PCR, along with the known standards, to quantitate gene
expression. Cycle number and annealing temperature were optimized for
each gene to obtain maximum sensitivity and to maintain linearity.
These quantitative assays for KGF and KGFR mRNAs are extremely
sensitive (<10-11 g/sample) and have intraassay
variabilities of 11.3% and 10.2%, respectively. As is shown in Figs. 2 and 3,
each assay is linear over several orders of magnitude (0.1–1000
fg/sample). Each assay was validated by demonstrating parallel curves
between the appropriate RNA samples and standards (Figs. 2A and 3A).
All samples were normalized for the constitutively expressed 1B15 mRNA,
as determined by the same procedure. This normalization corrects for
the number of cells, amount and integrity of initial mRNA, and small
differences in the efficiency of reverse transcription between samples.
Inset representative gels are shown, to demonstrate that minimal
background is detected and the same products are detected in the
standard DNA and sample RNA. The results are shown in Figs. 2C and 3C.
The steady-state levels of KGF mRNA in theca cells were highest in
large follicles and lowest in medium follicles (Fig. 2B). A small (but
significant) difference in KGF mRNA expression was observed between
theca cells from small and medium follicles. Steady-state levels of
KGFR were dramatically higher in granulosa cells from large follicles
than in small or medium follicles (Fig. 3B). In addition, steady-state
levels of KGF mRNA in theca cells (0.3–0.8 fg KGF/fg 1B15) were two
orders of magnitude higher than the levels of KGFR mRNA in granulosa
cells (0.001–0.003 fg KGFR/fg 1B15). These results demonstrate that
the KGF and KGFR genes are developmentally regulated during normal
follicular development. Both KGF (in theca cells) and KGFR (in
granulosa cells) were highest in large follicles. The IB15
normalization corrected for cell numbers and has previously been shown
not to vary in expression levels between cells at different stages of
development (data not shown). Because KGF has previously been shown to
stimulate granulosa cell proliferation (8), expression of KGF and KGFR
may be important for granulosa cell proliferation in large
follicles.
|
|
. Both
estrogen and hCG stimulated KGF mRNA in bovine theca cells. Similar
results were obtained with 10-7 M and
10-6 M estradiol and 50 mg and 100 mg CG,
indicating optimal stimulation (data not shown). These results are the
first to demonstrate regulation of the KGF gene in the ovary. The
regulation of KGF mRNA by estrogen and LH supports the hypothesis that
these hormones promote follicular growth by stimulating the local
production of KGF. The potential regulation of ovarian KGF expression
by other factors remains to be elucidated.
|
.
KGF significantly reduced basal levels of aromatase activity in bovine
and rat granulosa cells. In addition, KGF reduced the ability of FSH to
stimulate aromatase activity. Because KGF has previously been shown to
stimulate granulosa cell proliferation (8), this negative effect on
aromatase activity is probably the indirect effect of promoting cell
cycle entry. A similar effect has been observed with transforming
growth factor (TGF-) in the ovary (45). These results
demonstrate that KGF negatively regulates granulosa cell function by
decreasing aromatase activity.
|
. KGF had no effect on basal levels of
progesterone production but dramatically inhibited the ability of hCG
to stimulate progesterone. Therefore, KGF can inhibit both aromatase
activity and progesterone production in granulosa cells. These results
demonstrate that KGF has an inhibitory effect on the functional
differentiation (i.e. aromatase activity and progesterone
production) of granulosa cells. These differentiated functions are
indirectly decreased because of the previously shown ability of KGF to
stimulate granulosa cell.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The developing ovarian follicle is one of the most rapidly
proliferating normal tissues known in vivo, and granulosa
cell growth accounts for the majority of this follicle expansion (9).
The number of granulosa cells increase at least 10,000-fold during
development from preantral to ovulatory follicles, and the rate of
proliferation is highest in large follicles (47). Locally produced KGF
is proposed to be important for this growth process. Expression of KGF
in theca cells, and KGFR in granulosa cells, was observed throughout
follicular development (Fig. 1). Levels of KGF and KGFR mRNAs were
highest in large follicles (Figs. 2 and 3). Large follicles contain
large numbers of proliferating granulosa cells. The high expression of
KGF and KGFR in large follicles suggests that KGF actions may be
involved in this cellular proliferation. KGF has previously been shown
to stumunalte granulosa cell growth (8). These results demonstrate that
expression levels of KGF and KGFR mRNA are developmentally regulated
during follicular development. Additional studies are necessary to
determine whether expression of KGF protein is also developmentally
regulated in the ovarian follicle. It remains possible that mRNA levels
of these genes vary during follicular development, whereas protein
levels may not change. However, the observation that gene expression of
both KGF and KGFR is highest in large follicles suggests that increased
production of KGF is important in large follicles.
Endocrine hormones (i.e. gonadotropins and steroids), such
as estrogen and LH, stimulate follicular growth (10, 11, 12, 13, 14). The actions
of such hormones in the ovary are necessary for follicular development
and reproductive viability. Although these hormones increase follicular
growth in vivo, proliferative effects are not apparent on
purified cells in vitro. These observations led to the
hypothesis that estrogen and other hormones indirectly stimulate
follicular growth by influencing mesenchymal-epithelial cell
interactions in the ovary. This hypothesis is supported by the results
in Fig. 5. Both estrogen and hCG (an LH agonist) treatment increased
KGF mRNA levels in purified theca cells. These results suggest that KGF
is an estromedin (i.e. local mediator of estrogen action) in
the ovarian follicle. In addition, KGF may, in part, mediate the
actions of LH in the ovary. The regulation of KGF expression by
estrogen and LH helps explain the mechanism of hormone-induced growth
in the ovary. Endocrine hormones may stimulate follicular growth by
stimulating the local production of growth factors, such as KGF.
Factors that stimulate cellular proliferation generally inhibit
differentiated functions. Many growth factors stimulate DNA synthesis
in a particular cell by promoting entry of the cell into the cell cycle
(48, 49, 50, 51, 52). Progression of the cell into the cell cycle results in the
indirect effect of reducing the differentiated functions of the cell
(53, 54, 55, 56, 57, 58). KGF has been shown to stimulate granulosa cell proliferation
(8), but no information has been available on the effects of KGF on
granulosa cell differentiation. The current results suggest that KGF
can indirectly inhibit granulosa cell differentiated functions. KGF
reduced basal levels or hormone-stimulated levels of aromatase activity
and progesterone production. Rat granulosa cells were included in these
experiments because rat granulosa cells have a higher level of
aromatase expression in culture than bovine granulosa cells. Similar
effects were observed on bovine and rat granulosa cells. These
inhibitory effects of KGF on steroid production were similar to the
inhibitory actions of TGF- on granulosa cells (45). Roberts
demonstrated that TGF- also stimulates proliferation and inhibits
steroid production by bovine granulosa cells.
This study shows that KGF may be an important mediator of mesenchymal-epithelial cell interactions during ovarian follicular development. Results demonstrate that KGF in theca cells, and KGFR in granulosa cells, are expressed throughout follicular development and are more highly expressed in large follicles. Estrogen and LH are known to directly act on theca cells and stimulate follicular growth in vivo. This study demonstrated that both estrogen and hCG (an LH agonist) increase KGF steady-state mRNA levels in purified theca cells. Therefore, KGF, in part, mediates the actions of endocrine hormones, such as estrogen and LH, in the ovary. Other locally produced growth factors may also mediate the actions of these hormones in the ovary. The rapid proliferation of granulosa cells is a critical aspect of follicular development and is proposed to be controlled through mesenchymal-epithelial cell interactions between theca cells and granulosa cells. Future studies to investigate the significance of KGF on follicle growth and development have been initiated and will examine inhibiting the actions of KGF in ovary organ cultures to assess effects on follicle development. The current study helps to establish that KGF, in part, mediates hormone-induced follicular growth and helps gain an understanding of factors that control mesenchymal-epithelial cell interactions in the ovary.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received May 8, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  D. Schams, V. Steinberg, M. Steffl, H. H D Meyer, and B. Berisha Expression and possible role of fibroblast growth factor family members in porcine antral follicles during final maturation Reproduction, July 1, 2009; 138(1): 141 - 149. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Abir, B. Fisch, X.Y. Zhang, C. Felz, G. Kessler-Icekson, H. Krissi, S. Nitke, and A. Ao Keratinocyte growth factor and its receptor in human ovaries from fetuses, girls and women Mol. Hum. Reprod., February 1, 2009; 15(2): 69 - 75. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Portela, P. B. D. Goncalves, A. M. Veiga, E. Nicola, J. Buratini Jr., and C. A. Price Regulation of Angiotensin Type 2 Receptor in Bovine Granulosa Cells Endocrinology, October 1, 2008; 149(10): 5004 - 5011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J Buratini Jr., M.G.L Pinto, A.C Castilho, R.L Amorim, I.C Giometti, V.M Portela, E.S Nicola, and C.A Price Expression and Function of Fibroblast Growth Factor 10 and Its Receptor, Fibroblast Growth Factor Receptor 2B, in Bovine Follicles Biol Reprod, October 1, 2007; 77(4): 743 - 750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Drummond, M. Tellbach, M. Dyson, and J. K. Findlay Fibroblast Growth Factor-9, a Local Regulator of Ovarian Function Endocrinology, August 1, 2007; 148(8): 3711 - 3721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Berisha, M. Steffl, W. Amselgruber, and D. Schams Changes in fibroblast growth factor 2 and its receptors in bovine follicles before and after GnRH application and after ovulation Reproduction, February 1, 2006; 131(2): 319 - 329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J Buratini Jr, A B Teixeira, I B Costa, V F Glapinski, M G L Pinto, I C Giometti, C M Barros, M Cao, E S Nicola, and C A Price Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles Reproduction, September 1, 2005; 130(3): 343 - 350. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I Demeestere, J Centner, C Gervy, Y Englert, and A Delbaere Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents Reproduction, August 1, 2005; 130(2): 147 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Perry, M. F. Smith, M. C. Lucy, J. A. Green, T. E. Parks, M. D. MacNeil, A. J. Roberts, and T. W. Geary From the Cover: Relationship between follicle size at insemination and pregnancy success PNAS, April 5, 2005; 102(14): 5268 - 5273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. E. Nilsson and M. K. Skinner Bone Morphogenetic Protein-4 Acts as an Ovarian Follicle Survival Factor and Promotes Primordial Follicle Development Biol Reprod, October 1, 2003; 69(4): 1265 - 1272. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Taniguchi, T. Harada, Y. Sakamoto, N. Yamauchi, S. Yoshida, T. Iwabe, and N. Terakawa Activation of Mitogen-Activated Protein Kinase Pathway by Keratinocyte Growth Factor or Fibroblast Growth Factor-10 Promotes Cell Proliferation in Human Endometrial Carcinoma Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 773 - 780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Doraiswamy, J. A. Parrott, and M. K. Skinner Expression and Action of Transforming Growth Factor Alpha in Normal Ovarian Surface Epithelium and Ovarian Cancer Biol Reprod, September 1, 2000; 63(3): 789 - 796. [Abstract] [Full Text]
|
|
|
|
|
|
J. A. Parrott, R. Mosher, G. Kim, and M. K. Skinner Autocrine Interactions of Keratinocyte Growth Factor, Hepatocyte Growth Factor, and Kit-Ligand in the Regulation of Normal Ovarian Surface Epithelial Cells Endocrinology, July 1, 2000; 141(7): 2532 - 2539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ka, T. E. Spencer, G. A. Johnson, and F. W. Bazer Keratinocyte Growth Factor: Expression by Endometrial Epithelia of the Porcine Uterus Biol Reprod, June 1, 2000; 62(6): 1772 - 1778. [Abstract] [Full Text]
|
|
|
|
|
|
H. Yada, K. Hosokawa, K. Tajima, Y. Hasegawa, and F. Kotsuji Role of Ovarian Theca and Granulosa Cell Interaction in Hormone Productionand Cell Growth During the Bovine Follicular Maturation Process Biol Reprod, December 1, 1999; 61(6): 1480 - 1486. [Abstract] [Full Text]
|
|
|
|
 |
|
 P Gillis, U Savla, O. Volpert, B Jimenez, C. Waters, R. Panos, and N. Bouck Keratinocyte growth factor induces angiogenesis and protects endothelial barrier function J. Cell Sci., January 6, 1999; 112(12): 2049 - 2057. [Abstract] [PDF]
|
|
|
|
|
|
J. A. Parrott and and M. K. Skinner Developmental and Hormonal Regulation of Hepatocyte Growth Factor Expression and Action in the Bovine Ovarian Follicle Biol Reprod, July 1, 1998; 59(3): 553 - 560. [Abstract] [Full Text]
|
|
|
|
|
|
J. A. Parrott and M. K. Skinner Thecal Cell-Granulosa Cell Interactions Involve a Positive Feedback Loop among Keratinocyte Growth Factor, Hepatocyte Growth Factor, and Kit Ligand during Ovarian Follicular Development Endocrinology, May 1, 1998; 139(5): 2240 - 2245. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
) and a fresh theca cell
total RNA sample (•) are shown. Raw data are represented as arbitrary
units ± SEM (as read from the PhosphorImager) and are
directly proportional to actual counts per minute. An inset of a
representative gel is shown for both standard KGF subclone DNA (std)
and sample RNA (RNA). B, Analysis of steady-state KGF mRNA levels in
theca cells from small, medium, and large follicles. Levels of KGF mRNA
were determined and normalized to levels of 1B15 mRNA (fg KGF mRNA/fg
1B15 mRNA). Data are presented as the mean ± SEM of
duplicate determinations from seven different sets of theca cell RNA.
An ANOVA was performed, and significant differences between follicle
sizes were determined using the Tukey-Kramer HSD test.
Bars with different superscript letters
differ from each other (P < 0.05). C, Representative
electrophorectic gel of standard and RNA sample (TC, theca cell) with
the 306-bp PCR product indicated.
) and
a fresh granulosa cell total RNA sample (
) are shown. Raw data are
represented as arbitrary units ± SEM (as read from
the PhosphorImager) and are directly proportional to actual counts per
minute. An inset of a representative gel is shown for both standard
KGFR subclone DNA (std) and sample RNA (RNA). B, Analysis of
steady-state KGFR mRNA levels in granulosa cells from small, medium,
and large follicles. Levels of KGFR mRNA were determined and normalized
to levels of 1B15 mRNA (fg KGFR mRNA/fg 1B15 mRNA). Data are presented
as the mean ± SEM of duplicate determinations from
four different sets of granulosa cell RNA. An ANOVA was performed, and
significant differences between follicle sizes were determined using
the Tukey-Kramer HSD test. Bars with different
superscript letters differ from each other
(P < 0.05). C, Representative electrophoretic gel of
standard and RNA samples (GC, granulosa cell) with the 359-bp PCR
product indicated.
) of KGF. Additionally, cells were treated with
FSH as a positive control. Bovine granulosa cells can be isolated in
large numbers, but rat granulosa cells express higher levels of
aromatase in culture. Aromatase activity was determined on day 1 of
culture and normalized per microgram DNA (cpm/µg DNA). Data are
presented as mean ± SEM of triplicate determinations
from at least three different experiments. An ANOVA was performed, and
significant differences between treatments were determined using the
Tukey-Kramer HSD test. Bars with different
superscript letters differ from each other
(P < 0.05).