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; S. K. Han et al., 2005; S. Y.
Han, McLennan, Czieselsky, & Herbison, 2015; Seminara et al., 2004). It
is therefore surprising that observed patterns in Kiss1expression 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 reducedRfrp3 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 (Randy J. Nelson, Marinovic, Moffatt,
Kriegsfeld, & Kim, 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% of ad libitum in this study (Paul,
Pyter, Freeman, Galang, & Prendergast, 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, Tamarin, & Callard, 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, Stieglitz, Ruf, Heldmaier,
& Reiter, 1991) and Prairie voles, Microtus ochrogaster (R J
Nelson, Frank, Smale, & Willoughby, 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, Spencer, Bartol, & Hayashi, 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, in review). 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 (Guerra et
al., 2010; Hanon et al., 2008; Nakao et al., 2008). The observedTshβ 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 T3 levels 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, Druart,
Hazlerigg, Beltramo, & Dardente, 2020). The two-fold higher Dio2levels 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 (Coppola, Meli, & Diano, 2005; Diano, Naftolin,
Goglia, & Horvath, 1998). This might be an acute effect, which
disappears when food is restricted for longer periods as in our study
(i.e. 35 days). Stable Tshβ 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 (Paul et
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 that Rfrp3is a hypothalamic biomarker of ambient temperature, independent of
nutritional status in mice (Jaroslawska, Chabowska-Kita, Kaczmarek, &
Kozak, 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 ofRfrp3 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;
Rasri-Klosen, Simonneaux, & Klosen, 2017; Sáenz De Miera et al., 2014),
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 (Paul et
al., 2009). Since whole coronal sections were used in the study of Paul
et al. 2009, thalamic and cortical areas contribute to the detectedKiss1 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 (Vincent van der
Vinne et 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.