Response to exogenous kisspeptin varies according to sex and reproductive condition in Siberian hamsters (Phodopus sungorus)

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Abstract

Most animals experience marked changes in reproductive status across development that are regulated by changes in the hypothalamo–pituitary–gonadal (HPG) axis. The upstream mechanisms regulating this axis remain less well understood. The neuropeptide kisspeptin serves as a positive regulator of reproduction; the precise actions of kisspeptin on the HPG axis in animals of differing developmental and seasonal reproductive states, however, remain unresolved. Further, sex differences in response to kisspeptin have not been fully explored. In Experiment 1, we investigated whether sensitivity to a broad range of kisspeptin doses differed in adult male and female Siberian hamsters held on reproductively inhibitory or stimulatory photoperiods. In Experiment 2, we asked whether the response to kisspeptin differed across stages of reproductive development. Males and females displayed elevated luteinizing hormone (LH) in response to kisspeptin; however, the sexes differed in this response, with males showing greater LH responses to kisspeptin than females. Hamsters responded to kisspeptin across all stages of reproductive development, although the magnitude of this response differed between animals of differental ages and between the sexes. Males showed significant increases in LH at an earlier developmental age than females; females also showed blunted LH responses during early adulthood whereas males remained relatively constant in their response to kisspeptin. These findings suggest that reproductively active and inactive hamsters are responsive to kisspeptin, but that the sexes differ in their responsiveness. Collectively, these data provide further insight into the basic actions of kisspeptin in the regulation of reproduction and provide a potential mechanism for the regulation of differential reproductive responses between the sexes.

Research highlights

► Reproductively responsive and non-responsive hamsters demonstrate comparable luteinizing hormone (LH) responses to injections of exogenous kisspeptin. ► The magnitude of the LH response differed depending on the age of the animals. ► Sex differences were observed in response to kisspeptin that varied depending on reproductive condition of the animals. ► These findings suggest a modest sex difference in the sensitivity to kisspeptin that is dependent on the reproductive state of the animals.

Introduction

All vertebrates experience marked changes in reproductive physiology during the developmental transitions from a sexually immature, pre-pubertal state to a post-pubertal, reproductively-active state. In addition to these relatively permanent developmental changes in reproductive status, seasonally breeding animals must also transition between reproductively active and inactive states on an annual basis. These seasonal changes in reproduction have previously been likened to a form of “seasonal puberty” (Ebling and Foster, 1990).

Changes in the activity of the hypothalamo–pituitary–gonadal (HPG) axis are responsible for regulating both pubertal and seasonal transitions in reproductive function. The fundamental role that the hypothalamic hormone gonadotropin-releasing hormone (GnRH) plays in regulating reproduction and fertility has been unequivocally established (Herbison, 2005, Levine, 2003). Most animals do not display continuous reproduction, however, and must encode and integrate salient internal and environmental signals (e.g., energy stores, photoperiod), and subsequently adjust GnRH release to regulate seasonal reproductive timing (Baker, 1938, Bronson, 1989, Ebling, 2005, Wingfield, 2008). Specifically, most rodents respond to short “winter-like” day lengths by down-regulating HPG activity and inhibiting reproduction; reproductive function is restored after prolonged exposure to long “summer-like” day lengths. The upstream mechanisms that integrate these signals to regulate HPG activity, however, are less well understood. Further, it remains unclear whether such regulatory mechanisms act in a similar manner during both developmental and seasonal reproductive transitions (Clarke and Pompolo, 2005, Ebling and Foster, 1990, Lehman et al., 1997). Seasonally breeding animals, therefore, serve as an excellent model system to address these questions (Ebling and Cronin, 2000, Ebling and Foster, 1990).

Recently, the neuropeptide kisspeptin has been identified as a potential regulator of both developmental and photoperiodic changes in reproduction (Caraty et al., 2007, de Roux et al., 2003, Funes et al., 2003, Greives et al., 2007, Mason et al., 2007, Revel et al., 2006, Seminara et al., 2003, Shahab et al., 2005, Smith et al., 2007). Kisspeptin acts as a potent positive regulator of GnRH release in all mammals studied to date and is the endogenous ligand for the kisspeptin receptor (Kiss1R)(previously called GPR54) (Gottsch et al., 2009). The importance of kisspeptin in normal reproductive maturation has been demonstrated by the observation that mutations in either the Kiss1 gene or the gene for its cognate receptor Kiss1R render the animal unable to reach puberty (d’Anglemont de Tassigny et al., 2007, de Roux et al., 2003, Funes et al., 2003); animals expressing these mutations display pre-pubertal reproductive morphology and physiology for the remainder of their lives. Additionally, in non-human primates and rodents, Kiss1 gene expression and kisspeptin protein are up-regulated in the hypothalamus during reproductive pubertal development (Clarkson and Herbison, 2006, Han et al., 2005, Keen et al., 2008, Navarro et al., 2004, Shahab et al., 2005). Furthermore, kisspeptin content in the hypothalamus of rodents changes in response to manipulations of sex steroids as well as changes in photoperiod in seasonally breeding rodents (Greives et al., 2008a, Greives et al., 2007, Mason et al., 2007, Revel et al., 2006, Smith et al., 2007, Smith et al., 2008, Smith et al., 2005a, Smith et al., 2005b). These findings highlight a potential role for kisspeptin to serve as an integrative signal to the HPG axis; kisspeptin responds to relevant internal and environmental signals and alters activity of the HPG axis.

Although kisspeptin has been shown to regulate GnRH release in mammals (Clarke et al., 2009, Crown et al., 2007, Fernandez-Fernandez et al., 2006, Greives et al., 2008b, Herbison, 2007, Kriegsfeld, 2006), its basic functions are still being determined. For example, it remains unclear whether kisspeptin activates the HPG axis similarly in developmentally non-reproductive (i.e., pre-pubertal) animals as it does in seasonally non-reproductive (i.e., short-day) animals (Caraty et al., 2007, Greives et al., 2007, Messager et al., 2005), and whether kisspeptin acts in a similar fashion in both sexes, where the costs of activating or maintaining reproductive physiology may differ substantially (Ball and Ketterson, 2008). Interestingly, Kiss1 gene expression differs between male and female rats in one hypothalamic nucleus, the anteroventral periventricular nucleus (AVPV) (Kauffman et al., 2007), and female Siberian hamsters display reduced levels of the pituitary gonadotropin luteinizing hormone (LH) compared with males following repeated injections of a single dose of kisspeptin (Greives et al., 2007, Mason et al., 2007). These findings support the idea that kisspeptin and its downstream effects may differ between the sexes in certain contexts.

In the current study, we examined the ability of the HPG axis of male and female Siberian hamsters (Phodopus sungorus) to respond to an injection of exogenous kisspeptin in differing photoperiod-induced reproductive states (Experiment 1) and at different time points across reproductive development (Experiment 2). Additionally, in both experiments we investigated potential sex differences in sensitivity to kisspeptin. By comparing the actions of kisspeptin across reproductive conditions, this study will help elucidate the potential role of kisspeptin as a key mechanism regulating activity of the GnRH neuronal system.

Section snippets

Animals and housing

All animals were obtained from a breeding colony maintained at Indiana University. All animals were group-housed at weaning with same-sex siblings in a long-day photoperiod (L:D 16:8). Breeders and pre-weaned offspring were housed together in large polypropylene cages (45 × 23 × 15 cm) until weaning at 18 days of age; weaned and adult individually housed animals were housed in smaller polypropylene cages (27.8 × 17.5 × 13.0 cm). Temperature was kept constant at 20 ± 2 °C and relative humidity was maintained

Experiment 1: effect of photoperiod on HPG axis sensitivity to exogenous kisspeptin

The GLM revealed significant main effects of sex (F1,154 = 15.80, p  0.001), injection dose (F4,154 = 40.95, p  0.001) and photoperiod (F1,154 = 8.51, p  0.01). A significant interaction between injection dose and sex was also revealed (F4,154 = 4.35, p  0.01); all other interactions were not significant (p > 0.05 in all cases) (Fig. 1). To probe the above effects, separate ANOVAs were performed with the sexes split to investigate the effect of differing doses and photoperiod and their interaction on

Discussion

Overall, significant activation of the reproductive neuroendocrine axis (as measured by serum LH) in response to kisspeptin injections was observed in both long- and short-day hamsters and across all stages of reproductive development; however, the magnitude of this response differed depending on the age of the animals. Additionally, sex differences were observed in response to kisspeptin; males and females displayed different patterns of LH responses to an intermediate dose of kisspeptin

Acknowledgments

The authors thank Drs. Melissa-Ann Scotti and Lance Kriegsfeld for assistance and feedback on the study design and interpretation. The authors also wish to thank Drs. Ellen Ketterson and Devin Zysling, and Emily Chester and Jacqueline Ho for comments and discussion on earlier presentations of the data, and Nick Garcia, Stefanie Frommeyer, and Jill Lodde for assistance. This work was supported by a Society for Integrative and Comparative Biology Grant-in-Aid and NIH/T32 training grant HD049336-0

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