Epidermal growth factor and transforming growth factor-alpha stimulate the proliferation of mouse uterine stromal cells

Growth factors produced in the uterine endometrium are considered to be involved in the proliferation of the mouse uterine stromal cells induced by estradiol-17beta (E(2)) and progesterone (P). The effect of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), one of EGF-related growth factors, on the proliferation of mouse uterine stromal cells was studied in a serum-free culture. The growth of the uterine stromal cells was measured by MTT assay. EGF was found to increase the number of uterine stromal cells in a dose-dependent manner. The DNA-replicating cells were investigated using the immunocytochemical detection of bromodeoxyuridine (BrdU)-labeled cells. EGF and TGF-alpha increased the percentage of BrdU-labeled cells in a dose-dependent manner. Administration of the combination of E(2) (10(-9) M) and P (10(-7) M) for 2 days increased the percentage of BrdU-labeled cells 2.3-fold. The stimulatory effect of EGF, TGF-alpha and the combination of E(2) and P on DNA replication in the uterine stromal cells was repressed by RG-13022 (10(-5) M, the inhibitor of the EGF receptor tyrosine kinase). RT-PCR analysis of EGF-receptor-, TGF-alpha-, and EGF-mRNA was carried out in the cultured uterine stromal cells, and revealed the expression of those mRNAs. These data supported the hypothesis that uterine endometrial stromal growth induced by sex steroids required the EGF family of ligands such as EGF and TGF-alpha, both produced in the stromal cells, acting for DNA synthesis through EGF receptors.     

Nucleolar Sik-similar protein (Sik-SP) is required for the maintenance of uterine estrogen signaling mechanism via ERα

Sik-similar protein (Sik-SP), a small nucleolar ribonucleoprotein, has been shown to be primarily involved in ribosome biogenesis. However, its role in the hormone-directed nuclear receptor signaling is largely unknown. Here, we provide novel evidence that Sik-SP is required for appropriate regulation of estrogen receptor (ER)α-mediated estradiol-17β (E2)-dependent uterine physiologic responses in mice. Studies by Western blotting using the newly developed antibodies for Sik-SP showed that this protein is up-regulated in both the ovariectomized wild-type and ERα null uteri by E2. Immunohistochemical analyses in uterine sections showed that this protein is induced in the epithelial and stromal cells. Coimmunoprecipitation studies revealed that E2 directs molecular interaction between Sik-SP and ERα. Furthermore, gel-mobility shift and chromatin immunoprecipitation analyses provided evidence that Sik-SP is recruited with ERα to estrogen-responsive uterine gene promoters. Overexpression of Sik-SP in vitro demonstrated a role for Sik-SP in cellular growth and viability. In a primary uterine epithelial-stromal coculture system, E2 exhibited early induction of Sik-SP in both the epithelial and stromal cells. Interestingly, suppression of Sik-SP in this coculture model, for the stromal but not epithelial cells, caused perturbation of E2-dependent proliferation in the epithelial cell layer. Similarly, in vivo uterine suppression of Sik-SP also caused inhibition of epithelial cell proliferation and aberrant prolongation of water imbibition in the late phase by E2. Finally, studies showed that Sik-SP is physiologically important during the onset of implantation by E2. In conclusion, Sik-SP, an early E2-responsive nucleolar protein, is necessary to induce E2-dependent ERα-mediated appropriate physiologic responses in the uterus.     

Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract

Regulation of progesterone receptor (PR) by estradiol-17beta (E(2)) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E(2) induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor alpha (ERalpha) knockout (ERKO) mice showed that ERalpha is essential for E(2)-induced PR expression in both vaginal epithelium and stroma, and for E(2)-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E(2) was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E(2) only in wild-type epithelium and/or stroma. Hence, in vagina, E(2) induces PR directly via ERalpha within the tissue. Conversely, E(2) down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E(2) requires stromal, but not epithelial, ERalpha. In vitro, isolated uterine epithelial cells retained a high PR level with or without E(2), which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E(2) down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERalpha.     

Hoxa-10 regulates uterine stromal cell responsiveness to progesterone during implantation and decidualization in the mouse.

Hoxa-10 is an AbdominalB-like homeobox gene that is expressed in the developing genitourinary tract during embryogenesis and in the adult uterus during early pregnancy. Null mutation of Hoxa-10 in the mouse causes both male and female infertility. Defective implantation and decidualization resulting from the loss of maternal Hoxa-10 function in uterine stromal cells is the cause of female infertility. However, the mechanisms by which Hoxa-10 regulates these uterine events are unknown. We have identified two potential mechanisms for these uterine defects in Hoxa-10(-/-) mice. First, two PGE2 receptor subtypes, EP3 and EP4, are aberrantly expressed in the uterine stroma in Hoxa-10(-/-) mice, while expression of several other genes in the stroma (TIMP-2, MMP-2, ER, and PR) and epithelium (LIF, HB-EGF, Ar, and COX-1) are unaffected before implantation. Further, EP3 and EP4 are inappropriately regulated by progesterone (P4) in the absence of Hoxa-10, while PR, Hoxa-11 and c-myc, three other P4-responsive genes respond normally. These results suggest that Hoxa-10 specifically mediates P4 regulation of EP3 and EP4 in the uterine stroma. Second, since Hox genes are implicated in local cell proliferation, we also examined steroid-responsive uterine cell proliferation in Hoxa-10(-/-) mice. Stromal cell proliferation in mutant mice in response to P4 and 17beta-estradiol (E2 was significantly reduced, while epithelial cell proliferation was normal in response to E2. These results suggest that stromal cell responsiveness to P4 with respect to cell proliferation is impaired in Hoxa-10(-/-) mice, and that Hoxa-10 is involved in mediating stromal cell proliferation. Collectively, these results suggest that Hoxa-10 mutation causes specific stromal cell defects that can lead to implantation and decidualization defects apparently without perturbing epithelial cell functions.     

Epithelial-stromal tissue interaction in paramesonephric (Müllerian) epithelial differentiation.

During organogenesis, the middle to caudal portion of Müllerian epithelium differentiates into uterine and vaginal epithelia in females. Functional differentiation of uterine and vaginal epithelia occurs in adulthood, and is regulated by 17beta-estradiol (E(2)) and progesterone. In this report, the roles of mesenchyme/stroma in differentiation of uterine and vaginal epithelia were studied in tissue recombination experiments. At birth, Müllerian epithelium was negative for uterine and vaginal epithelial markers. Tissue recombinant experiments showed that uterine and vaginal gene expression patterns were induced in neonatal Müllerian epithelium by the respective mesenchymes. Differentiated adult uterine and vaginal epithelia did not change their original gene expression in response to heterotypic mesenchymal induction. In the adult vagina, E(2) induced expression of involucrin, a CCAAT/enhancer-binding protein beta and cytokeratin 1 via estrogen receptor alpha (ERalpha). Tissue recombination experiments with wild-type and ERalpha knockout mice demonstrated that epithelial gene expression is regulated by E(2) via epithelial-stromal tissue interactions. Uterine/vaginal heterotypic tissue recombinations demonstrated that functional differentiation of uterine and vaginal epithelia required organ-specific stromal factors. In contrast, stromal signals regulating epithelial proliferation appeared to be nonspecific in the uterus and vagina.     

The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen

The activation function-1 (AF-1) domain of the estrogen receptor alpha (ERalpha) in stromal cells has been shown to be required for epithelial responses to estrogen in the mouse uterus. To investigate the role of the stroma in estrogenic responses of human uterine epithelium (hUtE), human/mouse chimeric uteri composed of human epithelium and mouse stroma were prepared as tissue recombinants (TR) that were grown in vivo under the renal capsule of female nude mouse hosts. In association with mouse uterine stroma (mUtS), hUtE formed normal glands surrounded by mouse endometrial stroma and the human epithelium influenced the differentiation of stroma into myometrium, such that a histologically normal appearing uterine tissue was formed. The hUtE showed a similar proliferative response and increase in progesterone receptors (PR) in response to 17beta-estradiol (E2) in association with either human or mUtS, as TRs. However, under identical endocrine and micro-environmental conditions, hUtE required 5-7 days exposure to E2 rather than 1 day, as shown for mouse uterine epithelium, to obtain a maximal proliferative response. Moreover, this extended length of E2 exposure inhibited mouse epithelial proliferation in the presence of mouse stroma. In addition, unlike the mouse epithelium, which does not proliferate or show regulation of PR expression in response to E2 in association with uterine stroma derived from mice that are null for the AF-1 domain of ERalpha, hUtE proliferates and PR are up-regulated in response to E2 in association genetically identical ERalpha knock-out mouse stromal cells. These results clearly demonstrate fundamental differences between mouse and human uterine epithelia with respect to the mechanisms that regulate estrogen-induced proliferation and expression of PR. Moreover, we show that genetically engineered mouse models could potentially aid in dissecting molecular pathways of stromal epithelial interactions in the human uterus.     

Polarized uterine epithelial cells preferentially present antigen at the basolateral surface: role of stromal cells in regulating class II-mediated epithelial cell antigen presentation

To study Ag presentation in the female reproductive tract, DO11.10 TCR transgenic mice specific for the class II MHC-restricted OVA(323-339) peptide and non-transgenic BALB/c mice were used. We report here that freshly isolated uterine epithelial cells, uterine stromal, and vaginal APCs present OVA and OVA(323-339) peptide to naive- and memory T cells, which is reduced when cells are incubated with Abs to CD80 and 86. To determine whether polarized primary epithelial cells present Ags, uterine epithelial cells were cultured on cell inserts in either the upright or inverted position. After reaching confluence, as indicated by high transepithelial resistance (>2000 ohms/well), Ag presentation by epithelial cells incubated with memory T cells and OVA(323-339) peptide placed on the basolateral surface (inverted) was 2- to 3-fold greater than that seen with epithelial cells in contact with T cells and peptide on the apical surface (upright). In contrast, whereas freshly isolated epithelial cells process OVA, polarized epithelial cells did not. When epithelial cells grown upright on inserts were incubated with T cells and OVA(323-339) peptide, coculture with either hepatocyte growth factor or conditioned stromal medium increased epithelial cell Ag presentation (approximately 90% higher than controls). These studies indicate that uterine stromal cells produce a soluble factor(s) in addition to a hepatocyte growth factor, which regulates epithelial cell Ag presentation. Overall, these results demonstrate that polarized epithelial cells are able to present Ags and suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to regulate Ag presentation in the uterus.     

Effect of mouse uterine stromal cells on epithelial cell transepithelial resistance (TER) and TNFalpha and TGFbeta release in culture

Recognizing that uterine stromal cells regulate several uterine epithelial cell function(s), the current study was undertaken to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uterine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high transepithelial resistance (TER > 1000 ohms/well), preferentially release transforming growth factor-beta (TGFbeta) into the basolateral chamber ( approximately 70% > apical) and tumor necrosis factor-alpha (TNFalpha) into the apical compartment ( approximately 30% > basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, coculture for 24-48 h increased epithelial cell TER ( approximately 70% higher than control) and decreased TNFalpha release into both the apical and basolateral chambers ( approximately 30%-50%). In contrast, TGFbeta release was not affected by the presence of stromal cells. In other studies, the effects of stromal cells on epithelial cell TER and TNFalpha release persisted for 5-7 days following the removal of stromal cells and were also seen in coculture studies in which conditioned stromal media (CSM) was placed in the basolateral chamber. These studies indicate that uterine stromal cells produce a soluble factor(s) that regulates epithelial cell TER and release of TNFalpha without effecting TGFbeta release. These results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to maintain uterine integrity and epithelial secretory function.     

Expression and action of hepatocyte growth factor in bovine endometrial stromal and epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that acts on various epithelial cells. The objectives of this study were to determine whether HGF altered the proliferation and prostaglandin (PG) secretion of bovine endometrial stromal and epithelial cells in vitro. We also observed HGF and HGF receptor (c-met) mRNA expression in cultured bovine endometrial stromal and epithelial cells by RT-PCR. Stromal and epithelial cells obtained from cows in early stage of the estrous cycle (days 2-5) were cultured in DMEM/Ham's F-12 supplemented with 10% calf serum. The cells were exposed to HGF (0-10 ng/ml) for 2, 4, or 6 days. HGF significantly increased the total DNA in epithelial (P < 0.05), but not stromal cells. In another experiment, when the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA containing HGF 0-100 ng/ml and the cells were cultured for 24 hr. The HGF stimulated PGF2alpha secretion in epithelial, but not stromal cells. RT-PCR revealed that mRNA of HGF is expressed only in stromal cells, and that c-met mRNA is expressed in both stromal and epithelial cells. These results suggest that HGF plays roles in the proliferation and the regulation of secretory function of bovine endometrial epithelial cells in a paracrine fashion.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Vaginal and uterine stroma maintain their inductive properties following primary cullture

Summary Vaginal and uterine stromal (VS and UtS) cells have been cultured in a collagen gel matrix, and the ability of the cells to retain their identity and interact normally with epithelia after culture was examined. Stromal explant from 2-d-old mice were plated onto an extracellular matrix covered with collagen, and maintained in Ham’s F12∶DMEM (1∶1) containing 15% fetal bovine serum. The fibroblastic stromal cells invaded and eventually filled the overlying collagen during the 4-wk growth period, and the total DNA of the UtS and VS cultures increased 3.5- and 4-fold, respectively. To assess the ability of the cultured stroma to perform its normal functions after the in vitro period, recombinations of cultured stroma and fresh epithelia were preparaed and transplanted under the renal capsule of female hosts and grown for 4 wk. The epithelium in recombinants of cultured VS + vaginal epithelium (VE) and cultured UtS + uterine epithelium (UtE) was histologically normal and proliferated in response to estrogen. Cultured stroma also instructively induced heterologous epithelium; VS induced UtE to undergo vaginal differentiation, and UtS induced VE to undergo uterine differentiation. These results indicate that UtS and VS retain their identity and do not irreversibly dedifferentiate in culture. Stromal cells grown in a colagen gel matrix form a functional stroma; they interact normally with epithelium after culture and express normal permissive and instructive inductive functions when reassociated with epithelium and grown in vivo. This work was supported by grants HD 17491, AM/CA 16570, CA 05388, and 5 F32 HD06580 from the National Institute of Health, Bethesda, MD, and a grant from the UCSF Academic Senate.     

Hedgehog signaling plays roles in epithelial cell proliferation in neonatal mouse uterus and vagina

Both the uterus and vagina develop from the Müllerian duct but are quite distinct in morphology and function. To investigate factors controlling epithelial differentiation and cell proliferation in neonatal uterus and vagina, we focused on Hedgehog (HH) signaling. In neonatal mice, Sonic hh (Shh) was localized in the vaginal epithelium and Indian hh (Ihh) was slightly expressed in the uterus and vagina, whereas all Glioma-associated oncogene homolog (Gli) genes were mainly expressed in the stroma. The expression of target genes of HH signaling was high in the neonatal vagina and in the uterus, it increased with growth. Thus, in neonatal mice, Shh in the vaginal epithelium and Ihh in the uterus and vagina activated HH signaling in the stroma. Tissue recombinants showed that vaginal Shh expression was inhibited by the vaginal stroma and uterine Ihh expression was stimulated by the uterine stroma. Addition of a HH signaling inhibitor decreased epithelial cell proliferation in organ-cultured uterus and vagina and increased stromal cell proliferation in organ-cultured uterus. However, it did not affect epithelial differentiation or the expression of growth factors in organ-cultured uterus and vagina. Thus, activated HH signaling stimulates epithelial cell proliferation in neonatal uterus and vagina but inhibits stromal cell proliferation in neonatal uterus.     

Epidermal growth factor stimulates proliferation of mouse uterine epithelial cells in primary culture

Epidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9+/- 8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-I-induced growth.     

Antagonistic effects of estradiol dipropionate and progesterone on the histology of the vagina and uterus of the mouse

Administration of estradiol dipropionate (20 micrograms/day; 7 days) to ovariectomized mice caused heavy epithelial proliferation and intense cornification in the vagina and cellular as well as glandular proliferation in uterine tissues. Endometrial hypertrophy with cystlike appearance of uterine glands was seen in response to a long-term (14 days) administration of estradiol dipropionate. Daily injection of progesterone (2 mg; 7 days) to ovariectomized mice resulted in desquamating mucosa, without any trace of vaginal cornification, and the presence of dense uterine connective tissue in the stromal region with typical uterine glands. However, treatment of estradiol depropionate in combination with progesterone at 1:100 dose ratio for 7 days produced vaginal histology similar to that in proestrus and uterine histology equivalent to the ovariectomized condition. The results revealed that progesterone antagonized the estrogenic effects and also that estradiol dipropionate antagonized the effects of progesterone. The effects of the two female sex steroids (estradiol dipropionate and progesterone) in vivo appeared to be more potent in the uterus than in the vagina.     

Epithelial-mesenchymal interactions in uterus and vagina alter the expression of the cell surface proteoglycan, syndecan

The cell surface proteoglycan, syndecan, exhibits molecular and histological dimorphism in the mouse uterus and vagina. In the mature vagina, syndecan is localized at the surfaces of the basal and intermediate cells of the stratified epithelium and has a modal molecular mass of ca. 92 kDa. The uterus expresses a larger form of syndecan (ca. 110 kDa) which is detected at the basolateral surfaces of the simple columnar epithelial cells. We have investigated whether epithelial-mesenchymal interactions influence the expression of syndecan in these organs by analyzing tissue recombinants composed of mouse epithelium and rat mesenchyme or vice versa with monoclonal antibody 281-2, which recognizes mouse syndecan. In tissue recombinants composed of newborn mouse uterine epithelium and rat vaginal stroma, the uterine epithelium was induced to form a stratified vaginal epithelium which expressed syndecan in same the pattern and mass typical of vaginal epithelium. Likewise, rat uterine stroma induced newborn mouse vaginal epithelium to undergo uterine development, and this epithelium exhibited a uterine pattern of syndecan expression. Although stromal cells normally express little syndecan in most adult organs, analysis of recombinants composed of mouse stroma and rat epithelium revealed that both uterine and vaginal mouse stromata synthesized syndecan that was larger (ca. 170-190 kDa) than the epithelial syndecans. A quantitative increase in the amount of stromal syndecan was evident when stroma was grown in association with epithelium in comparison to stroma grown by itself. These data suggest that epithelial-mesenchymal interactions influence the amount, localization, and mass of both epithelial and stromal syndecan.     

Effects of follicle-stimulating hormone and 17beta-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Effects of follicle-stimulating hormone and 17β-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.