Discussion
Our data demonstrate that photoperiodic responses driving gonadal activation can be modified by negative energy balance. Food scarcity seems to act in part via the pars tuberalis to downregulate local levels of TSH, which leads in turn to suppression of gonadal growth, especially in Common voles. Tundra vole males additionally seem to use the hypothalamic Kiss1 system to control reproduction when food is scarce at long photoperiods. Kiss1 is one of the main drivers for GnRH neuron activation (De Roux et al. 2003; Seminara et al.2004; Han et al. 2005, 2015). It is therefore surprising that observed patterns in Kiss1 expression are not reflected in GnRH expression. Although, reproductive organ mass in Tundra vole females is reduced at high workloads, no effects at the level of candidate genes have been observed. In general, low temperature enhances the inhibitory effects of reduced energy intake on gonadal size. Within the hypothalamus we show that reduced Rfrp3 levels may cause decreased reproductive organ mass observed at low temperature.
Here, we chose to investigate the reproductive effects of a negative energy balance in young animals, since voles can reach sexual maturity within 40 days depending on environmental conditions. A negative energy balance under long photoperiod exerts similar effects on testis size of Common (Fig. 1A) and Tundra voles (Fig. 1D) as in Deer mice,Peromyscus maniculatus (Nelson et al. 1997). The lack of this effect in Siberian hamsters, Phodopus sungorus , may be explained by the fact that food was minimally reduced to 80-90% ofad libitum in this study (Paul et al. 2009). Photoperiod seems to be the driving factor for gonadal development in animals under positive or neutral energy balance, or even with a moderate negative energy balance. A further reduction in food intake counteracts the stimulating effects of long photoperiods on gonadal development, leading to small testes, ovaries and uterus (Fig. 1). Although, large testes are generally associated with high spermatogenic activity and high androgen levels, vole testicular weight may drop in summer while spermatogenic activity remains high (Adams et al. 1980; Wang et al.2019). Therefore, testicular weight is a reliable indicator for fertility in spring, but less so in summer. Here, voles were exposed to spring photoperiod transitions, therefore, we assume that in our study testes mass is a reliable predictor for fertility. Temperature did not affect testicular weight under long photoperiods when food is availablead libitum (Fig. 1A,D). This finding is consistent with prior reports in Siberian hamsters (Steinlechner et al. 1991) and Prairie voles, Microtus ochrogaster (Nelson et al.1989).
Lowering ambient temperature under high feeding related workloads further increases metabolic demands in females, as confirmed by reduced uterine size (Fig. 1C,F). Ovaries of Syrian hamsters (Mesocricetus auratus ) at low temperature or in the absence of light did not change in size, but had fewer follicles and corpora lutea (Reiter 1968). This indicates that ovary mass is a bad indicator for hormonal secretory activity. Here, we did not perform histological analysis on ovaries, therefore, this data should be interpreted with caution. On the other hand, small uteri at low temperature are related to reduced height of secretory epithelium and the number of endometrial glands (Reiter 1968). This confirms that the decline in uterine weight at high workload and low temperature, is the result of incomplete development of uterine glands. This may lead to infertility, since uterine glands are essential for pregnancy (Cooke et al. 2013).
Energetically challenged voles do not enter torpor, as observed in house mice (Hut et al. 2011), but average body temperature is decreased by ~0.5°C, yielding limited energy savings (Nieminenet al. 2013; van der Vinne et al. 2015; van Rosmalen and Hut, submitted). This results in reduced reproductive investment, because all ingested energy is needed for maintaining organ function crucial to survive. Reproductive organ development may persist as ambient temperature and food resources are sufficient for lactation and pup growth.
Our data show that hypothalamic gene expression is related to modify the PNES axis under energetically challenging conditions to reduce gonadal activation. The short duration of pineal melatonin release under long photoperiods lead to increased pars tuberalis Tshβ , which serves a pivotal role in the PNES (Hanon et al. 2008; Ono et al.2008). The present study reveals that the photoperiodic inducedTshβ signal can be downregulated by a negative energy balance in Common vole males at both temperatures, Common vole females only at 10°C and in Tundra vole males at both temperatures (Fig. 2A,B,C). Thus, reduced food availability decreases Tshβ mRNA at the level of the pars tuberalis, either by decreasing transcription or by increasing post-transcriptional processes. This indicates that a negative energy balance can modify photoperiodic responses at the level of the pars tuberalis or even more upstream in the photoperiodic-axis, primarily in common voles.
Pars tuberalis derived TSH binds locally to TSH receptors (TSHr) in the tanycytes, where it systematically leads to increased DIO2 (Hanonet al. 2008; Nakao et al. 2008; Guerra et al.2010). The observed Tshβ suppression caused by a negative energy balance is not reflected in tanycyte Dio2 expression (Fig. 2A-D). On one hand, this suggests that TSH modulates central T3levels and ultimately gonadal development, via pathways parallel to the DIO2/DIO3 system. On the other hand, sex steroid feedback on gene expression in the tanycytes, but not in the pars tuberalis, as observed in ewes, could provide an explanation for unaltered Dio2 levels (Lomet et al. 2020). The two-fold higher Dio2 levels in this study compared to our previous experiments might be explained by the fact that here animals were born at SP and transferred to LP at weaning, whereas our previous study used constant LP conditions (van Rosmalen et al. 2020). This effect of maternal photoperiodic programming on tanycyte gene expression has previously been confirmed (Sáenz de Miera et al., 2017; van Rosmalen et al., in preparation). In addition, two to three-day fasted rats show elevated Dio2 mRNA levels in tanycytes (Diano et al. 1998; Coppola et al.2005). This might be an acute effect, which disappears when food is restricted for longer periods as in our study (i.e. 35 days). StableTshβ and Dio2 levels at different temperatures at low workload under long photoperiods in spring programmed voles are confirmed by in situ hybridization in our prior experiments (van Rosmalen et al., in preparation). This indicates that our brain dissections in combination with RT-qPCR are a reliable method to assess gene expression at the level of the pars tuberalis and the tanycytes.
At the level of the posterior hypothalamus, where the DMH/VMH are located, low temperature induced a small reduction in Rfrp3 (i.e.Npvf ) expression, but consistent with Siberian hamsters (Paulet al. 2009), no effect of food scarcity was detected (Fig. 2A-D). In seasonal rodents, Rfrp3 synthesis appeared to be primarily regulated by photoperiod (for review, see Angelopoulou et al., 2019), but here we show that Rfrp3 may also be an important regulator of the PNES to adaptively respond to ambient temperature changes. This finding is consistent with previous reports, showing thatRfrp3 is a hypothalamic biomarker of ambient temperature, independent of nutritional status in mice (Jaroslawska et al.2015). Moreover, our findings are consistent with a field study in wild Brandt’s voles, Lasiopodomys brandtii , in which elevatedRfrp3 levels were observed during the warmest part of the year (June-August) (Wang et al. 2019). Since RFRP3 is expected to mediate reproductive-axis function, our findings suggest that downregulation of Rfrp3 by low temperature may be responsible for decreased reproductive organ mass observed at low temperature under high feeding related workloads (Fig. 1A-F).
We next focused on the hypothalamic Kiss1-system, because of its potential role in the integration of photoperiodic and metabolic cues controlling reproductive activity (Caro et al. 2013; Hut et al. 2014; Simonneaux 2020). Hypothalamic Kisspeptin neurons act on GnRH neurons driving gonadotropin release which promotes gonadal development (for review, see Simonneaux, 2020). Kiss1 expression in the posterior hypothalamus, where the ARC is located, was not affected by either food or temperature (Fig. 2A-D). ARC kiss1 expression can be reversed by strong negative sex steroid feedback (Greives et al. 2008; Sáenz De Miera et al. 2014; Rasri-Klosen et al.2017), which may explain similar Kiss1 and Gnrh levels in different experimental groups (Fig. 2). In Siberian hamsters, food restriction causes a decrease in ARC Kiss1 expression (Paulet al. 2009). Since whole coronal sections were used in the study of Paul et al. 2009, thalamic and cortical areas contribute to the detected Kiss1 signal, whereas we exclusively used hypothalamic tissue.
It seems important to note that the role of Kisspeptin in PNES regulation may be less generalizable over different species. For instance, reversed photoperiodic effects on ARC Kiss1 expression have been shown in Syrian versus Siberian hamsters (Klosen et al.2013). Furthermore, hypothalamic Kiss1 expression in Common voles is extremely low (Fig. 2A, B, E, F). These findings suggest that Kisspeptin systems have species-specific functions in regulating reproduction. Although Kiss1 in the posterior hypothalamus was not affected, food scarcity caused downregulation of Kiss1expression in the anterior hypothalamus of Tundra vole males (Fig. 2G). Although, this effect may be caused by positive sex steroid feedback on POA Kiss1 expression (Ansel et al. 2010), direct effects of metabolic cues cannot be excluded. Contrary to our expectations, anterior hypothalamic Kiss1 was not affected by temperature. Common voles exhibit extremely low Kiss1 levels under all conditions. This indicates that the Kiss1-system is not involved in metabolic neuroendocrine control of reproduction in Common voles and it is conceivable that Rfrp3 in this species has taken over the role of Kiss1 . Interestingly, Tundra voles have a functional Kiss-system, which may explain the greater reduction in reproductive organ mass at a negative energy balance. It would be important to investigate how gene-silencing and overexpression (Tshβ ,Kiss1 and Rfrp3 ) in specific hypothalamic regions may affect reproductive development, to disentangle causal relationships between hypothalamic gene expression and reproductive responses related to energy balance.
Differences in responses to food scarcity between common and tundra voles, suggest that tundra voles use food as a more important external cue to time reproduction. This seems to be in line with the hypothesis that tundra voles may use an opportunistic breeding strategy, while common voles use breeding strategy that is more driven by photoperiod (van Rosmalen et al. 2020). At northern latitudes, where tundra voles are abundant, voles live for a large part of the year under snow covers where light input is blocked. Reproducing whenever food is available, either as leaves in summer and autumn or as roots during winter and spring, may be a favorable breeding strategy in such environments.
Our findings show that a negative energy balance induced by food scarcity and ambient temperature, modifies long day responses in the PNES: In general, food scarcity regulates the photoperiodic regulatedTshβ response in the pars tuberalis (primarily in common voles), and the Kiss1 response in the anterior hypothalamus (exclusively in tundra vole males), whereas temperature regulates Rfrp3 in the posterior hypothalamus. Shutting off the photoperiodic-axis when food is scarce or temperatures are low, is an adaptive response that favors individual somatic maintenance and survival at the expense of reproductive organ development and current reproductive output. In addition, delaying reproductive onset will yield energy savings, which results in less required foraging time and reduced exposure to predation, which will further increase individual survival and the probability of successful future reproductive attempts (van der Vinneet al. 2019). Defining the mechanisms through which metabolic cues modify photoperiodic responses will be important for a better understanding of how annual cycling environmental cues shape reproductive function and plasticity in life history strategies.