Gonads or body? Differences in gonadal and somatic photoperiodic growth response in two vole species

Organisms use intrinsic annual timing mechanisms to adaptively prepare behavior, physiology and morphology for the upcoming season. In temperate regions, decreased ambient temperature is associated with reduced food availability during winter, which will impose increased energetic challenges that may, dependent on the species, prevent the possibility of successfully raising offspring. Annual variation in ambient temperature shows large fluctuations between years, with considerable day-to-day variations, whereas annual changes in photoperiod provide a consistent year-on-year signal for annual phase. This has led to convergent evolutionary processes in many organisms to use day length as the most reliable cue for seasonal adaptations.

In mammals, the photoperiodic neuroendocrine system (PNES) measures photoperiod and subsequently drives annual rhythms in physiology and reproduction (Fig. 1) (for review, see Dardente et al., 2018; Hut, 2011; Nakane and Yoshimura, 2019). The neuroanatomy of this mechanism has been mapped in detail, and genes and promoter elements that play a crucial role in this response pathway have been identified in several mammalian species (Dardente et al., 2010; Hanon et al., 2008; Hut, 2011; Masumoto et al., 2010; Nakao et al., 2008; Ono et al., 2008; Sáenz De Miera et al., 2014; Wood et al., 2015), including the common vole (Król et al., 2012).

Voles are small grass-eating rodents with a short gestation time (i.e. 21 days). They can have several litters a year, while their offspring can reach sexual maturity within 40 days during spring and summer. Overwintering voles may, however, delay reproductive activity by as much as 7 months (Wang et al., 2019). In small rodents, photoperiods experienced early in development determine growth rate and reproductive development. Photoperiodic reactions to intermediate day lengths depend on prior photoperiodic exposure (Hoffmann, 1978; Horton, 1984, 1985; Horton and Stetson, 1992; Prendergast et al., 2000; Sáenz de Miera et al., 2017; Stetson et al., 1986; Yellon and Goldman, 1984). By using information about day length early in life, young animals will be prepared for the upcoming season. Presumably, crucial photoperiod-dependent steps in PNES development take place in young animals to secure an appropriate seasonal response later in life (Sáenz de Miera et al., 2017, 2020; Sáenz De Miera, 2019; van Dalum et al., 2020). In Siberian hamsters, photoperiodic programming takes place downstream of melatonin secretion at the level of Tshr, with expression increased in animals born under short photoperiod (SP), associated with subsequent increases in thyroid-stimulating hormone (TSH) sensitivity (Sáenz de Miera et al., 2017).

Primary production in the food web of terrestrial ecosystems is temperature dependent (Robson, 1967; Peacock, 1976; Malyshev et al., 2014). Small herbivores may therefore show reproductive development either as a direct response to temperature increases (opportunistic response), or as a response to photoperiod that forms an annual proxy for seasonal temperature changes (photoperiodic response), or a combination of the two (Caro et al., 2013). Microtus species adjust their photoperiodic response such that reproduction in spring starts when primary food production starts (Baker, 1938).

Photoperiodically induced reproduction should start at longer photoperiods in more northern populations, as a specific ambient spring temperature at higher latitudes coincides with longer photoperiods compared with lower latitudes (Hut et al., 2013). To adapt to late spring arrival at northern latitudes, a lower sensitivity to photoperiod, and therefore a longer critical photoperiod, is expected to be necessary in northern species. This is crucial to arrest reproductive development until arrival of spring. Moreover, (epi)genetic adaptation to local annual environmental changes may create latitudinal differences in photoperiodic responses and annual timing mechanisms.

Microtus is a genus of voles found in the northern hemisphere, ranging from close to the equator to arctic regions, which makes it an excellent genus to study latitudinal adaptation of photoperiodic responses (for review, see Hut et al., 2013). In order to understand the development of the PNES for vole species with different paleogeographic origins, we investigated photoperiodic responses at different time points during development by exposing northern [tundra or root vole, Microtus oeconomus (Pallas 1776)] and southern vole species [common vole, Microtus arvalis (Pallas 1778)] to constant short or long photoperiods in the laboratory. Animals from our two vole laboratory populations originate from the same latitude in the Netherlands (53°N) where both populations overlap. This is for the common vole the center (mid-latitude) of its distribution range (38–62°N), while our laboratory tundra voles originate from a postglacial relict population at the southern boundary of its European geographical range (48–72°N). Assuming that the latitudinal distribution range is limited by seasonal adaptation, it is expected that latitudinal adaptation is optimal at the center of the distribution and suboptimal towards the northern and southern boundaries. Although this assumption remains to be confirmed at genetic and physiological levels, it does lead to the expectation that the PNES of the common vole is better adapted to the local annual environmental changes of the Netherlands (53°N, distribution center) than that of the tundra vole which is at its southern distribution boundary. Because lower latitudes have higher spring temperatures at a specific photoperiod (Hut et al., 2013), we hypothesize that gonadal activation through PNES signaling occurs under shorter photoperiods in common voles than in tundra voles.

All experimental procedures were carried out according to the guidelines of the animal welfare body (IvD) of the University of Groningen, and all experiments were approved by the Centrale Commissie Dierproeven of the Netherlands (CCD, license number: AVD1050020171566). The Groningen common vole breeding colony started with voles (M. arvalis) obtained from the Lauwersmeer area (Netherlands, 53°24′N, 6°16′E) (Gerkema et al., 1993), and was occasionally supplemented with wild caught voles from the same region to prevent the laboratory population from inbreeding. The Groningen tundra vole colony started with voles (M. oeconomus) obtained from four different regions in the Netherlands (described in Van de Zande et al., 2000). Both breeding colonies were maintained at the University of Groningen as outbred colonies and provided the voles for this study. All breeding pairs were kept in climate-controlled rooms, at an ambient temperature of 21±1°C and 55±5% relative humidity and housed in transparent plastic cages (15 cm×40 cm×24 cm) provided with sawdust, dried hay, an opaque PVC tube and ad libitum water and food (standard rodent chow, no. 141005; Altromin International, Lage, Germany). Over the last 4 years, our captive laboratory populations are housed under long photoperiod (LP) conditions (16 h:8 h light:dark) and switched to SP (8 h:16 h light:dark) for ∼2 months at least twice a year.

The voles used in the experiments (61 males, 56 females) were both gestated and born under either LP or SP. In the center of the distribution range of M. arvalis, 16 h:8 h light:dark in spring occurs on 17 May, and 8 h:16 h light:dark occurs on 13 January. In the center of the distribution range of M. oeconomus, 16 h:8 h light:dark in spring occurs on 1 May, and 8 h:16 h light:dark occurs on 1 February. Maximum and minimum photoperiods experienced by M. arvalis and M. oeconomus at the center of their distributional ranges are 17 h:7 h and 7.5 h:16.5 h light:dark, and 19 h:5 h and 6 h:18 h light:dark, respectively. Pups were weaned and transferred to individual cages (15 cm×40 cm×24 cm) when 21 days old but remained exposed to the same photoperiod as during both gestation and birth. All voles were weighed at post-natal days 7, 15, 21, 30, 42 and 50 (Fig. 2).

In order to follow development, animals were killed by decapitation, with prior sedation by CO₂, 17±1 h after lights off (Tshβ expression peaking in pars tuberalis) (Masumoto et al., 2010), at 15, 21, 30 and 50 days old. Brains were removed with great care to include the stalk of the pituitary containing the pars tuberalis. The hypothalamus with the pars tuberalis was dissected as described in Prendergast et al. (2013), the optic chiasm at the anterior border, and the mammillary bodies at the posterior border, and laterally at the hypothalamic sulci. The remaining hypothalamic block was cut dorsally 3–4 mm from the ventral surface. The extracted hypothalamic tissue was flash-frozen in liquid N₂ and stored at −80°C until RNA extraction. Reproductive organs were dissected and cleaned of fat, and wet masses of paired testis, paired ovary and uterus were measured (±0.0001 g).

Total RNA was isolated from the dissected part of the hypothalamus using TRIzol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). In short, frozen pieces of tissue (∼0.02 g) were homogenized in 0.5 ml TRIzol reagent in a TissueLyser II (Qiagen, Hilden, Germany) (2×2 min at 30 Hz) using tubes containing a 5 mm RNase free stainless-steel bead. Subsequently, 0.1 ml chloroform was added for phase separation. Following RNA precipitation by 0.25 ml of 100% isopropanol, the obtained pellet was washed with 0.5 ml of 75% ethanol. Depending on the size, RNA pellets were diluted in an adequate volume of RNase-free H₂O (range 20–50 µl) and quantified on a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA concentrations were between 109 and 3421 ng µl⁻¹ and ratio of the absorbance at 260/280 nm was between 1.62 and 2.04. After DNA removal by DNase I treatment (Invitrogen), an equal quantity of RNA from each sample was used for cDNA synthesis using RevertAid H minus first-strand cDNA synthesis reagents (Thermo Scientific). Reverse transcription (RT; 40 µl) reactions were prepared using 2 µg RNA, 100 µmol l⁻¹ Oligo(dT)₁₈, 5× reaction buffer, 20 U µl⁻¹ RiboLock RNase Inhibitor, 10 mmol l⁻¹ dNTP Mix and RevertAid H Minus Reverse Transcriptase (200 U µl⁻¹). Concentrations used for RT reactions can be found in the supplementary information (Table S1). RNA was reversed transcribed using a thermal cycler (S1000; Bio-Rad, Hercules, CA, USA). Incubation conditions used for RT were: 45°C for 60 min followed by 70°C for 5 min. Transcript levels were quantified by real-time qPCR using SYBR Green (KAPA SYBR FAST qPCR Master Mix, Kapa Biosystems). Twenty microliter (2 μl cDNA +18 μl Mastermix) reactions were carried out in duplo for each sample using 96-well plates in a Fast Real-Time PCR System (CFX96, Bio-Rad). Primers for genes of interest were designed using Primer-BLAST (NCBI) and optimized annealing temperature (T_m) and primer concentration. All primers used in this study were designed based on the annotated Microtus ochrogaster genome (NCBI:txid79684, GCA_000317375.1), and subsequently checked for gene specificity in the genomes of the common vole (Microtus arvalis) and the tundra vole (Microtus oeconomus), which were published by us on NCBI (NCBI:txid47230, GCA_007455615.1 and NCBI:txid64717, GCA_007455595.1) (Table S2). Thermal cycling conditions used can be found in the Table S3. Relative mRNA expression levels were calculated based on the ΔΔCT method using Gapdh as the reference (housekeeping) gene (Pfaffl, 2001).

Sample size (N=4) was determined by a power calculation (α=0.05, power=0.80) based on the effect size (d=2.53) of an earlier study, in which gonadal mass was assessed in female voles under three different photoperiods (Król et al., 2012). Effects of age, photoperiod and species on body mass, reproductive organs and gene expression levels were determined using a type I two-way ANOVA. Tukey's honestly significant difference post hoc pairwise comparisons were used to compare groups at specific ages. Statistical significance was determined at P<0.05. Statistical results can be found in the Table S4. All statistical analyses were performed using RStudio (version 1.2.1335) (http://www.R-project.org/), and figures were generated using the ggplot2 package (Wickham, 2016).

Photoperiod during gestation did not affect birth weight in either species (Fig. 3A,B). Both tundra vole males and females grow faster under LP compared with SP conditions (males, F_1,303=15.0, P<0.001; females, F_1,307=10.2, P<0.01) (Fig. 3A,B). However, no effect of photoperiod on body mass over time was observed in common vole males or females (males, F_1,243=2.1, not significant (n.s.); females, F_1,234=0.6, n.s.) (Fig. 3A,B).

Common vole males show faster testis growth under LP compared with SP [testis, F_1,33=17.01, P<0.001; gonoadosomatic index (GSI), F_1,33=32.2, P<0.001] (Fig. 3C,E). This photoperiodic effect on testis development is less pronounced in tundra voles (testis, F_1,35=8.3, P<0.01; GSI, F_1,35=9.3, P<0.01) (Fig. 3C,E).

Common vole female gonadal mass (i.e. paired ovary+uterus) is slightly higher at the beginning of development (until 30 days old) under SP compared with LP conditions (F_1,17=10.4, P<0.01) (Fig. 3D), while the opposite effect was observed in tundra voles (F_1,36=9.0, P<0.01) (Fig. 3D). For both species, these photoperiodic effects disappeared when gonadal mass was corrected for body mass (common vole, F_1,17=2.5, n.s.; tundra vole, F_1,36=2.3, n.s.) (Fig. 3F). Interestingly, gonadal mass significantly increased in 30- to 50-day-old LP common vole females (F_1,5=7.7, P<0.05) (Fig. 3D), but not in tundra voles (F_1,11=2.2, n.s.) or under SP conditions (common vole, F_1,7=0, n.s.; tundra, F_1,7=1.0, n.s.).

Melatonin binds to its receptors in the pars tuberalis where it inhibits Tshβ expression. In males of both species, Mtnr1a (Mt1, melatonin receptor) expression in the hypothalamic block with preserved pars tuberalis was highly expressed, but unaffected by photoperiod or age (photoperiod, F_1,43=0.08, n.s.; age, F_3,42=0.94, n.s.) (Fig. 4A). In females, Mtnr1a expression increases approximately 2-fold with age in both species (F_3,40=9.04, P<0.001) (Fig. 4B), but no effects of photoperiod were observed (F_1,40=1.59, n.s.).

In males and females of both species, Tshβ expression is dramatically elevated under LP throughout development (tundra vole males, F_1,27=49.3, P<0.001; common vole males, F_1,27=21.3, P<0.001; tundra vole females, F_1,30=63.7, P<0.001; common vole females, F_1,22=60.9, P<0.001) (Fig. 4C,D). Furthermore, a clear peak in Tshβ expression is observed in 21-day-old LP common vole males, while such a peak is lacking in tundra vole males. However, Tshβ expression in tundra vole males remains similar over the course of development under LP conditions. In females, photoperiodic responses in Tshβ expression did not differ between species (F_1,40=0.02, n.s.).

TSHβ binds to its receptor (TSHr) in the tanycytes around the third ventricle. In tundra vole males and females, Tshr expression is higher under SP compared with LP (males, F_1,27=23.7, P<0.001; females, F_1,30=6.2, P<0.05) (Fig. 4E,F), while photoperiodic-induced changes in Tshr expression are smaller in common vole males and females (males, F_1,27=23.7, P<0.01; females, F_1,22=4.3, P<0.05) (Fig. 4E,F). Photoperiodic responses in Tshr expression are significantly larger in tundra vole males compared with common vole males (F_1,42=8.17, P<0.01) (Fig. 4E).

In males of both species, the largest photoperiodic effect on Dio2, which is increased by TSHβ, is found at weaning (day 21), with higher levels under LP compared with SP (F_1,42=14.7, P<0.001) (Fig. 4G). Interestingly, Dio3 is lower in these animals (F_1,42=4.8, P<0.05) (Fig. 4I), leading to a high Dio2:Dio3 ratio under LP at the beginning of development (F_1,42=8.5, P<0.01) (Fig. 4K). We found a similar pattern in females, with higher Dio2 under LP compared with SP at the beginning of development (i.e. day 15) (F_3,10=8.9, P<0.01) (Fig. 4H).

In males of both species, no effects of photoperiod on Eyes Absent 3 (Eya3, transcription factor for the Tshβ promoter) (F_1,42=1.72, n.s.), Kisspeptin (Kiss1, hypothalamic gene involved in reproduction) (F_1,42=2.96, n.s.) and Neuropeptide VF precursor (Npvf, Rfrp3, hypothalamic gene involved in seasonal growth and reproduction) (F_1,42=0.61, n.s.) expression were found (Fig. S1A,C,E). In females, both Kiss1 (F_3,40=4.82, P<0.01) and Npvf are higher under LP dependent on age (F_3,40=3.51, P<0.05) (Fig. S1D,F), but there were no effects of photoperiod on Eya3 (F_1,40=0.30, n.s.) (Fig. S1B).

A positive correlation between the levels of Tshβ and Dio2 expression was found only at the beginning of development (15 days, F_1,25=12.6, P<0.01; 21 days, F_1,28=4.0, P<0.1; 30 days, F_1,30=0.1, n.s.; 50 days, F_1,23=0.1, n.s.) (Fig. 5A–D). Moreover, no significant relationship between Dio2 and Dio3 expression was found (Fig. 5E–H).

This study demonstrates different effects of constant photoperiod on the PNES in two different vole species: the common vole and the tundra vole. Overall, somatic growth is photoperiodically sensitive in the tundra vole while gonadal growth is photoperiodically sensitive in the common vole. Hypothalamic Tshβ, Tshr, Dio2 and Dio3 expression are highly affected by photoperiod and age, and some species differences were observed in the magnitude of these effects. Although the differences found between both vole species may provide interesting information on variation in annual timing, the data should be interpreted with caution because we cannot exclude relaxation of natural selection in our laboratory colonies.

These data demonstrate that photoperiod early in life affects pup growth in the tundra vole (Fig. 3A), and reproductive development in common vole males (Fig. 3C,E). In females, a similar photoperiodic effect on somatic growth is observed as in males. Tundra vole females grow faster under LP compared with SP, while there is no difference in growth rate between LP and SP in the common vole (Fig. 3B). In the tundra vole, somatic growth is plastic, whereas in the common vole, gonadal growth is plastic. Garden dormouse (Eliomys quercinus) born late in the season grow and fatten twice as fast as early born animals (Stumpfel et al., 2017), in order to partly compensate for the limited time before winter onset. This overwintering strategy might be favorable for animals with a short breeding season (i.e. at high latitude), and may also be used in tundra voles as they gain weight faster when raised under LP (i.e. late in the season) compared with SP (i.e. early in the season). Southern arvicoline species have longer breeding seasons (Tkadlec, 2000), and therefore have more time left to compensate body mass when born late in the season. Therefore, somatic growth rate may depend to a lesser extent on the timing of birth in southern species as observed in common voles raised under SP or LP.

Common vole female gonadal mass is slightly higher under SP compared with LP at the beginning of development (Fig. 3D,F). In contrast, in Siberian hamsters, uterus mass is increased after 3 weeks of constant LP exposure, which continued throughout development (Ebling, 1994; Phalen et al., 2010). In common voles, female gonadal mass increased from day 30 to day 50 in LP animals, whereas gonadal mass in SP females remained the same (Fig. 3D,F). Also, tundra vole female gonadal mass is not increased in this period of development under both LP and SP conditions. Puberty onset, based on gonadal mass, in common voles is later compared with Siberian hamsters (Phalen et al., 2010), while earlier compared with tundra voles. Therefore, LP common voles increase gonadal mass earlier in development (i.e. >30 days old) compared with LP tundra voles (i.e. >50 days old), in order to increase reproductive activity and prepare for pregnancy. An alternative hypothesis is that the tundra vole may sense 16 h:8 h light:dark not as too short for spring stimulation of reproduction, but rather as too long to switch off reproduction in autumn. These results suggest that tundra vole females have a different reproductive onset compared with common vole females under constant photoperiods. However, based on our data we cannot conclude whether the timing of the breeding season is different between those species, as we did not use naturally changing photoperiods to simulate different seasons. This can be tested by exposing voles to a broader range of different photoperiod regimes, mimicking spring and autumn photoperiod conditions in the laboratory. Our data show that the common vole invests more energy in gonadal growth, whereas the tundra vole invests more energy in body mass growth independent of gonadal growth under LP. This suggests that both body mass growth and gonadal development are plastic and can be differentially affected by photoperiod, perhaps through different mechanisms. In Siberian hamsters, the growth hormone (GH) axis is involved in photoperiodic regulation of body mass (Dumbell et al., 2015; Scherbarth et al., 2015). Our results indicate a different role for the GH axis in seasonal body mass regulation in tundra voles and common voles.

Common vole males show a clear photoperiodic response in both hypothalamic gene expression and gonadal activation. Genes in the female PNES are strongly regulated by photoperiod, which is not reflected in gonadal growth. In tundra voles, PNES gene expression profiles change accordingly with photoperiod, although the gonadal response is less sensitive to photoperiod, which is similar to the photoperiodic response observed in house mice (Masumoto et al., 2010). Because the tundra vole is more common at high latitudes, where they live in tunnels covered by snow in winter and early spring, photoperiodic information might be blocked during a large part of the year for these animals (Evernden and Fuller, 1972; Korslund, 2006). For this reason, other environmental cues, such as metabolic status, may integrate in the PNES in order to regulate the gonadal response and therefore timing of reproduction.

In both vole species, Tshβ expression is higher under LP conditions during all stages of development (Fig. 4C,D), which is in agreement with previous studies in other mammals, birds and fish (for review, see Dardente et al., 2014; Nakane and Yoshimura, 2019). We sampled 17 h after lights off, when Tshβ expression is peaking. EYA3 is a transcription factor that binds to the Tshβ promoter, which promotes transcription. Perhaps we sampled too late in order to find photoperiodic-induced changes in Eya3 expression (Fig. S1A,B), as in mice Eya3 peaks 12 h after lights off under LP conditions (Masumoto et al., 2010).

TSH binds to its receptor in the tanycytes around the third ventricle. Although less pronounced in common voles, elevated Tshr expression under SP (Fig. 4E,F) may be caused by low Tshβ levels in the same animals (Fig. 4C,D). In a previous study, a similar relationship between Tshr and Tshβ expression in the pars tuberalis and medial basal hypothalamus of Siberian hamsters was observed (Sáenz de Miera et al., 2017). In our study, the ependymal paraventricular zone (PVZ) around the third ventricle of the brain and the pars tuberalis are both included in samples for RNA extraction and qPCR, therefore we cannot distinguish between these two brain areas. Brains were collected 17 h after lights off, when Tshr mRNA levels in the pars tuberalis and PVZ are predicted to be similar based on studies in sheep (Hanon et al., 2008). Similar circadian expression patterns are expected in brains of seasonal long-day breeding rodents. Therefore, the observed increase in Tshr expression in SP voles, of both species and sexes (Fig. 4E,F), may relate to high TSH density in the tanycytes lining the third ventricle, which might lead to increased TSH sensitivity later in life. The high Tshr expression in voles developing under SP (Fig. 4E,F) may favor a heightened sensitivity to increasing TSH and photoperiods later in life. This in turn would promote increased DIO2 and decreased DIO3 levels in spring. Interestingly, photoperiodic responses on Tshr are more pronounced in tundra voles than in common voles, suggesting that tundra voles are more sensitive to TSH protein when raised under SP. However, TSH is a dimer of gonadotropin α-subunit (αGSU) and TSHβ, and we did not measure αGSU levels in this study.

Our laboratory vole populations are originally from the same latitude in the Netherlands (53°N) where both populations overlap. This is for the common vole the center (mid-latitude) of its distribution range, while our laboratory tundra voles are from a relict population at the lower boundary of its geographical range, which is an extension for this species to operate at southern limits. For this reason, local adaptation of the PNES may have evolved differently in the two species. The elevated Tshr expression and therefore the possible higher sensitivity to photoperiod in tundra voles raised under SP, might favor photoperiodic induction of reproduction earlier in the spring. This might be a strategy to cope with the extremely early spring onset at the low latitude for this relict tundra vole population.

Interestingly, the large peak in Tshβ expression (Fig. 4C) that is only observed in 21-day-old LP common vole males may be responsible for the drastic increase in testis mass when animals are 30 days old. Faster testis growth in LP common vole males (Fig. 3C) might be induced by the 2- to 3-fold higher Tshβ levels compared with LP tundra vole males (Fig. 4C). However, these data have to be interpreted with caution as the current study only considered gene expression levels and did not investigate protein levels.

The reduced Tshr expression under LP early in life (Fig. 4E,F) may be induced by epigenetic mechanisms, such as increased levels of DNA methylation in the promoter of this gene, which will reduce its transcription. A role for epigenetic regulation of seasonal reproduction has been proposed based on studies of the adult hamster hypothalamus (Stevenson and Prendergast, 2013). In order to study the effects of photoperiodic programming in development, DNA methylation patterns of specific promoter regions of photoperiodic genes at different circadian time points need to be studied in animals exposed to different environmental conditions earlier in development.

The photoperiodic-induced Tshβ and Tshr expression patterns are only reflected in the downstream Dio2:Dio3 expression differences at the beginning of development (Fig. 4K,L), suggesting that this part of the pathway is sensitive to TSH at a very young age. However, Dio2 and Dio3 are also responsive to metabolic status, which can change as a consequence of changing DIO2:DIO3 levels. Tundra and common vole females show similar photoperiodic-induced Tshβ patterns, while photoperiodic responses on Tshr are larger in tundra voles. The higher Tshr levels in tundra voles may be responsible for the higher Dio2, and lower Dio3 levels in tundra vole females compared with common vole females. However, the photoperiodic-induced differences in gene expression levels between species are not reflected in female gonadal mass, indicating that additional signaling pathways are involved in regulating ovary and uterus growth. In males, Dio2:Dio3 patterns are mainly determined by photoperiod, while different photoperiodic responses between species are lacking.

Dio2 and Tshβ expression correlate only at the beginning of development (i.e. at 15 days old) (Fig. 5A–D). These results are partly in agreement with the effects of constant photoperiod on hypothalamic gene expression in the Siberian hamster, showing induction of Dio2 at birth when gestated under LP, and induction of Dio3 at 15 days old when exposed to SP (Sáenz de Miera et al., 2017). Furthermore, it is thought that Dio2:Dio3 expression profiles will shift due to both photoperiodic and metabolic changes rather than by constant conditions. Also, negative feedback on the Dio2:Dio3 system might be induced by changes in metabolic status. In wild populations of Brandt's voles (Lasiopodomys brandtii), seasonal regulation of these genes show elevated Dio2:Dio3 ratios in spring under natural photoperiods, suggesting a crucial role for those genes in determining the onset of the breeding season in wild populations (Wang et al., 2019).

In females, both Kiss1 and Npvf expression is higher under LP dependent on age (Fig. S1D,F), whereas in males no effects of photoperiod on these genes are found (Fig. S1C,E). Other studies report inconsistent photoperiodic and seasonal effects on arcuate nucleus Kiss1 expression in different species, which may be related to a negative sex steroid feedback on Kiss1-expressing neurons (for review, see Simonneaux, 2020). For this reason, sex- and species-dependent levels of steroid negative feedback on both Kiss1- and Rfrp-expressing neurons in the caudal hypothalamus are expected.

In conclusion, our data show that somatic growth is photoperiodic sensitive in the tundra vole while gonadal growth is photoperiodic sensitive in the common vole. Our finding that the SP-induced Tshr expression is more pronounced in the developing hypothalamus of the tundra vole, may lead to the expectation that programming of TSH sensitivity is an important regulator of the PNES in this species. Reproductive development seems to be more dominated by photoperiodic responses in the common vole than in the tundra vole. It is not excluded that the PNES of the tundra vole has lost its photoperiodic capacity and instead has adopted responses to other environmental variables in its post-glacial relict population at the southern edge of its distribution. This raises the possibility that the tundra vole has a stronger response to other environmental cues (e.g. temperature, food, snow cover). Both vole species develop their PNES differently, depending on photoperiod early in development, indicating that they use environmental cues differently to time reproduction.

We would like to thank Saskia Helder for her valuable help in animal care. We thank L. van de Zande for his critical comments on the paper.