The TGF-β Family in the Reproductive Tract.

in granulosa cells also support this hypothesis, as these mice show dosage-depen- dent fertility defects (Pangas et al. 2007). T emporal regulation of activin A expression appears to be necessary for proper germ-cell dif- ferentiation (Mithraprabhu et al. 2010). Activin A is highly expressed in the testis during the first week of postnatal life; however, as spermatogenic differentiation progresses, the levels of activin A decrease along with high expression of its inhib- itors, follistatin and “BMP and activin mem- brane bound inhibitor” (BAMBI) (Barakat et al. 2012). In an in vivo coculture of spermato- gonia and germ cells from day 4 or day 8 testes,c- kit mRNA expression, which marks germ-cell differentiation, was not affected by activin A on postnatal day 4, but was significantly reduced in germ cells on postnatal day 8, indicating an in- hibitory role of activin A in germ-cell matura- tion. Activin A also plays a role in male embry- onic germ-cell and Sertoli-cell proliferation (Mendis et al. 2011). In Inhba 2/2 testes at E13.5 and E15.5, the number of proliferating Sertoli cells is significantly decreased compared to wild-type mice, likely as a result of impaired cell-cycle machinery activation. Similar defects in Sertoli cell proliferation are seen following conditional deletion ofInhba in fetal Leydig cells of Amhr2 cre/þ;Inhbaflox/- (Archambeault and Y ao 2010). Thus, activin signaling is crucial dur- ing fetal and postnatal testis development, affect- ing proliferation of Sertoli cells during embryonic development and germ-cell differen- tiation during postnatal life. A strikingly different phenotype is seen in male and female mice that lack a functional inhibin a gene (Matzuk et al. 1992). All male and female Inha 2/2 mice develop sex-cord stromal tumors by 4 – 6 wk of age and die within 3 – 4 mo of age (Fig. 6C). Inha 2/2 mice die from a cachexia-like wasting syndrome, likely caused by the high levels of activin produced in lieu of inhibin dimers. The wasting syndrome is modulated by the activin type II receptor, ActRII (Coerver et al. 1996), because mice dou- bly homozygous null for Inha and Acvr2 do not develop cachexia and live longer. Gonadectomy does not rescue the lethality of the Inha 2/2 mutation because these mice subsequently de- velop adrenal tumors (Matzuk and Bradley 1994). However, in males, the gonadal tumor phenotype can be rescued by expression of in- hibin A using a mifepristone-inducible system (Pierson et al. 2000). AMH and its type II receptor AMHRII also have a role in postnatal testis physiology. In the adult gonad, Amh and Amhr2 are expressed in Sertoli cells of the male and the granulosa cells of the female. Male mice defective in Amh or Amhr2 have Leydig cell hyperplasia suggesting that AMH suppresses Leydig cell proliferation (Behringer et al. 1994; Mishina et al. 1996). Male mice derived from genetic crosses between Inha 2/2 and Amh2/2 or Amhr22/2 mice, dis- play Leydig cell tumors by 1 wk of age (Matzuk et al. 1995a), suggesting an interaction between the two pathways in Leydig cell function. In the testis, Bmp8a, Bmp8b, Bmp7, and Bmp4 are expressed in male germ cells, and mice deficient in expression of any of these Figure 5. (Continued) In response, gonadotrope cells produce follicle-stimulating hormone (FSH) and lutein- izing hormone (LH), which control male and female gonadal function. FSH is stimulated by locally expressed activins that signal through the type II receptor, ACVR2 (ActRII). The gonad produces inhibin and estradiol (E2) that repress FSH production. Multiple TGF-b-related proteins are produced in the ovary. (B) Follicle develop- ment proceeds from primordial to antral stages, which culminates in the release of an oocyte during ovulation, which is then available for fertilization. Following ovulation, the remaining cells of the follicle differentiate into the corpus luteum. Follicular blocks seen in various mouse models are indicated. ( C) Conditional inactivation of Bmp2 or Alk2 with Pgr cre/þ results in decidualization defects and female sterility. Conditional inactivation of Bmpr2 with Pgrcre/þ results in female infertility because of intrauterine growth restriction, abnormal spiral artery remodeling, and deficient uNK infiltration. Conditional deletion of nodal in Nodalflox/flox-Pgrcre/þ females results in subfertility as a result of intrauterine growth restriction and placental defects. uNK, Uterine natural killer cells; SA, spiral arteries; F , fetus; T , trophoblasts; L, uterine lumen. D. Monsivais et al. 12 Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from genes have male reproductive phenotypes. Male Bmp8b2/2 mice are infertile because of apo- ptosis of spermatocytes, which eventually re- sults in germ-cell depletion (Zhao et al. 1996). The phenotype of male Bmp8a 2/2 mice is less severe, and about half of adult male Bmp8a2/2 mice show germ-cell degeneration (Zhao et al. 1998). Male Bmp8a 2/2 mice also show degen- eration of the epididymal epithelium, demon- strating a role for BMPs in epididymal function as well (Zhao et al. 1998). The phenotype of male Bmp8a 2/2 mice can be enhanced when one copy ofBmp7 is removed (Zhao et al. 2001). Defects in fertility are also seen in male Bmp4þ/2 mice in a C57BL /6 background; these males have lower fertility because of germ-cell loss, decreased sperm counts, and motility and epididymal defects (Hu et al. 2004). In the somatic cell compartment, there is as yet little genetic evidence for a role of BMPs in adult Sertoli and Leydig cell function. How- ever, mouse Sertoli cells express the BMP type II receptor BMPRII, and the type I receptors ALK- 2, ActRIB /ALK-4, and BMPRIA /ALK-3, en- coded by Acvr1, Acvr1b, and Bmpr1a, respec- tively ( Bmpr1b expression was not examined) Granulosa Cumulus Theca Oocyte SFD PrF AnF G Gdf9 –/– Gdf9 –/– Inha–/–;Gdf9 –/– Fshb –/– Oo Oo Gr Th Acvr2 –/–WT E Oviduct Ovary Uterus Inha –/–WT F Tumor BAC H I Figure 6. Ovarian phenotypes of mice with targeted gene inactivations. ( A) Schematic of the cell types in an antral stage follicle. The central oocyte is surrounded by cumulus cells. A fluid-filled antral cavity separates the cumulus cells from the mural granulosa cells. Outside a basement membrane are the steroidogenic thecal cells. (B) Gross anatomy of a normal female reproductive tract. (C) Ovarian tumors of an adult Inha 2/2 mouse. (D) Histology of a wild-type adult female ovary showing multiple follicular stages inducing primary (PrF), second- ary (SF) and antral (AnF) follicles. (E) Fshb 2/2 ovary arrests before the formation of an antral cavity similar to the Acvr22/2 ovary shown in panel F.( G,H ) Gdf92/2 ovaries have follicles arrested at the primary stage and oocytes that grow abnormally large. (I ) Follicles from Inha2/2;Gfd9 2/2 female mice grow to the multilaminar stage and develop a theca that is absent in Gdf92/2 follicles ( panel H ). Panels are not shown to scale. Oo, Oocyte; Gr, granulosa cells; Th, theca. The TGF-b Family in the Reproductive Tract Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 13 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from (Puglisi et al. 2004), suggesting that these cells are competent to respond to BMP ligands. TGF-b FAMILY SIGNALING IN OV ARIAN FUNCTION During ovary formation, somatic cells, which eventually form the granulosa cells, surround oocytes to form nongrowing primordial ovari- an follicles. These “pregranulosa cells” are de- rived in part from cells that ingress from the coelomic epithelium in multiple waves that be- gin in late gestation and cease a few days after birth (Mork et al. 2011). In the mouse, primor- dial follicle formation begins just before birth such that newborn mouse ovaries contain most- ly germ-cell cysts and a few primordial follicles. Subsequent folliculogenesis coordinates oocyte development, granulosa cell proliferation, and thecal cell steroidogenesis (Fig. 6A). Initially, these events appear to be orchestrated primarily by the oocytes (Eppig et al. 2002; Matzuk et al. 2002), although subsequent development is likely bidirectional. T wo key signaling proteins are growth and differentiation factor (GDF)-9 and BMP-15 oocyte-specific members of the TGF-b family (McGrath et al. 1995; Dong et al. 1996; Dube et al. 1998; Elvin et al. 2000). GDF-9 and BMP-15 are essential for fertility in many animals including mice, humans, and sheep (Dong et al. 1996; Galloway et al. 2000; Wilson et al. 2001; Juengel et al. 2002; Di Pas- quale et al. 2004). In mice, Gdf9 and Bmp15 are coexpressed during folliculogenesis starting ap- proximately at the newborn stage and until the late antral stage in adults (Dube et al. 1998; Elvin et al. 1999b; Rajkovic et al. 2004). Female Gdf9 2/2 mice have follicles arrested at the pri- mary stage with a single layer of granulosa cells, no associated thecal cell layer and defects in oocyte meiotic competence and growth (Fig. 6G,H) (Dong et al. 1996; Carabatsos et al. 1998). Granulosa cells from Gdf9 2/2 ovaries show increased expression of a number of genes, in- cluding the gene encoding inhibina (Elvin et al. 1999b). Excess inhibin is partly responsible for follicular arrest, and lack of inhibina expression in Gdf9 2/2 mice, in Inha2/2;Gdf92/2 mice allows follicles to progress to the multilaminar stage, although the follicles are abnormal (Fig. 6I) (Wu et al. 2004; Myers et al. 2013). The ovaries of Inha2/2;Gdf92/2 mice contain ad- ditional defects not found in Gdf92/2 mice. For example, a distinct theca layer forms in dou- ble mutant mice but does not become function- al (i.e., steroidogenic). This is not understood, but may relate to the loss of LH receptor expres- sion in thecal cells (Wu et al. 2004). Interesting- ly, Inha 2/2 ovaries show precocious follicle de- velopment, with overgrowth of granulosa cells, more follicles in the growth phase, and an in- crease in activin expression (Myers et al. 2009, 2013; Nagaraja et al. 2010). Deletion of theGdf9 gene in an Inha 2/2 genetic background partly rescues some of the follicle growth at the initial stages of Inha 2/2 follicle development, per- haps in part because of decreased levels of acti- vin B in the double mutant ovaries. However, at later follicle stages, the double mutant ovaries look similar to Inha2/2 mutant ovaries, with high levels of activin and suppressed levels of kit ligand, which promote granulosa cell prolifera- tion although suppressing oocyte growth. These data suggest that GDF-9 and activins have se- quential and partly nonredundant roles in fol- liculogenesis (Myers et al. 2013). GDF-9 and BMP-15 play important roles in cumulus cells (i.e., the cells directly adjacent to the oocyte), during ovulation. During ovula- tion, cumulus cells become embedded in a hya- luronan-rich matrix that binds them to the oo- cyte. Extracellular matrix synthesis is critical for proper ovulation and fertilization (Hizaki et al. 1999; V arani et al. 2002; Fulop et al. 2003) and oocytes are known to induce cumulus expan- sion in the presence of FSH (Buccione et al. 1990). Through analysis in cell culture, mouse GDF-9 has been shown to play a key role in this process by inducing the expression of genes nec- essary for cumulus expansion including those encoding hyaluronan synthase 2 ( Has2), pros- taglandin-endoperoxide synthase 2 ( Ptgs2, Cox2), pentraxin 3 ( Ptx3), and tumor necrosis factor a-induced protein 6 ( Tnfaip6), while re- pressing others, such as those encoding lutein- izing hormone /choriogonadotropin receptor (Lhcgr) and urokinase-type plasminogen acti- D. Monsivais et al. 14 Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from vator ( Plau) (Elvin et al. 1999a; Matzuk 2000; V arani et al. 2002). The phenotypes of double crosses of Gdf9 and Bmp15 mutant mice, in comparison with the single gene deficiencies, indicate an interaction of the two proteins during late stages of follicle development and ovulation. Although Bmp152/2 females are subfertile, female Gdf9þ/-;Bmp152/2 mice are infertile in a 129 /SvEv inbred background, and subfertile in a mixed hybrid background (C57Bl/6/129/SvEv) (Y an et al. 2001). Eggs of Gdf9 þ/-;Bmp152/2 mice lack associated cu- mulus cells, both within follicles and in ovi- ducts, suggesting that the cumulus cell-oocyte communication has been disrupted, and that cumulus cells no longer produce a stable extra- cellular matrix during ovulation. Additional oocyte and cumulus cell defects have been found in Gdf9 þ/-;Bmp152/2 mice. The ability of cumulus cells to undergo expan- sion can be tested using a cell culture assay: cumulus-oocyte complexes that have the oocyte removed (oocytectomized; OOX) undergo ex- pansion when cocultured with fully grown oo- cytes in the presence of FSH (Buccione et al. 1990; V anderhyden et al. 1992). Wild-type oo- cytes expand wild-type OOX complexes, but oocytes from Gdf9 þ/-;Bmp152/2 mice cannot expand wild-type OOX complexes to the same degree (Su et al. 2004). Wild-type oocytes also have a reduced ability to expand Gdf9þ/-; Bmp152/2 OOX complexes. Because the addi- tion of wild-type oocytes cannot rescue the de- fect in mutant cumulus cells, the problem is likely intrinsic to the cumulus cells. These data support the hypothesis that GDF-9 and BMP- 15 are necessary for proper development of cu- mulus cells throughout many stages of follicu- logenesis (Su et al. 2004). In sheep, homozygous or heterozygous mu- tations in the BMP15 gene result in sterile (ho- mozygous) or superfertile (heterozygous) fe- males (Davis et al. 1991; Galloway et al. 2000). Additionally, mutations in GDF9 and ALK6/ BMPR1B, which encode a type I receptor for BMP-15, also affect fertility in sheep (Otsuka et al. 2011). GDF-9 and BMP-15 are known to cooperate and heterodimerize, although the functional roles of the GDF-9:BMP-15 hetero- dimers have been poorly defined (Liao et al. 2003). BMP heterodimers were first isolated from bovine bone and shown to potently induce bone formation in vivo (W ang et al. 1988). Compared to BMP homodimers, BMP hetero- dimers show enhanced activity (Guo and Wu 2012). Accordingly, GDF-9:BMP-15 hetero- dimers also show enhanced bioactivity (Peng et al. 2013a). Studies using recombinant human and mouse GDF-9:BMP-15 heterodimers show that, relative to their respective homodimers, the heterodimer potently induces cumulus cell expansion. This is also reflected in the striking increases of the cumulus cell expansion genes Ptx3, Has2, and Ptgs2. Furthermore, these stud- ies point to species-specific differences in the bioactivity of these ligands; mouse GDF-9 ho- modimers strongly activate cumulus cell expan- sion, although human GDF-9 homodimers are inactive. Similarly, mouse BMP-15 homo- dimers are inactive when compared to mouse GDF-9 homodimers, although human BMP-15 homodimers have low bioactivity (Peng et al. 2013b). These results show that the heterodimer conformation of these key oocyte-secreted fac- tors may be a potent modulator of ovarian func- tion in mice and humans. Currently, a recom- binantly produced heterodimer of human BMP-15 and GDF-9 termed “cumulin, ” is being tested to boost success rates of some artificial reproductive technologies such as in vitro mat- uration of oocytes (Mottershead et al. 2015). Few mutations causal for infertility in hu- mans have been identified. BMP15, located on the X chromosome, is a candidate gene for fe- male infertility. A human mutation in BMP15 from two sisters causes hypergonadotropic ovarian failure and ovarian dysgenesis (Di Pas- quale et al. 2004). Loss of function mutations in NOBOX, a gene encoding a key transcription factor that regulates GDF9 expression (Rajkovic et al. 2004), are found at high frequency in women with primary ovarian insufficiency, also called premature ovarian failure (Bouilly et al. 2011), which affects approximately 1% of women under the age of 40 and is associated with infertility. Because of their role in PGC function and gonadal development, further ex- amination of members of the BMP family will The TGF-b Family in the Reproductive Tract Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 15 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from be necessary to establish any pathophysiology associated with improper germ-cell develop- ment in humans. Many other BMP ligands are expressed in the ovary during folliculogenesis, and there is much to be learned about the in vivo contribu- tions of the BMP system to female fertility. Both Bmp4 and Bmp7 are expressed in thecal cells, but homozygous null mutations in either gene in mice results in embryonic or postnatal lethal- ity, respectively, and these have yet to be ana- lyzed by conditional mouse models in the ovary. Inactivation of Bmp6, expressed in the oocyte, does not result in an overt reproductive pheno- type (Solloway et al. 1998). Instead, Bmp6 2/2 female mice have subtle fertility defects, includ- ing a 22% decrease in litter size and reduced numbers of ovulated oocytes with decreased developmental competence (Sugiura et al. 2010). Bmp2 has been reported to be expressed in rat granulosa cells (Erickson and Shimasaki 2003), but Bmp2 2/2 embryos die at E7.5 – E9.0 (Zhang and Bradley 1996), leaving questions unanswered regarding its specific reproductive function in granulosa cells. AMH plays a role in follicle development in the adult female. Female mice that lack AMH expression are fertile with normal litter sizes, and show premature infertility (Durlinger et al. 1999). Ovaries from adult Amh 2/2 mice show an approximately threefold greater num- ber of small growing follicles and concomitant loss of primordial follicles compared to wild- type littermates (Durlinger et al. 1999). Com- bined with cell culture evidence that recombi- nant AMH inhibits primordial follicle growth (Durlinger et al. 2002), it has been suggested that AMH plays an important role in the re- cruitment phase of folliculogenesis (Visser et al. 2006). This has also led to the proposal that AMH levels might be useful markers of ovarian reserve in humans during aging or dis- ease (van Rooij et al. 2002; Visser et al. 2006). In a study of female patients undergoing in vitro fertilization, serum AMH levels correlate highly with number of antral follicles and oocytes re- trieved, and reduced serum AMH is associated with a poor response during in vitro fertiliza- tion (van Rooij et al. 2002). In other studies, serum levels of AMH predict menopause (van Rooij et al. 2004, 2005). Detailed studies in mice show that, although AMH levels do not change in individual growing follicles of aging mice, serum AMH levels decline with age (Kevenaar et al. 2006). In addition, in mice, there is a cor- relation between growing follicles and numbers of primordial follicles, as well as a strong corre- lation between primordial follicles and serum AMH levels (Kevenaar et al. 2006). Thus, the use of serum AMH holds significant promise for monitoring fertility during the female re- productive lifespan (Visser et al. 2006, 2012; Anderson et al. 2012). The inhibin-activin system in female mice is critical for both follicle development and regu- lation of the estrous cycle (Fig. 5). FSH is a dimeric protein consisting of an a and b sub- unit, and FSH is required for antral follicle de- velopment in female mice (Kumar et al. 1999; Burns and Matzuk 2002). Pituitary-derived activins regulate the expression of the FSH b subunit ( Fshb), thereby controlling FSH pro- duction (Carroll et al. 1989, 1991; Bilezikjian et al. 2004). The phenotypes of Fshb 2/2 and Acvr22/2 female mice are very similar; both mouse models have impaired follicle develop- ment before the full development of the antral cavity (Fig. 6). In addition, the follicular block in both Fshb 2/2 and Acvr22/2 female mice can be rescued by treatment with exogenous gonadotropins, suggesting that lack of FSH is the principal cause of the phenotype. Mice con- ditionally null for Smad4 in gonadotrope cells (i.e., the FSH- and LH-producing cells in the anterior pituitary), are hypogonadal and FSH- deficient, although subfertile (Fortin et al. 2014a). However, Smad4 deletion in combina- tion with deletion of Foxl2, which encodes a transcription factor known to regulateFshb (Vi- dal et al. 1998; Corpuz et al. 2010; Lamba et al. 2010), results in sterility with no FSH produc- tion (Fortin et al. 2014b). These mice highlight the importance of TGF-b family signaling (like- ly activin) in FSH synthesis. Activin and TGF- b signal through Smad2 and Smad3. Smad2 2/2 embryos fail to gastru- late, have defective mesoderm formation and anteroposterior axis defects, and die at embry- D. Monsivais et al. 16 Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from onic day E6.5 – E8.5 (Nomura and Li 1998; W al- drip et al. 1998). Mice engineered to express either a splice variant of Smad2 that lacks exons 3, or engineered to replace the Smad2 coding sequence with Smad3, are viable and fertile, suggesting that the short form of Smad2, or Smad3, can regulate all essential Smad2 func- tions (Dunn et al. 2005). By comparison, Smad3 2/2 mice are viable (Zhu et al. 1998; Y ang et al. 1999b), but a targeted allele that re- moves exon 8 (encoding the carboxy-terminal phosphorylation site) has reproductive defects (T omic et al. 2002, 2004). Female Smad32/2 mice have reduced fertility, reduced numbers of large pre-antral and antral follicles, and a larger primordial follicle pool during adult- hood (T omic et al. 2002). Serum analysis in these mice shows high levels of FSH and low levels of estradiol, and, in ovaries, reduced ex- pression of inhibin a, cyclin D2, estrogen recep- tor-b but not of the FSH receptor (T omic et al. 2004). The low estradiol level and decreased in- hibin expression are consistent with the loss of antral follicles and the subsequent lack of neg- ative feedback control in the pituitary. However, Smad2 and Smad3 have been shown to com- pensate in mouse knockout models, and are also important for pituitary function. Thus, there was a need to generate additional condi- tional mutants using loxP-flanked alleles and Amhr2 cre/þ to address these issues (see next par- agraph on conditional mutations). Single con- ditional deletion of Smad2 or Smad3 in granu- losa cells results in normal fertility of female mice (Li et al. 2008). In contrast, combined de- letion of Smad2 and Smad3 in granulosa cells

in reproductive defects. These mice show disrupted follicle development, increased follic- ular atresia, reduced ovulation, and attenuated cumulus cell expansion, consistent with their function in activin and GDF-9 signaling (Li et al. 2008). T o circumvent the embryonic or perinatal lethality of homozygous null mutants, many researchers conditionally inactivate genes using the cre/loxP recombination system (Sauer and Henderson 1989). Several mouse lines express Cre recombinase in various cell types in repro- ductive tissues (T able 2). In female reproduc- tion, the Amhr2 cre/þ mouse is useful to direct recombination of floxed alleles in granulosa cells of the postnatal ovary (Jorgez et al. 2004). Amhr2cre/þ mice have been used to gen- erate mice with a conditional allele for Bmpr1a (Alk3) (see above), Smad4 (see below) or Fst. Conditional inactivation of theFst gene in gran- ulosa cells ( Fst cK O) results in decreased fertil- ity, increased levels of FSH and LH, and reduced testosterone (Jorgez et al. 2004). In many ways, the phenotype of adult female mice with a con- ditionally inactivated Fst gene is reminiscent of premature ovarian failure in women. Premature ovarian failure is characterized by amenorrhea, infertility, and elevated gonadotropins in wom- en before the age of 40. Its etiology is unknown but is likely heterogeneous. Therefore, these mice may be seen as a model to study premature ovarian failure. The mechanism of their rapid follicular loss is unknown but possibly related to unregulated activin activity in early follicular recruitment and growth (Jorgez et al. 2004). The roles of the TGF- b family in follicle development have also been analyzed by a con- ditional inactivation of Smad4 in the ovary (Pangas et al. 2006). These mice are initially subfertile, but are infertile at 4 – 6 mo of age. Folliculogenesis is disrupted because granulosa cells in growing follicles undergo precocious differentiation (i.e., luteinization), in response to stimulation by pituitary gonadotropins. In addition, severe cumulus cell defects occur dur- ing folliculogenesis and cumulus expansion, confirming the critical role of the TGF-b family in the function of these cells. SMAD4 is a tumor suppressor gene in humans (Hahn et al. 1996), and mice with conditionally inactivated Smad4 in the epidermis and mammary gland develop carcinomas (Li et al. 2003; Y ang et al. 2005; Qiao et al. 2006). However, no tumor development is seen as a result of lack of ovarian Smad4 expres- sion, and conditional loss of Smad4 results in a phenotype that is very similar to the phenotype resulting from inactivation of both Smad2 and Smad3, which also results in premature granu- losa cell luteinization (Li et al. 2008). Possibly, granulosa cell tumor formation is prevented by the premature luteinization inSmad4 2/2 gran- ulosa cells (Pangas 2012a). The TGF-b Family in the Reproductive Tract Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 17 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from Although mice with inactivation of Smad4 or inactivation of Smad2 and Smad3 in ovarian granulosa cells show similarities in their phe- notype, inactivation of genes encoding Smad1 and Smad5, which transduce signals for AMH and BMPs, results in a phenotype that is completely different. Smad1 and Smad5 are coexpressed in mouse granulosa cells, and de- letion of both genes is required to produce a phenotype in granulosa cells (Pangas et al. 2008). Single deletion of either Smad1 or Smad5 in granulosa cells has no effect on fe- male reproductive function, suggesting func- tionally redundant activities in particular cell types (Pangas et al. 2008). Functional redun- dancy between Smad1 and Smad5 may also occur during Mu¨llerian duct regression in the mouse (Orvis et al. 2008). Combined deletion of Smad1 and Smad5 using the Amhr2 cre/þ allele causes development of granulosa cell tu- mors in females or Sertoli – Leydig cell tumors in males in 100% of the mice (Pangas et al. 2008). These tumors develop around 8 wk of age and progress to metastatic tumors at older ages leading to decreased survival (Middle- brook et al. 2009). Granulosa cell tumor devel- opment is also seen in granulosa cell-specific deletion of the genes encoding the BMP type I receptors, Bmpr1a and Bmpr1b (Edson et al. 2010), suggesting that loss of the BMP-activat- ed Smad signaling is likely the cause of the tumors. These tumors phenocopy the juvenile form of granulosa cell tumors in humans based on histological, molecular, and hormonal Table 2. Mouse lines expressing Cre recombinase for analyses of reproductive function Cre Line Promoter Type Tissue References Amh-Cre Anti-Mu¨llerian hormone T ransgene Sertoli cells (male), granulosa cells (female) Le´cureuil et al. 2002 Amhr2-Cre Anti-Mu¨llerian hormone receptor 2 Knock-in Granulosa cells (female), Leydig cells (male), Mu¨llerian duct Jamin et al. 2002; Jorgez et al. 2004 Alpha GSU-Cre a Subunit glycoprotein hormone T ransgene Gonadotropes, thyrotropes of anterior pituitary Cushman et al. 2000 Cyp19-cre Aromatase T ransgene Granulosa cells (antral follicles) Fan et al. 2008 Gdf9-iCre Growth differentiation factor-9 T ransgene Oocytes Lan et al. 2004 GnRH-iCre Gonadotropin- releasing hormone T ransgene GnRH neurons Shimshek et al. 2002 GnRHR-cre Gonadotropin- releasing hormone receptor Knock-in Gonadotropes of the anterior pituitary W en et al. 2008 Inha-Cre Inhibin a T ransgene Sertoli, Leydig cells (male), granulosa, theca (female) Jorgez et al. 2006 Pgr-Cre Progesterone receptor Knock-in Anterior pituitary gland, uterus, oviduct, ovary, mammary gland Soyal et al. 2005 Prm1-Cre Protamine-1 T ransgene Spermatogenic cells O’Gorman et al. 1997 PrP-Cre-ER T Prion protein (inducible) T ransgene Spermatogonia, spermatocytes W eber et al. 2003 Sycp1-Cre Synaptonemal complex protein-1 T ransgene Spermatogonia Vidal et al. 1998 Tnap-cre Tissue-nonspecific alkaline phosphatase Knock-in Primordial germ cells Kehler et al. 2004 Zp-Cre Zona pellucida 3 T ransgene Oocytes Lewandoski et al. 1997; de Vries et al. 2000 D. Monsivais et al. 18 Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from characteristics (Middlebrook et al. 2009). One characteristic shared by the Smad12/2; Smad52/2 granulosa cell tumors and primary samples of human juvenile granulosa cell tu- mors, is the activation of Smad2 and Smad3 signaling. Although the role of active Smad2 and Smad3 signaling in granulosa cell tumors has yet to be determined, a number of mouse mutants suggest that in granulosa cells, Smad2 and Smad3 signaling promotes cell proliferation and tumor growth. For instance, loss of activin or loss of Smad2 and Smad3 in the somatic cells of the follicle results in infertility, but not tumor formation (Pangas et al. 2007; Li et al. 2008). In contrast, loss of inhibin expression causes sex- cord stromal tumors producing supraphysio- logical levels of activin (Matzuk et al. 1992, 1994; Li et al. 2007a,b). Combined with the phe- notypes of granulosa cell Smad mutants, it is likely that signaling by Smad1 and /or Smad5 in response to BMPs or AMH, and Smad2 and/or Smad3 in response to TGF-b or activin, controls the balance of cell proliferation and differentiation in granulosa cells. Disruption of this balance can lead to either formation of ovarian tumors (i.e., in the Inha 2/2 or condi- tional Smad12/2;Smad52/2 mice), or infertil- ity (i.e., afterSmad2 and Smad3as well asSmad4 conditional inactivation). In support of this hypothesis, triple conditional inactivation of Smad1, Smad5, and Smad4, which should addi- tionally inhibit all Smad4-dependent signaling within the Smad1 and Smad5 granulosa cell tumors, remarkably slows tumor growth and metastasis development (Mansouri-Attia et al. 2014). TGF-b FAMILY SIGNALING IN THE UTERUS AND PREGNANCY The endometrial layer of the uterus is continu- ously remodeled throughout the menstrual cy- cle in response to ovarian hormones and various growth factors to coordinate embryo implanta- tion (Fig. 5C) (Cha et al. 2012). Estradiol (E 2) induces the proliferative phase of the endome- trium, while progesterone (P 4) and several growth factors stimulate endometrial stromal cell decidualization during the secretory phase of the cycle (Cha et al. 2012). Decidualization, a process of stromal cell differentiation, prepares the uterus for pregnancy by transforming the stromal cells of the endometrium into secretory cells that nurture the embryo during early preg- nancy (Ramathal et al. 2010). Ultimately, the endometrium regresses through menstruation in the absence of pregnancy, or continues de- cidualizing if pregnancy occurs. In the mouse, decidualization is also coordinated by growth factors and steroid hormones, but occurs only in response to an implanting embryo (Ramathal et al. 2010). In mice, artificial decidualization can be induced with a mechanical stimulus to the uterine wall followed by estrogen and pro- gesterone treatments that mimic pregnancy (Fig. 7B,C) (Lydon et al. 1995). Both mice and humans establish hemochorial placentation, where the trophoblasts are in direct contact with the maternal blood, making the mouse a suitable model to study pregnancy (Schlafke and Enders 1975; Carson et al. 2000). V arious components of TGF- b family sig- naling pathway are expressed in the uterus, and their expression changes throughout pregnancy. TGF-b ligands and their receptors are expressed at the fetal – maternal interface (T amada et al. 1990; Selick et al. 1994; Godkin and Dore 1998). Tgfb2 mRNA is expressed in the luminal and glandular epithelium during the mouse peri-implantation period, and in decidual cells following implantation (Das et al. 1992). In mice, Tgfb3 transcripts are detected in myome- trial cells and in the vascular smooth muscle cells during the peri- and postimplantation pe- riods (Das et al. 1992). Furthermore, E 2 or di- ethylbesterol (DES) treatments increase the ex- pression of uterine Tgfb1, Tgfb2 , and Tgfb3 (Das et al. 1992; T akahashi et al. 1994). Similar dynamic expression patterns of TGF- b1, - b2, and - b3 are observed in the human uterus. TGFB1, TGFB2, and TGFB3 mRNAs are detect- ed in the three cell types of the uterus, with maximal expression observed in the luminal and glandular epithelium during the late prolif- erative to early secretory phase, suggesting pro- gesterone-mediated regulation (Chegini et al. 1994). TGFB1, TGFB2, and TGFB3 expression is also dynamically expressed during pregnancy, The TGF-b Family in the Reproductive Tract Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 19 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from and TGF-b is localized to cytotrophoblasts, in- termediate trophoblasts, and villious cytotro- phoblasts, as well as in the stroma and glands of the maternal decidua (Selick et al. 1994). An increasing number of mouse models have examined the in vivo contribution of TGF-b signaling in the female reproductive tract using conditional gene deletion strategies (T able 2) to overcome the embryonic lethality of global mutations. T ypically, these models use the deleter strain,Amhr2 cre/þ, to target the uter- ine myometrium and stroma (i.e., mesenchymal cells originating from the Mu ¨llerian duct), or they use the progesterone receptor cre (Pgrcre/þ) to target the uterine endometrium. Female mice with conditional deletion of Tgfbr1 (Alk5), thus lacking the expression of TGF-b type I receptor, or ALK-5, generated using Amhr2 cre/þ are ster- ile. The primary defect appears to be in the ovi- duct and uterine myometrium. Mutant ovi- ducts develop oviductal diverticula, which could impede sperm transport and /or capaci- tation, and reduce transit of embryos through the oviduct (Li et al. 2011). These mutant mice also show defective smooth muscle develop- ment in the uterus, with uterine cyst formation and abnormal masses of tissue forming at 8 months of age. Thus, Tgbr1 has a key role in the development of the muscular architecture of the oviduct and uterus (Li et al. 2011; Gao et al. 2014, 2015). Tgfbr1 has also been conditionally inacti- vated using Pgr cre/þ to target cells in the en- dometrial compartments of the uterus (Peng et al. 2015b). Conditional deletion of Tgfbr1 with Pgr cre/þ results in subfertility because of defects throughout gestation, including abnor- mal embryo implantation, decreased uterine natural killer (uNK) cell infiltration, defective uterine spiral artery remodeling, and placental p p Type 1 Type 2 ALK-2 BMPR2 ACVR2A ACVR2B BMPsA BC DE Smad4 Smad4 CEBPB Smad1/5/8 Smad1/5/8 Smad1/5/8 Endometrial decidualization E8.5 E8.5 ALK2 ckO dh DecidualizedControl Control uh Progesterone receptor p p p p Figure 7. Bone morphogenetic protein (BMP) signaling via ALK-2 promotes endometrial stromal cell decidu- alization in the mouse and human uterus. (A) BMPs signal through the BMP type I receptor, ALK-2 and activate Smad1, Smad5 or Smad8 to increase CEBPB expression. C/EBPb then increases Pgr ( progesterone receptor) expression, which is necessary for endometrial stromal cell decidualization. ( B,C ) Gross anatomy of a normal uterus and a uterus after an artificial decidual stimulus. Decidualization is assessed by increased uterine weight and histologically by the presence of proliferative and decidual cells. Following the induction of an artificial decidual stimulus that mimics embryo implantation, the stromal cells of a healthy uterus proliferate and decidualize into specialized secretory cells. ( D,E) Conditional deletion of Acvr1/Alk2 or Bmpr2 with Pgrcre/þ

in mouse sterility. E8.5 uteri of control and Alk2flox/flox-Pgrcre/þ mice. Alk2flox/flox-Pgrcre/þ females show postimplantation and decidualization defects. uh, Undecidualized horn; dh, decidualized horn. D. Monsivais et al. 20 Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from defects (Peng et al. 2015b). Thus, observations from these two different mouse models (Tgfbr1 flox/bgal-Amhr2cre/þ and Tgfbr1 flox/bgal- Pgrcre/þ) show the unique roles of Tgfbr1 in the different cell types of the female reproduc- tive tract. Nodal signaling is important during the process of embryo implantation and through- out pregnancy in the mouse (Park and Dufort 2011). Conditional inactivation of Nodal in progesterone receptor– expressing tissues (with Pgr cre/þ) results in subfertility and postimplan- tation defects, such as intrauterine growth restriction (Park et al. 2012). Placental defects include abnormal giant cell morphology that extends into the mesometrial and conceptus sites. Additionally, females with conditional in- activation of Nodal have a defective and hemor- rhagic decidua basalis (i.e., the endometrial cells that will become the maternal part of the placental) at day 10.5 of pregnancy, with de- creased proliferation and increased apoptosis (Park et al. 2012). Recent studies suggest that uterine Acvr1b (encoding the activin A receptor, type IB or ALK-4) is the nodal receptor throughout gestation (Peng et al. 2015a). Fe- male mice with conditional inactivation of Acvr1b with Pgr cre/þ are subfertile and show defects during gestation that include intrauter- ine growth restriction and placental abnormal- ities (Peng et al. 2015a). Reminiscent of the Nodal flox/flox-Pgrcre/þ females, mice with condi- tional Acvr1b deletion display abnormal tro- phoblast giant cell expansion and decreased placental spongiotrophoblast and labyrinth lay- ers. These studies show the interplay between nodal and its receptor, ALK-4, in placental de- velopment and throughout gestation. Activin signaling also uses ALK-4, but the role of activin in the uterus is unclear. No genes have been conditionally inactivated to specifi- cally study activin function in vivo. Activin ex- pression is detected in primary human placen- tal cells, and activin stimulates the production of gonadotropin-releasing hormone (GnRH), progesterone, and human chorionic gonado- tropin (hCG) (Petraglia et al. 1989; Debieve et al. 2000). Activin A expression increases in the serum and placentas of preeclamptic wom- en, and is associated with increased apoptosis at the maternal– fetal interface (T able 3) (Yu et al. 2012). In addition, genome-wide association studies indicate linkage to chromosome 2q22 in families affected with preeclampsia or eclamp- sia, and point to AC VR 2A(AC VR 2) as the can- didate susceptibility gene (T able 3) (Moses et al. 2006; Fitzpatrick et al. 2009; Roten et al. 2009). Several mouse models have been generated to examine BMP signaling in the uterus using Pgr cre/þ, including Bmp2, the type II receptor Bmpr2, and the type I receptors Acvr1 and Bmpr1a, as well as a global knockout of Bmpr1b (T able 1). These mouse models show critical BMP roles at multiple stages, including implan- tation, decidualization, and placentation. Con- ditional inactivation of Bmp2 using Pgr cre/þ re- sulted in the first model for BMP signaling in the uterus (Lee et al. 2007). Female mice with conditional Bmp2 inactivation were infertile, with impaired uterine decidualization (Lee et al. 2007). In contrast, conditional ablation of the BMP type II receptor, Bmpr2 with Pgr cre/þ shows defects in the later stages of ges- tation (Nagashima et al. 2013). These midges- tation defects include defective spiral artery re- modeling and placental hemorrhage, which leads to restricted embryo growth and fetal death. Females with conditional ablation of Bmpr2 are sterile (Nagashima et al. 2013). Con- ditional deletion of Acvr1 (the gene encoding ALK-2) using Pgr cre/þ shares some similarity with the Bmp2-Pgrcre/þ model, with defects in implantation and decidualization (Clementi et al. 2013). The defects may be due in part to loss of induction of the gene encoding the key transcription factor, CCAA T /enhancer– bind- ing protein, b (CEBPB) by Smad1 and Smad5 (Clementi et al. 2013). CEBPb activates proges- terone receptor expression, and both are re- quired for endometrial cell decidualization (Fig. 7) (Mantena et al. 2006). The earliest pregnancy phenotype for mouse models of the BMP signaling pathway was discovered through conditional deletion of the BMP type I receptor Bmpr1a (ALK3) us- ing Pgr cre/þ (Monsivais et al. 2015). Female mice with conditional Bmpr1a inactivation are sterile with a nonreceptive endometrium char- The TGF-b Family in the Reproductive Tract Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 21 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from acterized by elevated microvilli density in the luminal uterine epithelium, enhanced response to estrogen, and decreased sensitivity to proges- terone. Furthermore, mice with Bmpr1a condi- tional inactivation showed an integration of BMP- and progesterone-mediated signaling at the promoter of Klf15 (Monsivais et al. 2015). KLF15 is a critical transcription factor during the window on implantation that programs the luminal epithelium of the endometrium into a state receptive for embryo implantation (Ray and Pollard 2012). Mouse models for canonical Smad signaling have only been examined in the reproductive tract using Amhr2 cre/þ to conditionally inacti- vate the Smad gene of interest. Many of these models do not appear to show reproductive tract defects, including single conditional dele- tion of Smad1, Smad2, Smad3, Smad4, and Smad5 (Pangas et al. 2006, 2008; Li et al. 2008). Surprisingly, triple conditional deletion of Smad1, Smad5, and Smad4 using Amhr2 cre/þ not only affects granulosa cell tumor develop- ment (see previous section), but also causes ste- rility because of defects in the development of the oviduct and uterus (Rodriguez et al. 2016). These mice have abnormal smooth muscle de- velopment with the oviduct that may disrupt the transport of embryos or sperm (Rodriguez et al. 2016). These mice also show defects within myometrium and endometrium, resulting in pregnancy loss by midgestation (Rodriguez et al. 2016). Thus, inactivation of ligands, recep- tors and the downstream effectors of the BMP signaling pathway show its fundamental role in the development and function of the female reproductive system. TGF-b FAMILY SIGNALING AND FEMALE REPRODUCTIVE TRACT DISEASES Endometriosis is an estrogen-dependent gyne- cological disease that affects about 10% of wom- en of reproductive age, and is characterized by extrauterine growth of endometrial tissues (Giudice and Kao 2004; Bulun 2009). Endome- triosis is associated with chronic pelvic pain and infertility and has only a few effective thera- peutic options (V alle and Sciarra 2003; Giudice 2010). Although most therapies include sup- pression of ovarian hormones using oral contraceptives, GnRH-analogs, or aromatase inhibitors as nonhormonal interventions are desirable (V alle and Sciarra 2003). Several stud- Table 3. Alterations in the TGF- b family signaling pathways in female reproductive diseases Gene Disease Description References ACVR2A Preeclampsia GW AS identifies ACVR2A as the candidate susceptibility gene in families affected with preeclampsia Moses et al. 2006; Fitzpatrick et al. 2009; Roten et al. 2009 BMP6 Endometriosis BMP-6 is expressed in ectopic endometriotic lesions Athanasios et al. 2012 BMPR1B Endometriosis Deactivating polymorphism in miR-125b enhances BMPR1B production in women with endometriosis Chang et al. 2013 INHBA Preeclampsia W omen with preeclampsia have increased serum and placental levels of activin A Yu et al. 2012 LEFTY Infertility Lefty is overexpressed in the endometrium of women with unexplained infertility T abibzadeh et al. 2000 TGFB1 Endometriosis W omen with endometriosis have elevated peritoneal fluid concentrations of TGFB1 Oosterlynck et al. 1994; Kupker et al. 1998; Sotnikova et al. 2010 TGFB3 Uterine fibroids TGFB3 mRNA is increased in uterine fibroids Arici and Sozen 2000 TGFB1, TGFB2, TGFB3 Uterine fibroids T reatment with leuprolide acetate decreases TGFB1, TGFB2, and TGFB3 mRNA in uterine fibroids Dou et al. 1996 D. Monsivais et al. 22 Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from ies indicate a correlation between TGF-b signal- ing and progression of endometriosis. Com- pared to healthy women, TGF- b levels are ele- vated in the peritoneal fluid of women with endometriosis (Oosterlynck et al. 1994; Kupker et al. 1998); elevated TGF- b induces fibrosis, increasing the formation of endometriotic ad- hesions in the peritoneum (Chegini 2008). TGFB2 mRNA levels are increased in the endo- metriotic lesions of women and in a rat model of endometriosis (Sotnikova et al. 2010). Further evidence shows that TGF- b signaling increases endometriotic lesion growth by transcriptional activation of KLF11 (Correa et al. 2016). Genet- ic evidence shows that a deactivating polymor- phism in miR-125b results in enhanced BMPRIB expression in women with endometri- osis (Chang et al. 2013). Furthermore, BMP-6 expression is increased in endometriotic lesions (Athanasios et al. 2012), suggesting a role for BMP signaling pathways in endometriosis. TGF-b signaling has also been widely ex- plored in the pathogenesis of uterine fibroids. Uterine fibroids, or leiomyomas, are benign smooth muscle tumors of the uterus that are associated with uterine bleeding, anemia, pelvic discomfort, and recurrent pregnancy loss (Okolo 2008; Bulun 2013). Compared to nor- mal myometrial tissue, uterine fibroids have in- creased extracellular matrix deposition with dis- organized collagen fibril formation (Leppert et al. 2004). Increased collagen deposition is characteristic of other diseases with altered TGF-b signaling such as pulmonary fibrosis, scleroderma, and liver cirrhosis (Border and No- ble 1994; Blobe et al. 2000). Compared to healthy myometrium, TGFB3 expression is in- creased in uterine fibroids and correlates with increased fibronectin and increased cell prolif- eration (Arici and Sozen 2000). TGF- b3 also increases the expression of versican in myome- trial and leiomyoma-derived cells (Norian et al. 2009). V arious studies have tested the prolifera- tive effects of TGF- b on cultured myometrial and leiomyoma-derived smooth muscle cells (T ang et al. 1997; Arici and Sozen 2000; Lee and Nowak 2001). These studies show that TGF-b signaling increases leiomyoma cell pro- liferation (T ang et al. 1997), and this response is affected by the phase of the menstrual cycle (Arici and Sozen 2003). In other studies, TGF- b3, but not TGF- b1 or TGF- b2, significantly stimulates leiomyoma cell proliferation (Lee and Nowak 2001). The GnRH agonist leuprolide acetate significantly decreases TGFB1, TGFB3, and TGFBR2 expression in leiomyoma tissues (Dou et al. 1996). Overall, these studies show that TGF- b increases extracellular matrix for- mation and cell proliferation in uterine fibroids.

In vivo studies using mouse models have con- tributed significantly to our understanding of the physiology and pathophysiology of the re- productive system that may not have been dis- covered through other experimental analyses. Such was the case for the novel tumor suppres- sor functions of inhibin a, because inhibin was best known for its role in regulating pituitary gonadotrope function. Mice null for Gdf9 and Bmp15, oocyte-specific members of the TGF- b family, highlight the importance of the oocyte in controlling follicular growth; both have be- come candidate genes for infertility research. Mutations in BMP15 have been identified that cause ovarian failure in women (Di Pasquale et al. 2004). In addition, GDF-9 and BMP-15 may form highly potent heterodimers, which may have clinically useful activities. However, most of the TGF- b family proteins and their signaling systems control critical stages in em- bryonic development or the function of multi- ple organ systems. As such, mice with targeted gene inactivation display embryonic or perina- tal lethality that precludes their use in studying their reproduction function. T o circumvent this issue, mice with conditional gene inactivation have been generated. Bmp2 and Acvr1 condi- tional inactivations result in early pregnancy failure, and conditional deletion of Bmpr2 or Nodal disrupts signaling pathways during late pregnancy. T ranslational studies using human tissues, cells, and genome-wide association studies also implicate abnormal TGF- b signal- ing in female reproductive diseases, such as en- dometriosis, uterine fibroids, and preeclampsia. In the end, mice with tissue-specific gene inac- The TGF-b Family in the Reproductive Tract Cite this article as Cold Spring Harb Perspect Biol 2017;9:a022251 23 on September 6, 2026 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from tivation and mice with global gene inactivation ultimately provide a basis for understanding in- fertility and disease in humans. ACKNOWLEDGMENTS Studies on TGF-B signaling pathways have been supported by the Eunice Kennedy Shriver Na- tional Institute of Child Health and Human Development grants R01-HD033438 and R01- HD032067 (to M.M.M.) and R01-HD085994 and R01-HD076980 (to S.A.P) and by the In- stitutional Research and Academic Career De- velopment A ward (IRACDA) K12-GM084897 (to D.M.). D.M. holds a Postdoctoral Enrich- ment Program A ward from the Burroughs W ell- come Fund.

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