Research paper
Temperature affects sexual maturation through the control of kisspeptin, kisspeptin receptor, GnRH and GTH subunit gene expression in the grass puffer during the spawning season

https://doi.org/10.1016/j.ygcen.2016.11.012Get rights and content

Highlights

  • The kiss2 and kiss2r along with gnrh1 were significantly decreased at both low and high temperature.

  • The fshb and lhb were significantly decreased in both low and high temperature.

  • Plasma levels of cortisol were significantly increased in low temperature but not in high temperature.

  • Anomalous low and high temperature may shut down the reproduction through suppression of kisspeptin/GnRH1/GTH system.

Abstract

Water temperature is an environmental factor of primary importance that influences reproductive function in fish. To understand the molecular and physiological mechanisms underlying the regulation of reproduction by temperature, we examined changes in expression of genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r) and three gonadotropin-releasing hormones (gnrh1, gnrh2 and gnrh3) in the brain and genes encoding gonadotropin (GTH) subunits (gpa, fshb and lhb) in the pituitary of grass puffer exposed to a low temperature (14 °C), normal temperature (21 °C) and high temperature (28 °C) for 7 days. In addition, the plasma levels of cortisol were examined after exposed to three temperature conditions. The gonadosomatic index was significantly decreased in both low and high temperature conditions. The levels of kiss2 and kiss2r mRNAs were significantly decreased at both low and high temperature conditions compared to normal temperature (control) condition. gnrh1 but not gnrh2 were significantly decreased in both temperature conditions, while gnrh3 showed a decreasing tendency in low temperature. Consequently, the levels of fshb and lhb mRNAs were significantly decreased in both low and high temperature conditions. Interestingly, the plasma levels of cortisol were significantly increased in low temperature but remain unchanged in high temperature, suggesting that the fish were under stress in the low temperature conditions but not in the high temperature conditions. Taken together, the present results indicate that anomalous temperature have an inhibitory effect on reproductive function through suppressing kiss2/kiss2r/gnrh1/fshb and lhb expression and these changes may occur in a normal physiological response as well as in a malfunctional stress response.

Introduction

Among the environmental factors, temperature plays an important role in the control of reproduction in vertebrates. Temperature directly affects molecular, biochemical, and physiological processes, especially in the ectothermic vertebrates (Pankhurst and King, 2010, Strussmann et al., 2010). In teleost fish, gonadal development, spawning and gonadal regression are greatly influenced by water temperature (Pankhurst and King, 2010, Wang et al., 2010, Okuzawa and Gen, 2013). Both anomalous increase and decrease in water temperature inhibit gonadal development, maturation and spawning. The increase in water temperature suppresses fish reproduction, decreases fish abundance and even induces extinction (Soria et al., 2008, Portner and Peck, 2010).

Ovarian development and maturation have been shown to be inhibited in above or below the temperature limit in a number of fish species (Wang et al., 2010). A decrease in water temperature showed limited accumulation of yolk and uncompleted or delayed final oocyte maturation in the blue gourami (Cheal and Davis, 1974) and mummichog (Cerda et al., 1996). Low temperature inhibited ovulation, reduced fecundity and size of vitellogenic oocytes in Prochilodus argenteus (Arantes et al., 2011). Both low and high temperature prevented gonadal development in the pikeperch (Hermelink et al., 2011). In the goldfish, ovulation induced by gonadotropin-releasing hormone (GnRH) was delayed with an increase in temperature (Rottmann and Shireman, 1985). However, the molecular and endocrine mechanisms that underlie these effects are poorly understood.

In fish, like other vertebrates, the central regulation of reproduction occurs through hypothalamus-pituitary-gonadal (HPG) axis. GnRH synthesized in the hypothalamus, regulates the synthesis and release of two gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), from the pituitary, which stimulate gonadal development and secretion of sex steroid hormones (Yaron et al., 2003, Zohar et al., 2010). It is now witnessed that kisspeptin plays an important role in the control of reproduction through regulating GnRH secretion in mammals. In teleost fish, kisspeptin and its receptor (Kissr, GPR54) system has been shown to have a stimulatory role in the control of reproduction (Parhar et al., 2004, Chang et al., 2012, Tena-Sempere et al., 2012, Zmora et al., 2012, Ohga et al., 2014, Espigares et al., 2015, Park et al., 2016). However, recently, it was demonstrated that the kisspeptin/Kissr system was dispensable for reproduction in zebrafish using gene knockout models (Tang et al., 2015). Furthermore, GnRH1 neurons do not co-express Kissr in medaka (Kanda et al., 2013). These results suggest that the physiological pathways that control reproduction are complex in fish, and kisspeptin may have a stimulatory role on GnRH neurons directly and/or indirectly via interneurons. It should be also noted that genes for kisspeptin and Kissr were diversified during the evolution of teleost fish, increasing the complexity of the kisspeptin/Kissr system (Kitahashi et al., 2009, Ogawa and Parhar, 2013). In the present study, however, the grass puffer (Takifugu niphobles), which has only a single set of genes for kisspeptin (kiss2) and Kissr (kiss2r) was utilized as a research model. Our previous studies on their expression in the hypothalamus and pituitary strongly support the concept that Kiss2 has an important role in the control of reproduction like other fishes mentioned above (Ando et al., 2013, Ando et al., 2014, Shahjahan et al., 2010).

The effects of temperature on the HPG axis have been examined in regard to GnRH and GTH subunit gene expression. Increase and decrease in water temperature (4 °C) suppresses gnrh3 mRNA levels in the blue gourami (David and Degani, 2011, Levy et al., 2011). The expression of gnrh1 in the brain and lhb in the pituitary was suppressed by high water temperature in the red seabream (Okuzawa and Gen, 2013). Elevated temperature also reduced fshb and lhb mRNA levels in the pejerrey (Soria et al., 2008). However, there is a paucity of information concerning the effect on kisspeptin, except one study in zebrafish, in which low and high temperature decreased kiss2 and gnrh3 mRNA levels (Shahjahan et al., 2013), and there is a clear need of further study to understand the effects of temperature on kisspeptin and its receptor gene expression.

The grass puffer shows unique reproductive physiology that is synchronized with seasonal, lunar and daily cycles. During the spawning season from spring to early summer, spawning occurs only during spring tide every two weeks (Motohashi et al., 2010, Ando et al., 2013). The fish usually aggregate at a certain seashore location for spawning 2–3.5 h before high tide at dusk, and spawning continues for 1–2 h before high tide during the rising tidal phase. Therefore, temperature as well as light, tide and time are considered to be important factors for the control of reproduction in the grass puffer (Ando et al., 2013). During the spawning periods, gnrh2, kiss2, kiss2r, lpxrfa and lpxrfa-r showed daily and circadian fluctuations in expression in the hypothalamus in association with the expression of melatonin receptor genes (Ikegami et al., 2009, Shahjahan et al., 2011, Ando et al., 2013, Ando et al., 2014), indicating the possible role of circadian clock and the pineal hormone, melatonin, in the control of reproduction of this species.

In our previous study, we found that gnrh1, kiss2 and kiss2r in the brain, and fshb and lhb in the pituitary showed seasonal fluctuations with gonadal development and regression (Shahjahan et al., 2010, Shahjahan et al., 2010, Ando et al., 2013). As a result, we hypothesized that a rise in water temperature in summer may trigger the end of the spawning season in the grass puffer. High water temperature leads to termination of the spawning period in the bitterling (Shimizu and Hanyu, 1982), honmoroko (Okuzawa et al., 1989) and pejerrey (Soria et al., 2008). Likewise, low temperature may postpone the commencement of spawning in Prochilodus argenteus (Arantes et al., 2011). Therefore, in the present study, we aimed to investigate the effects of low and high temperature on the expression of genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r), three gonadotropin-releasing hormones (gnrh1, gnrh2 and gnrh3) and three gonadotropin subunits (gpa, fshb and lhb) in the grass puffer during the spawning period. We also examined the plasma levels of cortisol as an indicator of stress in fish during the experimental periods.

Section snippets

Fishes

Mature male grass puffer were collected at spawning grounds in Sado, Niigata in June for the 1st experiment, and in Kawana, Shizuoka in July for the 2nd experiment. The water temperature of sampling sites was 18 and 21 °C in Sado and Kawana, respectively. The fish were transferred to the Sado Marine Biological Station, Niigata University, Japan, and reared in indoor tanks (500 L) with flow of seawater under natural photoperiod (LD 14:10) for 2 weeks. The water temperature during the

Changes in GSI and expression of kiss2 in the 1st experiment

In the 1st experiment, the GSI showed no distinct changes in response to temperature changes in any exposure periods (Fig. 1A). The absolute amounts of kiss2 mRNA in the diencephalon/midbrain sample were examined by real-time PCR assay. In the day-3, no changes were observed in any temperature regime, while in the day-7, a decreasing tendency were observed in low temperature (11 °C), whereas an increasing tendency were observed in high temperature (25 °C) compared to the normal temperature (18 °C)

Discussion

To know the role of temperature in the regulation of reproductive function, we investigated the effects of exposure to low and high temperature on the expression of genes encoding kisspeptin, kisspeptin receptor and three GnRHs in the brain and genes encoding GTH subunits in the pituitary of sexually mature male grass puffer. In parallel to GSI, the kiss2 and kiss2r mRNA levels were significantly decreased by the low and high temperature exposures, concomitant with the decrease in gnrh1, fshb

Acknowledgments

We are grateful to Dr. Satoshi Awata, Mr. Tomonobu Uryu and Mr. Yoichiro Kato for their help in collecting fish. This study was supported by MEXT/JSPS KAKENHI grants (245700659 and 16H04812 to H.A.).

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