Hormone Signaling in the Testis Chapter 16 Hormone Signaling in the Testis © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16.1 Schematics of the structure of the testis and seminiferous tubule. (A) The testis consists of lobules that originate from and then return to the rete testes (left side of the testis shown). The rete testis provides a passageway for sperm and fluid to the epididymis. (B) A diagrammatic cross-section through two seminiferous tubules shows germ cells at different stages of development embedded in Sertoli cells. Maturing spermatozoa are shown in the lumen of the tubules. Peritubular myoid (PTM) cells line the outside of the seminiferous tubule. Leydig cells (LC) and blood vessels are located in the interstitial space between seminiferous tubules. (C) A single Sertoli cell and associated germ cells. A region of tight and adherens junctions that forms the blood–testis barrier between Sertoli cells is denoted with an arrowhead. Sertoli cells extend from the basement membrane to the lumen of the seminiferous tubule. The cytoplasm of a Sertoli cell surrounds germ cells during all stages of germ cell development. Spermatogonia lie on the basement membrane of the seminiferous tubule. As germ cells progress through their developmental steps (spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, round spermatids, and elongated spermatids), they move toward the lumen and are released as spermatozoa. Source: Reproduced with permission from Ref. 6. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16.2 The cell associations comprising the XIV stages of the cycle of the rat seminiferous epithelium. Each column numbered with a Roman numeral shows the cell types present in one of the cellular associations found in cross-sections of seminiferous tubules. The cellular associations or stages of the cycle succeed one another in time in any given area of the seminiferous epithelium in the rat. Following cellular association XIV, cellular association 1 reappears, so that the sequence starts over again. The stages of the cycle were identified by means of 14 of the 19 steps of spermiogenesis (numbers 1–19). These steps were defined by the changes observed in the nucleus and in the acrosomic structure (the acrosome and head cap are seen applied to the surface of the nucleus). Letters: A1, A2, A3, and A4 represent four generations of type A spermatogonia; In: intermediate spermatogonia: B: type B spermatogonia; the subscript m next to a spermatogonium indicates occurrence of mitosis; P1: preleptotene spermatocyte; L: leptotene spermatocyte; Z: zygotene spermatocytes; P: pachytene spermatocyte; Di: diakinesis of primary spermatocytes; and 11: secondary spermatocyte. The type As, Apr, and Aal undifferentiated spermatogonia, present in all stages of the cycle that give rise to type A1 differentiated spermatogonia, were not included in this drawing. Source: From Ref. 11 with permission. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16.3 Diagram of the progression of germ cell associations (stages of the semiferous epithelial cycle) as a wave along the seminiferous tubule in rat and human. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16.4 Hormone action. (A) Protein and peptide hormones (H in a diamond) usually bind to membrane-spanning receptors and then alter receptor structure to activate kinase activity, cause dimerization with other receptors, or recruit proteins to the receptor. Often, a series of adaptor proteins are recruited and/or kinases activated that result in changes to cell physiology, cell structure, and/or the exocytosis of regulatory factors. Signals can also be sent to the nucleus to alter transcription factors (TFs) or other members of the gene regulatory machinery to alter gene expression. (B) Lipophilic hormones such as steroids (H in a circle) pass through the plasma membrane to bind receptors (R in a circle) present in the nucleus or in the cytoplasm that then translocate to the nucleus. In the nucleus, the lipophilic hormone–receptor complex acts to alter gene expression. Some lipophilic hormone–receptor complexes are also capable of activating kinases to alter cell function. HRE: hormone response element. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16. 5 LH-mediated signaling in a Leydig cell FIGURE 16.5 LH-mediated signaling in a Leydig cell. The major pathway regulated by LH binding to its receptor is the activation of adenylate cyclase (AC) via Gs proteins. The resulting increase in cAMP causes the activation of protein kinase A (PKA) that can directly regulate transcription factors such as CREB or GATA4 to increase the transcription of genes required for steroidogenesis, including StAR. LH stimulation can also cause the activation of the MAP kinase pathway (from Ras to ERK), in part via PKA-mediated activation of the epidermal growth factor receptor (EGFR). Activation of ERK increases the expression and phosphorylation of StAR and promotes the proliferation of Leydig cells. Ca2+ influx into Leydig cells increases in response to LH and results in the activation of the Nur77 transcription factor that induces StAR transcription. LH-mediated increases in phospholipase C (PLC) and then protein kinase C (PKC) activity facilitate the activation of ERK and result in the amplification of ERK, Ca2+, and PKA stimulation of StAR activity. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16. 6 Human androgen receptor (AR) structure FIGURE 16.6 Human androgen receptor (AR) structure. The eight exons of the human AR are located on chromosome X bands q11–12. Exon 1 encodes the amino terminal domain (NTD), exons 2 and 3 encode the DNA-binding domain (DBD), and exons 4 through 8 encode the hinge region and ligand-binding domain (LBD). Also shown is the proline-rich domain (PRD) used to interact with Src kinase, as well as the transcription activating function domains 1, 2, and 5 (AF1, AF2, and AF5). Amino acid residues that separate the domains are shown below the AR protein. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16. 7 Testosterone-signaling pathways FIGURE 16.7 Testosterone-signaling pathways. (1) The classical testosterone-signaling pathway. Testosterone diffuses through the plasma membrane and binds with the AR. The AR undergoes an alteration in conformation that allows it to be released from heat shock proteins in the cytoplasm. AR then is able to translocate to the nucleus, where it binds to specific DNA sequences called androgen response elements (AREs). AR binding to an ARE allows the recruitment of co-activator and co-repressor proteins that alter the expression of genes to alter cellular function. (2) The nonclassical kinase activation pathway: testosterone interacts with the classical AR that then is able to recruit and activate Src, which causes the activation of the EGF receptor via an intracellular pathway. The EGF receptor then activates the MAP kinase cascade, most likely through Ras, resulting in the sequential activation of RAF and MEK and then ERK that activates p90Rsk-kinase, which is known to phosphorylate CREB on serine 133. As a result, CREB-regulated genes such as lactate dehydrogenase A (LDH-A), early growth response 1 (Egr1), and CREB can be induced by testosterone. (3) The nonclassical Ca2+ influx pathway: testosterone interacts with a receptor in the plasma membrane that has characteristics of a Gq-coupled G protein–coupled receptor (GPCR). Phospholipase C (PLC) is activated to cleave PIP2 into IP3 and DAG. Lower concentrations of PIP2 inhibit K+ ATP channels, causing membrane depolarization and Ca2+ entry via L-type Ca2+ channels. Source: Reproduced with permission from Ref. 124. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16. 8 Androgen signaling within the testis FIGURE 16.8 Androgen signaling within the testis. Androgen receptor (AR) is not expressed in germ cells, and as such, mediation of the testosterone signal on germ cells is via somatic cells that express AR. The cell-specific roles of AR signaling have been established through conditional gene targeting, revealing a complex paracrine signaling system at work in the testis that can be summarized as follows: LH enters the testis, binding LHCGR to stimulate testosterone production by Leydig cells (LC) (a). Testosterone binds AR in Sertoli cells (SC), promoting development of the blood–testis barrier (b) and supporting postmeiotic germ cell development (c). Unknown paracrine factors stimulated by SC-AR signal to control the final Leydig cell numbers (d). Stimulation of AR in PTM cells promotes PTM development and function (e), and the release of unknown paracrine-signaling molecules that support SC maturation and function, which in turn supports spermatogenesis (f). Furthermore, stimulation of AR in PTM cells also feeds back to promote differentiation of adult Leydig cells (g). Testosterone signaling to AR in vascular smooth muscle (VSM) promotes testicular vasomotion and fluid regulation in the interstitium (h), whereas AR activation in vascular smooth muscle cells supports overall Leydig cell function (i). E: endothelial cells, surrounding an arteriole; Spg: spermatogonia; Spc: spermatocyte; Spd: spermatid. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16. 9 FSH-signaling pathways FIGURE 16.9 FSH-signaling pathways. Initially, FSH binding to the FSH receptor causes receptor-coupled G proteins (Gs) to activate adenylate cyclase and increase intracellular cAMP levels. Multiple factors can be activated by cAMP in Sertoli cells, including PKA that can phosphorylate a number of proteins in the cell and also regulate the expression and activity of numerous transcription factors, including CREB. FSH also causes Ca2+ influx into Sertoli cells that is mediated by cAMP and perhaps PKA modification of surface Ca2+ channels. Depolarization of the cell is also involved in Ca2+ influx. Elevated Ca2+ levels can activate calmodulin (CaM) and CaM kinases that have multiple potential downstream effects, including the phosphorylation of CREB. FSH activates or represses the MAPK cascade and ERK kinase in Sertoli cells in a developmental stage–specific manner. Inhibition of the pathway is not well characterized. Activation is most likely via cAMP interactions with guanine nucleotide exchange factors (GEFs) and activation of Ras-like G proteins followed by sequential activation of RAF, MEK, and ERK kinases. ERK is capable of activating CREB and other transcription factors, including SRF, and c-jun (not shown). In granulosa cells, FSH also activates the p38 MAPK. FSH and cAMP also likely act through GEFs to activate PI3K and then phosphoinositide-dependent protein kinase (PDK1) and PKB in Sertoli cells. Studies of granulosa cells identified Forkhead transcription factor (Forkhead), SGK (glucocorticoid-induced kinase), and GSK-3 (glycogen synthase kinase-3) as additional downstream targets of the PI3K pathway. FSH also mediates the induction of PLA2, the subsequent release of arachidonic acid, and the activation of eicosanoids such as PGE2 that may act as intracellular or extracellular signaling agents. Source: Modified from Ref. 229. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16.10 The expression patterns of FSHR and AR during the cycle of the seminiferous epithelium in rats. The relative levels of expression for FSHR (solid line) and AR (dashed line) are shown throughout the 14 stages (I–XIV) of the seminiferous epithelium cycle in the rat. Source: Adapted from data provided by Refs 107,366. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

FIGURE 16. 11 Summary of hormone signaling in the testis FIGURE 16.11 Summary of hormone signaling in the testis. Hormones derived from the circulation, including follicle-stimulating hormone (FSH) and thyroid hormone (TH), act on Sertoli cells to regulate Sertoli cell proliferation and support spermatogenesis. LH acts on Leydig cells to promote testosterone production. Osteocalcin and insulin-like growth factor 1 (IGF1) increase testosterone production, whereas glucocorticoids and leptin oppose testosterone production. Testosterone acts upon Leydig, peritubular myoid (PTM), and Sertoli cells to support the survival, development, attachment, and release of germ cells as well as maintain the blood–testis barrier (BTB). Estrogen produced by Leydig cells and Sertoli cells provides additional signals required to maintain fertility. Sertoli and Leydig cells produce activin that increases the duration of Sertoli cell proliferation and supports spermatogonia proliferation. In response to hormonal and paracrine signals, Sertoli cells produce glial cell line–derived neurotrophic factor (GDNF) to maintain spermatogonial stem cells, as well as activin, kit ligand, and retinoic acid that support the differentiation and proliferation of spermatogonia and entry into meiosis. Inhibin produced by Sertoli cells prevents uncontrolled Sertoli cell growth and provides feedback information to the pituitary gland to regulate FSH secretion. Less well-characterized signals are transmitted between PTM and Sertoli cells. © 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition