1. Chapter 21 © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition Follicular Development: Mouse, Sheep, and Human Models

2. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition 2 FIGURE 21.1 Classification of mouse follicles. From left to right, postnatal follicle development begins at the primordial stage to the antral stage. Prior to antrum formation, follicle growth is independent of pituitary gonadotropins. Following ovulation, granulosa cells undergo lutein- ization to become the corpus luteum. Pederson classification (Types 2–8) is shown for each stage. Corresponding oocyte diameters for each type are also indicated. A subset of genes implicated at various stages of follicular development is also listed when initial gross disruption is observed in respective mouse knockouts. *Amh and Foxo3 knockout mice show accelerated transition from primordial to primary follicles.

3. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition 3 FIGURE 21.2 Folliculogenesis in the mouse. Light micrographs depicting follicle stages are shown in panels (A)–(F) above the timeline. Follicle development begins embryonically when primordial germ cells migrate to the developing gonad. By E11–13, the PGCs undergo mitotic division, and by E13.5 they begin meiosis. Oocytes will arrest in meiotic prophase I until ovulation. Panels (A) and (D): Newborn ovaries contain oocytes in germ cell cysts (GCs) and are beginning to form primordial follicles (PFs). Panels (B) and (E): By 7 days after birth, primary follicles (PrFs) and secondary follicles (SFs) have developed. Primary follicles contain a growing oocyte and are surrounded by cuboidal granulosa cells. These further develop into secondary follicles by proliferation of granulosa cells and the acquisition of a thecal cell layer (Th). A 4–5-day estrous cycle begins at 4 weeks of age, and pituitary gonadotropins are required to develop follicular stages beyond the secondary stage. Panels (C) and (F): Adult ovaries contain multiple-stage follicles, including antral- stage follicles (AnF) and corpora lutea from previous ovulations (CL). Granulosa cells from antral follicles are divided into two types: mural granulosa cells (Gr) that line the follicle wall, and cumulus granulosa cells (Cu) that surround the oocyte (Oo). GC: germ cell cyst; PF: primordial follicle; PrF: primary follicle; SF: secondary follicle; AnF: antral follicle; CL: corpus luteum; Oo: oocyte; Gr: granulosa cell; Cu: cumulus granulosa cell; Th: thecal cell. Micrographs not shown to scale.

4. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition 4 FIGURE 21.3 Ovarian histology for various mouse knockout models. Light micrographs of adult ovarian sections from knockout models. Panel (A): Adult wild-type ovaries demonstrate follicles from many stages. Panel (B): Adult Fsh −/− mutant ova- ries lack antral follicles. Panel (C): 14-day-old Nobox −/− ovaries contain only follicular nests and degenerating oocytes. Panel (D): Esr1 −/− Esr2 −/− double-mutant ovaries containing many advanced follicle types as well as struc- tures that resemble male seminiferous tubules (boxed and shown at higher magnification in panel (G)). Panel (E): Gdf9 −/− null ovaries arrest at the primary follicle stage. Panel (F): Mag- nification of boxed Gdf9 −/− follicles shown in panel (E). Panel (H): Double-mutant Gdf9 −/− Inha −/− ovary demonstrates partial rescue of the Gdf9 −/− phenotype. Panel (I): Higher magnification of the Gdf9 −/− Inha −/− showing multilaminar follicles and a thecal cell layer. PrF: primary follicle; SF: secondary follicle; AnF: antral follicle; Oo: oocyte; Gr: granulosa cell; Th: thecal cell; DF: degenerating follicle; FN: follicular nest. Micrographs not shown to scale.

5. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition 5 FIGURE 21.4 Early folliculogenesis. Germ cell cysts break down after birth, and primordial follicles form. Germ cell–specific factors consid- ered part of the same pathway are shown in a lighter font. The factors are listed at the site of proximate action as derived from mouse transgenic and ovarian culture studies. LIF: leukemia inhibitory factor; KITL: kit ligand; FGF2: fibroblast growth factor; AMH: anti- Müllerian hormone; FGF7: fibroblast growth factor 7; NGF: nerve growth factor; NT4/5: nerve growth factor receptors; NOBOX: newborn ovary homeobox gene; FIGLA: factor in the germline alpha; FOXL2: forkhead box L2; GDF9: growth differentiation factor 9; BMP15: bone morphogenetic protein 15; BDNF: neurotrophic factor; NT4/5: neurotrophin 5; FOXO3A: forkhead transcription factor; LHX8: LIM homeodomain 8; SOHLH1: spermatogen- esis and oogenesis helix loop helix 1; SOHLH2: spermatogenesis and oogenesis helix loop helix 1.

6. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition 6 FIGURE 21.5 Summary of the core signaling pathway used by ligands of the TGFβ family. The TGFβ family signals through a membrane-bound receptor complex consisting of two type II and two type I receptors, which activate the intra- cellular SMAD transcription factors. Additional co-receptors and regulatory proteins are not shown. Activin signaling can be antagonized by inhibin (see the text for discussion). Activin signals through activin receptor type IIA (ACVR2A) or IIB (ACVR2B). BMPs may also signal through ACVR2A, but they also use the bone morphogenetic protein receptor type II (BMPR2). BMPR2 also binds GDF9. Once the ligand is bound, the type II receptor phosphorylates (P) and activates the type I receptor (ALK4 or ALK7 for activin, ALK5 for GDF9, and ALK1, ALK2, ALK3, or ALK6 for the BMPs). ALK5 has been shown to be a type I receptor for GDF9 in vitro but not in vivo; therefore, additional type I receptors (indicated by “ALK?”) are likely also involved. Activated type I receptors then phosphorylate a restricted set of SMADs at a C-terminal motif. ALK4/5/7 phosphorylate SMAD2 and SMAD3, while ALK1/2/3/6 phosphorylate SMAD1/5/8. The receptor- restricted SMADs then form a trimeric complex with SMAD4, accumulate in the nucleus, and mediate gene transcription along with additional co-factors.

7. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition 7 FIGURE 21.6 Gross anatomy of ovar- ian tumor and cyst formation in several mouse models. Panel (A): Reproductive tract of an adult female mouse. Panel (B): Female Inha −/− mice develop ovarian tumors with 100% penetrance. Panel (C): Double-knockout Inha −/− Acvr2 −/− mice develop tumors similar to those with Inha −/− despite the lack of activin signaling through ACVR2. Panel (D): Bilateral fluid-filled cysts in a Gdf9 −/− Bmp15 −/− double- knockout ovary.