Elsevier

Molecular and Cellular Endocrinology

Volume 411, 15 August 2015, Pages 223-231
Molecular and Cellular Endocrinology

Gonadotropin and kisspeptin gene expression, but not GnRH, are impaired in cFOS deficient mice

https://doi.org/10.1016/j.mce.2015.04.033Get rights and content

Highlights

  • cFOS is necessary for gonadotropin-beta gene expression in vivo.

  • cFOS is superfluous for GnRH neuronal migration, cell number, and mRNA levels.

  • cFOS is unnecessary for granulosa cell proliferation or steroidogenesis.

  • cFOS is essential for antral follicle development and spermiogenesis.

  • cFOS is indispensable for normal kisspeptin neuron number in females in vivo.

Abstract

cFOS is a pleiotropic transcription factor, which binds to the AP1 site in the promoter of target genes. In the pituitary gonadotropes, cFOS mediates induction of FSHβ and GnRH receptor genes. Herein, we analyzed reproductive function in the cFOS-deficient mice to determine its role in vivo. In the pituitary cFOS is necessary for gonadotropin subunit expression, while TSHβ is unaffected. Additionally, cFOS null animals have the same sex-steroid levels, although gametogenesis is impeded. In the brain, cFOS is not necessary for GnRH neuronal migration, axon targeting, cell number, or mRNA levels. Conversely, cFOS nulls, particularly females, have decreased Kiss1 neuron numbers and lower Kiss1 mRNA levels. Collectively, our novel findings suggest that cFOS plays a cell-specific role at multiple levels of the hypothalamic–pituitary–gonadal axis, affecting gonadotropes but not thyrotropes in the pituitary, and kisspeptin neurons but not GnRH neurons in the hypothalamus, thereby contributing to the overall control of reproduction.

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.

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