Introduction

Temperature plays an important role in reproductive physiology of vertebrates including mammals, fish, and birds (Hansen 2009; Boulangé-Lecomte et al. 2014; Sengar et al. 2017). In mammals, high temperature disrupts spermatogenesis and oocyte development, oocyte maturation, early embryonic development, and foetal and placental growth and lactation (Hansen 2009; Boulangé-Lecomte et al. 2014). High temperature also affects reproductive cycles in birds; increase in environmental temperature has resulted in shifting of the egg-laying timings (Visser et al. 2009). Similarly, in case of fish, temperature has varied effects on reproduction, depending upon the species; higher temperature favor the spring-spawning species, while lower temperature stimulates reproduction in autumn spawners (Pankhurst and Munday 2011). High temperature stress is the major environmental concern in the climate change regime, and assessing its impact on reproductive physiology of fish species is important as selection of species that can withstand higher temperatures with relatively lower impact on their reproductive performance would be required for sustainable aquaculture (Mohanty et al. 2010).

A number of experimental studies have shown that increase in temperature could affect reproduction in fish; however, the nature of these effects would depend on the period and amplitude and would vary from species to species (Pankhurst and King 2010; Pankhurst and Munday 2011). Puntius sophore is a micronutrient dense fish with a wide distribution in the tropical region, and it is being seen as an important tool for fighting hidden hunger and malnutrition (Mahanty et al. 2014). Thus, efforts are being made by aquaculturists to standardize its breeding and increase its production through aquaculture (Mohanty et al. 2018a, b). The breeding biology of Puntius has been reported earlier, and it has been seen that the fish is a seasonal breeder; the spawning period is marked by the gradual increase in the gonado-somatic index (GSI) during the month of March and July which then gradually starts decreasing (Choudhury et al. 2015; Hasan et al. 2018).

In this backdrop, the present study was carried out to investigate the impact of short-term (7 days) and long-term (60 days) high temperature stress on the reproductive physiology of this important species through expression analysis of a number of hsp genes (hsp90, hsp70, hsp60), heat-shock factor 1 (hsf1), vitellogenin (vtg), pleuripotency marker genes (sox-2, oct-4 and nanog), and dmrt-1. Expression of heat-shock protein (hsp) and vitellogenin (vtg) genes were carried out as there are numerous reports which depict the important roles played by these genes in maintenance of gonadal integrity. Besides, hsp70 has also been reported as a possible marker for assessing the egg quality (Kohn et al. 2015; Sullivan et al. 2015). Like the hsps, the pleuripotency markers, SRY (sex-determining region Y) -box 2 (sox-2), octamer-binding transcription factor 4 (oct-4), and nanog are also considered as markers for assessing the quality of oocyte (Zuccotti et al. 2011).

Materials and methods

Ethics statement

The study including sample collection, experimentation, and sacrifice met the ethical guidelines including adherence to the legal requirements of the study country. The study was approved by the Institute Animal Ethics Committee (IAEC) of Central Inland Fisheries Research Institute vide approval no. CIFRI/IAEC-17/03.

Sample collection after short- and long-term thermal exposure

P. sophore (weight 4.22 ± 0.5 g, length 6.46 ± 0.56 cm) were collected from aquaculture ponds and were acclimatized under laboratory conditions for 30 days in acrylic tanks of 30 l capacity fitted with thermostats. Fishes were fed once daily by providing laboratory prepared feed. Feed was prepared using soybean oil cake (290 g Kg−1), mustard oil cake (524 g Kg−1), fish meal (50 g Kg−1), vitamin–mineral premix (20 g Kg−1), and edible vegetable oil (15 g Kg−1). The protein and fat contents of the feed were 34% and 5.8%, respectively. P. sophore is found in almost all freshwater ecosystems in the tropical countries where the water temperature varies between 25 and 30 °C baring the peak summers. Therefore, the fishes were acclimatized at water temperature of 27 ± 0.2 °C prior to the heat-stress treatment. The fishes were randomly assigned to three experimental groups; one of the group maintained at 27 °C during the experimental regime served as the control. In the other two groups, the temperature was raised from 27 °C at the rate of 2 °C/h using a thermostat and temperature was maintained at 36 °C for 7 days and 60 days, respectively. A photoperiod of 12 h light and 12 h dark was maintained throughout the experimental regime. The water temperature was monitored using a calibrated thermometer.

The critical thermal maximum temperature (CTMax) of P. sophore has been found to be ranging between 39 and 41.5 °C depending upon the acclimatization temperature and other factors (Mahanty et al. 2016a, b, 2017). Therefore, a sublethal temperature close to the CTMax of the fish (36 °C) was chosen for this experimental study.

P. sophore is a prolific breeder, and its breeding biology has been studied by a number of researchers (Choudhury et al. 2015; Hasan et al. 2018). It has been reported that the fish spawns during the months March to July (Choudhury et al. 2015; Hasan et al. 2018). In the present study, gonads of mature fishes were collected during the pre-spawning period after completion of the exposure period. Nine male and 9 female fishes from each group were euthanized with tricaine, MS-222, 200 mg/ml, prior to dissection and collection of tissues in RNA later (R0901, Sigma).

RNA extraction and cDNA synthesis

RNA was extracted from tissue samples (nine samples from each experimental group, weighing approximately 50–70 mg of tissues) using RiboZol (HiMedia, India) according to the manufacturer’s protocol. RNA integrity was confirmed by determining their RNA integrity no. (RIN) by a Bioanalyzer (Agilent 2100), and samples with RIN values > 6 were processed for further analysis. RNA samples were treated with the DNase1 (NEB, UK) as per the manufacturer’s recommended protocol to remove DNA contamination. One microgram of DNase treated total RNA was reversely transcribed using M-MLV reverse transcriptase (New England Biology, UK) according to manufacturer’s protocols.

Primer synthesis

Expression analysis of four hsp genes: hsp90, hsp70, hsp60, and hsf1; three pleuripotency marker genes: oct-4, nanog, and sox-2 were carried out in both testis and ovary of P. sophore. Primers for the hsps 90, 70, and 60 were adapted from Mahanty et al. 2016b. Primers for oct-4 and sox-2 were adapted from Wagner and Podrabsky (2015). Primers for nanog and vtg were adapted from Zhendong (2009) and Henry et al. (2009), respectively. Lyophilized primers were procured from Integrated DNA Technologies (USA) and were reconstituted using nuclease-free water. Information of the sequences of the primers, annealing temperature, and accession numbers are listed in Table 1. The specificity of the primer sets was confirmed by the presence of a single band of appropriate size obtained after PCR amplification.

Table 1 Primer sets used for RT-qPCR analysis
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qPCR analysis

The real-time PCR amplifications were carried out using SYBR Green detection chemistry. cDNA were run in triplicates on a 96-well reaction plates with the CFX Connect real-time PCR (Bio-Rad, UK). Twenty microliter of reaction mixture contained 10 μl of VeriQuest SYBR Green Mix (Bio-Rad, UK), 1.0 μl of each 10 μM of primers and 5 μl of diluted cDNA as template and 3 μl of RNase/DNase-free sterile water (Thermo Scientific, USA).

The following amplification programs were used in all RT-qPCR reactions: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 15 s, and 95 °C annealing and extension for 45 s at optimized temperatures for specific candidate genes. The specificity of each amplification reaction was verified by a melting curve analysis after 40 cycles. No template controls (NTC) were included for each primer pair for cross-checking any possible contamination of assay reagents.

PCR efficiency of the genes was determined by a standard curve analysis of cDNA samples according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline (Bustin et al. 2009). A series of 10-fold dilution of three replicates of cDNA was made to determine the gene-specific PCR amplification efficiency for each primer pair used in qPCR experiments. Standard curve was derived from the E values by the formula E = 10–1/slope. The mean efficiency values were obtained for each tissue samples and were used to adjust the quantitative cycle (Ct) values for further analysis.

Expression analysis of ten candidate reference genes was earlier carried out, and eef1 and b2mg were found to be the most suitable reference genes in ovary and testis, respectively (Mahanty et al. 2017). Thus, these two genes were used for normalization of expression of the target genes (hsps, sox-2, oct-4, nanog, vtg, dmrt-1) in the respective tissues. The comparative Cq (delta Cq) method was used to calculate the changes in gene expression as a relative-fold difference between the control and treated sample. MIQE guidelines were followed for the qPCR experiments (Bustin et al. 2009).

Statistical analysis

Fold changes of gene expression are expressed in comparison with the control. One way analysis of Variance (ANOVA) followed by Tukey’s test was employed to compare the variation between the experimental groups (p < 0.05).

Results

Expression of hsp genes in gonadal tissues

In ovary, the expression of hsp70 and hsf1 was found to be downregulated in both the heat-stressed groups (both short-term and long-term). hsp60 was found to be significantly upregulated, whereas the expression of hsp90 was found to be downregulated in the 7-day heat-exposed fishes. Expressions of both hsp60 and hsp90 were found to be returning to the basal level in the fish exposed to heat stress for 60 days.

Similar to ovary, in testis also, the expressions of hsp70 and hsf1 were found to be downregulated in both the heat-stressed groups. But unlike the ovarian tissues, in testis, expressions of hsp60 and hsp90 were found to be downregulated in both the heat-stressed groups and no signs of recovery were found in the 60-day heat-exposed groups (Fig. 1).

Fig. 1
figure 1

Expression profile of hsp genes in gonadal tissues of Puntius sophore heat stressed for 7 and 60 days. The hsps were found to be downregulated in the gonadal tissues of heat-stressed fish except hsp60 in ovary. (*) above the bars indicate significant difference in the values in comparison to control (p < 0.05)

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Expression of pleuripotency marker genes (sox-2, nanog, oct-4)

In both ovary and testis, significant downregulation in expression of sox-2, nanog, and oct-4 was observed in the heat-stressed groups. There was a gradual decrease in expression of these genes with increase in time period of heat exposure (Fig. 2).

Fig. 2
figure 2

Expression profile of pleuripotency marker genes sox2, oct4, and nanog in gonadal tissues of Puntius sophore heat stressed for 7 and 60 days. Downregulation in expression of the pleuripotency marker genes was observed in the gonads of heat-stressed fishes indicating loss of pleuripotency of gonadal cells. (*) above the bars indicate significant difference in the values in comparison to control (p < 0.05)

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Expression of vtg and dmrt-1

Expression of vtg remained unaltered in both ovary and testis of fishes heat stressed for 7 days while significant downregulation was observed in gonadal tissues of fishes exposed to heat stressed for 60 days. In ovary, the expression of dmrt-1 (double sex mab-3–related transcription factor 1) was found to be downregulated in the heat-stressed groups, while in testis, the expression of dmrt-1 was upregulated in the fishes heat stressed for 7 days but found to be returning to the basal level in the 60 days exposed fishes (Fig. 3).

Fig. 3
figure 3

Expression profile of sex determining gene dmrt1 and gene involved in vitellogenesis vtg in gonadal tissues of heat-stressed Puntius sophore. dmrt1 was found to be downregulated in ovary and upregulated in the testis of heat-stressed Puntius sophore indicating that high temperature might favor production of higher number of males. (*) above the bars indicate significant difference in the values in comparison to control (p < 0.05)

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Discussion

Expression of hsps

Hsps play important roles in maintaining physiological integrity by stabilizing, refolding the denaturing proteins, and facilitating the proteolysis of the denatured proteins (Sottile and Nadin 2017; Wang et al. 2015; Zunino et al. 2016; Mahanty et al. 2016a, b). The hsps are known to be expressed in various cell types including gonadal cells, and studies suggest that some of the hsps play fundamentally important roles during early development. Hsps are known to be expressed in normal cells, but their expression is increased during stressed condition. However, in case of gonadal (ovary) tissues, a number of studies have shown the decrease in abundance of the hsps as a marker of poor egg quality (Kohn et al. 2015; Chapman et al. 2014). Kohn et al. (2015) have reported a decrease in abundance of hsp70 in poor-quality eggs in Polyprion oxygeneios. Chapman et al. (2014) identified hsp90 transcripts among a subset of 233 mRNA species whose abundance had predictive value for egg quality in striped bass. “Egg quality” has been defined as the ability of the egg to be fertilized and subsequently develop into a normal embryo (Bobe and Labbe 2010), and poor-quality eggs are those which have very less ability to get fertilized and subsequently develop into a viable embryo (Bobe 2015). In the present study also, we found that downregulation in the expression of hsp70, and hsf1 could be indicative of deteriorative changes in the ovarian functionalities that could lead to production of poor quality eggs. Along with that, significant alterations in expression of hsp90 and hsp60 were observed in ovary of fishes exposed to heat stress for 7 days (down- and upregulations, respectively) which were found to be returning to basal level after 60 days of heat exposure. This could be an indication of acclimatization to warm temperature after a relatively longer period of time.

In testes, all the hsps were found to be downregulated in the heat-stressed groups. Studies have shown that downregulation of hsps in testes lead to apoptosis of testicular cells (He et al. 2010). The downregulation in expression of hsps could be because of the progression of testicular cells to apoptotic pathways. Similar to the present findings, Domingos et al. (2013) have shown downregulation of hsps in testes of fishes during warmer seasons of year.

Expression of pleuripotency marker genes

The sox-2, oct-4, and nanog are transcription factors that regulate transcription of proteins of the leukemia inhibitory factor (LIF) signaling pathway (Magnani and Cabot 2009). These three transcription factors contribute to a complex molecular network necessary for maintenance of cellular pluripotency. Pleuripotent stem cells are necessary for the maintenance of many adult tissues including the gonads (Greenspan et al. 2015). Gonads are the only organs capable of transmitting genetic materials to the offspring and are composed of both somatic and germline stem cells (Liu et al. 2009). However, pleuripotency of the gonadal stem cells and their self-renewal capability largely depend on their microenvironment and any misregulation in the signaling pathways in this microenvironment can lead to depletion of different stem cell pools (Greenspan et al. 2015). In this context, we analyzed the expression profile of three pleuripotent stem cell marker genes sox-2, oct-4, and nanog in both testis and ovary of fish exposed to heat stress. All these genes were found to be downregulated in both ovary and testis of P. sophore exposed to heat stress which indicates depletion in the number of stem cells; the stem cell could possibly be either undergoing apoptosis, or these cells might be losing their pleuripotency.

Expression of vtg and dmrt-1

Vtg gene synthesizes vitellogenin which is the precursor protein of yolk. Vitellogenin is a protein that is generally synthesized in female fishes; however, in presence of endocrine disruptive chemicals (EDC), it can be synthesized in male fish also (Hara et al. 2016). Vitellogenin has been found to be a marker of egg quality, and its downregulation in ovary is indicative of poor egg quality (Kohn et al. 2015) and one of the markers used for presence of endocrine disruptive chemicals (Endocrine disruptor screening and testing advisory Committee 1998; Marin and Matozzo 2004; Klaper et al. 2006). In the present study, we observed downregulation in expression of vtg in ovary of fish exposed to heat stress but its expression was inconsistent in testis. The downregulation of vtg in ovary indicates that, like EDCs, high temperature stress could also disrupt the endocrine functions and can hamper the egg production process. This inference has been made on the basis of the downregulation of vtg in the gonads of heat-stressed fish. However, the possibility that high temperature acts as an endocrine disruptor merits further investigation.

Sex in fish is plastic and in several species can be influenced by environmental factors (Díaz and Piferrer 2015). Thus, we studied the expression of dmrt-1 in both the gonads of fish exposed to high temperature stress to know whether high temperature supports masculinization or feminization. In case of zebrafish, which is also a cyprinid like Puntius, dmrt-1 plays an important role in male sex determination and testis development (Webster et al. 2017; Lambeth et al. 2014). Webster et al. have shown that the mutations in dmrt-1 gene result in complete sex reversal from male to female in zebrafish. It not only plays a role in genotypic sex determination but also plays important functions in environmental sex determination especially in temperature-dependent sex determination (Fernandino et al. 2008). The expression of dmrt1 was upregulated in testes and downregulated in ovary of the heat-stressed fishes which could be possibly because of a masculinization supportive role of high temperature. In many fish species, temperature-dependent sex determination (TSD) has been observed along with genetic sex determination (GSD) which varies among the species (Baroiller et al. 2009). In some species like tilapia, high temperature above 32 °C favors feminization (Baroiller et al. 2009), whereas in species like Danio rerio, Oryzias latipes, and Onchorhynchus nerka, high temperature favors masculinization. The present study suggests that high temperature could favor masculinization in P. sophore like its close relative D. rerio (Ospina-A lvarez and Piferrer 2008). However, fish in general have a weak genetic component of sex determination and the mechanisms of sexual differentiation may vary among fish species. Thus, this merits further investigation to see if dmrt-1 is involved in sex determination in Puntius also and if the upregulation in its expression has any male favoring effect.

The present study showed that even though expression of some of the genes had recovering tendency with prolonged period of temperature stress, most of the genes had altered expression indicating a negative impact on the reproductive physiology of the fish. As observed in the present study, anomalous temperature has been found to be having an inhibitory effect on reproductive physiology of the fish Takifugu niphobles through suppression in expression of a number of genes like kisspeptin and gnrh (Shahjahan et al. 2017). Similarly, Díaz and Piferrer (2017) have reported that temperature has a masculinizing effect which is overridden by estrogen exposure.

Conclusion

The present study showed that with increasing temperature, there was a downregulation of hsps indicating that high temperature can affect the quality of gametes produced. Similarly, the downregulation of pleuripotency marker genes in the heat-stressed fish suggests that the gonads could lose their pleuripotent cells thereby affecting the gonadal integrity. This study suggests that the reproductive physiology and health of the nutrient dense P. sophore could be negatively affected with rise in environmental temperature.