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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 99164-4234
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 99164-4234. E-mail: skinner{at}mail.wsu.edu
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Abstract |
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Introduction |
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The KL [also named stem cell factor (14), mast cell factor (15), or steel factor] and its tyrosine kinase receptor c-kit are encoded at the steel (Sl) and white spotting (W) loci of the mouse, respectively (16, 17, 18, 19, 20, 21, 22). Both SI and W mutations cause defects in melanogenesis, gametogenesis, and hematopoiesis at several stages of embryonic development and adult life (23, 24, 25, 26, 27, 28). During male and female embryonic development, KL and c-kit are essential for germ cell migration (29, 30, 31, 32, 33). In the adult, KL and c-kit are important for follicular development in the ovary and survival/proliferation of type A spermatogonia in the testis (6, 34). Several KL and c-kit mutations have been described with many gonadal phenotypes. The ovaries of adult mice carrying the steel-panda (Slpan), steel-contrasted (Slcon), or steel-t (Slt) mutations contain predominantly small follicles whose development is arrested at the stage that theca cells organize around the follicles (35, 36, 37). This lack of follicle development and subsequent infertility have been widely attributed to a defect in granulosa cell-oocyte interactions via KL/c-kit. However it is possible that these mutations arrest follicular development by disrupting granulosa cell-theca cell interactions. Terada et al. (38) found that ovaries from suckling Sl/Slt mice do not produce androgens in response to LH, suggesting a defect in theca cells. Kuroda et al. (37) suggested a possible stromal cell/theca cell defect in the same mutant mice. These studies raised the possibility that granulosa cell-derived KL may promote follicular development by directly regulating theca cell function.
The current study used bovine ovaries to examine the potential role of KL to directly regulate theca cell growth and functional differentiation. Experiments are also presented that evaluated the regulation of KL and c-kit messenger RNA (mRNA) expression during follicular development. These experiments establish KL as the first granulosa cell-derived growth factor that stimulates theca cell function in the absence of gonadotropins. These observations help gain an understanding about the local feedback mechanisms that regulate mesenchymal-epithelial cell interactions (i.e. theca-granulosa cell) in the ovary.
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Materials and Methods |
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Growth assays
Cell growth was analyzed by quantitating
[3H]thymidine incorporation into newly synthesized DNA.
Theca cells were plated at subconfluent densities (<1 million
cells/cm2) in 0.5 ml DMEM containing 0.1% calf serum.
After 24 h, the cells were treated with no growth factor
(control), 10–50 ng/ml KL, or 50 ng/ml EGF as a positive control.
Cells were plated for 24 h, then treated for an additional 24
h or 48 h. After treatment, 0.5 ml DMEM containing 2 µCi
[3H]thymidine was added to each well, and the cells were
incubated for 4 h at 37 C followed by sonication. The quantity of
[3H]thymidine incorporated into DNA was determined as
previously described (41). Data were normalized to total DNA per well
using an ethidium bromide procedure, described previously (40). Under
these subconfluent culture conditions, approximately 0.5–1.5 µg DNA
was detected per well. Values of [3H]thymidine
incorporation were generally greater than 2 x 103
cpm/µg DNA.
Steroid assays
Steroid production by theca cells was determined by quantitating
androstenedione and progesterone accumulation in the culture medium.
Fresh theca cells were plated at subconfluent densities (
0.5–1
million cells/cm2) or at confluent densities ( 3–4
million cells/cm2) in 1 ml serum-free Ham’s F-12 medium
containing 0.1% BSA. Cells were immediately treated with
10-7 M estradiol, 100 ng/ml hCG, or 50 ng/ml
KL and cultured for 72 h. At the end of the culture period, the
medium was collected and assayed for androstenedione and progesterone
using the RSL 125I-androstenedione kit and the ImmunoChem
125I-progesterone kit, respectively (ICN, Costa Mesa, CA).
The sensitivities of the steroid assays are 0.01 ng/ml for
androstenedione and 0.01 ng/ml for progesterone. The cells were
cultured for an additional 4 h in 0.5 ml DMEM medium containing
0.1% calf serum and 2 µCi [3H]thymidine to determine
whether or not the cells were proliferating. Under subconfluent culture
conditions, [3H]thymidine incorporation values were
generally greater than 2 x 103 cpm/µg DNA, indicating that the
cells were readily entering the cell cycle. Under confluent culture
conditions, [3H]thymidine incorporation values were
generally less than 2 x 102 cpm/µg DNA, indicating
that the cells were contact inhibited and not proliferating. The
observation that [3H]thymidine incorporation in confluent
cell cultures was at least 10-fold less than in subconfluent cell
cultures validates the use of these culture conditions as nongrowth
permissive and growth permissive, respectively. All steroid data were
normalized to total DNA per well. Under these culture conditions,
approximately 0.5–2.5 µg DNA (subconfluent densities) and 6–10 µg
DNA (confluent densities) was detected per well.
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)
size 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 (42).
Alternatively total RNA was prepared using the RNA-Stat 60 kit
(Tel-Test, Friendswood, TX). For qualitative analysis of gene
expression, 10 µg total RNA was reverse transcribed with Moloney
murine leukemia virus reverse transcriptase (Gibco BRL) at 37 C for
1 h using oligo(dT)12–18 primers (Gibco BRL). This
complementary DNA (cDNA) template was amplified by PCR using specific
bovine primers for KL, c-kit, or the constitutively expressed gene
cyclophilin (IB15). The KL primers were 5'-GGA CAA GTT TTC GAA TAT TTC
TGA AGG CTT GAG TAA TTA TTG-3' (5' primer, 42-mer) and 5'-GGC TGC AAC
AGG GGG TAA CAT AAA TGG TTT TGT GAC ACT GAC-3' (3' primer, 42-mer),
which generated a specific 315-bp KL PCR product from bovine granulosa
cells. These KL primers are designed to specifically amplify the longer
KL transcript, which codes for the secreted form of the factor.
Experiments using a different 3' KL primer that amplifies both the
soluble (KL1) and membrane-bound (KL2) forms of KL demonstrated that
bovine granulosa cells primarily express the soluble form of KL (Fig. 1B
). This alternative KL primer is
described below in the quantitative RT-PCR method. The c-kit primers
were 5'-GTT CAT GTG TTA CGC CAA TAA CAC TTT TGG ATC AGC AAA-3' (5'
primer, 42-mer) and 5'-TTC AGT CCC TTT TAA TCT GGT TAG ATG AAG TTC
ATT-3' (3' primer, 36-mer), which generated a specific 298-bp c-kit PCR
product from bovine theca and stromal cells. The primers for
cyclophilin (IB15) 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 each primer, 100
µM deoxynucleotide triphosphates (dNTPs), 1.5
mM Mg++, and 1.25 U Taq polymerase
in 50 µl total volume. PCR products were visualized by UV
illumination (312 nm) of 2% agarose gels stained with ethidium
bromide.
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Quantitative RT-PCR assays
Steady state levels of KL, c-kit, and IB15 mRNAs were measured
using a specific quantitative RT-PCR assay for each gene. The primers
used in this quantitative analysis of KL, c-kit, and IB15 were the same
as described above except for KL (see below). Before RT, 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 four different
primers including IB15), 0.1 mM dNTPs, 10 mM
DTT, 40 U RNase inhibitor (Promega, Madison, WI), and 200 U Moloney
murine leukemia virus reverse transcriptase (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 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 (43). Plasmid DNAs containing
bovine KL, c-kit, or IB15 subclones were used to generate standard
curves from 1 ag/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 µCi
32P-labeled deoxycytidine triphosphate (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 KL and c-kit data
were normalized for IB15.
The primers used in this quantitative amplification of KL, c-kit, and
IB15 were the same as described above except for a single KL primer. A
smaller 3'-primer was used for KL that facilitates the proper melting
and annealing of the primer during RT: 5'-AGG CCC CAA AAG CAA ACC CGA
TCA CAA GAG-3' (3' primer, 30-mer). When combined with the above 5'-KL
primer, this primer amplifies both the soluble (KL1) and membrane-bound
(KL2) forms of KL. This KL primer set generated a specific 452-bp KL
PCR product from bovine granulosa cells that codes for the longer,
soluble form of KL (example shown in Fig. 1B
). A small but detectable
amount of the shorter, membrane-bound form of KL was also expressed by
granulosa cells (368-bp PCR product, Fig. 1B). Optimal cycle number for
amplification was determined for each assay to achieve maximum
sensitivity while maintaining linearity (i.e. logarithmic
phase of PCR reactions). Both KL and c-kit quantitative PCR products
were amplified for 31 cycles, while the IB15 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 8.9% (KL), 6.0%
(c-kit), and 6.5% (IB15).
Statistical analysis
All data were analyzed by a JMP 3.1 statistical analysis program
(SAS Institute Inc., Cary, NC). Effects of KL, growth factor, or
hormones on [3H]thymidine incorporation into DNA (Fig. 2) or steroid production (Figs. 3 and 4)
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 [E0]-size (Type I)
error rate across the hypothesis tests (44). Effects of follicle size
on steady state KL or c-kit mRNA levels (Figs. 5 and 6)
were analyzed by a one-way ANOVA as described above. Significant
differences between small, medium, and large size follicles were
determined using the Tukey-Kramer HSD (honestly significant difference)
test, which protects the significance tests of all combinations of
pairs (45). These multiple comparisons tests are recommended for
multiple comparisons with control (Dunnett’s) or multiple comparisons
of all pairs (Tukey-Kramer HSD) (46).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). These
KL primers are designed to specifically amplify the soluble form of KL,
which has been shown to be the predominant form expressed in rat and
human ovaries (47, 48, 49). Experiments using primers designed to detect
both the soluble (KL1) and membrane-bound (KL2) forms of KL confirmed
that bovine granulosa cells predominantly express the soluble form of
the factor (Fig. 1B). The 298-bp c-kit PCR product was observed in
theca cell RNA from small, medium, and large size follicles, as well as
stromal-interstitial cell RNA (Fig. 1A). These PCR products were
subcloned into the Bluescript plasmid, sequenced, and confirmed to be
bovine KL and c-kit cDNA (data not shown). Negative controls with the
omission of RNA or reverse transcriptase resulted in no PCR product
being detected, demonstrating that the signal did not arise from
contaminating DNA (data not shown). These results are the first to
directly demonstrate receptor c-kit mRNA expression in theca cells from
ovarian follicles. These results establish that bovine granulosa cells
express KL and bovine theca cells express the receptor c-kit.
The ability of KL to regulate the growth of bovine theca cells was
examined through an analysis of [3H]thymidine
incorporation into DNA. After initial plating at subconfluent
densities, freshly isolated theca cells were cultured in the absence or
presence of recombinant KL for 20 h followed by
[3H]thymidine for an additional 4 h. KL was found to
stimulate the growth of bovine theca cells in a dose-dependent manner
(Fig. 2). Preliminary studies to examine the effects of KL on theca
cell proliferation used a 72-h treatment with KL followed by a DNA
assay. Data suggested that KL may increase total DNA, but these results
require further investigation (data not shown). EGF (50 ng/ml) was used
as a positive control. These results suggest that KL is a theca cell
growth factor that is at least as effective as EGF in stimulating theca
cell growth. These results demonstrate that KL directly regulates theca
cell growth.
To further evaluate the potential role that KL may have in regulating
theca cells, the effect of KL on theca cell steroid production was
examined. Initially theca cell steroid production was examined under
subconfluent culture conditions (growth-permissive conditions). Under
these conditions KL had no significant effect on androstenedione or
progesterone accumulation in the culture medium (Fig. 3). These results
are consistent with the role of KL as a theca cell growth factor that
can promote entry of the cells into the cell cycle under these culture
conditions. As previously demonstrated (4, 40), both estrogen
(10-7 M) and hCG (100 ng/ml) stimulated
androstenedione production, whereas only hCG stimulated progesterone
production (Fig. 3). Similar experiments were performed to examine
theca cell androstenedione and progesterone production at confluent
densities (non-growth-permissive conditions). Theca cells do not
readily enter the cell cycle under these conditions due to cellular
contact inhibition. KL clearly stimulated androstenedione accumulation
in the culture medium when theca cells were cultured under confluent
culture conditions (Fig. 4A). The magnitude of this KL stimulation was
similar to the effects of estrogen and hCG. Preliminary studies showed
that the combined effects of KL and hormones were the same as either KL
or hormones alone, suggesting a maximum level of stimulation was
obtained (data not shown). The combined actions of KL and hormones on
theca cell androgen production require further investigation. KL had no
significant effect on theca cell progesterone production at any cell
density (Figs. 3B and 4B). These results demonstrate that KL can
directly regulate theca cell differentiation as measured by
androstenedione production. These results establish KL as the only
known growth factor to directly regulate theca cell steroid production
in the absence of gonadotropins.
The potential regulation of KL in granulosa cells and c-kit in theca
cells during follicular development was evaluated by analyzing total
RNA samples from small (<5 mm), medium (5–10 mm), and large (>10 mm)
size follicles. Sensitive quantitative RT-PCR assays were developed for
both bovine KL and c-kit. Under specific amplification conditions these
assays used the bovine KL and c-kit PCR products shown in Fig. 1 as a
template to generate standard curves. Samples consisting of total RNA
from freshly isolated granulosa cells or theca 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. For each gene cycle,
number and annealing temperature were optimized for maximum sensitivity
and linearity. These quantitative assays for KL and c-kit mRNAs are
extremely sensitive (<10-12 g/sample) and have intraassay
variabilities of 8.9% and 6%, respectively. As is shown in Figs. 5 and 6, 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. 5A and 6A).
All samples were normalized for the constitutively expressed
cyclophilin mRNA (IB15) as determined by the same procedure. This
normalization corrects for the amount of initial mRNA, as well as small
differences in the efficiency of RT between samples. The results are
shown in Figs. 5B and 6B. The steady state levels of KL mRNA in
granulosa cells is higher in large size follicles than in small or
medium size follicles (Fig 5B). There are no significant differences in
the steady state levels of theca cell c-kit mRNA between small, medium,
and large size follicles (Fig 6B). In addition the steady state levels
of KL mRNA in granulosa cells (0.4–0.8 fg KL/fg IB15) are an order of
magnitude higher than the levels of c-kit mRNA in theca cells
(0.02–0.04 fg c-kit/fg IB15). These results demonstrate that the KL
gene is developmentally regulated during normal follicular development
and may be particularly important for theca cell function in large
size follicles. The c-kit gene was shown to be constitutively
expressed throughout follicular development.
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Discussion |
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(TGF-) and -ß (TGF-ß), which regulate granulosa cell function
(2, 3, 55). However the role of locally produced substances from
granulosa cells that feedback to regulate theca cell function has not
been studied extensively. KL appears to be such a substance. Previous
studies have demonstrated that granulosa cells in developing ovarian
follicles express KL, which may be important for granulosa cell-oocyte
interactions (5, 6, 7, 35, 36). Many of these previous studies used
in situ and immunohistochemical techniques to demonstrate
receptor c-kit expression in growing and full-grown oocytes. It was
apparent in these studies that differentiated theca cells and possibly
undifferentiated stromal-interstitial cells express the c-kit receptor
and therefore may also respond to granulosa cell-derived KL (6, 7, 12).
To examine the direct action of KL on theca cell function, it is
necessary to isolate purified theca cells. The bovine ovary is large
enough and available in sufficient quantities to isolate large numbers
of purified theca cells (40). Also the bovine ovary is
endocrinologically similar to the human and is monoovulatory. This
model system is used in the current study to establish that KL can
directly regulate theca cell function. KL is established as the only
known granulosa cell-derived growth factor that can stimulate theca
cell androstenedione production in the absence of gonadotropins. These
results demonstrate that KL is an important regulator of theca cell
function and may help regulate local mesenchymal-epithelial cell
interactions. Although the expression patterns of KL and c-kit in the ovary have been studied in the rat, mouse, and human, no information about the expression patterns in the bovine ovary has been published. Bovine granulosa cells from small, medium, and large size follicles were found to express the KL gene. In addition bovine granulosa cells were found to primarily express the soluble form of KL (KL1) rather than the membrane-bound form of the factor. Bovine theca cells from small, medium, and large size follicles, as well as stromal-interstitial cells, were found to express the receptor c-kit. This study is the first to directly demonstrate c-kit mRNA expression in purified theca cells. The observation that KL and c-kit mRNA’s were observed in small, medium, and large size follicles demonstrates that the KL and c-kit genes are expressed throughout ovarian follicular development.
KL (also called stem cell factor, mast cell growth factor, or steel factor) can have a wide range of activities on germ cells, melanocytes, mast cells, and primitive hematopoietic cells of the myeloid, erythroid, and lymphoid cell lineages (23). Many of these multipotent stem cells alter their developmental program and differentiate in response to KL. It also appears that KL can cause many of these cell types to proliferate. Results from this study show that KL can also stimulate the growth of theca cells as measured by [3H]thymidine incorporation into DNA. The dose-response curve for KL on theca cells is similar to other cells examined (14, 15, 34, 56, 57). This observation establishes KL as the first granulosa cell-derived growth factor that can act in a paracrine manner to stimulate theca cell growth. Although IGF-I is produced in the ovary and can act on theca cells, the high circulating levels of insulin as well as the presence of IGF binding proteins in follicular fluid may limit the regulatory role of locally produced IGF-I on theca cells (58). Theca cells surround the outer layer of granulosa cells and provide the structural integrity of the follicle. The stimulation of theca cell growth by KL may be important for the formation of a thick theca interna/externa layer around healthy developing follicles. Disruption of this theca cell layer may result in abnormal follicular development. Further evaluation of the role of KL during follicular development will require an analysis of KL actions on theca cell proliferation in small, medium, and large size follicles.
The ability of KL to regulate the cellular differentiation of several
target tissues (i.e. mast cells, hematopoietic cells, and
melanocytes) suggests that KL may also regulate the differentiated
function of theca cells in the ovary. The steroidogenic capacity of
theca cells is a direct reflection of functional differentiation.
Therefore theca cell androstenedione and progesterone production was
examined. Many growth factors stimulate DNA synthesis (i.e.
growth) in a particular cell by promoting entry of the cell into the
cell cycle (59, 60). Progression of the cell into the cell cycle
results in the indirect effect of reducing the differentiated functions
of the cell (61, 62). For example, TGF- acts as a growth factor for
theca cells and can reduce theca cell androstenedione and progesterone
production in vitro (55). KL was a growth factor for theca
cells under subconfluent culture conditions (i.e. not
contact inhibited) that allow the cells to readily enter the cell cycle
(growth-permissive conditions). As expected, both androstenedione and
progesterone production by bovine theca cells was unaffected in
response to KL. This action of KL is consistent with the ability of KL
to act as a theca cell mitogen similar to TGF-, but it does not
exclude the possibility that KL can also stimulate theca cell steroid
production when the cells are not growing. Therefore, theca cells were
cultured under confluent conditions (i.e. contact inhibited)
that do not allow the cells to enter the cell cycle
(non-growth-permissive conditions). Interestingly KL alone stimulated
androstenedione production by bovine theca cells under these
conditions. Because estrogen and hCG do not directly act as growth
factors for theca cells, these hormones stimulated theca cell steroid
production at subconfluent and confluent cell densities. These results
establish KL as the only identified growth factor made by granulosa
cells that can directly stimulate theca cell androstenedione
production. Granulosa cell-derived activin, inhibin, and follistatin
can affect theca cell steroid production, but these factors can only
augment the actions of hormones such as LH. KL stimulated
androstenedione production directly. The observation that KL stimulates
androstenedione but not progesterone suggests that KL may promote a
follicular phase (i.e. high androstenedione, low
progesterone production) rather than a luteal phase differentiated
state of theca cells. Further evaluation of the effects of KL on theca
cell differentiation during follicular development will require the
isolation of theca cells from individual follicles from different
stages of development.
The potential roles of KL/c-kit during normal follicular development was investigated by analyzing the regulation of KL and c-kit mRNAs in small, medium, and large size follicles. Steady state KL mRNA levels in granulosa cells were significantly higher in large size follicles than in small or medium size follicles. Steady state receptor c-kit mRNA levels in theca cells did not significantly vary among small, medium, or large size follicles. During normal follicular development, larger follicles produce increasing amounts of steroids. Eventually a single dominant follicle is selected to ovulate. The observation that KL mRNA is highest in granulosa cells from large follicles suggests that KL may be important for increased theca cell steroid production and the selection of the dominant follicle. The concentration of KL in the follicles remains to be determined. Therefore, abnormal expression/action of KL during follicle development may dramatically alter ovarian function. Overexpression of KL may result in increased numbers of developing follicles and unusually high levels of androgen production. Such events may eventually cause polycystic ovary syndrome. Abnormally low levels of KL may not be sufficient to select a dominant follicle or support later stages of follicular development. Although the current analysis of KL and c-kit used pools of small, medium, and large size follicles, the quantitative RT-PCR assays that were developed are sensitive enough to analyze KL and c-kit mRNA expression in individual follicles. Elucidation of the regulation of KL and c-kit in individual healthy, atretic, and dominant follicles may be useful in understanding the cellular mechanisms that control follicular development and dominant follicle selection.
This study shows that kit ligand is an important local regulator of ovarian follicular development. The direct actions of KL on theca cells provides new insight into the mechanisms by which KL acts in the ovary. The actions of granulosa-derived KL on theca cell growth and androstenedione production provide a feedback mechanism that may regulate mesenchymal-epithelial cell interactions in the ovary (i.e. theca cell-granulosa cell interactions). These cell-cell interactions may be essential for normal ovarian follicular development and reproductive function. Several mutations at the Steel locus in mice (Slpan, Slcon, and Slt) cause ovarian follicular arrest at very early stages of follicular development. Follicular development is arrested in these mutant mice at the time that theca cells are being recruited to differentiate from the surrounding stromal cells. At least two previous studies have suggested that Slt mutations in mice may disrupt theca cell function (37, 38), but no direct examination of this hypothesis has been previously possible. Terada et al. (38) reported that suckling Sl/Slt mice do not produce androgens in response to LH, suggesting a theca/stromal cell malfunction. The current study helps explain why KL mutations arrest follicular development and inhibit androgen production in the ovary. The ability of KL to recruit a variety of stem cell populations to proliferate and differentiate raises the possibility that KL may also recruit undifferentiated stromal cells to differentiate into theca cells in the ovary. This recruitment of stromal cells to theca cells is an essential aspect of early primordial follicle development. This study has established the importance of KL for theca cell function during ovarian follicular development. The potential role of KL during primordial follicle development remains to be elucidated.
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Acknowledgments |
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Footnotes |
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Received January 30, 1997.
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S. Lenie, R. Cortvrindt, T. Adriaenssens, and J. Smitz A Reproducible Two-Step Culture System for Isolated Primary Mouse Ovarian Follicles as Single Functional Units Biol Reprod, November 1, 2004; 71(5): 1730 - 1738. [Abstract] [Full Text] [PDF]
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J. Wang and S. K. Roy Growth Differentiation Factor-9 and Stem Cell Factor Promote Primordial Follicle Formation in the Hamster: Modulation by Follicle-Stimulating Hormone Biol Reprod, March 1, 2004; 70(3): 577 - 585. [Abstract] [Full Text] [PDF]
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) and a fresh granulosa cell total RNA sample (•) are
shown. Raw data are represented as arbitrary units ±
SEM (as read from PhosphorImager) and are directly
proportional to actual cpm. B, Analysis of steady state KL mRNA levels
in granulosa cells from small, medium, and large size follicles. Each
sample was run in duplicate and normalized to levels of cyclophilin
(IB15) mRNA. Data are presented as level of KL mRNA per IB15 mRNA,
represented as mean ± SEM from four different sets of
granulosa cell RNA. An ANOVA was performed, and significant differences
between follicle sizes were determined using Tukey-Kramer HSD test.
Bars with different superscript letters
differ from each other (
) and a fresh theca cell total RNA sample (
) are shown. Raw data
are represented as arbitrary units ± SEM (as read
from PhosphorImager) and are directly proportional to actual cpm. B,
Analysis of steady state c-kit mRNA levels in theca cells from small,
medium, and large size follicles. Each sample was run in duplicate and
normalized to levels of cyclophilin (IB15) mRNA. Data are presented as
level of c-kit mRNA per IB15 mRNA, represented as mean ±
SEM from at least 10 different sets of theca cell RNA. No
differences were seen between sizes as determined by Tukey-Kramer HSD
test.