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Molecular Endocrinology, doi:10.1210/me.2005-0462
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Molecular Endocrinology 20 (6): 1406-1422
Copyright © 2006 by The Endocrine Society

Premature Luteinization and Cumulus Cell Defects in Ovarian-Specific Smad4 Knockout Mice

Stephanie A. Pangas, Xiaohui Li, Elizabeth J. Robertson and Martin M. Matzuk

Departments of Pathology (S.A.P., X.L., M.M.M.), Molecular and Cellular Biology (M.M.M.), and Molecular and Human Genetics (M.M.M.), Baylor College of Medicine, Houston, Texas 77030; and the Wellcome Trust Center for Human Genetics (E.J.R.), University of Oxford, Oxford OX3 7BN, United Kingdom

Address all correspondence and requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Stuart A. Wallace Chair and Professor, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SMAD4 is a central component of the TGFß superfamily signaling pathway. Within the ovary, TGFß-related proteins play crucial roles in controlling granulosa cell growth, differentiation, and steroidogenesis. To study the in vivo roles of SMAD4 during follicle development, we generated an ovarian conditional knockout of Smad4 using the cre/loxP recombination system. Smad4 ovarian-specific knockout mice are subfertile with decreasing fertility over time and multiple defects in folliculogenesis. Regulation of steroidogenesis is disrupted in the Smad4 conditional knockout, leading to increased levels of serum progesterone. In addition, severe cumulus cell defects are present both in vivo and when assayed in vitro. These findings demonstrate that disrupting signaling through SMAD4 in the ovarian granulosa cells leads to premature luteinization of granulosa cells and eventually premature ovarian failure, thereby demonstrating key in vivo roles of TGFß superfamily signaling in the timing of granulosa cell differentiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AN INTEGRATED DEVELOPMENTAL program directs the growth of oocytes and their associated somatic cells within the ovarian follicle (1, 2). During the reproductive cycle, primordial follicles progress through a series of coupled developmental stages that results in growth of the oocyte, proliferation and differentiation of the granulosa cell layers, and development of a defined theca. Early stages of follicle growth are independent of the pituitary gonadotropins, FSH and LH (3, 4, 5), and are driven by intraovarian factors. In adult female mice, a rhythmic production of hormones from the pituitary gland governs the final stages of follicle development and ovulation (1, 6). After an ovulatory peak of LH, follicular granulosa and thecal cells undergo terminal differentiation to become corpora lutea, whose major function is the production of progesterone required for estrous cyclicity and maintenance of pregnancy (7).

The TGFß superfamily ligands are critical for female fertility and ovarian function (8, 9, 10). Members of this protein family signal through membrane-bound serine-threonine kinase receptors, called type II and type I receptors, which upon activation by ligand binding, phosphorylate intracellular SMAD proteins (11). Type I receptors phosphorylate a distinct set of receptor-associated SMADs (R-SMADs) (12); the activin/TGFß type I receptors use SMAD2 and SMAD3, whereas the bone morphogenetic proteins (BMPs) signal through SMADS 1, 5, and 8. In addition, the capacity for the TGFß-related ligands to signal is thought to require SMAD4, the common SMAD, that partners with the R-SMADs to form the core of a transcriptional complex (13). Understanding the role of SMAD4 function in the ovary in vivo has proven difficult because mice null for Smad4 die at embryonic d 6.5 and arrest before gastrulation (14, 15). Therefore, to study SMAD4 function in the adult or in specific cell types, it is necessary to generate conditional knockout mice. Conditional knockouts for Smad4 have been used to study Smad4 in the early embryo (16), adult brain (17), mammary gland (18), and epidermis (19, 20).

During development of the reproductive system, the TGFß family proteins have many diverse functions in multiple cell types (8). Primordial germ cell specification and proliferation requires BMPs and their downstream SMADs (8, 21). Sex determination involves Müllerian-inhibiting substance and follistatin, an antagonist of activin and BMPs (22, 23). Postnatal ovarian folliculogenesis depends upon growth and differentiation factor 9 (GDF9) and BMP15 production from oocytes (24, 25, 26). Additionally, loss of inhibin {alpha} (Inha) in mice causes gonadal cancer (27), and loss of granulosa cell-expressed follistatin results in premature ovarian failure (28). Even so, the in vivo roles of many of the TGFß superfamily ligands, particularly the intraovarian role of activin and BMPs, is unknown in the adult ovary because null mutations in many of the BMPs, activins, their regulatory proteins, and signaling systems have embryonic or perinatal lethal phenotypes. Therefore, much of the TGFß superfamily function in ovarian cells has been derived from in vitro culture analysis.

To study the roles of TGFß superfamily activity in follicle development in vivo, we ablated Smad4 expression using the cre/loxP conditional knockout system. Recombination of a Smad4 conditional allele (16) was generated through expression of Cre recombinase from the anti-Müllerian hormone type II receptor (Amhr2) locus (28, 29). Cre expression mimics the expression of Amhr2, and in adult female ovaries, Cre recombinase is highly expressed in granulosa cells of developing follicles (28, 30, 31). Loss of SMAD4 severely affects fertility of female mice and causes reproductive senescence by 4–6 months of age. There is a significant increase in the number of atretic preantral follicles concordant with a decrease in antral follicles. Importantly, granulosa cells from Smad4 conditional knockout (cKO) females abnormally express a number of luteal markers after stimulation with pregnant mare serum gonadotropin (PMSG), suggesting that, in the absence of TGFß-related signaling, granulosa cells undergo precocious luteinization in response to gonadotropin stimulation. These data suggest that one of the primary functions of the TGFß superfamily in vivo is to regulate the timing of terminal differentiation of granulosa cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeted Disruption of Smad4 in the Ovary
We generated a conditional knockout of Smad4 in the ovary using the cre/loxP system by crossing a mouse strain carrying the previously described conditional (Smad4Flox) and null (Smad4–) alleles of Smad4 (16) to the Amhr2cre knock-in mouse line (Amhr2cre/+) (29) (Fig. 1AGo). The conditional allele contains loxP sites flanking the first coding exon (exon 2) of Smad4 and recombination results in a null allele (16). The ovarian expression pattern of Amhr2cre allele has been previously described (28, 31) and used in adult female mice to direct granulosa cell-specific deletion of follistatin (28) and steroidogenic factor 1 (31) conditional alleles, as well as expression of a stable form of ß-catenin (30). Using the ROSA26 reporter mouse strain (32), previous studies have shown Cre-mediated recombination in Amhr2cre/+ mice occurs predominantly in granulosa cells of developing follicles but not in primordial or primary follicles (28). Although some studies have reported low levels of Cre recombinase in theca (28), others have only seen expression in granulosa cells (31).


Figure 1
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  		Fig. 1. Generation of the Smad4 cKO in the Ovary

A, Schematic of the conditional (Flox) and null alleles of Smad4. LoxP sites (open triangles) flank the first ATG-containing exon (exon 2). Recombination by Cre recombinase results in a null allele. PCR analysis using primers (F1, F2, R1) for genotyping as indicated (B) Smad4 transcript levels measured in granulosa cells by quantitative PCR in wild-type, Smad4Flox/– control and Amhr2cre/+;Smad4Flox/– experimental mice. Levels of Smad4 (RQ) are shown relative to the wild type. C, Immunohistochemistry of SMAD4 in 3-wk-old control (Smad4F/–) and (D) experimental (Amhr2cre/+;Smad4Flox/–) ovaries. Brown staining indicates immunoreactivity and nuclei are counterstained blue. Loss of SMAD4 occurs in granulosa cells of developing follicles after the two-layer secondary stage (SF) (D). Note similar immunostaining in the epithelium lining the oviduct, which does not express Cre, and serves as a control for staining intensity between ovaries. Gr, Granulosa; Oo, oocyte; Th, theca; AnF, antral follicle; PF, primordial follicle; PrF, primary follicle.

 
We verified loss of Smad4 transcript in granulosa cells of control (Smad4Flox/–) and conditional knockout (cKO) (Amhr2cre/+;Smad4Flox/–) by real-time quantitative PCR using primers within exon 2, which is deleted during recombination (Fig. 1AGo). In Smad4 cKO granulosa cells, Smad4 mRNA levels are 87% lower than wild type (Fig. 1BGo) and approximately half the levels of heterozygous (Smad4Flox/–) control mice (Fig. 1BGo). Even though Smad4 transcript levels in the cKO granulosa cells are markedly decreased, these data suggest that granulosa cells still contain a low level of Smad4 mRNA expression. Therefore, we examined the expression pattern of SMAD4 protein in control and conditional knockout mice by immunohistochemistry. In the control Smad4Flox/– ovaries, SMAD4 immunoreactivity was detectable in granulosa cells of all follicles stages from primordial to antral, in oocytes of preantral follicles, in thecal cells of early antral follicles, and in the ovarian surface epithelium (Fig. 1CGo). This is in agreement with published localization studies on human (33) and rat (34) ovaries. In addition, granulosa cells of control mice had a uniform staining pattern with little variation within and between follicles (Fig. 1CGo). In contrast, SMAD4 immunoreactivity is markedly reduced in the Smad4 cKO ovary and varied with follicle stage (Fig. 1DGo). In Smad4 cKO ovaries, follicles up to the two layer [early secondary; stage 4 (Ref. 35)] have oocytes and granulosa cells that contain equivalent levels of immunostaining as the controls (Fig. 1Go, C and D). This is expected based on the lack of Cre recombinase at these stages (28). The ovarian surface epithelium also had similar staining between the control and the Smad4 cKO ovaries, suggesting that recombination does not occur in this cell type either. The most severe loss of SMAD4 was within granulosa and thecal cells. Granulosa cells from large secondary (+3 granulosa cell layers) and early antral follicles in the Smad4 cKO ovary had markedly reduced expression from the control. Thecal cells from early antral follicles of the Smad4 cKO ovaries also did not show any immunoreactivity for SMAD4 (Fig. 1DGo). Levels of SMAD4 immunoreactivity in granulosa cells, although low, were not uniform and could vary within the same follicle as well as between follicles (Fig. 1DGo). Similar variation in recombination has been noted in other studies using the Amhr2cre deleter strain and suggests that not all developing follicles undergo equivalent recombination (30). Thus, within the same ovary, some follicles may be null for SMAD4, whereas others may be considered hypomorphic.

Smad4 cKO Female Mice Have a Severe Reduction in Fertility
To establish the fertility of the Smad4 cKO mice, Smad4Flox/– (control) and Amhr2cre/+;Smad4Flox/– (Smad4 cKO; experimental) females were bred to wild-type males continuously for 6 months. Because the average number of pups per litter of the control mice (Smad4Flox/–) (10.4 ± 0.7) was not different from the average of eight pups per litter for our mixed hybrid C57BL/6J;129S5/SvEvBrd mouse colony (4), the Smad4Flox/– mice were used as control mice for this study. A significant difference was seen in the numbers of pups born to the Smad4 cKO mice as compared with control mice (Fig. 2Go and Table 1Go). Smad4 cKO experimental mice gave birth to an average of 1.3 ± 0.4 pups per litter over the 6-month period (Fig. 2BGo and Table 1Go). This is significantly decreased from the controls (P < 0.01). In control mice, an approximately equal number of pups were generated every 3–4 wk for the entire 6-month period (Fig. 2AGo). In contrast, at 3 months of age, 25% of the breeding Smad4 cKO mice were infertile; by 6 months, 50% of the females were infertile (Fig. 2BGo). Therefore, the average number of litters per month was significantly decreased in the Smad4 cKO experimental mice (Table 1Go). Thus, Smad4 cKO females display severely disrupted fertility.


Figure 2
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  		Fig. 2. Fertility of Smad4 cKO Female Mice over 6 Months Ovary

Seven control (Smad4Flox/–) (solid line) and seven experimental (Amhr2cre/+;Smad4Flox/–) (dashed line) mice were bred continuously to wild-type males and the number of pups born was counted over a 6-month period. A, Data are shown as total accumulated pups at each month of breeding. Control mice (Smad4Flox/–) have a uniform accumulation of pups at each month, whereas Amhr2cre+;Smad4Flox/– experimental cKO female mice have fewer pups and become infertile by 4–6 months of age. B, Fertility data shown as mean pups per litter for eight breeding pairs over during 6 months. Control (Smad4Flox/–) (solid line) averaged 10.4 ± 0.4 pups during the entire 6-month period. Experimental (Amhr2cre/+;Smad4Flox/–) (dashed line) averaged 1.3 ± 0.2 pups/litter. Fertility begins to rapidly decline in the experimental mice at 3 months of age.

 

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  		Table 1. Fertility Testing of Smad4Flox/– Controls and Mutant Females

 
Serum data were collected from mice at 5–6 and 12 wk of age. No differences were detected in levels of FSH, LH, estradiol, progesterone, or androstenedione at 5–6 wk of age (data not shown). At 12 wk of age, at the beginning of the onset of infertility, no statistically significant differences were seen for FSH, LH, estradiol, or androstenedione (Table 2Go); however, a statistically significant 3-fold increase in serum progesterone was found in 12-wk-old female Amhr2cre/+;Smad4Flox/– experimental mice.


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  		Table 2. Serum Hormone Data for 12-Week-Old Smad4Flox/– Amhr2cre-Negative and -Positive Females

 
Histological Analysis of 12-Week-Old Smad4 cKO Ovaries
Given the decline in fertility and the increase in serum progesterone of Smad4 cKO mice at 12 wk of age, ovaries from 12-wk-old mice were analyzed histologically for changes in follicular development. No gross changes in size were noticeable between the control and knockout mice. Control ovaries have follicles at all stages and exhibit several corpora lutea per section (Fig. 3Go, A and B). Histologically, most Smad4 cKO ovaries contained follicles of all stages (Fig. 3Go, C–E). Comparable numbers of primordial and primary follicles per area were found between the control and Smad4 cKO ovaries. Most developing follicles contained a thecal cell layer that was morphologically indistinguishable from control mice. However, multiple defects were visible histologically within granulosa cells of the cKO ovaries. Often, Smad4 cKO ovaries contained small follicles with luteinizing cells surrounding either oocytes or oocyte remnants (Fig. 3Go, D and E). Also prominent were atretic preantral follicles, with and without oocytes (Fig. 3EGo). Large follicle-like structures that did not contain an antrum but enclosed a trapped oocyte (Fig. 3EGo) also were found in the Smad4 cKO ovaries. Quantification of follicle numbers in ovaries from Smad4 cKO mice reflected these histological observations and revealed more atretic preantral follicles and fewer large antral follicles (Fig. 3FGo). These data suggest that both preantral and antral stage follicle development was disrupted in the Smad4 cKO ovary.


Figure 3
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  		Fig. 3. Histological Analysis of Control and Experimental Smad4 cKO Ovaries

Ovaries were collected from 12-wk adult female Smad4Flox/– control (A and B) and Amhr2cre/+;Smad4Flox/– experimental cKO mice (C–E) and stained with PAS and hematoxylin. Two different experimental mouse ovaries are shown in panels (C) and (E). A and B, Normal histology of control mice showing all stages of follicular development, including primary follicles (PrF), SF, antral follicles (AF), and corpora lutea (CL). Boxed area is shown in panel B at a higher magnification. C and D, Ovary of an Amhr2cre/+;Smad4Flox/– female; boxed area is shown at a higher magnification in panel D. A large number of oocyte remnants as indicated by PAS-positive material are visible within the stroma (arrowheads in D) surrounded by luteinized cells. Areas of luteinized cells in the interstitial tissue (arrows in panel D) are also shown at higher magnification. E, Ovary from a Smad4 cKO mouse demonstrating a large luteinized follicle with a trapped oocyte (arrow) and oocytes in preantral atretic follicles with granulosa cell defects (arrowheads), including loss of the oocyte, and luteinization. Gr, Granulosa cells; Th, theca. Black bars, 100 µm. F, Quantification of follicle numbers per surface area with the following follicle categories: PF, primordial and primary follicles; PrF, preantral follicles; APrF, atretic preantral follicles; AnF, antral follicles; AAnf, atretic antral follicles; CL, corpora lutea.

 
Smad4 cKO Female Mice Have Decreased Ovulation Rates
The lower number of developing antral follicle suggests that defects in estrous cyclicity might be present. To test the ability of the Smad4 cKO mice to ovulate, mice were pharmacologically superovulated, and ovulation rates were assessed to determine whether the defect could be rescued. Eight- to 10-wk-old female mice were treated with exogenous gonadotropins and mated to known fertile wild-type males. Oocytes and one-cell embryos were collected from oviducts. A statistically significant reduction in the number of oocytes collected from the oviducts is seen in experimental mice as compared with controls (Table 3Go). On average, the experimental mice ovulate approximately half as much as control mice. These data indicate that there are intraovarian defects in preovulatory follicles, either during development or at ovulation, which cannot be overcome by treatment with ovulatory doses of gonadotropins.


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  		Table 3. Superovulation of 8- to 10-Week-Old Smad4Flox/– Amhr2cre-Negative and -Positive Females

 
Smad4 cKO Antral Follicles from Immature Mice Prematurely Luteinize
To examine preovulatory follicle development, we used PMSG stimulation of immature mice. At 3 wk of age, mice are sexually immature, and follicle development in untreated females does not proceed beyond the preantral/early antral stage due to lack of support by the pituitary gonadotropins. No histological differences were noted in control and experimental ovaries of uninjected 21-d-old mice (Fig. 4Go, A and B). At this age, both genotypes show primordial, preantral, and early antral stage follicle development. To examine antral follicle development, we injected immature mice with PMSG and examined ovarian histology 44–46 h after injection. Forty-four to 46 h of PMSG stimulation induces follicle maturation and antral development in control mice (Fig. 4CGo). In experimental Smad4 cKO ovaries, PMSG induces follicle development, and some antral follicles can be seen (Fig. 4DGo). However, many of the large antral follicles underwent premature luteinization (Fig. 4DGo). The luteinizing follicular structures were never seen in control ovaries treated with PMSG. Additional defects include oocytes trapped within the luteinizing granulosa cells of the Smad4 cKO ovaries (Fig. 4EGo) and other smaller degenerating follicles with oocytes devoid of cumulus cells (Fig. 4FGo). Thus, in Smad4 cKO ovaries, instead of inducing preovulatory development, 44–46 h of PMSG stimulation induced formation of large luteinized structures similar to corpora lutea that trap oocytes within them.


Figure 4
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  		Fig. 4. Histology of Control and Experimental Smad4 cKO Ovaries during PMSG Stimulation Ovary

Uninjected ovaries from immature 3-wk-old female Smad4Flox/– control (A) and Amhr2cre/+;Smad4Flox/– cKO experimental mice (B) are shown. Both show similar follicle development. C, Ovary collected from a 3-wk-old female Smad4Flox/– control mouse after 46 h of PMSG treatment shows multiple developing large antral follicles (AnF) (arrows). D–F, Ovary collected from 3-wk-old female Amhr2cre/+;Smad4Flox/– experimental mouse. Some normal large antral follicle development is seen (arrows), but other antral follicles undergo premature luteinization (arrowheads). E, Trapped oocyte (arrow) within a luteinizing follicle (LF); F, oocytes without cumulus cells in degenerating follicles (arrowhead) from a Amhr2cre/+;Smad4Flox/– experimental mouse are shown at higher magnification. Scale bar, 100 µm.

 
Smad4 cKO Female Mice Have Defects in Cumulus Expansion
Because cumulus cell defects were visible histologically in Smad4 cKO ovaries (Figs. 3EGo and 4FGo), we examined cumulus cell expansion during superovulation in vivo. Control and experimental mice were injected with PMSG for 44 h, followed by injection with human chorionic gonadotropin (hCG). Ovaries were harvested 6 h later for histological analysis (Fig. 5Go). During normal ovulation, cumulus cells within the preovulatory follicle undergo expansion after the LH surge. Cumulus cells surrounding oocytes in preovulatory follicles of control mice undergo expansion (Fig. 5AGo). In contrast, during 6 h of hCG treatment, some follicles from Smad4 cKO experimental mice show oocytes that lack cumulus cells (Fig. 5BGo). In some of the antral follicles, dispersed cumulus cells are seen within the antral cavity. Some smaller secondary stage follicles also show oocytes without associated granulosa cells (Fig. 5BGo, arrowhead), similar to the 12-wk-old uninjected Smad4 cKO ovaries (Fig. 3EGo), and other follicles have trapped oocytes (Fig. 5BGo, arrow) as is seen in PMSG-stimulated Smad4 cKO ovaries (Fig. 4EGo). These data indicate that there are multiple disruptions in cumulus cell physiology, including defects during follicle development and expansion.


Figure 5
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  		Fig. 5. Histology of Control and Experimental Smad4 cKO Ovaries during Superovulation Ovary

Ovaries were collected from adult female Smad4Flox/– control (A) and Amhr2cre/+;Smad4Flox/– cKO experimental mice (B) after 44 h of PMSG and 6 h of hCG treatment and sections were stained with PAS and hematoxylin. A, Normal cumulus expansion is seen in control mice (arrow, Cu, cumulus; Oo, oocyte). B, Naked oocytes are seen in large antral follicles (Oo, arrow) and in secondary follicles (arrowhead) of experimental mice. Dispersed cumulus cells can be seen within the antral cavity. An oocyte is also trapped within a luteinized follicle (arrow, lower left). Both images taken at the same magnification (x100).

 
To test whether Smad4 cKO cumulus cells undergo expansion in vitro, we collected intact cumulus-oocyte complexes (COCs) from large antral follicles of control and experimental Amhr2cre/+;Smad4Flox/– ovaries after 44 h of PMSG treatment. Consistent with a defect in antral follicle development, approximately half as many intact COCs were recovered from large antral follicles of the Smad4 cKO ovaries (Fig. 6AGo) as compared with the controls. All intact COCs were collected and cultured for 15–18 h in the presence of FSH and serum because these conditions stimulate cumulus expansion in vitro (36). No differences were seen between different genotypes of COCs cultured in serum alone, and the cumulus masses remained intact surrounding the oocyte with some cells migrating onto the surface of the culture dish (Fig. 6Go, C and E). Control COCs undergo normal expansion when cultured for 15 h in serum and FSH and radiate spherically outward from the oocyte (Fig. 6Go, B and D). In contrast, COCs from Smad4 cKO ovaries showed reduced or absent cumulus expansion compared with the controls (Fig. 6Go, B, E, and F). The cumulus cells remained closely associated with the oocyte, or otherwise underwent a disorganized expansion that was far reduced compared with the COCs isolated from Cre-negative mice (Fig. 6Go, compare D and F). The disorganized cumulus expansion phenocopies that seen in other knockout mouse models, including Gdf9+/–;Bmp15–/– double mutant mice (37).


Figure 6
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  		Fig. 6. Cumulus Expansion Defects in Control and Experimental Smad4 cKO Ovaries during in Vitro Culture

Immature 3-wk mice were injected with PMSG and intact COCs collected 44 h later by puncturing large antral follicles. A, The average number of intact COCs collected from Smad4 cKO ovaries (Amhr2cre/+;Smad4Flox/–) as compared with controls (Smad4Flox/–) was significantly reduced. **, P < 0.01. B, Intact COCs treated with serum (gray bars) or serum and FSH (black bars) for 18 h show reduced expansion in the cKO. Bars with different letters indicate statistical significance (P < 0.05). C–F, Micrographs of COCs during in vitro cumulus expansion. Control COC (C and D) and Smad4 cKO COCs (E and F) treated with serum or serum and FSH for 18 h. Control COCs undergo a typical cumulus cell expansion when treated with serum and FSH (D), whereas Smad4 cKO COCs have an abnormal expansion (arrow) or do not undergo expansion (F). Magnification (x50) is the same for all panels C–F. Each experiment was performed independently in triplicate.

 
Molecular Changes in Granulosa Cells from Immature Smad4 cKO Mice
To understand the defects in granulosa cells during gonadotropin stimulation, we verified expression of genes that are important granulosa cell differentiation markers during folliculogenesis and luteinization. Granulosa cells were collected from 44-h PMSG primed immature 3-wk-old control and experimental females, and mRNA transcript levels were determined by quantitative real-time PCR. These cells are predominantly mural granulosa cells but also contain cumulus cells. A number of ovarian-expressed genes showed significant changes (Fig. 7Go and Table 4Go). Fshr and Lhcgr are necessary to mediate the actions of the pituitary gonadotropins during the antral stages of follicle development. Fshr is slightly down-regulated in granulosa cells from PMSG-stimulated immature mice, but Lhcgr transcripts are surprisingly up-regulated approximately 5-fold (Fig. 7Go, A and B, and Table 4Go). During rodent follicle development, Lhcgr is expressed in theca of preantral and antral follicles, mural granulosa cells of tertiary follicles, and in corpora lutea; it is absent in oocytes, granulosa cells from preantral follicles, periantral granulosa cells from tertiary follicles, and cumulus cells (38, 39). Immature control mice stimulated with PMSG for 44 h follow this pattern (Fig. 8Go, A and B). However, as indicated by quantitative PCR, PMSG stimulated Smad4 cKO ovaries contain high levels of Lhcgr, inappropriately expressed in throughout the granulosa cells in antral follicles (Fig. 8Go, C and D). Unlike the control ovaries, expression of Lhcgr is observed in granulosa cells of small early antral follicles that are luteinizing, as well as large antral follicles (Fig. 8Go, C and D). However, expression of Lhcgr remains suppressed in cumulus cells in Smad4 cKO ovaries.


Figure 7
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  		Fig. 7. Gene Expression Changes in Granulosa Cells from Immature Control and Experimental Smad4 cKO Mice Ovary

Real-time PCR analysis of mRNA from 3-wk-old control (Smad4Flox/–) and Smad4 cKO experimental (Amhr2cre/+;Smad4Flox/–) granulosa cells. Granulosa cells were collected from three independent control and four cKO ovaries stimulated with PMSG. Mean and SE are shown. Significant changes were seen in the relative quantity (RQ) of Fshr, Lhcgr, Star, Cyp11a1, Hsd17b7, Ptgfr, and Sfrp4. Other genes examined are shown in Table 4Go. Control values were set to equal 1. *, P < 0.05; **, P < 0.01.

 

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  		Table 4. Summary of QPCR Results for Gene Expression in Granulosa Cells Derived from 3-Week-Old Smad4 Control and cKO Female Mice

 

Figure 8
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  		Fig. 8. Localization of Lhcgr in Ovaries from Immature Control and Experimental Smad4 cKO Mice Treated with PMSG Ovary

In situ hybridization of ovaries from 3-wk-old control (Smad4Flox/–) and Smad4 cKO experimental (Amhr2cre/+;Smad4Flox/–) were stimulated for 44 h with PMSG. A, C, and D, Bright-field images; B, D, and E, the respective dark-field images. A and B, In situ hybridization of an antisense Lhcgr probe in control ovaries is limited to thecal (Th) cells of early antral follicles (EAF) and theca and mural granulosa (MuGr) of large preovulatory follicles (indicated by double asterisks). Expression is absent in cumulus cells (Cu) and oocytes (Oo). C and D, Experimental Smad4 cKO ovaries have strong hybridization of the antisense Lhcgr probe in granulosa cells in luteinizing follicles (LF) but similar levels of hybridization to the theca (Th) of EAF. As in the controls, cumulus cells (Cu) do not express Lhcgr. E and F, Hybridization of a sense Lhcgr probe to the experimental Smad4 cKO ovary shows little background signal. All dark-field images were captured at the same exposure and magnification. Scale bar, 200 µm.

 
The production of inhibin and activin is necessary for control of the estrous cycle and ovarian function, and Smad4 cKO experimental mice show slightly increased levels of Inha expression in granulosa cells as compared with the control mice, but normal levels of Inhba and Inhbb (Table 4Go). The cell cycle-positive regulatory gene, Ccnd2 (cyclin D2) and the cell cycle inhibitory gene, Cdkn1b (p27Kip1), are unchanged between granulosa cells obtained from control andexperimental mice (Table 4Go). In addition, the Ink4 class cyclin-dependent kinase inhibitor, Cdkn2b (p15), a corpus luteum marker, is not expressed in granulosa cells from either genotype (Table 4Go). However, a number of genes related to steroidogenesis and luteinization were significantly up-regulated in the Smad4 cKO experimental granulosa cells. Cyp11a1, Hsd3b1, and Hsd17b7 were up-regulated 4.8-, 1.3-, and 6.3-fold, respectively (Fig. 7Go and Table 4Go). In addition, the expression of Star, necessary for transport of cholesterol to the mitochondrial membrane, was up-regulated 11.8-fold (Fig. 7Go and Table 4Go). Two additional luteal markers, secreted frizzled-related sequence protein 4, Sfrp4 (40), and Ptgfr, the prostaglandin F2{alpha} receptor (41), were up-regulated 22.3- and 18.4-fold, respectively. Therefore, it appears that Smad4 cKO granulosa cells inappropriately up-regulate a subset of luteal markers in response to gonadotropin stimulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The TGFß superfamily controls a variety of intraovarian reproductive processes (8, 9, 10). We disrupted these signaling pathways in the ovary by conditional deletion of Smad4, the common signaling SMAD, using the cre/lox P recombination system. Loss of SMAD4 is observed in both granulosa cells and thecal cells. Adult female Smad4 cKO mice show severe deficits in fertility, including significant reductions in the number of pups generated per litter, litters born per month, and oocytes ovulated during superovulation protocols. These were accompanied by a significant increase in serum progesterone levels. Morphometric analysis indicated that these ovaries had defects in follicle development, including increased atretic preantral follicles and fewer antral follicles. In addition, after treatment with PMSG, granulosa cells of immature females prematurely luteinize and oocytes within them become trapped or lose cumulus-oocyte contacts. These data led us to analyze the expression of a number of genes associated with luteinization. In granulosa cells of PMSG stimulated immature female Smad4 cKO mice, luteal markers such as Cyp11a1, Hsd17b7, Star, Sfrp4, and Ptgfr were significantly up-regulated. These data indicate that one of the primary functions of members of the TGFß superfamily in vivo is to establish the timing of the luteinization program of granulosa cells during follicle development as hypothesized from studies in vitro (10, 42).

Initial stages of follicle development are not disrupted in the Smad4 cKO ovaries. Several TGFß-related ligands have been implicated in postnatal primordial and primary follicle development in mice, including GDF9 and AMH (24, 43). In the Smad4 cKO ovaries, follicle development before the early secondary stage type 4 (35) (i.e. those with two layers of granulosa cells) is unaffected, as indicated by equivalent follicle counts. In addition, follicles up to the secondary follicles stage demonstrate similar levels of SMAD4 immunoreactivity between control and Smad4 cKO ovaries. These results are likely due to limited or absent cre expression in granulosa cells at the primordial and primary stages (28). Therefore, this model is not useful for studying signaling by TGFß-related ligands that act during the postnatal primordial and primary stages, and additional mouse lines expressing Cre recombinase in earlier granulosa cells is warranted to study these critical stages.

Adult Smad4 cKO ovaries contain significant numbers of atretic preantral follicles. Loss of oocyte-granulosa cell communication has been hypothesized to be a major component of granulosa cell luteinization, and it is well known that granulosa cells spontaneously luteinize when placed into culture in vitro (44), although this is prevented by the addition of oocytes (45). Because two-layer follicles are unaffected and do not express Cre recombinase, loss of oocyte-granulosa cell communication may be occurring in the Smad4 cKO during development of preantral follicles but only after the early secondary stage. Due to unequal recombination, preantral follicles hypomorphic for Smad4 will escape atresia, but then additional defects appear at the antral stage including abnormal luteinization of mural granulosa cells and defects in expansion of cumulus cells during gonadotropin stimulation.

Lhcgr acquisition is a key event in the ability of antral granulosa cells to undergo luteinization (46, 47). The well-described gradient in expression of Lhcgr (e.g. no expression in cumulus cells, low expression in periantral granulosa cells, and high expression in mural granulosa cells) (38, 39, 48) suggests suppression by a factor in the follicular fluid (49) and a luteinization inhibitor has long been proposed to be present in follicular fluid (44, 50). Because oocyte removal from a preovulatory follicle results in spontaneous luteinization of granulosa cells (51), the oocyte is the likely source of this factor. In addition, oocyte-secreted factors such as GDF9 prevent mural granulosa cell luteinization (52, 53, 54, 55, 56, 57). GDF9 also suppresses Lhcgr (56, 57). In Smad4 cKO mural granulosa cells, levels of Lhcgr increase over the controls and the gradient of Lhcgr expression in periantral granulosa cells is lost, clearly indicating a primary regulation through the SMAD4 pathway. Surprisingly, however, Lhcgr remains unexpressed in cumulus cells, similar to the controls. In contrast, other studies have shown that removal of the oocyte in toto from cumulus-oocyte complexes results in Lhcgr up-regulation in the remaining cumulus cells (49). One explanation for these findings is that some genes (in this instance, Lhcgr) have a dose-dependent requirement for SMAD4 during TGFß family signaling: when ligand concentrations are low, SMAD4 is required to potentiate the cellular response; when ligand concentrations are high enough, the R-SMADs bypass SMAD4. Such a dose-dependent requirement for SMAD4 has been hypothesized to be functioning during embryo development in mice and other organisms (16). In the ovarian follicle, cumulus cells receive the highest dose of GDF9 from the oocyte, and therefore may be able to repress Lhcgr without SMAD4 by only using the R-SMADs. Because periantral granulosa cells receive lower levels of GDF9, they would require SMAD4 to repress Lhcgr. Hence, in the Smad4 cKO, periantral granulosa cells show an increase in expression of Lhcgr (i.e. Lhcgr repression is SMAD4-dependent in these cells), whereas cumulus cells do not (i.e. Lhcgr repression is SMAD4 independent in these cells). An alternative explanation is that another oocyte-secreted factor outside the TGFß superfamily may be responsible for Lhcgr suppression in cumulus cells but not periantral granulosa cells.

Morphometric analysis of ovaries from 12-wk-old Smad4 cKO experimental mice demonstrates that antral follicle development was perturbed. This may indicate an impaired estrous cycle, although serum FSH, LH, estradiol, and androstenedione levels were equivalent in the 3-month-old control and Smad4 cKO mice. Normal levels of androstenedione suggest that theca cell function is intact, as does the similar expression of Lhcgr in thecal cells of control and cKO ovaries. The higher progesterone levels may be responsible for the premature infertility beginning at 3 months of age because high progesterone levels can inhibit follicle development and increase atresia in large preantral follicles (58). However, this cannot account for the defects in antral follicle development in younger mice because progesterone levels are unchanged in the Smad4 cKO when measured at 5–6 wk of age. Thus, there are likely multiple causes for the decreased fertility before 3 months of age. This includes the inappropriate response of immature granulosa cells to PMSG. At 21 d of age, antral follicle development in the Smad4 cKO ovaries is only partially rescued by treatment with exogenous gonadotropins. Instead of developing large tertiary follicles by 44 h of PMSG stimulation, large antral follicles luteinize and oocytes within them become trapped. Therefore, fewer eggs are ovulated. It is likely that these luteinized structures are only short-lived because random cycling Smad4 cKO ovaries do not contain large numbers of corpora lutea or large numbers of similar-sized luteinized follicles.

During the LH surge, mural granulosa cells respond to LH and initiate terminal differentiation that results in the formation of the corpus luteum and production of progesterone. Several cell cycle genes are regulated in granulosa cells during the LH surge including Ccnd2 and Cdkn1b (59). Granulosa cells during ovulation and luteinization down-regulate Ccnd2 and up-regulate Cdkn1b, and disruptions in these genes results in female infertility (60, 61, 62, 63). In corpora lutea, another cell cycle inhibitor, Cdkn2b (p15Ink4b), is also highly expressed (64). In immature Smad4 cKO granulosa cells, no change is seen in Ccnd2 or Cdkn1b, and similar to the control, Cdkn2b is undetectable. Thus, given the similar sizes of control and Smad4 cKO ovaries after PMSG stimulation, Smad4 cKO granulosa cells are able to proliferate in response to PMSG, and continue to express a number of granulosa cell markers associated with granulosa cell proliferation including Fshr, Cyp19a1, Inha, Inhba, Inhbb, and Ccnd2. Only minimal changes are seen only for Inha and Fshr [i.e. Fshr decreases slightly (1.3-fold) in the Smad4 cKO granulosa cells]. In contrast to data in rat granulosa cells, which require a positive signal from SMADs, specifically Smad2/3, to induce Ccnd2 and Inha (65), Smad4 cKO granulosa cells express both of these markers similarly to controls. This may suggest that induction of Ccnd2 and Inha by FSH and activin in mice are not regulated by the SMAD pathway in vivo or are SMAD4-independent events. Whereas Smad4 cKO granulosa cells maintain a proliferative expression profile, a number of luteal markers are overexpressed including Lhcgr, Cyp11a1, Star, Sfrp4, and Ptgfr, whereas the differentiation markers remain intact (e.g. Cdkn1b) or are not expressed (e.g. Cdkn2b). Thus, growth arrest and differentiation are uncoupled in Smad4 cKO granulosa cells indicating that SMAD-mediated signaling is critical for maintaining an undifferentiated state. An uncoupling of proliferation and differentiation is also seen in Cdkn1b knockout mice, where luteal cells continue to proliferate after hormonal stimulation, but arrest does not occur until several days later (66).

The BMP family regulates steroidogenic enzymes and their associated proteins, such as StAR. Oocyte-derived BMP6 and BMP15 modulate FSH action on rat granulosa cells in vitro by inhibiting FSH-induced mRNA for Star and Cyp11a1 (67). Thecal cell-derived BMP7 promotes estradiol production and inhibits progesterone synthesis by increasing FSH-induced aromatase (Cyp19a1) expression and inhibiting Star mRNA in rats (68). We did not detect any change in Cyp19a1 in mouse Smad4 cKO granulosa cells. However, Star and Cyp11a1 are highly up-regulated in the Smad4 cKO ovaries (i.e. 12- and 5-fold, respectively). Thus, loss of signaling by SMAD4 results in the increased expression of these genes. Other increases were seen in Hsd17b7, a gene normally associated with corpora lutea in mice and rats (69, 70). Star and Cyp11a1 are known luteal markers with high expression in corpora lutea (71). Quantitative PCR also revealed increases in other genes associated with luteinization such Sfrp4 and Ptgfr (40, 41). Sfrp4 expression is regulated by both prolactin and LH, but regulation of these genes by members of the TGFß superfamily is unknown (40, 72). It is possible that up-regulation of these genes is a consequence of luteinization and not a direct effect of absence of SMAD4.

Additionally, Smad4 cKO ovaries demonstrate considerable defects in cumulus cell expansion and development by multiple criteria. Defects appear both before and after the LH surge. Oocytes lack cumulus cells in follicles of Smad4 cKO ovaries, and Smad4 cKO cumulus cells undergo abnormal expansion during in vivo and in vitro assays. Previously, GDF9 and BMP15 have been shown to be important determinants of cumulus cell physiology (25, 37, 73), and GDF9 has been labeled as the cumulus expansion-enabling factor as recombinant GDF9 can substitute for the oocyte activity in in vitro experiments (56, 74, 75). Recent studies, however, argue that GDF9 cannot account for all of the cumulus expansion activity of fully grown oocytes in in vitro assays (76). In contrast, the data presented in this study demonstrate the importance of SMAD4 and, by extension, TGFß superfamily signaling in cumulus expansion. The most likely cause of the cumulus cell defects during expansion in Smad4 cKO cumulus-oocyte complexes is loss of GDF9 signaling through SMAD4 because no other oocyte-expressed TGFß superfamily protein demonstrates the same cumulus expansion activity as GDF9 and neither oocyte-expressed BMP15 nor BMP6 stimulates cumulus expansion in vitro (56, 77). Furthermore, although TGFß induces cumulus expansion in cocultures of fully grown oocytes and oocytectomized cumulus-oocyte complexes, neutralizing antibodies to TGFß fail to inhibit cumulus expansion (74).

In summary, members of the TGFß superfamily have been implicated in multiple granulosa cell processes, including proliferation, differentiation, and regulation of steroidogenesis. SMAD4 is a tumor suppressor gene in humans (78), and mice conditionally null for Smad4 in the epidermis and mammary gland develop carcinomas (18, 19, 20). Therefore, we were surprised that no tumors developed in the Smad4 cKO ovary. In contrast, our data on the Smad4 cKO ovary suggest that one of the primary defects of SMAD4 loss is premature luteinization of granulosa cells, possibly preventing granulosa cell tumor development, as is observed in inhibin {alpha} knockout mice that have high activin levels (27, 79). Consistent with this absence of tumors, little effect was seen on the expression of nonluteal and proliferation markers such as Fshr and Ccnd2 in the Smad4 cKO granulosa cells. Additional defects are seen in cumulus cell physiology, highlighting the importance of the TGFß superfamily in this cell type. Still unknown is whether there exists a division of labor between the activins, BMPs, and GDFs or whether they act redundantly with respect to luteinization. Future studies are necessary to understand the temporal and spatial regulation of independent TGFß superfamily signaling pathways that ultimately determine the timing of granulosa cell differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of Smad4 Conditional Knockout Mice
The Smad4 conditional allele (Smad4RobCA) (designated throughout as Smad4Flox) and Smad4 null allele (Smad4RobN) (designated throughout as Smad4–) have been described (16, 29) and were maintained on a C57BL/6J;129S5/SvEvBrd mixed hybrid background. Mice were genotyped from tail genomic DNA using PCR primers as described (16, 29). Smad4+/– mice were bred to Amhr2cre/+ mice (28, 29) to generate Amhr2cre/+;Smad4+/– mice. Amhr2cre/+;Smad4Flox/– mice were generated by crossing Amhr2cre/+;Smad4+/– to Smad4Flox/Flox mice. Expression of Amhr2cre/+ in granulosa cells has been validated previously (28). All experimental animals were maintained in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory animals.

Fertility Studies of Smad4Flox/– and Amhr2cre/+;Smad4Flox/– Female Mice
Eight to 10 individually housed female mice of each genotype were bred at 6 wk of age to wild-type C57BL/6J;129S5/SvEvBrd hybrid males with known fertility. The number of litters and number of pups were recorded over a 6-month period.

Serum Analysis
Mice were anesthetized by isoflurane inhalation (Abbott Laboratories, North Chicago, IL), and blood was collected through cardiac puncture. Serum was separated using Microtainer tubes (Becton Dickinson, Franklin Lakes, NJ) and stored at –20 C until assayed. FSH, LH, estradiol, progesterone, and androstenedione measurements were made by the University of Virginia Ligand Core Facility (Specialized Cooperative Centers Program in Reproductive Research NICHD/NIH U54 HD28934) as described (28). Assay information is available at (http://www.healthsystem.virginia.edu/internet/crr/ligand.cfm). The mouse FSH RIA has a sensitivity of 2.0 ng/ml, and an average intraassay coefficient of variation (CV) of 10.1 and interassay CV of 13.3%. The mouse LH sandwich immunoradiometric assay has a sensitivity of 0.07 ng/ml, and an average intraassay CV of 4.7% and interassay CV of 13.5%. The progesterone RIA has a sensitivity of 0.10 ng/ml and an average intraassay CV of 4.5% and interassay CV of 6.9%. The androstenedione RIA has a sensitivity of 0.1 ng/ml, and an average intraassay CV of 4.9%, and an interassay CV of 11.9%. The estradiol RIA has a sensitivity of 10 pg/ml, and an intraassay CV of 5.33%, and an interassay CV of 12.1%. When necessary, serum samples were diluted in PBS to fall within the detectable range.

Histology and Follicle Counts
For each mouse, one ovary was collected in 10% formalin for histological analysis. The other ovary was stored at –80 C in RNA Later (Ambion, Austin, TX) for subsequent RNA isolation. For histological analysis, tissues were fixed overnight, processed, and embedded in paraffin using standard protocols. Four- to 6-µm sections were stained using the periodic acid Schiff reaction (PAS) and hematoxylin. Follicle classification was based on Pedersen and Peters (35). At least three ovaries were analyzed for each genotype. Follicle counts were carried out as described (80, 81). Briefly, six ovaries of each genotype were serially sectioned at 8 µm, and every 10th section was kept. Follicles were counted from five of the largest sections, and normalized to the total area of the section. Counts and area were collected using the AxioVision 4.0 software (Carl Zeiss, Jena, Germany). Statistical analysis was performed using JMP version 5.1 software (SAS Institute, Cary, NC).

Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed, paraffin-embedded 4-µm-thick sections using the Mouse-on-Mouse Basic and Vectastain ABC kits (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. A mouse monoclonal antibody (B8) against SMAD4 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoreactivity was visualized by diaminobenzidine and counterstained in hematoxylin. For direct comparisons, control and experimental ovary sections were placed on the same slide and processed together.

Isolation of Oocytes and Embryos after Superovulation
Superovulation experiments were carried out as described (82). Eight- to 10-wk-old female mice were given ip injections of 5 IU PMSG (VWR, West Chester, PA) for 46 h, followed by injection with 5 IU Pregnyl (Organon, West Orange, NJ) and bred to known fertile wild-type males. Eighteen hours later, eggs and one-cell embryos were recovered from the oviduct, collected into M2 medium (Invitrogen, Carlsbad, CA) and cultured overnight in M16 medium (Invitrogen). Numbers of unfertilized eggs and embryos were counted after 24 h. Data were collected from three independent experiments.

In Vitro Cumulus Expansion Assays
Four- to 5-wk-old female mice were given ip injections of 5 IU PMSG for 44 h. Cumulus-oocyte complexes (COCs) were isolated into DMEM/F12 containing 1% BSA, 1x penicillin/streptomycin (Invitrogen) by puncturing large antral follicles with forceps. Intact COCs were collected by mouth pipetting, and washed once in collection medium, then transferred into DMEM/F12 containing 10% heat-inactivated fetal bovine serum. COCs were placed into chamber slides (Nunc, Rochester, NY) and treated with 200 ng/ml FSH. The degree of cumulus expansion was scored after 15 h culture according to an arbitrary scale of 0 (no expansion) to 4 (full expansion). A minimum of three animals of each genotype was used for each experiment, and three independent experimental repeats were performed.

Granulosa Cell Collection
Twenty-one to 23-d-old control and experimental mice were given ip injections of 5 IU PMSG for 44 h. A minimum of three control and experimental animals were used. Granulosa cells were harvested by puncturing large antral follicles as described (83). Granulosa cells were collected into DMEM/F12 (Invitrogen) containing 0.3% BSA, 10 mM HEPES, and 10 U/ml penicillin and streptomycin. Ovarian debris and oocytes were removed by filtered cells through a 40 µm nylon mesh filter (Nalgene, Rochester, NY). Granulosa cells were collected by centrifugation and processed for RNA using the QIAGEN (Valencia, CA) RNAeasy kit. Granulosa cells represent a mixture of both mural and cumulus cells and are referred to, generically, as "granulosa cells" in the text.

In Situ Hybridization
In situ hybridization was performed as previously described (83). Briefly, freshly dissected ovaries from 21-d females injected for 44 h with PMSG were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Six-micrometer-thick sections were cut and pretreated as described (83). Sections from control and experimental ovaries were placed on the same slide for direct comparisons. A riboprobe was generated from a plasmid containing a 750-bp portion of the mouse Lhcgr gene as previously described (57). After hybridization, dehydrated sections were dipped in NTB emulsion (Kodak, Rochester, NY) and exposed at 4 C for variable times, counterstained with Meyer’s hematoxylin, and mounted for photography.

Quantitative Real-Time PCR (QPCR)
Two hundred micrograms of total RNA were reverse-transcribed in a 50-µl reaction using 250 U Superscript III reverse transcriptase (Invitrogen) and oligo-deoxythymidine primers. Samples were diluted 50-fold, and 5 µl were used for each QPCR. Real-time QPCR was performed on the ABI Prism 7500 Sequence Detection System (ABI, Foster City, CA) using predesigned Taqman Assays-On-Demand (ABI) PCR primer and probe sets and mouse Gapd as an endogenous control. The following Taqman assays were used: Ccnd2, Mm00438071; Cdkn2b, Mm00483241;Ckn1b, Mm00438167;Gja1, Mm00439105; Gja4, Mm00433610; Cyp11a1, Mm00490735; Cyp17a1, Mm00484040; Cyp19a1, Mm00484049; Fshr, Mm00442819; Grem1, Mm00488615; Hsd17b7, Mm00501703; Hsd3b1, Mm00476184; Inha, Mm00439683; Inhba, Mm00434338; Lhcgr, Mm00442931; Ptgfr, Mm00436055; Star, Mm00441558; Gapd (4352339E, primer limited). Sfrp4 cDNA were amplified using Sybr Green Master Mix (ABI) with the primers 5'-CCTGCCAGTGTCCACATATCC and 5'-GCAATTTTCAAGAAGCATCATCCT. Smad4 exon 2 was amplified using Sybr Green Master Mix (ABI) and the primers 5'-CACTGCCTTCAAAAGATCAAAATTAC and 5'-TGGTGTATTTGTTATGAGCATATTGTCCAT. Taqman PCR was performed using the TaqMan Universal PCR Master Mix (ABI) in 20 µl. Smad4 (exon 2) and Sfrp4 primers were designed using the Primer Express software (ABI). Ten-fold serial dilutions were used to determine amplification efficiency for each primer set. The reaction conditions were as follows: 10 min hold at 95 C followed by 40 cycles of: 15 sec at 95 C, (denaturation); 1 min at 60 C (annealing/extension). Each sample was analyzed in duplicate from a minimum of three animals per genotype. Two nontemplate control (ribonuclease-free water) samples were included on each plate for each primer-probe set. The relative amount of transcript was calculated by the {Delta}{Delta}CT method as described by Applied Biosystems using the ABI 7500 System Software (version 1.2.3) and normalized to the endogenous reference (Gapd). The calibrator sample was randomly chosen from the wild-type samples. The average and SE was calculated, and the relative amount of target gene expression for each sample was plotted in Excel (Microsoft).

Statistical Analysis
Statistical analysis was carried out using the JMP version 5.1 statistical package. Statistical differences were tested using the nonparametric Mann-Whitney U for single comparisons or Kruskal-Wallis analysis of ranks test for multiple comparisons. Nonparametric tests gave identical results to parametric tests but were used because sample sizes were small. P values smaller than 0.05 were considered statistically significant. Statistics were performed on no less than three independent experiments.


   ACKNOWLEDGMENTS
 
We thank Dr. Herman Dierick for critical reading of the manuscript and the University of Virginia Ligand Core Facility [Specialized Cooperative Centers Program in Reproduction Research National Institute of Child Health and Human Development/National Institutes of Health (NIH) U54 HD28934] for hormone assays. We thank Dr. Richard Behringer for the Amhr2cre mice.


   FOOTNOTES
 
This work was supported by NIH Grants HD32067 (to M.M.M.), HD44156 (to E.J.R.), and a Ruth L. Kirschstein National Research Service Award 5F32HD46335 (to S.A.P.).

Disclosure Statement: S.A.P., X.L., E.J.R., and M.M.M. have nothing to declare.

First Published Online March 2, 2006

Abbreviations: BMPs, Bone morphogenetic proteins; cKO, conditional knockout; COCs, cumulus-oocyte complexes; CV, coefficient of variation; GDF9, growth and differentiation factor 9; hCG, human chorionic gonadotropin; PAS, periodic acid Schiff reaction; PMSG, pregnant mare serum gonadotropin; QPCR, quantitative real-time PCR; SF, secondary follicle.

Received for publication November 17, 2005. Accepted for publication February 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Matzuk MM, Burns K, Viveiros MM, Eppig J 2002 Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296:2178–2180[Abstract/Free Full Text]
  2. Matzuk MM, Lamb DJ 2002 Genetic dissection of mammalian fertility pathways. Nat Med 8(Suppl 1):S41–S49
  3. Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 15:201–204[CrossRef][Medline]
  4. Ma X, Dong Y, Matzuk MM, Kumar TR 2004 Targeted disruption of luteinizing hormone ß-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility. Proc Natl Acad Sci USA 101:17294–17299[Abstract/Free Full Text]
  5. Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur M, Sassone-Corsi P 1998 Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acad Sci USA 95:13612–13617[Abstract/Free Full Text]
  6. Richards JS, Russell DL, Ochsner S, Hsieh M, Doyle KH, Falender AE, Lo YK, Sharma SC 2002 Novel signaling pathways that control ovarian follicular development, ovulation, and luteinization. Recent Prog Horm Res 57:195–220[Abstract/Free Full Text]
  7. Niswender G, Nett T 1994 The corpus luteum and its control in infraprimate species. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press; 781–816
  8. Chang H, Brown CW, Matzuk MM 2002 Genetic analysis of the mammalian TGF-b superfamily. Endocr Rev 23:787–823[Abstract/Free Full Text]
  9. Pangas SA, Matzuk MM 2004 Genetic models for transforming growth factor ß superfamily signaling in ovarian follicle development. Mol Cell Endocrinol 225:83–91[CrossRef][Medline]
  10. Shimasaki S, Moore RK, Otsuka F, Erickson GF 2004 The bone morphogenetic protein system in mammalian reproduction. Endocr Rev 25:72–101[Abstract/Free Full Text]
  11. Massagué J 1998 TGF-ß signal transduction. Annu Rev Biochem 67:753–791[CrossRef][Medline]
  12. Chen YG, Hata A, Lo RS, Wotton D, Shi Y, Pavletich N, Massague J 1998 Determinants of specificity in TGF-ß signal transduction. Genes Dev 12:2153–2163[Abstract/Free Full Text]
  13. Attisano L, Wrana JL 2000 Smads as transcriptional co-modulators. Curr Opin Cell Biol 12:235–243[CrossRef][Medline]
  14. Sirard C, de la Pompa JL, Elia A, Itie A, Mirtsos C, Cheung A, Hahn S, Wakeham A, Schwartz L, Kern SE, Rossant J, Mak TW 1998 The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo. Genes Dev 12:107–119[Abstract/Free Full Text]
  15. Yang X, Li C, Xu X, Deng C 1998 The tumor suppressor SMAD4/DPC4 is essential for epiblast proliferation and mesoderm induction in mice. Proc Natl Acad Sci USA 95:3667–3672[Abstract/Free Full Text]
  16. Chu GC, Dunn NR, Anderson DC, Oxburgh L, Robertson EJ 2004 Differential requirements for Smad4 in TGFß-dependent patterning of the early mouse embryo. Development 131:3501–3512[Abstract/Free Full Text]
  17. Zhou YX, Zhao M, Li D, Shimazu K, Sakata K, Deng CX, Lu B 2003 Cerebellar deficits and hyperactivity in mice lacking Smad4. J Biol Chem 278:42313–42320[Abstract/Free Full Text]
  18. Li W, Qiao W, Chen L, Xu X, Yang X, Li D, Li C, Brodie SG, Meguid MM, Hennighausen L, Deng CX 2003 Squamous cell carcinoma and mammary abscess formation through squamous metaplasia in Smad4/Dpc4 conditional knockout mice. Development 130:6143–6153[Abstract/Free Full Text]
  19. Qiao W, Li AG, Owens P, Xu X, Wang XJ, Deng CX 2006 Hair follicle defects and squamous cell carcinoma formation in Smad4 conditional knockout mouse skin. Oncogene 25:207–217[Medline]
  20. Yang L, Mao C, Teng Y, Li W, Zhang J, Cheng X, Li X, Han X, Xia Z, Deng H, Yang X 2005 Targeted disruption of Smad4 in mouse epidermis results in failure of hair follicle cycling and formation of skin tumors. Cancer Res 65:8671–8678[Abstract/Free Full Text]
  21. Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP, Hogan BL 1999 Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 13:424–436[Abstract/Free Full Text]
  22. Behringer RR, Finegold MJ, Cate RL 1994 Müllerian-inhibiting substance function during mammalian sexual development. Cell 79:415–425[CrossRef][Medline]
  23. Yao HH, Matzuk MM, Jorgez CJ, Menke DB, Page DC, Swain A, Capel B 2004 Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis. Dev Dyn 230:210–215[CrossRef][Medline]
  24. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM 1996 Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383:531–535[CrossRef][Medline]
  25. Yan C, Wang P, DeMayo J, DeMayo F, Elvin J, Carino C, Prasad S, Skinner S, Dunbar B, Dube J, Celeste A, Matzuk M 2001 Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol Endocrinol 15:854–866[Abstract/Free Full Text]
  26. McNatty KP, Juengel JL, Wilson T, Galloway SM, Davis GH, Hudson NL, Moeller CL, Cranfield M, Reader KL, Laitinen MP, Groome NP, Sawyer HR, Ritvos O 2003 Oocyte-derived growth factors and ovulation rate in sheep. Reprod Suppl 61:339–351[Medline]
  27. Matzuk MM, Finegold MJ, Su J-GJ, Hsueh AJW, Bradley A 1992 {alpha}-Inhibin is a tumor-suppressor gene with gonadal specificity in mice. Nature 360:313–319[CrossRef][Medline]
  28. Jorgez CJ, Klysik M, Jamin SP, Behringer RR, Matzuk MM 2004 Granulosa cell-specific inactivation of follistatin causes female fertility defects. Mol Endocrinol 18:953–967[Abstract/Free Full Text]
  29. Jamin SP, Arango NA, Mishina Y, Hanks MC, Behringer RR 2002 Requirement of Bmpr1a for Mullerian duct regression during male sexual development. Nat Genet 32:408–410[CrossRef][Medline]
  30. Boerboom D, Paquet M, Hsieh M, Liu J, Jamin SP, Behringer RR, Sirois J, Taketo MM, Richards JS 2005 Misregulated Wnt/ß-catenin signaling leads to ovarian granulosa cell tumor development. Cancer Res 65:9206–9215[Abstract/Free Full Text]
  31. Jeyasuria P, Ikeda Y, Jamin SP, Zhao L, De Rooij DG, Themmen AP, Behringer RR, Parker KL 2004 Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function. Mol Endocrinol 18:1610–1619[Abstract/Free Full Text]
  32. Soriano P 1999 Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71[CrossRef][Medline]
  33. Pangas SA, Rademaker AW, Fishman DA, Woodruff TK 2002 Localization of the activin signal transduction components in normal human ovarian follicles: implications for autocrine and paracrine signaling in the ovary. J Clin Endocrinol Metab 87:2644–2657[Abstract/Free Full Text]
  34. Drummond AE, Le MT, Ethier JF, Dyson M, Findlay JK 2002 Expression and localization of activin receptors, Smads, and ß glycan to the postnatal rat ovary. Endocrinology 143:1423–1433[Abstract/Free Full Text]
  35. Pedersen T, Peters H 1968 Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil 17:555–557[Medline]
  36. Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ 1990 FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte. Dev Biol 138:16–25[CrossRef][Medline]
  37. Su YQ, Wu X, O’Brien MJ, Pendola FL, Denegre JN, Matzuk MM, Eppig JJ 2004 Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol 276:64–73[CrossRef]