Gonadotropin and kisspeptin gene expression, but not GnRH, are impaired in cFOS deficient mice
Introduction
cFOS is a basic leucine-zipper protein which forms a heterodimer with the cJUN isoform, thus forming an AP1 transcription factor that binds the TPA-response element in the promoter of target genes. cFOS is an immediate-early gene that is activated rapidly and transiently in most cell types. It is induced by a variety of growth factors, cytokines, neurotransmitters, and hormonal signals, as well as environmental stimuli. In turn, cFOS controls a diverse array of cellular processes, including cell proliferation, differentiation, survival, and death. Of all of the eclectic variations of cellular functions in which it is involved, the primary role(s) of cFOS, in any given tissue, is dependent on the cell type and stimuli (Shaulian, Karin, 2002, Wagner, Eferl, 2005). We have shown that although pituitary gonadotropes express receptors for EGF or insulin, which in other cells induce cFOS, in the gonadotrope only GnRH induces cFOS and through it GnRH-target genes (Ely et al., 2011). Thus, cFOS although versatile, has cell-specific and stimulus-specific function in each cell type.
To study the roles of cFOS in vivo, two different cFOS deficient mice were created and their phenotypes were analyzed in mouse strains with mixed backgrounds (129/SvJ × C57Black6J). Similar abnormalities were reported for both mouse strains lacking cFOS (Johnson et al, 1992, Wang et al, 1992). cFOS nulls are born at the proper Mendelian ratio, demonstrating that cFOS is not necessary for embryonic development. However, after 4 weeks of age, cFOS null mice exhibit growth retardation, osteopetrosis, and ultimately, hematopoiesis deficiency. Although cFOS null mice exhibit impairments in peripheral organs such as bone and hematopoietic system, the alteration in the central nervous system (CNS) are cell-specific (Benes et al, 2013, Fleischmann et al, 2003, Yasoshima et al, 2006). For example, adult mice lacking cFOS in the CNS exhibited normal general and emotional behavior, but were specifically impaired in hippocampus-dependent spatial and associative learning tasks (Fleischmann et al., 2003). Johnson et al. reported infertility in both sexes, though the cause was not examined (Johnson et al., 1992). Since cFOS null mice are viable, redundancy poses a question and the potential complementary role of a closely related protein, FOSB, was also examined in vivo. Unlike cFOS null mice, FOSB deficient mice were reported to be healthy, viable, fertile, and had a normal life expectancy (Brown et al., 1996). Thus, cFOS is crucial for fertility, whereas FOSB is not. Moreover, intact, functional FOSB is unable to substitute for the loss of cFOS with regards to the reproductive system. This necessitates further examination of the roles of cFOS in modulating the hypothalamic–pituitary–gonadal axis.
cFOS is rapidly induced in gonadotrope cells following GnRH treatment, both in vivo (Padmanabhan et al., 1995) and in model cell lines (Cesnjaj et al, 1994, Wurmbach et al, 2001). cFOS mediates GnRH induction of the FSHβ gonadotropin subunits by binding to the AP1 site in the proximal mouse FSHβ promoter (Coss et al., 2004). Furthermore, cFOS is involved in synergistic induction of FSHβ by GnRH and activin, which is specific for FSHβ and may play a role in differential expression of gonadotropin subunits (Coss et al., 2007). Specific decrease of cFOS protein turnover may contribute to the rise in FSHβ transcription during the time of low GnRH pulse frequency (Reddy et al., 2013). Induction of the GnRH receptor by GnRH is also dependent on cFOS binding to both the AP1 site, to mediate GnRH responsiveness (White et al., 1999), and to the GRAS element where, through interaction with SMAD proteins and FOXL2, mediates the synergy between GnRH and activin (Ellsworth et al, 2003, Norwitz et al, 2002).
In the brain, cFOS serves as a marker of neuronal activation and its expression is increased in GnRH neurons during the preovulatory LH surge and after kisspeptin treatment (Kauffman et al, 2007, Lee et al, 1992). cFOS expression in kisspeptin neurons also coincides with a preovulatory LH surge (Clarkson et al, 2008, Robertson et al, 2009). However, a role of cFOS in GnRH and kisspeptin neurons is still poorly understood. In the gonads, as well, cFOS is expressed in germ cells and granulosa and theca cells in females (Rusovici and LaVoie, 2003), and Sertoli cells in males (Araujo et al., 2009), but its target genes are not known. Thus, a role for cFOS in the testes and ovaries is not elucidated.
The involvement and necessity of cFOS at different levels of the reproductive axis is not well-addressed, and therefore, the underlying cause(s) of infertility in mice lacking cFOS remains unknown. Here, we examined several levels of the hypothalamic–pituitary–gonadal axis in cFOS null mice of both sexes, to ascertain if gene expression impairments exist within the reproductive axis at either the brain, pituitary, and/or gonadal levels.
Section snippets
cFOS-null mice
The cFOS-null mice were obtained from Jackson Laboratories, where Papaioannou laboratory deposited them, and back crossed to C57Bl6J for six generations. Animals were maintained under a 12-hour light, 12-hour dark cycle and received food and water ad libitum. All experiments were performed with approval from the University of California Animal Care and Use Committee and in accordance with the National Institutes of Health Animal Care and Use Guidelines using 5 and 6 weeks old animals. Genomic
Gonadotrope gene expression is lower in cFOS null mice
We determined previously that cFOS is a critical transcription factor through which GnRH induces FSHβ gene in the LβT2 gonadotrope model cell line (Coss et al., 2004). It is also involved in differential expression of gonadotropin subunits that are necessary for reproductive fitness (Coss et al, 2007, Reddy et al, 2013). In this study, we analyzed a role of cFOS in reproduction in vivo, using cFOS null animals. As reported before (Johnson et al., 1992), we observed that heterozygous crosses
Discussion
Despite extensive studies on regulation of the gonadotropin subunit genes using cell models and dispersed pituitary cultures, little is known about in vivo roles of intermediary immediate early genes, such as cFOS, that are proposed to be involved in GnRH induction of gonadotrope specific genes. Studies using cell lines indicate that cFOS is a direct target of GnRH signaling that, upon induction, activates FSHβ and GnRH receptor gene transcription (Coss et al, 2004, White et al, 1999). In the
Conclusions
We determined that cFOS plays a cell-specific role at multiple levels of the hypothalamic–pituitary–gonadal axis in vivo. In the pituitary, cFOS is required for gene expression in the gonadotrope, but not in thyrotrope. Furthermore, cFOS is necessary for spermatogenesis and ovulation, but not for early gametogenesis or sex-steroid hormone synthesis. In the brain, cFOS is essential for kisspeptin expression and kisspeptin neuron number specifically in females, but not for GnRH neuron migration
Acknowledgements
The authors thank Kristen Tolson and Matthew Poling for their help.
This research was supported by NIH grants R01 HD057549 and R21 HD058752 (to DC); R01 HD065856 (to ASK). University of Virginia, Ligand Core is supported by NIH NICHD U54-HD28934.
References (50)
- et al.
The restructuring of muscarinic receptor subtype gene transcripts in c-fos knock-out mice
Brain Res. Bull
(2013) - et al.
A defect in nurturing in mice lacking the immediate early gene fosB
Cell
(1996) - et al.
A novel AP-1 site is critical for maximal induction of the follicle-stimulating hormone beta gene by gonadotropin-releasing hormone
J. Biol. Chem
(2004) - et al.
The gonadotropin releasing hormone (GnRH) receptor activating sequence (GRAS) is a composite regulatory element that interacts with multiple classes of transcription factors including Smads, AP-1 and a forkhead DNA binding protein
Mol. Cell. Endocrinol
(2003) - et al.
Activation of the ovalbumin gene by the estrogen receptor involves the fos-jun complex
Cell
(1990) - et al.
Mechanisms involved in the homologous down-regulation of transcription of the follicle-stimulating hormone receptor gene in Sertoli cells
Mol. Cell. Endocrinol
(2001) - et al.
Estrogen receptor binding to DNA is not required for its activity through the nonclassical AP1 pathway
J. Biol. Chem
(2001) - et al.
Pleiotropic effects of a null mutation in the c-fos proto-oncogene
Cell
(1992) - et al.
Estrogen receptor pathways to AP-1
J. Steroid Biochem. Mol. Biol
(2000) - et al.
Gonadotropin regulation of c-fos and c-jun messenger ribonucleic acids in cultured rat granulosa cells
Mol. Cell. Endocrinol
(1992)
Direct binding of AP-1 (Fos/Jun) proteins to a SMAD binding element facilitates both GnRH- and activin-mediated transcriptional activation of the mouse GnRH receptor gene
J. Biol. Chem
Gonadotropin-releasing hormone receptor-coupled gene network organization
J. Biol. Chem
Expression of the proto-oncogene c-fos and the immunolocalization of c-fos, phosphorylated c-fos and estrogen receptor beta in the human testis
Histol. Histopathol
Differential stability of mRNAs coding for alpha and gonadotropin beta subunits in cultured rat pituitary cells
Mol. Cell. Endocrinol
Coordinate actions of calcium and protein kinase-C in the expression of primary response genes in pituitary gonadotrophs
Endocrinology
Synchronous activation of gonadotropin-releasing hormone gene transcription and secretion by pulsatile kisspeptin stimulation
PNAS
Pro-opiomelanocortin messenger RNA in hypothalamic neurons is increased by testosterone through aromatization to estradiol
Neuroendocrinology
Kisspeptin-GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge
J. Neurosci
p38 mitogen-activated kinase is critical for synergistic induction of the FSH beta gene by gonadotropin-releasing hormone and activin through augmentation of c-Fos induction and Smad phosphorylation
Mol. Endocrinol
Differential gene expression of granulosa cells after ovarian superstimulation in beef cattle
Reproduction
Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor alpha in kisspeptin neurons
Endocrinology
GnRH induces the c-Fos gene via phosphorylation of SRF by the calcium/calmodulin kinase II pathway
Mol. Endocrinol
Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene
PNAS
Impaired long-term memory and NR2A-type NMDA receptor-dependent synaptic plasticity in mice lacking c-Fos in the CNS
J. Neurosci
Patterns of gonadotrophin secretion associated with ovulation
Fed. Proc
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Present address: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.