Growth Factors and Reproduction

Growth Factors in Primordial Germ Cell Formation and Migration

Future components of the mammalian ovary and testis develop long before a distinct bipotential organ can be discerned ( Fig. 6.2 ). Growth factors are key for maintenance of the germline and their migration to the genital ridge following their specification outside the embryo proper. At the onset of gastrulation, PGC progenitors are induced in the epiblast in response to instructive signals from BMPs. This has been defined with great help with the development of mouse genetic models (summarized in Table 6.1 ). BMP4 and BMP8B secreted from the extraembryonic ectoderm, and BMP2 secreted from the visceral endoderm, are required for the early discrimination of PGC precursors from somatic cells of the embryo. These BMPs signal in a dosage-dependent manner to the epiblast cells through a BMP receptor serine-threonine kinase cascade that involves phosphorylation of the SMAD transcription factors, SMAD1 and SMAD5. These SMADs, along with the common SMAD partner, SMAD4, have been shown to function in PGC specifications. BMP2, BMP4, and BMP8B act on the pluripotent epiblast cells in the posterior of the mouse embryo between embryonic day (E)5.5 to E6.0 to induce their competency to become PGC precursors. PGC specification also requires wingless/Int3 (Wnt3) signaling through β-catenin signaling to allow the epiblast cells to respond to BMP signaling. At approximately E6.25, six epiblast cells adjacent to the extraembryonic ectoderm express the BMP-regulated and PGC specification genes, PR-domain containing-1 ( Prdm1; also called Blimp1 ), and Prdm14 , and they commit to becoming true PGC precursors, the first such fate-committed cells of the embryo. By E7.5, approximately 40 founder PGCs are established.

Once PGC specification has occurred at E7.5 in the mouse embryo, PGCs express markers of pluripotency including POU (Pit-Oct-Unc) domain class 5 transcription factor 1 ( Pou5f1 ; also called Oct4 ), sex-determining region Y-box 2 (Sox2) , Nanog , as well as developmental pluripotency-associated-3 ( Dppa3 ; also called Stella ) and alkaline phosphatase (Alpl). Between E7.5 and E8.5, the PGCs demonstrate major chromatin changes, increasing the levels of trimethylation of lysine 27 on histone H3 (H3K27me3) and erasing the dimethylation (H3K9me2) marks, which are patterns that resemble the chromatin patterns of pluripotent stem cells. The H3K27me3 marks appear to repress the somatic cell gene expression program similar to ESCs. These changes cause PGC arrest at the G2 stage of the cell cycle, transiently causing the PGCs to become transcriptionally silent as they migrate from the base of the yolk sac along the hindgut to the genital ridge.

An extracellular matrix gradient, as well as various chemoattractants, are important for appropriate PGC migration. If too much extracellular matrix is deposited, PGCs show reduced migration. For example, suppression of TGF-β signaling by genetically removing the TGF-β type I receptor, Tgfbr1, leads to enhanced migration due to a reduction in the levels of TGF-β-induced collagen type 1 in the extracellular matrix. Alternatively, in vitro migration assays demonstrate that KITL, a growth factor that binds to KIT on PGCs, functions as an effective chemoattractant for PGC migration into the genital ridge. Consistent with the pleiotropic roles of the KITL/KIT pathway, these proteins not only function in PGC migration but also aid in PGC survival and proliferation. Noncanonical WNT signaling through the receptor tyrosine kinase orphan receptor (ROR2) also regulates PGC migration. Ror2 is expressed in PGCs and enhances their response to secreted KITL, which may enhance PGC chemotaxis toward KITL in the genital ridge during PGC migration. The PI3K/AKT and SRC kinase pathways are also involved downstream of KIT in the functioning of the PGCs.

Ovary Specification

PGCs maintain a genetic program of pluripotency and proliferate during their migration to the genital ridge and after colonization. At this time, the gonad is not sexually differentiated and is termed bipotential or indifferent . BMP signaling within the bipotential gonad is necessary to develop a niche for migrating PGCs. Deletion of the BMP type I receptor, Bmpr2a/Alk3 , in the genital ridge leads to the death of somatic cells within the mesonephric mesenchyme and reduced levels of Kitl in the coelomic epithelium, ultimately decreasing the number and migration of the PGCs. In mice, PGC proliferation ceases around E10.5 to 11.5, corresponding to 8 to 9 weeks of gestation in humans. Bmp7 is required for germ cell proliferation and Bmp7 null mice have reduced numbers of germ cells after E11.5. Activin may substitute for BMP7 in human embryos, since BMP7 is not expressed in the human ovary and BMP signaling alternatively promotes germ cell apoptosis. Activin increases germ cell survival and proliferation in human ovaries prior to the entry of PGCs into meiosis.

Signaling from the gonadal ridge is required for PGCs to become gametogenesis-competent in a process called germ cell licensing, which precedes meiotic entry Deletion of Gata4 from somatic cells does not affect PGC migration but does result in loss of expression of the germ cell expressed genes, Dazl and Mvh , which are normally upregulated at the time of licensing and arrival at the gonadal ridge. The signaling cues expressed from somatic cells of the gonadal ridge that regulate PGC licensing and gametogenesis-competent gene expression are not currently known. Systematic analysis of four major signaling pathways in first- and second-trimester human gonads suggests that the transition of PGCs to premeiotic germ cells involves cytokines such as KITL but is regulated in a sex-specific manner, with high levels of BMPs and canonical WNTs driving the transition in females while low levels of BMPs plus FGF, IGF1, and activin A function in males. Additional studies will be necessary to understand these signaling pathways in the mouse and human developing gonads at the very early stages of oogonia and prospermatogonia.

Differentiation of the bipotential gonad to form an ovary requires multiple signaling inputs, including the WNT/β-catenin pathway. Partial sex reversal is seen in XX mice null for Wnt4, the WNT pathway activator protein R-spondin 1 (Rspo1) , or follistatin (Fst) , an antagonist of activin and BMP signaling. Loss-of-function RSPO1 in XX patients results in complete female-to-male sex reversal, while duplication of the chromosome region containing WNT4 and RSPO1 causes XY male-to-female-sex reversal. Similarly, XY male-to-female sex reversal is found in mice with stable overexpression of β-catenin. β-catenin appears to be a central component of ovary specification, integrating signaling from the WNT and TGF-β family. Activation of WNT signaling allows stabilization and nuclear translocation of β-catenin, which interacts with several transcription factors of the TCF/LEF family to promote specific gene transcription. Increased levels of β-catenin prevent the expression of SOX9, a transcription factor that promotes testes development by inducing Sertoli cell differentiation. β-catenin also increases the expression of Fst , which shows a female-specific expression pattern at E11.5. Wnt4 -null or Fst -null ovaries develop a coelomic vessel similar to that found in testes, and germ cells are lost by birth. Wnt4 -null ovaries show an upregulation of Inhbb (activin B) and Fst -null ovaries also express Inhbb . Genetic crosses between Wnt4 or Fst -null mice to Inhbb null mice prevent development of the coelomic vessel and rescue normal ovary development, indicating that Inhbb expression (via suppression by Wnt4) or activity (via antagonism through follistatin) must be limited to maintain ovary specification. Bmp2 is also expressed in a female-specific pattern after E11.5, although Bmp2 -null mice die prior to this time period, and its function in the developing ovary has yet to be determined.

Formation of the Ovarian Reserve

During embryonic development, oocytes within the ovary are found as clusters, which are known as germ cell cysts (see Fig. 6.2 ). These clusters form by both aggregation and clonal division. ^, Meiotic progression in oocytes ceases at the diplotene stage of prophase I when germ cells are enclosed by somatic cells into individual follicles called primordial follicles. Oocytes in primordial follicles remain arrested until released to complete meiosis I during ovulation and form the “ovarian reserve,” the size and quality of which is thought to ultimately determine reproductive lifespan. Germ cell cyst breakdown occurs around birth in mice or during the second trimester in humans. In mice, the majority of germ cells within the cysts will die, with an estimated one-third remaining as primordial follicles. Massive germ cell loss also occurs in humans, with an estimated 1 million oocytes surviving at birth from approximately 6 million in the fetal human ovary.

The full mechanism by which oocytes become enclosed in primordial follicles has remained elusive, although multiple signaling inputs are likely required, particularly from those factors that mediate the interaction of oocytes with somatic cells ( Fig. 6.3 ). Signaling pathways that have been implicated in germ cell cyst breakdown are KITL, NGF, BDNF, Notch-Jagged, AMH, mTOR, and STAT. ^, One mechanism for primordial follicle formation is that Rac1, a Rho GTPase, promotes germ cell cyst breakdown by inducing nuclear translocation of signal transducer and activator of transcription 3 (STAT3) to activate transcription of oocyte-specific genes including Jagged1 , Nobox , Gdf9 , and Bmp15 in cultured mouse ovaries. Another mechanism suggests that mTOR signaling regulates KITL to facilitate germ cell nest breakdown and subsequent primordial follicle assembly. Genetic ablation of Tsc1 or Tsc2 , negative regulators of mTOR complex 1 (mTORC1) in oocytes, impairs the differentiation of pregranulosa cells and causes premature primordial follicle activation and follicular depletion by early adulthood. Suppression of mTORC1 is critical for maintaining the pool of follicles within the ovarian reserve. ^, Treatment of somatic cells with mTOR inhibitors impedes somatic cell invasion into germ cell cysts in ovaries cultured in vitro . Therefore, mTOR signaling is critical for bidirectional communication between somatic cells and oocytes to facilitate germ cell cyst breakdown and follicle assembly. ^, Improper germ cell cysts breakdown can manifest as multiple oocytes within follicles (called polyovular follicles ), although their biologic effects on fertility are not certain. Mice with mutations in the TGF-β family, such as ovarian activin βA (Inhba) deficiency, inhibin alpha subunit overexpression, and knockout mice for oocyte-expressed Bmp15 , often show increases in polyovular formation. In addition, polyovular follicle formation can be generated by exposure of neonatal mice to estrogen and estrogen-like compounds, which may suppress ovarian activin expression, thus disrupting the regulation of germ cell cyst breakdown. The regulation of estrogen signaling to maintain normal germ cell cyst breakdown appears to be conserved with other species, as studies in pregnant monkeys given oral bisphenol A (BPA; an endocrine-disruptor) have offspring with increased numbers of multioocyte follicles, though its role on activin A has not been determined in these animals.

There are two different origins for somatic cells that surround the primordial follicle and eventually become granulosa cells. During sexual differentiation, an initial population of bipotential precursor cells forms under the influence of the WNT4/RSPO1/β-catenin pathway to upregulate the forkhead box 2 transcription factor, FOXL2. These cells contribute to the population of medullary follicles that become the first set of ovarian follicles. ^, A second population of pregranulosa cells ingresses from the proliferating coelomic epithelium and will eventually become primordial follicles of the ovarian reserve, which are recruited during adult life. ^, In the testis, regulation of the transcription factor Sox9 by the testis-determining factor SRY is required for Sertoli cell differentiation. In the ovary, FOXL2 maintains granulosa cell differentiation, in part by suppressing the expression of Sox9 . Mutations in FOXL2 are associated with several human diseases. The FOXL2 gene is mutated in women with blepharophimosis ptosis epicanthus syndrome (BPES), one form of which is associated with primary ovarian failure. Mice null for Foxl2 are sterile with small ovaries and defects in primordial follicle assembly and growth ( Fig. 6.4 ). FOXL2 interacts with different transcription factors, including SMAD2 and SMAD3, to control gene transcription in nonovarian cell types, such as regulating FSHβ , FST , and gonadotropin-releasing hormone receptor synthesis in anterior pituitary cells. In the developing mouse ovary, BMP2 and FOXL2 also cooperatively upregulate Fst gene expression. A somatic missense mutation in FOXL2 is associated with almost all adult granulosa cell tumors, a rare class of ovarian tumors. This mutation causes an amino acid substitution in the DNA-binding domain of FOXL2, although its functional consequence has not been fully clarified. The role of two different populations of primordial follicles in ovary development has not been fully characterized. During puberty, a large number of primordial follicles is culled in a hormonally driven process dependent upon the proapoptotic protein BCL-2 modifying factor (BMF) between postnatal day 40 and 50. These may represent the earliest wave of follicle development that is used to establish the timing of puberty and the reproductive cycle.

Ovarian Folliculogenesis

• TGF-β superfamily members, GDF9, BMP15, AMH, activin, and inhibin function to regulate ovarian follicle growth and development.

• Oocyte-secreted growth factors (GDF9 and BMP15) and EGF-like family members (amphiregulin, epiregulin, and betacellulin) modulate cumulus cell expansion and oocyte maturation.