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 specification. 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. 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.

Timeline for female germline development from fertilization to birth in wild-type mice.

Days post coitum are shown in the top panel with key landmarks shown below. PGC , Primordial germ cell. Left lower panel, histology of a newborn mouse ovary (postnatal day 0). Oocytes are organized into germ cell cysts (dashed lines) containing multiple oocytes, with a few forming primordial follicles (arrows) . Right lower panel, histology section of a postnatal day 3 mouse ovary. Most germ cell cysts have undergone breakdown to form primordial follicles, which in turn activate to form primary follicles (arrowheads) .

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 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. The PI3K/AKT and SRC kinase pathways are also involved downstream of KIT in the functioning of the PGCs.

Ovary Specification

PGCs 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 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 proliferation in human ovaries prior to the entry of PGCs into meiosis.

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 a stable overexpression of β-catenin. β-catenin appears to be a central component to 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 a number of 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 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 prevents development of the coelomic vessel and rescues 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,” 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 mechanism for how 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 germ 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 shows 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 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 in vitro cultured ovaries. Therefore mTOR signaling is critical for bidirectional communication between somatic cells and oocytes to facilitate germ cell cyst break down 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.

Key pathways in mice regulating germ cell cyst breakdown to form the ovarian reserve.

Oocytes in germ cell cysts (left) in the developing ovary undergo breakdown to either form primordial follicles (upper pathway) or die (lower pathway) . The number of dormant primordial follicles makes up what is called the true “ovarian reserve.” Several key signal transduction pathways that regulate these processes are shown. AMH, Anti-müllerian hormone.

The origin of the somatic cells that surround each primordial follicle and eventually become granulosa cells of the follicle has been controversial. Sertoli cells and granulosa cells may arise from ingressing cells of the coelomic epithelium during gonad development, although the timing of their specification may be different. In the testis, regulation of the transcription factor Sox9 by the testis-determining factor SRY is required for Sertoli cell differentiation. In the ovary, forkhead box 2 (Foxl2) acts to maintain granulosa cell differentiation, in part by suppressing the expression of Sox9 . Mutations in the FOXL2 genes are associated with several human diseases. The FOXL2 gene is mutated in women with blepharophimosis ptosis epicanthus syndrome (BPES), which in one form 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.

Schematic of ovarian follicle development.

Dormant primordial follicles are recruited into the growing pool and initially form “primary” follicles as granulosa cells (GCs) transition to a cuboidal epithelium around the oocyte and proliferate. Multiple layers of granulosa cells (GCs) and acquisition of a thecal cell layer characterize “secondary” follicles. An antrum then forms, and the follicle undergoes further differentiation to the preovulatory or “Graafian” stage under the influence of pituitary gonadotropins. Blocks in follicle development for various mouse knockout models are shown as discussed in the text. The arrow s next to a gene name indicate a more rapid transition.

Cumulus Expansion and Ovulation

Preantral follicle growth is followed by an antral follicle stage, which is defined by the presence of a fluid fill cavity, the antrum (see Fig. 6.4 ). Under the influence of pituitary gonadotropins, antral follicles are selected to undergo final differentiation, ovulation, and, subsequently, corpus luteum formation. Because of the presence of an antrum, preovulatory follicles contain two independent populations of granulosa cells: mural granulosa cells line the follicle wall and are closest to the theca, basement membrane, and vasculature, while cumulus cells surround the oocyte and are linked initially with the oocyte for their functions ( Fig. 6.5 ). The growth factor contributions of the mural and cumulus cell and their relevance to ovulation are most apparent at the time of the LH surge. The LH receptor (Lhcgr) is preferentially expressed only in mural granulosa cells, allowing them to respond to the LH surge, which initiates a cascade of events leading to oocyte meiotic resumption, cumulus expansion, follicle rupture, and terminal differentiation of the remaining granulosa and thecal cells to create the corpus luteum. Cumulus cell expansion, which is the production of a hyaluronan-rich extracellular matrix surrounding the oocyte, is initiated by the LH surge and is required for normal ovulation and fertilization. However, since the cumulus granulosa cells lack LHCGR and yet respond to the LH, this indicates an indirect effect of the LH surge on their fate.

Ovulation and cumulus expansion in the mouse.

Prior to the luteinizing hormone (LH) surge, preovulatory follicles contain an oocyte (Oo) surrounded by cumulus cells (CC) and separated from the mural granulosa cells by a fluid filled cavity called an antrum. After a surge of LH, the cumulus cells undergo expansion and become embedded in a hyaluronan-rich matrix, and the cumulus-oocyte complex is extruded from the follicle. An example of cumulus expansion in vitro using intact mouse cumulus-oocyte complexes and FSH treatment is shown in the right panels. Genes that are known to play critical roles in cumulus expansion are indicated.

How does the LH surge lead to induction of target genes in cumulus cells that are critical for cumulus expansion? Conti and colleagues have shown that the LH surge causes a rapid increase in EGF-like family members, amphiregulin (AREG), epiregulin, and betacellulin (BTC) specifically in mural granulosa cells of preovulatory follicles (see Fig. 6.5 ). These ligands are synthesized as membrane proteins and released from the cell surface into the antral fluid by proteolytic cleavage. These EGF-like proteins activate the EGF receptor (EGFR) on cumulus cells to exert the indirect effect of the LH surge to stimulate cumulus expansion and oocyte maturation. The effects of these EGF-like factors on cumulus expansion occur through upregulation of the genes encoding hyaluronan synthase 2 (Has2), tumor necrosis factor-induced protein 6 (Tnfaip6), pentraxin 3 (Ptx3), and prostaglandin synthase 2 (Ptgs2), whose products are essential for formation and stabilization of the extracellular matrix of the cumulus cells (see below). Transcripts for Areg , Ereg , and Btc are also detected in cumulus oocyte complexes after the surge, suggesting that an autocrine regulatory loop maintains high EGF-like growth factor expression in cumulus cells. These data are not only relevant to mice, since amphiregulin is abundant in human follicular fluid from patients undergoing in vitro fertilization.

Although EGF-like factors play an important role in cumulus expansion and oocyte maturation, oocyte-secreted growth factors are also essential for cumulus expansion. In vitro, expansion of isolated cumulus oocyte complexes can occur with the addition of EGF (as well as FSH and cAMP analogues). The oocyte is required for expansion, since oocytectomized cumulus oocyte complexes fail to expand when only FSH or EGF is added to the culture medium. Expansion of OOX complexes are rescued by coculture with denuded oocytes or conditioned medium from denuded oocytes, indicating the presence of oocyte growth factors is also involved in the expansion. As mentioned earlier, follicular development in Gdf9 KO mice is arrested at the primary follicle stage. However, recombinant GDF9 upregulates Has2 , Ptgs2 , Ptger2 , Tnfaip6 , and Ptx3 in granulosa cell culture systems. Injection of mouse oocytes with Gdf9 small interfering (si)-RNA, but not Bmp15 si-RNA, suppresses Has2 and Ptgs2 expression, and cumulus expansion when oocytectomized cumulus complexes are incubated with knockdown oocytes.

The cumulus cell matrix contains an essential meshwork of hyaluronan and proteins. Hyaluronan chains from the structural backbone of the cumulus matrix, and these chains are stabilized through interactions with other matrix proteins. TNFAIP6 catalyzes the formation of covalent cross-links between hyaluronan and the heavy chain of serum-derived inter-α-trypsin inhibitor (IαI), and PTX3 stabilizes the cumulus matrix through interactions with IαI. PTGS2 synthesizes PGE2 and prostaglandin EP2 receptor (PTGER2); it appears to function upstream of TNFAIP6. Although Has2 ovary-specific knockouts have not been generated, the importance of the regulation of the other EGF/GDF9/BMP15 regulated gene products in vivo in cumulus expansion is obvious. Targeted disruption of Ptgs2 , the prostaglandin E receptor 2 (Ptger2) , Tnfaip2 , or Ptx3 results in abnormal or absent cumulus expansion and extreme subfertility while Tnfaip6 -null females are sterile. Knockdown of Has2 in cultured cumulus oocyte complexes suppresses cumulus expansion. IαI biosynthesis in the liver requires alpha-1-microglobulin/bikunin light chain (encoded by Ambp ) for proper assembly and secretion, and Ambp -null females have impaired cumulus expansion and subfertility. Thus oocyte-secreted growth factors (GDF9 and BMP15) and EGF-like growth factors (AREG, EREG, and BTC) combine to induce an array of genes that are necessary for mammalian cumulus expansion and fertility.

Mutations in mice, sheep, and humans have been important in identifying key functions of GDF9, BMP15, and their downstream gene products in cumulus expansion. GDF9 signals through a heterodimeric complex of type 1 and a type 2 receptors to phosphorylate SMAD2/3 and complex with SMAD4 and conditional deletion of both Smad2 and Smad3 or Smad4 in granulosa cells using Amhr2-cre disrupts cumulus expansion in vivo and suppresses fertility (as mentioned earlier). Recent studies demonstrated that recombinant mouse and human GDF9:BMP15 heterodimers are more bioactive than GDF9 and BMP15 homodimers in cumulus expansion in vitro . Furthermore, GDF9:BMP15 heterodimers predominantly activate the SMAD2/3 pathway likely through a BMPR2-ALK4-ALK6 receptor complex in mouse and human granulosa cells. Naturally occurring mutations in sheep BMP15 and GDF9 or the BMP type I receptor, BMPR1B/ALK6 , modify ovulation rate in sheep ( Table 6.4 ). In most cases, heterozygous mutations in BMP15 or GDF9 increase ovulation rates, while homozygous null animals are sterile. Ovaries from homozygous mutant sheep appear similar to mouse Gdf9- null ovaries, with follicles arrested at the primary stage. Partial immuno-neutralization against GDF9, BMP15, or in combination in sheep will increase ovulation rate and number of lambs, indicating that both GDF9 and BMP15 regulate this process. Genetic variants have been identified in women with premature ovarian failure ( BMP15 or GDF9 ) or dizygotic twinning (GDF9).

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