| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Program in Developmental Biology (C.J.J., R.R.B., M.M.M.), Departments of Pathology (M.K., M.M.M.), Molecular and Human Genetics (M.M.M.), and Molecular and Cellular Biology (M.M.M.), Baylor College of Medicine, and Department of Molecular Genetics (S.P.J., R.R.B.) University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: Martin M. Matzuk, M.D., Ph.D., The 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 INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Activins and inhibins are dimeric protein members of the TGF-ß superfamily that share ßA and ßB subunits [activin A (ßA:ßA), activin B (ßB:ßB), activin AB (ßA:ßB), inhibin A (
:ßA), and inhibin B (:ßB)]. These proteins are named for their respective roles in stimulating and inhibiting the secretion of FSH from the pituitary (6). Follistatin interacts with activin at physiological pH; this affinity is similar to or exceeds that of activin binding to its receptor. The bound complex consists of one follistatin molecule bound to each of the two activin ß-subunits. Follistatin has low affinity for inhibins, which have only one ß-subunit (7, 8).
Follistatin also regulates members of the BMP subfamily by binding to them with lower affinities than for activin (5); however, this activity may be sufficient to allow follistatin to act as a regulator in the numerous systems expressing these factors. In Xenopus laevis embryos, follistatin is able to directly bind, inhibit, and neutralize BMP2, BMP4, and BMP7. The phenotype caused by overexpression of follistatin in early X. laevis embryos is similar to those achieved by overexpression of dominant negative BMP receptors (9, 10). Likewise, mouse BMP11 is able to induce dorsal mesoderm and neural tissue formation in X. laevis assays, and both effects are inhibited by follistatin (11). Follistatin also attenuates BMP15 stimulation of rat granulosa cell proliferation (12). These studies suggest that both ovarian activin and BMPs are potential targets for inhibition of ovarian-derived follistatin. Thus, knockout of follistatin in the ovary may affect both activin and BMP systems in the ovary because BMP4 and BMP7 are expressed in thecal cells; activin ßA and ßB and ALK3, ALK6, and BMPR2 are expressed in granulosa cells; and growth differentiation factor 9, BMP15, and BMP6 are expressed in oocytes (13, 14, 15).
To study the in vivo roles of follistatin (Fst), knockout mice were generated (16). Unfortunately, these mice die within hours of birth due to multiple defects including growth retardation, craniofacial defects, and decreased mass of the diaphragm and intercostal muscles, making it difficult to study the postnatal roles of follistatin in reproduction (16). Conversely, mice overexpressing follistatin are viable and develop to adulthood, but have extensive reproductive defects, including decreased testis size, an arrest in spermatogenesis in males, and a block in folliculogenesis in females (17). These findings, together with the abundance of follistatin mRNA in the ovary (18, 19), suggest that follistatin produces important effects within the gonads by regulating the local functions of activins and/or other TGF-ß family members.
Due to the lethality of the Fst knockout mice and to further study the postnatal functions of follistatin, we chose to produce mice in which the follistatin gene could be manipulated with the cre/loxP system. Mice carrying a follistatin allele flanked by loxP sites (FstFlox) were generated. Anti-Müllerian hormone receptor type II (Amhr2) knock-in mice were chosen for granulosa cell-specific expression of Cre recombinase. The Amhr2cre mice were hypothesized to be a key reagent to study the postnatal roles of granulosa cell genes in the ovary where Amhr2 and follistatin are highly expressed. These studies open new avenues of investigation using conditional mice to study the roles of essential granulosa cells expressed genes involved in the female reproductive system.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
). This construct was electroporated into the original HPRT-positive Fsttm1/+ ES cell line (FS3-C2) that is heterozygous for a null mutation at the Fst locus (16). We used 6-thioguanine for negative selection of the PgkHPRT cassette expressed in the ES cells and G418 for positive selection of the PgkNeo cassette in the targeting construct. Of the 36 ES cell clones that survived the double selection, 33 of them (92%) were correctly targeted at the Fst locus and contained two loxP sites. Two of these cell lines were used to produce chimeric male mice that were fertile and transmitted the FstFlox allele to F1 progeny. Heterozygous FstFlox mice were crossed to produce FstFlox homozygous mice. FstFlox/FstFlox mice are viable and fertile and were obtained at the expected Mendelian frequency. Intercrossing of heterozygotes (FstFlox/+) yielded 299 progeny including 81 wild-type mice (27%), 150 heterozygous (FstFlox/+) mice (50%), and 68 homozygous (FstFlox/ FstFlox) mice (23%) out of 39 litters analyzed with an average of 7.7 ± 0.37 pups per litter.
|
allele (Fig. 1B
). Thus, Cre recombinase can efficiently recombine the loxP sites flanking the Fst exons to produce a null allele.
Cre Activity in the Ovaries of Amhr2cre Knock-in Mice
Amhr2 together with its ligand anti-Müllerian hormone (Amh) are members of the TGF-ß family of signaling proteins expressed in the female gonads. AMH induces the regression of Müllerian ducts, precursors of the oviducts, uterus, and upper vagina by binding to AMHR2 expressed in the mesenchyme surrounding the ductal epithelium during embryogenesis. Amhr2 is expressed in Sertoli and Leydig cells in testes and granulosa cells in ovaries (21, 22, 23, 24, 25, 26, 27).
As a driver of Cre expression, the Amhr2cre knock-in mice were selected. Amhr2cre knock-in mice were generated with an insertion of a cre-neo cassette into the fifth exon of Amhr2 to genetically modify Amhr2-expressing tissues (28). The Amhr2cre mice were initially used to selectively disrupt the expression of Bmpr1a in the mesenchymal cells surrounding the Müllerian ducts, thereby identifying BMPR1A as a type I receptor for AMH-induced regression of the Müllerian ducts (28).
To monitor the Cre activity in the postnatal ovaries, the Amhr2cre mice were crossed to Gt(ROSA)26Sortm1Sor (R26R) mice that express ß-galactosidase activity only in cells that express Cre (29). After staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactosidase, we detected ß-galactosidase activity in the ovaries of Amhr2cre R26R double heterozygous females (Fig. 2A). ß-Galactosidase (ß-gal) activity was present in the ovary as early as embryonic d 17.5 (Fig. 2B). Throughout the postnatal ovary, ß-gal activity was present in granulosa cells of all secondary and small antral follicles, but was at lower or undetectable levels in granulosa cells of primordial and primary follicles (Fig. 2, C and D). These observations are consistent with the endogenous Amhr2 expression pattern. However, low Cre activity was also found in some theca cells and oocytes (Fig. 2D). In addition to the ovary, we checked for Cre activity in the endometrium and the muscular layer of the uterus. The endometrium was negative for Cre; on the other hand, the muscular layer of the uterus was Cre positive as expected because Amhr2 is expressed in the mesenchyme of the Müllerian duct that gives rise to the uterine musculature and not the endometrium (24). These results indicate that the Amhr2cre knock-in line of mice is a good cre deleter strain to ablate follistatin expression in the granulosa cells of the ovary.
|
To produce follistatin deficiency in the ovaries, Amhr2cre+; Fsttm1/+ mice were generated, and these mice were subsequently mated to FstFlox/FstFlox mice. All four expected genotypes were recovered from the above cross at close to the expected Mendelian frequency.
To determine the efficiency of the Cre recombinase of the Amhr2cre knock-in mice in ablating follistatin expression in the ovary, we performed Southern blot analysis of granulosa cells from immature females hormonally treated with 5 IU of pregnant mare’s serum gonadotropin (PMSG) for 48 h and Northern blot analysis of ovaries from adult untreated and hormonally stimulated mice.
Granulosa cells derived from six females (two Amhr2cre-; Fsttm1/FstFlox control and four Amhr2cre+; Fsttm1/FstFlox experimental mice) were isolated from the ovaries after PMSG stimulation, and DNA from these cells was analyzed by Southern blot (Fig. 3A). Whereas the Amhr2cre negative granulosa cells contained the FstFlox allele, the granulosa cells from all four Amhr2cre positive mice had the Fst (deleted) allele at equal intensity to the Fsttm1 allele, demonstrating efficient recombination of the loxP sites of the FstFlox allele and conversion to the Fst allele. Thus, these mice are follistatin null in their granulosa cells.
|
, Northern blot analysis of wild-type untreated or PMSG-treated ovaries using a follistatin cDNA probe identified a follistatin mRNA band of approximately 2.6 kb. However, no follistatin mRNA signal was detected in the mRNA derived from Amhr2cre+; Fsttm1/FstFlox ovaries, either untreated or PMSG treated. Thus, inactivation of the follistatin gene in granulosa cells abolishes follistatin mRNA expression in the entire adult ovary in the presence or absence of PMSG treatment.
Fertility Analysis of Follistatin Conditional Mice
To study the fertility of mice with follistatin-deficient gonads, male or female Amhr2cre-; Fsttm1/FstFlox (control mice) and Amhr2cre+; Fsttm1/FstFlox (experimental mice) were mated to wild-type female or male mice for a 6-month period. There was no significant difference in the fertility between the seven experimental males (9.62 ± 0.59 pups per litter and 0.96 ± 0.03 litter/month) and six control males (8.74 ± 0.82 pups per litter and 0.92 ± 0.04 litter/month) (Table 1). On the other hand, experimental females exhibited fertility defects; one of the 11 experimental female mice was infertile, whereas the remaining 10 experimental females displayed significantly reduced fertility. The number of litters per month for the experimental females (0.42 ± 0.07) was less than half of that of the control females (0.97 ± 0.01). The number of pups per litter for the experimental group (3.75 ± 0.35) was less than half of the values of control females (7.88 ± 0.52) (Table 1). Thus, ovarian follistatin plays an important role in female reproduction, and the absence of follistatin in the granulosa cells of the ovary reduces fertility.
|
).
|
); however, the experimental ovaries exhibited a decrease in the number of antral follicles and corpora lutea and an increase in accumulation of periodic acid Schiff (PAS)-positive material in the interstitium that was likely due to zona pellucida (ZP) remnants (Fig. 4D; see Ref.30) when compared with the ovaries of control mice (Fig. 4A). By 6 months of age, a marked contrast in control vs. experimental ovaries was observed. All four experimental mice analyzed had a reduction in the number of follicles (specifically the antral follicles), an abundance of ZP remnants, and a decrease in the number of corpora lutea (Fig. 4E) when compared with control mice of the same age with follicles at all the stages of folliculogenesis, few ZP remnants, and abundant corpora lutea (Fig. 4B). One of the 6-month-old experimental ovaries showed Sertoli cell tubule-like structures (data not shown).
Comparative histology of 8-month-old mice demonstrated remarkable differences. Ten of the 11 experimental mice analyzed had few follicles (<five primary or later follicles per cross-section of the ovary; Fig. 4, F and G). This is a significant decrease when compared with control mice of the same age that had more than 10 follicles at different stages of folliculogenesis, abundant corpora lutea, and few ZP remnants (Fig. 4C). The experimental ovaries showed an increase in ZP remnants when compared with control and younger experimental mice, indicating an active oocyte death process (Fig. 4, F and G). Another important finding was that seven of the 11 experimental mice had Sertoli cell tubule-like structures (Fig. 4, F–H), reminiscent of the ovarian tumors from inhibin-deficient mice (31, 32), mice overexpressing follistatin (17) and FSH (33), mice lacking both estrogen receptor and ß (34), and mice overexpressing AMH (35). Two of the experimental mice had single ovarian cysts (Fig. 4F), previously reported in mice lacking growth differentiation factor 9 (36). Therefore, these findings suggest that follistatin plays roles in oocyte survival and folliculogenesis due to the accelerated loss of oocytes and follicles with age in the experimental mice.
Consistent with the normal fertility of the mutant males, there was no significant difference in testes size, weight, or histology between the control and experimental mice (data not shown). These results indicate that either follistatin does not play an essential role in the testis or the Amhr2cre knock-in mouse model is not an appropriate cre deleter strain to abolish follistatin expression in the Sertoli cells of the testis where follistatin is expressed (37).
Ovarian and Oocyte Physiology
To assess whether follistatin plays a critical role in successful fertilization of the released ovum, 3-wk-old females were superovulated and then mated to stud males. Approximately 20 h after the human chorionic gonadotropin (hCG) injection, oocytes and one-cell embryos were isolated. Eighty-one percent of the oocytes from the control mice (Amhr2cre-; Fsttm1/ FstFlox) and 70% of the oocytes from the experimental mice (Amhr2cre+; Fsttm1/FstFlox) developed to two-cell embryos after 24 h culture (Table 2). No significant differences were found either in the number of oocytes released or the fertilization rates between the two groups, indicating that the number of oocytes ovulated and fertilized in the presence or absence of follistatin, at least under pharmacological conditions, were the same in these immature mice.
|
). A significant difference was found in the number of oocytes released as well as the number that developed to the two cell stage in the two groups. These findings indicate that adult mice lacking follistatin in the granulosa cells do not respond as well as the control mice to exogenous gonadotropins because they 1) release fewer oocytes and 2) these oocytes are less competent to be fertilized.
|
, A and B) even though it seemed that control mice have a better response to gonadotropin stimulation. One additional important finding in this experiment was the presence of abnormal follicle development (i.e. a follicle with three oocytes) in the ovary of one of the experimental mice (Fig. 5, B and C).
|
. There was a significant difference in the values of three of the four measured hormones, specifically in the oldest mice.
|
) had one of the highest serum levels of FSH (i.e. 23.63 ng/ml). The increase in the FSH values supports the histological findings that show a pattern resembling that of premature ovarian failure (POF). The levels of the gonadotropin LH were also evaluated. A significant difference between the LH levels of control and experimental mice in immature and adult animals was also discovered with the differences in the oldest mice being the most substantial. At 8 months of age, the experimental mice exhibited a value of 0.53 ± 0.07 ng/ml whereas the control mice had a level of 0.23 ± 0.07 ng/ml. These results of the gonadotropins (FSH and LH) indicate a dysfunction in the ovary and in the hypothalamic pituitary-ovarian feedback system, a finding that might be expected from the ovarian histology (Fig. 4). Estradiol is an estrogen produced in the granulosa cells of the ovary. Levels of estradiol were measured in experimental and control mice at 3 months, 6 months, and 8 months of age. Surprisingly, no significant differences between experimental and control mice at any age were found. The estradiol levels start to decrease in the experimental mice (15.82 ± 1.41 pg/ml) at 6 months of age when compared with the control mice (18.17 ± 3.99 pg/ml) of the same age. In the 8-month-old mice, the estradiol levels were reduced to 6.61 ± 1.11 pg/ml but we also noticed a decrease in the levels in the control mice (7.93 ± 0.99).
Half of the circulating levels of testosterone are produced in the ovary. Deficiencies in testosterone have been reported in women with premature ovarian failure (38). Consistent with studies in those women, we found a significant difference in the values of testosterone when we compared experimental (0.18 ± 0.02 pg/ml) and control (0.40 ± 0.11 pg/ml) mice at 6 months of age (Table 4). Also, at 8 months of age, a significant difference in testosterone levels were found in experimental mice (0.19 ± 0.01 pg/ml) vs. control mice (0.35 ± 0.07 pg/ml). No significant differences were found in the 3-month-old mice.
Study of the Ovarian Expression of TGF-ß Superfamily Members in the Amhr2cre+; Fsttm1/FstFlox Mice
Discrepancies between the phenotypes of transgenic mice deficient in either activin or activin type II receptor and the phenotype of transgenic mice overexpressing follistatin (17, 39) suggest that activins are not the only growth factors that may be affected by the absence of follistatin. Possible candidates include members of the TGF-ß family such as inhibins that share the same ß-subunits with activins and BMPs (e.g. oocyte-derived factor BMP15) that are inhibited by follistatin (12).
To determine whether mRNAs of any TGF-ß superfamily members are regulated by follistatin, the levels of activin ßA and ßB, inhibin , and Bmp15 mRNAs were measured by Northern blot analysis of Amhr2cre+; Fsttm1/Fstflox (experimental mice) and Amhr2cre-; Fsttm1/Fstflox (control mice) ovarian RNA. Initially individual sets of ovaries from 3-month-old females untreated or treated with 5 IU of PMSG for 48 h before ovary collection were used to assess whether the absence of follistatin in the gonads has an effect on these ovarian-expressed TGF-ß members. Regarding the levels of -subunit mRNA, no differences were found. The subunit was present in both experimental and control mice, both untreated and treated (Fig. 6C). However, some individual differences were found when the blots were probed with cDNA against activin ßA and ßB, and Bmp15 (data not shown). These results suggest that follistatin could have an effect on the expression of these genes. To corroborate these findings, Northern blot analysis using a pools of ovaries from four mice at 6 months of age was performed. The results from the Northern blot of pool of ovaries using cDNA probe against activin ßA and ßB and BMP15 revealed no significant differences between the ovaries of Amhr2cre-; Fsttm1/FstFlox (control) mice, and ovaries from Amhr2cre+; Fsttm1/FstFlox (experimental) mice (Fig. 6, A and B). These result indicate that follistatin does not seem to have a role in the transcriptional regulation of any of these ovarian TGF-ß members.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
). This system allowed for removal of all of the exons required for the synthesis of the functional follistatin protein when these mice were crossed to cre-expressing mice. As a cre deleter strain, the Amhr2cre knock-in mice were selected due to the high levels of Cre activity in granulosa cells of postnatal ovaries of these mice (Fig. 2).
No expression of ovarian follistatin was detected in experimental mice tested at 3 months of age (Fig. 3A). Also, Southern blot analysis of the granulosa cells of 21-d-old experimental ovaries showed the Fst allele and not the FstFlox allele (Fig. 3B), demonstrating that recombination occurs in essentially 100% of the granulosa cells. These results indicate that using a mouse to deliver cre under the control of the Amhr2 gene represents an excellent system for studying follistatin function in the granulosa cells of the postnatal ovary. These results also open new avenues of research for using conditional mutant mice to study the roles of other (ubiquitous) genes expressed in the granulosa cell of the ovary. Even though we obtained almost 100% recombination in the granulosa cells studied, the concern is always present that Cre activity will be insufficient to cause loxP recombination in all cells early in development, resulting in mosaicism. However, transplanting follistatin null ovaries into the bursa of wild-type mice creates a similar phenotype to the Fst conditional mutant mice. These mice are infertile, and minimal follicle formation in the ovaries is observed (Matzak, M., unpublished data). One advantage of using the conditional knockout mice as a model rather than ovarian transplant recipients is the opportunity to have an unlimited number of mice to use in the studies. Another advantage is that FstFlox mice will allow study of the in vivo roles of follistatin not only in the reproductive system but also in other systems. Areas of interest include the development of the skin, hair, and whiskers (16, 17, 40, 41, 42, 43), as well as kidney tubule morphogenesis, where Fst is highly expressed and its expression is reduced by injury (44), and bone and palate development, where it plays a role in the transition from cartilage to bone (16, 45).
The preliminary results of ovarian transplantation experiments in which the females were infertile and the fact that our experimental mice were also infertile or subfertile indicate the importance of follistatin in communication between different cell types of the follicle for its formation and maturation. The reason that only one of the experimental mice was completely infertile may be due to delayed postnatal deletion of the FstFlox allele in the subfertile mice. Mosaicism, in which gene inactivation is induced in only a fraction of the desired cells, has been reported in the other cre/loxP systems (46). Although a range of phenotypes was found with use of Amhr2cre knock-in mice (28), mosaicism appears minimal based on our Southern and Northern blot analyses (Fig. 3).
Our results show that mice with the deletion of the follistatin gene in ovarian granulosa cells exhibited a reduced number of litters per month and reduced litter size, and, in the most severe case, infertility. No defects in ovulation or fertilization were found with superovulation of immature mice (Table 2). However, 6-month-old experimental mice did show a significant reduction in ovulation and fertilization of oocytes, suggesting reductions in the number and quality of available oocytes (Table 3). Although five of the seven experimental mice responded to stimulation with exogenous gonadotropins to varying degrees, two mice did not respond.
Histological examination of experimental mice beyond 6 months of age reveals a drastic reduction in the number of the follicles (Fig. 4, E–G), corroborating our hypothesis that there is a reduction in the pool of available oocytes as a consequence of the loss of follistatin. Furthermore, some of the 8-month-old females did not have any ovarian follicles (Fig. 4F). These results indicate premature ovarian failure in mice lacking follistatin in the granulosa cells. One interesting histological finding in the experimental mice was the presence of Sertoli cell tubule-like structures (Fig. 4, F–H) noted in seven of the eleven 8-month-old and one of the four 6-month-old, experimental mice. Sertoli cell tubule-like structures have been reported in female mice lacking inhibin (31, 32), both estrogen receptors and ß (34), and in those overexpressing AMH, follistatin, and FSH (17, 33, 35). All of the above mentioned genes are expressed in granulosa cells, and their perturbation can cause changes in granulosa cell fate. Furthermore, experimental mice experience an increase in oocyte loss (demonstrated by an abundance of ZP remnants) (Fig. 4, D and G). When a follicle loses its oocyte, the follicle is no longer regulated by oocyte factors, and that subsequently results in an alteration in cell fate. Follicle development is a very sensitive process influenced by a variety of factors and disturbances of any of them, either by removal or excess, can cause a change in fate pattern. The Sertoli cell tubule-like structures are a consequence of an imbalance in the follicle equilibrium.
Ovarian failure in women may be diagnosed by high concentrations of the gonadotropins, FSH and LH, and a low concentration of estradiol. A rise in FSH is the most sensitive and the best early marker for ovarian failure (47). FSH levels in our experimental mice lacking ovarian follistatin were significantly increased in both immature and mature mice. Also LH levels were elevated in the experimental mice (Table 4). Interestingly, there was no significant difference in the level of estradiol between experimental and control mice in our study. Women with premature ovarian failure (POF) produce estrogen intermittently and may ovulate despite the presence of high gonadotropin levels (48). Ovaries of POF patients sporadically go through a temporary phase of low activity, which can return to normal later in life. For that reason, at least two hormone level measurements, taken some weeks apart, are necessary before a POF diagnosis is made (47, 49). This could be a possible explanation for the lack of variation in the estradiol levels of the experimental mice. Unfortunately, we can not take a second sample to confirm whether or not there is a significant reduction in the levels of estradiol in the experimental mice.
We also evaluated testosterone levels in experimental mice. Women with POF have the potential for loss of ovarian androgens due to the atrophy of the ovarian cortex (38, 50, 51, 52). In agreement with the human POF clinical findings, we found a significant decrease in testosterone in experimental mice beyond 6 months of age (Table 4). Thus, the hormonal profile of mice lacking ovarian follistatin resembles the hormonal profile of women with POF.
The quality and number of oocytes in the experimental mice are compromised as seen by a reduction in the ovulation and fertility rate in the 6-month-old experimental mice when in vivo fertilization experiments were performed (Table 3). Two of the seven experimental mice did not respond to the ovulation induction. Women with POF, who underwent attempts to induce ovulation using different regimens, have reduced ovulation rates (53, 54, 55). This result indicates that defects in granulosa cell function during follicle development can compromise not only ovulation but also oocyte competence.
Follistatin has been associated with polycystic ovary syndrome (PCOS) in women through genetic linkage studies. PCOS is an endocrine disorder characterized by reduced fertility, hyperandrogenism, and chronic anovulation (56). After the original study linking follistatin to PCOS, three different studies (including one from the same group) did not show any relation between mutations in the human follistatin gene and PCOS (57, 58, 59). We did not find any indication of hyperandrogenism or multiple ovarian cysts in our ovarian follistatin deletion mice. In contrast, the values of testosterone in 6- and 8-month-old female mice showed a significant reduction when compared with controls. Thus, loss of ovarian follistatin in mice fails to model several features of PCOS.
Our findings of early deterioration of ovarian function, increased gonadotropins, decreased testosterone, decreased number of follicles, and failure of follicles to be fertilized and ovulated in response to exogenous gonadotropins suggest that a mutation compromising follistatin in the granulosa cell of the ovary could be a cause of POF. POF is a condition causing amenorrhea, infertility, and elevated gonadotropin concentrations in women under the age of 40 yr. In some women, intermittent ovarian function has been reported and pregnancy can occur in 5–10% of patients subsequent to diagnosis. In most patients with POF and normal female chromosome constitution, no cause can be identified. The hypotheses of the causes of POF are: 1) failure to attain the appropriate primordial follicle pool, and 2) accelerated loss of oocytes and follicles. Our animal model provides us with an insight into a new candidate gene possibly involved in POF. Follistatin should be included in future POF genetic studies. If follistatin is found to be associated with POF in women, our animal model will be useful in elucidating the mechanism of this disease that has significant psychosocial sequelae and major health implications.
One model to explain our findings is that mouse follistatin is required in the antral follicle to neutralize atretic effects of activin and allow the follicle to progress to ovulation. This model is based on in vitro evidence that follistatin is able to bind and neutralize activins (8). Activin ßA is able to induce apoptosis in different cell lines including B9 (60) and immortalized ovarian surface epithelium (IOSE-29) (61). Furthermore, when recombinant activin is injected directly into the ovary of immature female rats, in the presence or absence of systemic PMSG, follicles showed morphological signs of atresia in which granulosa and theca cell layers were atrophied and oocytes were highly fragmented (62). In our mice, absence of follistatin may facilitate activin-induced follicle atresia and oocyte loss. Due to excess of activin and continuous loss of oocytes, the oocyte pool is subsequently reduced in 6-month-old experimental mice, leading to a loss of ovarian function. We have shown that activin subunits are highly expressed in the granulosa cells of antral and atretic follicles, and follistatin is highly expressed in the granulosa cells of preantral and antral follicles using in situ hybridization (13). These expression patterns in wild-type mice support the model that follistatin is required in the antral follicle to neutralize activin-induced atresia. We are currently producing and studying activin conditional knockout mice, which will help us to understand better the interrelationships of activins and follistatin in ovarian physiology.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
. Southern blot analysis of granulosa cells was performed with mural granulosa cells isolated from large antral follicles of 3-wk-old control and experimental mice treated with PMSG for 48 h before granulosa cell collection as described in Ref.63 and shown in Fig. 2A.
Genotyping of the Amhr2cre Allele and EIIacre and ROSA 26 Reporter (R26R) Transgene
Genotyping analysis of the Amhr2cre allele and EIIacre and R26R transgene was determined using PCR. The sequences of the primers for the Amhr2cre genotyping were 5'-cgcattgtctgagtaggtgt-3' and 5'-gaaacgcagctcggccagc-3'. The sequences of the primers for the EIIacre genotyping were 5'-ccgggctgccacgaccaa-3' and 5'-ggcgcggcaacaccattttt-3'. The sequences of the primers for the R26R genotyping were 5'-gcgttacccaacttaatcg-3' and 5'-tgtgagcgagtaacaacc-3'.
RNA Isolation and Northern Blot Analysis
Total ovarian RNA was isolated from individual mice by acid guanidium thiocyanate-phenol-chloroform extraction using the RNA STAT-60 reagent (Leedo Medical Laboratories, Houston, TX). Each RNA sample (12 µg) was used for electrophoresis and transferred to nylon membranes as described previously (13). Radioactive complementary cDNA probes were synthesized from the templates listed in Table 5 using [32P]dATP and the Strip-EZ kit (Ambion, Inc., Austin, TX). Autoradiography and phosphor imaging allowed for visualization and quantification of probe hybridization, respectively. Phosphor imaging plates were scanned and analyzed using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). A background level for each blot was determined and subtracted. Blots were stripped and reprobed for glyceraldehyde 3-phosphate dehydrogenase (Gapd), and phosphor imaging of the Gapd signal allowed us to correct each lane for RNA loading.
|
Serum Analysis
Mice were anesthetized by isoflurane inhalation (Abbott Laboratories, North Chicago, IL), and blood was recovered by closed cardiac puncture. Serum was separated by centrifugation in Microtainer tubes (Becton Dickinson, Franklin Lakes, NJ) and stored at -20 C before analysis. FSH, LH, estradiol, and testosterone measurements were made by the University of Virginia Ligand Core Facility (Specialized Cooperative Centers Program in Reproduction Research NICHD/NIH U54 HD28934). Estradiol and testosterone were measured using commercially prepared kits from Diagnostic Systems Laboratories [ULTRA ESTRADIOL (3rd Gen.) DA RIA (DSL-39100), sensitivity, 1.5 pg/ml; and FREE TESTOSTERONE RIA (DSL-4900) sensitivity, 0.25 pg/ml].
Mouse LH Sandwich Assay (MLHS).
LH was measured in serum by a modified supersensitive two-site sandwich immunoassay (1) using monoclonal antibodies MAB1 (no. 581B7) against bovine LH and TMA (no. 5303: Medix, Kauniainen, Finland) against the human LH-ß-subunit. The tracer antibody (no. 518B7 kindly provided by Dr. Janet Roser, Department of Animal Science, University of California, Davis) was iodinated by the chloramine T method and purified on Sephadex G-50 columns. The capture antibody (no. 5303) was biotinylated and immobilized on avidin-coated polystyrene beads (7 mm; Nichols Institute, San Juan Capistrano, CA). Mouse LH reference preparation provided by Dr. A. F. Parlow and the National Hormone and Peptide Program was used as standard. The assay has a sensitivity of 0.07 ng/ml.
Mouse FSH RIA.
Mouse FSH measurements were determined by RIA using reagents provided by Dr. A.F. Parlow and the National Hormone and Peptide Program and procedures validated earlier. Mouse FSH reference preparation was used for assay standards, and mouse FSH antiserum (guinea pig) AFP-1760191, diluted to a final concentration of 1:200,000, was used as a primary antibody. Secondary antibody was purchased from Antibodies, Inc. (catalog no. 51-534) and was diluted to a final concentration of 1:60. The assay has a sensitivity of 4.5 ng/ml and less than 0.5% cross-reactivity with other pituitary hormones.
Superovulation and Isolation of Oocytes/Embryos
Experimental and control female mice (19–21 d-old and 6 months old) were injected ip with PMSG (5 IU/mouse) and given hCG ip (5 IU/mouse) 48 h later. Mice were bred to C57/129 hybrid stud males. The following morning eggs and/or embryos were recovered in M2 medium, counted, and cultured in vitro for 24 h in M16 medium.
Histochemical Analysis
X-galactosidase (X-gal) staining on ovaries was performed by fixing the tissue in 2% paraformaldehyde in 1x PBS, pH 7.2, overnight, rinsing three times for 30 min at 4 C in rinse buffer (2 mM MgCl2; 0.1% sodium deoxycholate; 0.2% Nonidet P-40 in PBS, pH 7.2) and staining overnight at 37 C in X-gal staining solution (5 mM potassium ferricyanide; 5 mM potassium ferrocyanide; X-gal, 1 mg/ml in rinse buffer). After X-gal staining, tissues were washed in three changes of 1x PBS. Tissues were embedded in paraffin, and 5-µm tissue sections were stained with nuclear fast red and analyzed by light microscopy.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Abbreviations: AMH, Anti-Müllerian hormone; Amhr2, anti-Müllerian hormone receptor type II; BMP, bone morphogenetic protein; ES, embryonic stem; Fst, follistatin; ß-gal, ß-galactosidase; hCG, human chorionic gonadotropin; PAS, periodic acid Schiff; PCOS, polycystic ovary syndrome; PMSG, pregnant mare’s serum gonadotropin; POF, premature ovarian failure; X-gal, X-galactosidase; ZP, zona pellucida.
Received for publication July 31, 2003. Accepted for publication December 23, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-Inhibin is a tumor-suppressor gene with gonadal specificity in mice. Nature 360:313–319[CrossRef][Medline]
and ß. Science 286:2328–2331This article has been cited by other articles:
 |
|
 |
|
  B. S. Middlebrook, K. Eldin, X. Li, S. Shivasankaran, and S. A. Pangas Smad1-Smad5 Ovarian Conditional Knockout Mice Develop a Disease Profile Similar to the Juvenile Form of Human Granulosa Cell Tumors Endocrinology, December 1, 2009; 150(12): 5208 - 5217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Edson, A. K. Nagaraja, and M. M. Matzuk The Mammalian Ovary from Genesis to Revelation Endocr. Rev., October 1, 2009; 30(6): 624 - 712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Hernandez Gifford, M. E. Hunzicker-Dunn, and J. H. Nilson Conditional Deletion of Beta-Catenin Mediated by Amhr2cre in Mice Causes Female Infertility Biol Reprod, June 1, 2009; 80(6): 1282 - 1292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ren, R. G. Cowan, R. M. Harman, and S. M. Quirk Dominant Activation of the Hedgehog Signaling Pathway in the Ovary Alters Theca Development and Prevents Ovulation Mol. Endocrinol., May 1, 2009; 23(5): 711 - 723. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Boyer, M. Paquet, M.-N. Lague, L. Hermo, and D. Boerboom Dysregulation of WNT/CTNNB1 and PI3K/AKT signaling in testicular stromal cells causes granulosa cell tumor of the testis Carcinogenesis, May 1, 2009; 30(5): 869 - 878. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Li, S. A. Pangas, C. J. Jorgez, J. M. Graff, M. Weinstein, and M. M. Matzuk Redundant Roles of SMAD2 and SMAD3 in Ovarian Granulosa Cells In Vivo Mol. Cell. Biol., December 1, 2008; 28(23): 7001 - 7011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Hong, L. J. Luense, L. K. McGinnis, W. B. Nothnick, and L. K. Christenson Dicer1 Is Essential for Female Fertility and Normal Development of the Female Reproductive System Endocrinology, December 1, 2008; 149(12): 6207 - 6212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q.-Y. Sun, K. Liu, and K. Kikuchi Oocyte-Specific Knockout: A Novel In Vivo Approach for Studying Gene Functions During Folliculogenesis, Oocyte Maturation, Fertilization, and Embryogenesis Biol Reprod, December 1, 2008; 79(6): 1014 - 1020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-N. Lague, M. Paquet, H.-Y. Fan, M. J. Kaartinen, S. Chu, S. P. Jamin, R. R. Behringer, P. J. Fuller, A. Mitchell, M. Dore, et al. Synergistic effects of Pten loss and WNT/CTNNB1 signaling pathway activation in ovarian granulosa cell tumor development and progression Carcinogenesis, November 1, 2008; 29(11): 2062 - 2072. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Nagaraja, C. Andreu-Vieyra, H. L. Franco, L. Ma, R. Chen, D. Y. Han, H. Zhu, J. E. Agno, P. H. Gunaratne, F. J. DeMayo, et al. Deletion of Dicer in Somatic Cells of the Female Reproductive Tract Causes Sterility Mol. Endocrinol., October 1, 2008; 22(10): 2336 - 2352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Andreu-Vieyra, R. Chen, and M. M. Matzuk Conditional Deletion of the Retinoblastoma (Rb) Gene in Ovarian Granulosa Cells Leads to Premature Ovarian Failure Mol. Endocrinol., September 1, 2008; 22(9): 2141 - 2161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Boyer, L. Hermo, M. Paquet, B. Robaire, and D. Boerboom Seminiferous Tubule Degeneration and Infertility in Mice with Sustained Activation of WNT/CTNNB1 Signaling in Sertoli Cells Biol Reprod, September 1, 2008; 79(3): 475 - 485. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-Y. Fan, M. Shimada, Z. Liu, N. Cahill, N. Noma, Y. Wu, J. Gossen, and J. S. Richards Selective expression of KrasG12D in granulosa cells of the mouse ovary causes defects in follicle development and ovulation Development, June 15, 2008; 135(12): 2127 - 2137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. V. Ingman and R. L. Jones Cytokine knockouts in reproduction: the use of gene ablation to dissect roles of cytokines in reproductive biology Hum. Reprod. Update, March 1, 2008; 14(2): 179 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-Y. Lin, R. G. Craythorn, A. E. O'Connor, M. M. Matzuk, J. E. Girling, J. R. Morrison, and D. M. de Kretser Female Infertility and Disrupted Angiogenesis Are Actions of Specific Follistatin Isoforms Mol. Endocrinol., February 1, 2008; 22(2): 415 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Pangas, X. Li, L. Umans, A. Zwijsen, D. Huylebroeck, C. Gutierrez, D. Wang, J. F. Martin, S. P. Jamin, R. R. Behringer, et al. Conditional Deletion of Smad1 and Smad5 in Somatic Cells of Male and Female Gonads Leads to Metastatic Tumor Development in Mice Mol. Cell. Biol., January 1, 2008; 28(1): 248 - 257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Pangas, C. J. Jorgez, M. Tran, J. Agno, X. Li, C. W. Brown, T. R. Kumar, and M. M. Matzuk Intraovarian Activins Are Required for Female Fertility Mol. Endocrinol., October 1, 2007; 21(10): 2458 - 2471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Andreu-Vieyra, R. Chen, and M. M. Matzuk Effects of Granulosa Cell-Specific Deletion of Rb in Inha-{alpha} Null Female Mice Endocrinology, August 1, 2007; 148(8): 3837 - 3849. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.R. Barnett, C. Schilling, C.R. Greenfeld, D. Tomic, and J.A. Flaws Ovarian follicle development and transgenic mouse models Hum. Reprod. Update, September 1, 2006; 12(5): 537 - 555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Pangas, X. Li, E. J. Robertson, and M. M. Matzuk Premature Luteinization and Cumulus Cell Defects in Ovarian-Specific Smad4 Knockout Mice Mol. Endocrinol., June 1, 2006; 20(6): 1406 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Guigon and S. Magre Contribution of Germ Cells to the Differentiation and Maturation of the Ovary: Insights from Models of Germ Cell Depletion Biol Reprod, March 1, 2006; 74(3): 450 - 458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Boerboom, L. D. White, S. Dalle, J. Courty, and J. S. Richards Dominant-Stable {beta}-Catenin Expression Causes Cell Fate Alterations and Wnt Signaling Antagonist Expression in a Murine Granulosa Cell Tumor Model Cancer Res., February 15, 2006; 66(4): 1964 - 1973. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Moll, C. Millet, D. Noel, B. Orsetti, A. Bardin, D. Katsaros, C. Jorgensen, M. Garcia, C. Theillet, P. Pujol, et al. Chordin is underexpressed in ovarian tumors and reduces tumor cell motility FASEB J, February 1, 2006; 20(2): 240 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Roy and M. M Matzuk Deconstructing mammalian reproduction: using knockouts to define fertility pathways Reproduction, February 1, 2006; 131(2): 207 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Kumar Too Many Follistatins: Racing Inside and Getting Out of the Cell Endocrinology, December 1, 2005; 146(12): 5048 - 5051. [Full Text] [PDF]
|
|
|
|
|
|
X. Ma, A. Reyna, S. K. Mani, M. M. Matzuk, and T. R. Kumar Impaired Male Sexual Behavior in Activin Receptor Type II Knockout Mice Biol Reprod, December 1, 2005; 73(6): 1182 - 1190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Boerboom, M. Paquet, M. Hsieh, J. Liu, S. P. Jamin, R. R. Behringer, J. Sirois, M. M. Taketo, and J. S. Richards Misregulated Wnt/{beta}-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development Cancer Res., October 15, 2005; 65(20): 9206 - 9215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ndiaye, T. Fayad, D. W. Silversides, J. Sirois, and J. G. Lussier Identification of Downregulated Messenger RNAs in Bovine Granulosa Cells of Dominant Follicles Following Stimulation with Human Chorionic Gonadotropin Biol Reprod, August 1, 2005; 73(2): 324 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. McRae, H. M. Johnston, M. Mihm, and P. J. O'Shaughnessy Changes in Mouse Granulosa Cell Gene Expression during Early Luteinization Endocrinology, January 1, 2005; 146(1): 309 - 317. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-J. Lan, X. Xu, and A. J. Cooney Differential Oocyte-Specific Expression of Cre Recombinase Activity in GDF-9-iCre, Zp3cre, and Msx2Cre Transgenic Mice Biol Reprod, November 1, 2004; 71(5): 1469 - 1474. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
