The establishment and maintenance of spermatogenesis in mammals requires specialized networks of gene expression programs in the testis. The gonad-specific TAF4b component of TFIID (formerly TAF II 105) is a transcriptional regulator enriched in the mouse testis. Herein we show that TAF4b is required for maintenance of spermatogenesis in the mouse. While young Taf4b-null males are initially fertile, Taf4b-null males become infertile by 3 mo of age and eventually exhibit seminiferous tubules devoid of germ cells. At birth, testes of Taf4b-null males appear histologically normal; however, at post-natal day 3 gonocyte proliferation is impaired and expression of spermatogonial stem cell markers c-Ret, Plzf, and Stra8 is reduced. Together, these data indicate that TAF4b is required for the precise expression of gene products essential for germ cell proliferation and suggest that TAF4b may be required for the regulation of spermatogonial stem cell specification and proliferation that is obligatory for normal spermatogenic maintenance in the adult. Spermatogenesis is a complex process requiring the specialized function of multiple cell types including somatic and germ cells that collectively results in the continuous production of functional sperm in adult males. The unlimited production of male gametes is largely accomplished through the ability of spermatogonial stem cells to self-renew in the adult testis. These complex and multifaceted events are dependent on appropriate expression and action of specific genes at multiple stages of germ cell and testicular development (Matzuk and Lamb 2002;McLaren 2003). The precise temporal and spatial expression of specific transcription factors is also essential for proper execution of spermatogenesis (SassoneCorsi 1997). Emerging evidence now suggests that in addition to gonad-specific transcription factors, specialized components of the basal RNA Polymerase II machinery are also critical for the execution of gonad-specific programs of gene expression (Hochheimer and Tjian 2003).The TFIID complex is a core RNA polymerase complex that contains the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs) that function in core promoter recognition and activator-dependent RNA Polymerase II recruitment (Verrijzer and Tjian 1996). While most TFIID subunits are expressed and function broadly in most cell types, there are selective TFIID subunits that apparently have evolved to function in the specification of gonadal-specific programs of gene expression. In the mouse, TAF4b is a component of TFIID that is highly enriched in gonadal tissues and is required for ovarian follicle development (Freiman et al. 2001). TAF4b is similar in structure to its broadly expressed paralog TAF4 (TAF II 130). While TAF4 and TAF4b display overlapping expression patterns in certain cell types, TAF4b is essential for regulating the selective expression of ovarian-specific gene expression patterns required for female fertility (Freiman et al. 2001).Several other members of the basal transcription mac...

Steroidogenic factor-1 (SF-1) (NR5A1) is an orphan nuclear receptor that plays a premier role in ovarian organogenesis. Recent studies document mRNA expression of the structurally related factor NR5A2 (FTF, LRH-1, SF-2) in the adult ovary and more specifically in granulosa cells and luteal cells but not theca cells. Conversely, SF-1 was shown to be expressed at higher levels in theca/interstitial cells. These latter observations raised the possibility that FTF/LRH-1 may control target gene expression in granulosa cells of developing follicles. Using quantitative PCR our results show that FTF/LRH-1 message is expressed at higher levels in the ovary than in liver or other tissues analyzed. We show by in situ hybridization and LacZ expression in ovaries of transgenic mice bearing an FTF-promoter-LacZ fusion gene that FTF/LRH-1 is selectively expressed in granulosa cells of rat and mouse ovaries and is not present in theca cells or interstitial cells. However, by a variety of approaches, we showed that SF-1 mRNA and protein are expressed in greater amounts than FTF/LRH-1 in granulosa cells of follicles at all stages of development. Expression of SF-1 mRNA and protein in granulosa cells was verified by in situ hybridization, immunohistochemistry of ovarian sections, and immunocytochemistry of cultured rat granulosa cells. The significance of SF-1 in regulating target gene activation was supported by EMSA. An abundant granulosa cell protein binding to the SF-1-binding motif (CCAAGGTCA) present in the aromatase promoter and an FTF/LRH-1 motif (TGTCCTTGAACA) in the alpha-fetoprotein promoter was supershifted by two SF-1-specific antibodies but not by an FTF antibody. Conversely, with the same probes, a less abundant protein/DNA complex present in liver and ovarian cell extracts was shifted by an FTF antibody but not by the SF-1 antibodies. SF-1 and FTF/LRH-1 were differentially regulated in vivo by estradiol, FSH and prolactin. Collectively these data indicate that granulosa cells of small and preovulatory follicles express both SF-1 and FTF/LRH-1 and that each orphan receptor may regulate target gene expression in these cells.

FSH stimulates in ovarian granulosa cells diverse, differentiation-dependent responses that implicate activation of specific cellular signaling cascades. In these studies three kinases were investigated to determine their relationship to FSH, cAMP, and A kinase signaling: protein kinase B (PKB/Akt), serum and glucocorticoid-induced kinase (Sgk), and p38 mitogen-activated protein kinase (p38MAPK). The phosphorylation (activation) of these kinases was analyzed by using selective agonists/inhibitors: forskolin/H89 for cAMP-dependent protein kinase (A kinase), insulin-like growth factor I (IGF-I)/LY294002 and wortmannin for phosphatidylinositol-dependent kinase (PI3-K), and phorbol myristate (PMA)/GF109203X for diacylglycerol and Ca++-dependent kinases (C kinases). An inhibitor (PD98059) of MEK1, which regulates extracellular regulated kinases (ERKs), and SB203580, which inhibits p38MAPK, were also used. In addition, we analyzed the expression of the recently described, cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFI and GEFII) that impact Ras-related GTPases and Raf kinases, known regulators of various protein kinase cascades. We provide evidence that FSH, forskolin, and 8-bromo-cAMP stimulate phosphorylation of PKB by mechanisms involving PI3-K (LY294002/wortmannin sensitive) not A kinase (H89 insensitive), a pattern of response mimicking that of IGF-I. In contrast, FSH induction and phosphorylation of Sgk protein requires A kinase (H89 sensitive) but also involves PI3-K (LY294002 sensitive) as well as p38MAPK (SB203580 sensitive) pathways. PMA (C kinase) abolished FSH-mediated (but not IGF-I-mediated) phosphorylation of PKB at a step(s) upstream of PI3-K and independent of A kinase. Lastly, FSH-mediated phosphorylation of p38MAPK is negatively affected by A kinase and PI3-K, suggesting that it may be downstream of specific members of the cAMP-GEF/Rap/Raf pathway. We propose that cAMP activation of A kinase is obligatory for transcription of Sgk in granulosa cells whereas cAMP (IGF-I-like)-mediated phosphorylation (activation) of PKB and Sgk (via PI3-K), as well as p38MAPK, involves other cellular events. These results provide new and exciting evidence that cAMP acts in granulosa cells by A kinase-dependent and -independent mechanisms, each of which controls specific kinase cascades.

Follicular development is dependent on both intraovarian growth regulatory factors, such as IGF-I and estrogen, as well as the pituitary gonadotropins, FSH and LH. Recently, we have shown that FSH impacts the IGF-I pathway via stimulation of the PI3K cascade leading to phosphorylation of protein kinase B (PKB)/Akt and the PKB-related kinase, Sgk. This study was undertaken to determine if during ovarian follicular development FSH regulates putative targets of PKB and Sgk, namely specific Forkhead transcription factor family members. Using in vivo and in vitro mouse and rat models, we show 1) that FKHR [Forkhead homolog of rhabdomysarcoma = Forkhead box binding protein (Foxo1), FKHRL1 (Forkhead-like protein-1 = Foxo3), and AFX (a Forkhead transcription factor = Foxo4); all defined according to the Human and Mouse Gene Nomenclature Committee) are expressed in the rodent ovary and 2) that FSH regulates transcription of the FKHR gene as well as phosphorylation of FKHR protein. Specifically, FSH/PMSG (primarily via E2) enhance expression of the FKHR gene in granulosa cells of developing follicles. Furthermore, E2 enhances expression of other IGF-I pathway components (IGF-1Rbeta and Glut-1), and IGF-I enhances expression of ERbeta, indicating that these two hormones comprise an autocrine regulatory network within growing follicles. In contrast, FSH and LH/human CG (via cAMP, PKA, and PI3K pathways) terminate FKHR expression as granulosa cells differentiate to luteal cells. In naïve granulosa cells, both FSH and IGF-I stimulate rapid phosphorylation of FKHR at multiple sites causing its redistribution from the nucleus to the cytoplasm in a PI3K-dependent manner. However, the effects of FSH and IGF-I differ markedly in differentiated granulosa cells in which FSH (but not IGF-I) induces Sgk and enhances phosphorylation of FKHR, PKB, and Sgk. The elevated expression of FKHR in granulosa cells of growing follicles indicates that FKHR may be linked to the proliferation of granulosa cells and that its phosphorylation by FSH, IGF-I, and other factors may impact its functional activity in this process. Thus, as a target of FSH (cAMP), E2 and IGF-I signaling in granulosa cells, FKHR likely coordinates numerous cell survival mechanisms.

Protease cascades are essential for many biological events, including the LH-induced process of ovulation. ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin-like repeats-1) is expressed and hormonally regulated in the ovary by LH and the progesterone receptor. To determine whether other family members might be expressed and regulated in the rodent ovary, those closely related to ADAMTS1 (ADAMTS4 and ADAMTS5) were analyzed in the mouse ovary by reverse transcription-polymerase chain reaction as well as by Western blot, immunohistochemical, and immunocytochemical analyses using highly specific antibodies. Prior to ovulation, ADAMTS4 and ADAMTS5 were coexpressed in granulosa cells of most follicles, whereas ADAMTS5 was also present in granulosa cells of atretic follicles. Following ovulation, ADAMTS1 and ADAMTS4 (but not ADAMTS5) were expressed in multiple cell types, including those within the highly vascular ovulation cone that marks the site of follicle rupture, endothelial cells of newly forming corpora lutea, and cumulus cells within the ovulated cumulus cell-oocyte complex (COC). Versican, a substrate for ADAMTS1 and ADAMTS4, colocalized with these proteases and hylauronan on the cumulus cell surface. To further characterize induction of these proteases and associated molecules, COCs and granulosa cells were isolated from preovulatory follicles and treated with FSH. In expanded COCs and differentiated granulosa cells, FSH induced expression of ADAMTS4 and versican message and protein, whereas increased levels of ADAMTS1 protein was observed in the media of granulosa cells where it was stabilized by heparin in this in vitro system. These studies provide the first evidence that ADAMTS1, ADAMTS4, and ADAMTS5 are expressed in spatiotemporal patterns that suggest distinct as well as some overlapping functions that relate to the broad expression pattern of versican in granulosa cells of small follicles, expanded COCs, and endothelial cells of the mouse ovary.