| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ovary |
Departments of Obstetrics and Gynecology3
Physiology and Biophysics,4 University of Nebraska Medical Center, Omaha, Nebraska 68198-4515
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
follicle, follicle-stimulating hormone, follicular development, growth factors, ovary
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Similarly to GDF-9, kit mRNA has been detected in mouse primordial germ cells as early as Embryonic Day 7.5 (E7.5), and subsequently high expression is evident in primordial oocytes [11]. On the other hand, kit ligand or stem cell factor (SCF) has been localized in granulosa cells [11]. SCF from granulosa cells has been proposed to bind kit receptors in the oocytes, thus regulating the growth of the oocytes. Further, increased SCF expression in the granulosa cells occurs in mice with GDF-9 null mutation, which exhibit abnormal oocyte development [5], suggesting that GDF-9 negatively influences SCF production. Despite the important roles of GDF-9 and SCF in follicular development beyond the primary stage, information about their roles in the formation and subsequent development of primordial follicles is meager. We have demonstrated that neutralization of FSH in vivo during early ovarian morphogenesis in the hamster results in significant attenuation in the formation of primordial follicles, which can be effectively reversed by exogenous FSH-like hormones, such as eCG [12]. However, whether FSH action involves GDF-9 or SCF, or that they interact to induce somatic cell differentiation into granulosa cells, is not known. The objectives of the present studies were to determine whether GDF-9 and SCF influenced primordial follicle formation in fetal hamster ovary and whether FSH played a modulatory role in this process. These objectives were addressed using an in vitro organ culture of 15-day-old fetal hamster ovaries, which contained undifferentiated somatic cells, oocytes, and dividing oogonia without any evidence of folliculogenesis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adult golden male and female hamsters were purchased from SASCO (Madison, WI) and maintained in a climate-controlled room with 14L:10D with free access to food and water according to the Institutional Animal Care and Use Committee (IACUC) and the United States Department of Agriculture (USDA) guidelines. The use of hamsters for this study was approved by the IACUC. Females with at least three consecutive estrous cycles were mated with males on the evening of proestrous, and the presence of sperm in the vaginal smear the next morning was considered Day 1 of pregnancy. Hamster gestation lasts for 16 days, and pups are born on Day 16 of gestation. Dulbecco modified Eagle medium (DMEM) was purchased from Gibco-Invitrogen (Carlsbad, CA); human transferrin, selenium, and bovine insulin were purchased from Collaborative Research (Bedford, MA); linoleic acid, bovine serum albumin, and other fine chemicals were from Sigma Chemical Company (St. Louis, MO); Falcon tissue culture inserts and plates and solvents for histology were from Fisher Scientific Company (Pittsburgh, PA); SCF, basic fibroblast growth factor (FGF), and SCF antibody were from R&D Systems, Inc. (Minneapolis, MN); human recombinant GDF-9 was a gift from Dr. Aaron Hsueh, Department of OB/GYN, Stanford University, CA; ovine-FSH-20 was purchased from the National Pituitary Program, NIH; and GB4 plastic embedding medium and rings were purchased from EM Sciences (Fort Washington, PA).
Organ Culture of Prenatal Hamster Ovaries
Ovaries were collected from 15-day-old (D15G) fetal golden hamsters in DMEM (Sigma), pH 7.4, containing antibiotics (100 U/ml of penicillin G, 100 mg/ml of streptomycin sulfate, 0.25 mg/ml amphotericin B; GIBCO-BRL); 0.5% BSA; and 0.3 mg/ml phenol red at room temperature. They were cleaned of extraneous tissues; rinsed three times with DMEM without any supplement and twice with DMEM containing 1% ITS+ (final concentration: 100 ng/ml insulin, 6.25 µg transferrin, 6.25 ng selenium, 5.35 µg of linoleic acid/ml, 100 U/ml of penicillin G, 100 mg/ml of streptomycin sulfate, 0.25 mg/ml amphotericin B, and 5% BSA/ml) [13]; and cultured for 9 days at 37°C in a Haereus incubator (Life Scientific, St. Louis, MO) under 5% CO2 in air as described previously [14]. Medium was changed every 48 h with replacement of half of the complete medium. Ovaries were retrieved from the culture and processed for morphometric evaluation of follicular development.
Effect of SCF, FGF, and FSH on Primordial Folliculogenesis
In the first experiment, ovaries were cultured in the absence or presence of 50 ng/ml SCF or FGF, or 8 mIU of FSH, for 9 days. In the next experiment, ovaries were cultured in the presence of 2 µg of a goat-polyclonal anti-SCF antibody with or without FSH, or an equal amount of nonimmune goat IgG to check for the specificity of the antibody action. Ovaries were retrieved and processed for morphometric evaluation of folliculogenesis.
Dose-Response Effect of SCF and GDF-9, and the Effect of SCF Antibody on the GDF-9 Effect
Ovaries were cultured for 9 days in the presence of 10, 20, 50, or 100 ng/ml SCF or 10, 50, 100, or 200 ng/ml GDF-9. To verify the specificity of the GDF-9 effect, ovaries were also cultured in the presence of 200 ng/ml of inactive GDF-9, GDF-9 N-tagged.
In the next experiment, ovaries were cultured in the presence of suboptimal doses of GDF-9 and SCF, or high doses of GDF-9 and an SCF antibody. After the culture, ovaries were processed for morphometric evaluation of folliculogenesis.
Effect of FSH, SCF, or GDF-9 on In Vitro Folliculogenesis in Fetal Ovaries Exposed In Vivo to an Anti-FSH Antiserum
Pregnant hamsters were injected i.p. with 200 µl of a rabbit anti-FSH serum [12] on Day 12 of gestation. Fetal ovaries were collected on Day 15 of gestation and cultured in the absence or presence of 8 mIU ovine-FSH either throughout the culture period or during a specific period. In a separate experiment, ovaries were exposed to 1 or 10 ng/ml SCF with or without FSH, SCF antibody with or without FSH, and GDF-9 optimum dose or suboptimum dose with or without FSH. Ovaries were processed for morphometric evaluation of folliculogenesis.
Ontogeny of GDF-9 Protein in the Oocytes Growing In Vivo or In Vitro
To determine in vivo expression, ovaries were collected from fetal hamsters on Gestation Days 13–15 and on Postnatal Days 1–15. For in vitro expression, 15-day-old fetal ovaries were cultured for 9 days in the absence or presence of 8 mIU of FSH, and ovaries were retrieved every 48 h. Whereas in vivo-developed ovaries were placed directly in the OCT compound and snap frozen in liquid nitrogen-cooled methyl-isopentane bath, cultured ovaries were impregnated with 30% sucrose containing protease inhibitor cocktail (Sigma) for 15 min, followed by placement in OCT compound before freezing in liquid nitrogen-cooled isopentane.
Morphometric Evaluation of Folliculogenesis
Ovaries attached to the tissue culture grid were rinsed in phosphate buffered saline, pH 7.4 at room temperature, and fixed with Bouin fixative for 10 h followed by transfer to 70% ethanol for 24 h. After dehydration through ascending grades of ethanol, ovaries were embedded in GB4 plastic resin. After curing for at least 24 h, 3 µm sections were cut in a Leica automatic microtome (Leica Microsystems, Wetzlar, Germany); taken on superfrost glass slides; dried; stained with hematoxylin and eosin sequence; and mounted with DPX distyrene, tricresyl phosphate, and xylenes (BDH, Poole, England). Images were captured by a Leica DMR research microscope equipped with an Optronics MagnaFire digital camera (Optronics, Goleta, CA) and analyzed by Openlab image analysis software (Improvision, Lexington, MA).
Morphometric evaluation was done essentially as described previously [12]. Briefly, because primordial oocytes (i.e., clusters of oocytes without any definite somatic cell partners) dominated in the ovaries cultured up to 9 days (equal to 8 days of in vivo development [12]) and primordial follicles were forming for the first time, the total number of oocytes with a nucleolus, regardless of their follicular association, was counted in a given optical field. Next, the number of follicles, at various stages, corresponding to those oocytes was determined. The fields were chosen at random from the entire ovary, and 
300 oocytes for each ovary were counted. The proportion of follicles in an ovary was then expressed as percentage of oocytes. An oocyte surrounded by the cytoplasmic processes of at least one flattened parenchymal cell was considered a primordial follicle, whereas a primary follicle was defined as one surrounded by a complete layer of granulosa cells, of which the majority were cuboidal. Early secondary follicles contained more than one layer of cuboidal granulosa cells.
The average diameter of the oocytes and follicles was determined by measuring two perpendicular diameters. Follicular diameter included the enclosed oocyte.
Immunofluorescence Detection of GDF-9 Expression
Frozen ovaries were sectioned at 6 µm in a Leica automatic cryostat; taken on precleaned superfrost glass slides; and air dried and fixed in freshly prepared ice-cold 4% paraformaldehyde in PBS, pH 7.4, for 10 min. After rinsing three times in fresh PBS, sections were incubated with 10% normal donkey serum in PBS, pH 7.4 containing 0.05% Tween 20 at 4°C, for 1 h, followed by an overnight incubation at 4°C with goat polyclonal anti-GDF-9 IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Sections were incubated with nonimmune IgG to verify the specificity of the antibody reaction. Sections were rinsed three times in PBS at room temperature and incubated for 30 min at room temperature with a donkey anti-goat IgG conjugated with Alexa 488 (green fluorescence, Molecular Probes, Eugene, OR) and rinsed. Nuclei were stained with DAPI, mounted with Fluoromount G, and viewed under epifluorescence using a Leica DMR research microscope (North Central Instruments, Plymouth, MN) and digitized with an Optronics MagnaFire digital camera. For colocalization of GDF-9 and nuclear fluorescence signal, digital images were merged using Openlab Image analysis software. The capture time was set using sections exposed to preimmune rabbit IgG to subtract any background autofluorescence. For contrast, the blue color of DAPI was converted to red, whereas the GDF-9 signal remained green. GDF-9 fluorescence intensity was measured using NIH Image 1.6 image analysis software, and the data were presented as average optical density per pixel.
Statistical Analyses
All cultures and immunofluorescence localization were repeated at least three times using ovaries from different fetuses, and representative images were presented. Ovaries from untreated and FSH antiserum-treated groups were cultured in parallel. Immunofluorescence signal for each group of oocytes reflected an average of at least 20 oocytes from two different ovaries. All quantitative data were analyzed using two-way ANOVA with Scheffe post hoc test. The level of significance was P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The objectives were to determine whether SCF, basic FGF, or FSH influenced the formation of primordial follicles in fetal hamster ovaries growing in vitro, and whether FSH action involved SCF action. Morphologically 15-day-old fetal ovaries contained clusters (egg nest) of newly formed oocytes (following oogonial mitosis) surrounded by undifferentiated somatic cells and without any distinct follicular structure [12]. Because the formation of the very first cohort of primordial follicles and their subsequent growth occurred during organ culture [14], the influence of any of the factors on primordial folliculogenesis could be examined. Fetal ovaries were cultured in vitro for 9 days to match in vivo postnatal development up to Day 8 when distinct primordial and a few primary follicles could be detected (see Fig. 1A) [12]. In the absence of any exogenous factor, 16% of the oocytes were surrounded by immature, flattened granulosa cells forming the first cohort of primordial follicles (Figs. 1B and 2). The percentage of primordial follicles increased noticeably in response to SCF, but marked increase was noted in the presence of FSH (Fig. 2). In contrast, basic FGF at 50 ng/ml had no effect on primordial follicle formation. Whereas the percentage of primordial follicles was not altered by nonimmune goat IgG, SCF antibody not only suppressed the basal percentage of primordial follicles, but also markedly attenuated FSH-stimulated primordial follicle formation (Fig. 2). These results suggested that both SCF and FSH could stimulate primordial follicle formation in the hamster ovary, and at least part of the FSH effect might be due to SCF action.
|
|
SCF and GDF-9 Influence Primordial Folliculogenesis in the Fetal Hamster Ovary in Organ Culture
Because somatic cells differentiate into granulosa cells for the first time during the formation of the first batch of primordial follicles, it was of interest to determine the optimal dose of growth stimulators, which would mimic the in vivo growth situation. It was also of interest to determine whether GDF-9 and SCF would interact to regulate primordial follicle formation. Fetal ovaries at age 15 days contained oocytes in the prophase as well as in metaphase I of the first meiotic division, and clusters of oocytes were surrounded by undifferentiated somatic cells (see Fig. 1A). No morphologically defined follicle was apparent in the fetal ovaries. Morphologically distinct primordial follicles developed in the ovaries in vivo by Day 8 of postnatal (D8pn) life (see Fig. 1B) as well as in vitro after 9 days of culture (see Fig. 1C). SCF, at a 10 ng/ml dose level, significantly stimulated the formation of primordial follicles and their development into the primary stage (Figs. 1D and 3A); however, the effect was attenuated even when the dose was merely doubled (Fig. 3A). With further higher dosages, somatic cells started losing the nucleus and assumed a ‘ghost’ cell appearance (data not shown). Further, despite an increase in the percentage of primordial follicles, ovaries exposed to 10 ng/ml of SCF contained numerous pyknotic somatic cells (Fig. 1D), and the number of total oocytes per unit section area was reduced (data not shown). In contrast to SCF, GDF-9 dose-dependently stimulated the formation of the primordial follicles (Figs. 1E and 3B). Further, GDF-9, even at a low-dose level, stimulated the exit of the primordial follicles from the resting stage to the growing pool, resulting in the formation of primary follicles (Fig. 3B). Whereas 10 ng/ml GDF-9 stimulated the formation of a small percentage of the primary follicles with a slight decrease in the percentage of primordial follicles, higher doses increased the percentages of primordial and primary follicles (Fig. 3B), and even secondary follicles developed when the dose was 200 ng/ml (Figs. 1E and 3B). The maintenance of the steady percentage of primordial follicles despite a significant increase in the percentage of the primary stage suggested that at high dosage, GDF-9 not only stimulated the formation of primordial follicles but also their progression into preantral stages. N-tagged GDF-9 (GDF-9NT) had no effect on the basal percentage of primordial follicles (Fig. 3B), suggesting the specificity of the GDF-9 effect [15].
|
Combined suboptimal doses of GDF-9 and SCF significantly (P < 0.05) increased the percentage of primordial follicles without stimulating their progression in the primary stage (Fig. 3C), but each dose alone had no effect on the basal percentage of follicles (data not shown). Further, SCF antibody significantly attenuated the effect of 200 ng/ml GDF-9 on all stages of follicular development, but the inhibitory effect was more prominent for follicles at the primary and early secondary stages (Fig. 3C). These results indicated that SCF and GDF-9 might interact to bring about primordial follicle formation, and GDF-9 might influence the formation of SCF by the somatic cells.
The formation of primordial follicles in vivo coincided with an increase in the oocyte diameter (Fig. 4). The diameter of the primordial oocytes remained constant regardless of the treatment and was comparable with their in vivo counterparts (Fig. 4). However, primordial follicles formed in the control, SCF-, or GDF-9NT-treated ovaries and their oocytes were smaller compared with those developed in vivo or those developed under FSH in vitro (Fig. 4), indicating stunted growth of the oocytes and their accompanying granulosa cells. The disparity in oocyte and granulosa cell growth was more prominent for the primary follicles developed under the influence of 100 ng/ml of SCF (Fig. 4), and the results were identical regardless of SCF dosages (data not shown). In contrast, the diameters of the primordial and primary follicles developed in GDF-9-exposed ovaries and their enclosed oocytes were virtually identical to follicles at corresponding stages developed in vivo (Fig. 4). The diameter of primordial follicles developed in the presence of FSH was comparable with those developed in vivo, although no primary follicle was developed (Fig. 4).
|
Ontogeny of GDF-9 Expression in the Oocyte During Postnatal Ovary Development In Vivo and In Vitro
Although exogenous GDF-9 could stimulate the formation of primordial follicles, to establish a biological role in hamster folliculogenesis it was important to determine whether GDF-9 was present in the hamster oocytes and, if so, its developmental expression. Oocytes showing a distinct GDF-9 immunosignal were digitized for comparison across various groups. GDF-9 protein expression occurred for the first time on Postnatal Day 4, long before the formation of any primordial follicle, and was exclusively present in the oocytes (Figs. 5B and 6). Although GDF-9 expression was evident in the oocyte of primordial follicles by Postnatal Day 8, the intensity of the immunosignal decreased significantly (Figs. 5C and 6), suggesting that GDF-9 might have a more critical role in the formation of primordial and primary follicles, which might include oocyte growth after the completion of the prophase of the first meiotic division.
|
|
In contrast to in vivo expression, a low but detectable GDF-9 immunosignal was present in the oocytes after 2 days of organ culture (Figs. 5D and 6), and the expression increased significantly after 4 days of culture (Figs. 5F and 6), indicating a possible withdrawal of a suppressive mechanism, which was active in vivo. Interestingly, GDF-9 expression in the primordial oocytes increased by 15-fold after 2 days of culture with FSH, and the intensity was comparable with that of Postnatal Day 4 (Figs. 5E and 6). GDF-9 immunosignal in the oocytes increased further when ovaries were cultured for 4 days with FSH (Figs. 5G and 6). Whether the oocytes with modest to low immunosignal (Fig. 5G) were destined to be apoptotic, did not reach appropriate maturity, or did not develop communication with the somatic cells destined to form the granulosa cells remains unknown.
Role of FSH in Primordial Follicular Development
FSH appears to play an important role in vivo in hamster primordial follicle formation [12]. The rationale for this experiment was to determine whether elimination of FSH stimulation in vitro during the final days of fetal life would affect primordial follicle formation in vitro, and whether FSH, SCF, or GDF-9 would restore folliculogenesis. Ovaries exposed to FSH antiserum in vivo from the 12th through 15th days of fetal life had a significantly lower percentage of primordial follicles by Day 9 of culture compared with untreated 15-day-old fetal (D15G) ovaries cultured for the same period (Fig. 7). FSH, regardless of the duration of exposure, significantly improved primordial follicle formation. However, tonic FSH treatment for 9 days of FSH antiserum-exposed ovaries resulted in only 10% primordial follicle development (Fig. 7) compared with 30% primordial follicle development in untreated ovaries exposed to FSH in vitro (Fig. 2). Therefore, it was of interest to determine whether phasic FSH stimulation would improve the percentage of primordial follicle development to the level of untreated ovaries. Notably, the percentage of primordial follicles increased dramatically when FSH exposure was limited to the first 48 h, suggesting that precise temporal action of FSH might be critical for the formation of primordial follicles. SCF alone also reversed the antiserum inhibition of primordial follicle formation. Nevertheless, neither treatment was able to restore folliculogenesis to the level observed for non-antiserum-treated ovaries (compare with Figs. 2 and 3A). GDF-9 at the 200 ng/ml dose level was the only factor that almost completely reversed the antiserum effect on primordial follicle formation and partially reversed the formation of primary follicles, albeit to a lower extent compared with non-antiserum-exposed ovaries (Fig. 3B). Whereas a lower dose of GDF-9 could also partially reverse the formation of primordial follicles, it improved the effect of the continuous exposure of FSH, suggesting that GDF-9 might be involved in FSH-stimulated follicular development. A low dose of GDF-9, however, interfered with the SCF effect on primordial follicle formation. Previously, we demonstrated that s.c. injection of nonimmune rabbit IgG to pregnant hamsters did not interfere with the formation and subsequent growth of primordial follicles [12].
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A slight decrease in the percentage of primordial follicles concurrent with the first appearance of primary follicles seems to suggest that GDF-9, at a very low dose level, influences granulosa cell maturation while maintaining a steady formation of primordial follicles. The formation of primordial follicles and their progression into subsequent preantral stages accelerate with higher doses. It is equally possible that the progression of folliculogenesis in preantral stages depends on the critical mass of primordial follicles. A similar situation has been observed for 8-day-old postnatal hamster ovaries developed in vivo, where the first cohort of primary follicles (1%) is visible when the percentage of primordial follicles reaches 25% [12]. The marked increase in GDF-9 protein expression following FSH exposure in vitro suggests that FSH-induced primordial follicle formation may involve GDF-9. The precocious expression of GDF-9 in vitro also suggests that inhibitory mechanism(s) may exist in vivo. Although the exact nature of such inhibitory mechanisms is not yet known, Kezele and Skinner [19] have demonstrated that ovarian steroids suppress primordial to primary follicle transition without affecting the formation of primordial follicles in newborn rat ovaries in vitro. Whether ovarian steroids have any suppressive influence on GDF-9 expression or the mode and mechanisms whereby FSH influences GDF-9 expression in the hamster oocytes remains to be investigated. The absence of precocious formation of primordial follicles, despite an increase in the expression of GDF-9 by FSH, suggests that while an increased number of somatic cells can differentiate into granulosa cells and cause a net increase in the percentage of primordial follicles, the assembly of these cells around the naked oocytes requires a relatively long time, which cannot be influenced. Temporal studies using eCG in vivo [12] or GDF-9 in vitro (unpublished observation) support this view.
The increase in the percentage of primordial follicle formation with a low dose of SCF and with combined suboptimal doses of SCF and GDF-9, and a reduction in GDF-9-induced folliculogenesis by an SCF antibody, suggest that at least part of the GDF-9 action may be mediated by SCF. The improvement of the percentage of primordial follicles in FSH antiserum-exposed ovaries by the suboptimal dose of either SCF or GDF-9 further proves the importance of these growth factors in primordial folliculogenesis. Nilsson and Skinner [6] have demonstrated that GDF-9 increases the steady-state levels of kit-ligand or SCF mRNA in neonatal rat ovaries in vitro. Whether the same is true for the hamster is not yet known, but the inhibition of GDF-9-induced folliculogenesis by SCF antibody tends to corroborate the finding. SCF expression has been primarily detected in the granulosa cells adjacent to the oocytes [20] and has been shown to influence theca cell growth in the rat [21]. The results of the present study suggest that SCF primarily stimulates granulosa cell development, leading to disproportionate follicle growth. On the other hand, GDF-9 or FSH in vitro maintains proportionate development of the oocyte and granulosa cells in follicles similar to that which occur in vivo under normal developmental conditions; however, the SCF antibody attenuates the functions of either FSH or GDF-9. Taken together, it is apparent that the intensity of SCF stimulation must be very precisely coordinated with GDF-9 signaling for normal folliculogenesis.
The results of the present study corroborate our previous finding [12] about the supportive role of FSH in hamster primordial folliculogenesis. Because the antiserum prevented in vivo FSH action from the 12th to the 15th day of fetal life, somatic cells that have already processed the endogenous FSH signal by Gestation Day 12 continue forming primordial follicles in vitro without any exogenous hormonal support (untreated group). In contrast, somatic cells awaiting FSH action by Gestation Day 12 respond to in vitro FSH, differentiate into immature granulosa cells, and form primordial follicles leading to an increase in their percentage in the ovaries. However, lack of FSH action in vivo during the critical period of cytodifferentiation may also render a large population of somatic cells refractory to in vitro FSH; hence, the percentage of primordial follicles never increases beyond 16%, which is comparable with untreated ovaries in control cultures. The exact reason for the marked increase in the percentage of primordial follicles in FSH antiserum-exposed ovaries following in vitro FSH exposure for the first 48 h is not known. However, altering sensitivity of undifferentiated ovarian somatic cells to FSH during the perinatal period may explain the findings. For example, in FSH-deprived hamster ovaries, a temporal FSH signaling may recruit more somatic cells to differentiate into immature, flattened granulosa cells and their assembly to form primordial follicles, but a sustained FSH action may only support the assembly of immature granulosa cells around the oocyte. Full-length FSH-receptor mRNA (S.K. Roy, unpublished observation) and functional FSH-receptor [12] are present in the hamster ovary from fetal age 13 day onward.
In summary, results of these studies provide first and direct evidence to prove that GDF-9 can promote the formation of primordial follicles and their subsequent growth in neonatal hamster ovaries. Further, the results also suggest that FSH plays an important role in the differentiation of somatic cells into granulosa cells and their assembly around the primordial oocytes, at least in the hamster, by modulating the production of GDF-9 by the oocytes and the production of SCF by somatic cells. GDF-9 may be a major player mediating FSH action and influencing granulosa cell development via SCF.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Shyamal K. Roy, Departments of Obstetrics and Gynecology and Physiology and Biophysics, University of Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}unmc.edu
Received: 14 September 2003.
First decision: 2 October 2003.
Accepted: 21 October 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. Wang, E. R. Prossnitz, and S. K. Roy G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary Endocrinology, September 1, 2008; 149(9): 4452 - 4461. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Pinkas, B. Fisch, G. Rozansky, C. Felz, G. Kessler-Icekson, H. Krissi, S. Nitke, A. Ao, and R. Abir Platelet-derived growth factors (PDGF-A and -B) and their receptors in human fetal and adult ovaries Mol. Hum. Reprod., April 1, 2008; 14(4): 199 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J Spicer, P. Y Aad, D. T Allen, S. Mazerbourg, A. H Payne, and A. J Hsueh Growth Differentiation Factor 9 (GDF9) Stimulates Proliferation and Inhibits Steroidogenesis by Bovine Theca Cells: Influence of Follicle Size on Responses to GDF9 Biol Reprod, February 1, 2008; 78(2): 243 - 253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang and S. K. Roy Development of Primordial Follicles in the Hamster: Role of Estradiol-17{beta} Endocrinology, April 1, 2007; 148(4): 1707 - 1716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Gueripel, V. Brun, and A. Gougeon Oocyte Bone Morphogenetic Protein 15, but not Growth Differentiation Factor 9, Is Increased During Gonadotropin-Induced Follicular Development in the Immature Mouse and Is Associated with Cumulus Oophorus Expansion Biol Reprod, December 1, 2006; 75(6): 836 - 843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. G Knight and C. Glister TGF-{beta} superfamily members and ovarian follicle development. Reproduction, August 1, 2006; 132(2): 191 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L J Spicer, P Y Aad, D Allen, S Mazerbourg, and A J Hsueh Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells. J. Endocrinol., May 1, 2006; 189(2): 329 - 339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang and S. K. Roy Expression of Growth Differentiation Factor 9 in the Oocytes Is Essential for the Development of Primordial Follicles in the Hamster Ovary Endocrinology, April 1, 2006; 147(4): 1725 - 1734. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Lacham-Kaplan, H. Chy, and A. Trounson Testicular Cell Conditioned Medium Supports Differentiation of Embryonic Stem Cells into Ovarian Structures Containing Oocytes Stem Cells, February 1, 2006; 24(2): 266 - 273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, H. Newton, J. A. Spaliviero, C. M. Allan, B. Marshan, D. J. Handelsman, and P. J. Illingworth Gonadotropin Control of Inhibin Secretion and the Relationship to Follicle Type and Number in the hpg Mouse Biol Reprod, October 1, 2005; 73(4): 610 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Skinner Regulation of primordial follicle assembly and development Hum. Reprod. Update, September 1, 2005; 11(5): 461 - 471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Steckler, J. Wang, F. F. Bartol, S. K. Roy, and V. Padmanabhan Fetal Programming: Prenatal Testosterone Treatment Causes Intrauterine Growth Retardation, Reduces Ovarian Reserve and Increases Ovarian Follicular Recruitment Endocrinology, July 1, 2005; 146(7): 3185 - 3193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.L. Juengel and K.P. McNatty The role of proteins of the transforming growth factor-{beta} superfamily in the intraovarian regulation of follicular development Hum. Reprod. Update, March 1, 2005; 11(2): 144 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Yang, J. Wang, Y. Shen, and S. K. Roy Developmental Expression of Estrogen Receptor (ER) {alpha} and ER{beta} in the Hamster Ovary: Regulation by Follicle-Stimulating Hormone Endocrinology, December 1, 2004; 145(12): 5757 - 5766. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Britt, P. K. Saunders, S. J. McPherson, M. L. Misso, E. R. Simpson, and J. K. Findlay Estrogen Actions on Follicle Formation and Early Follicle Development Biol Reprod, November 1, 2004; 71(5): 1712 - 1723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-M. Zhang and S. K. Roy Downregulation of Follicle-Stimulating Hormone (FSH)-Receptor Messenger RNA Levels in the Hamster Ovary: Effect of the Endogenous and Exogenous FSH Biol Reprod, June 1, 2004; 70(6): 1580 - 1588. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||
