Reproductive phase-dependent variation, sexually dimorphic expression and sex steroids-mediated transcriptional regulation of lep and lepr in lymphoid organs of Channa punctata

The reproductive phase-dependent and sex-related differential expression of leptin (lep) and its receptor (lepr) in primary and secondary lymphoid organs of a highly nutritive economically important Channa punctata preempts the involvement of sex steroids in modulating intra-immuno-leptin system. This hypothesis was strengthened when plasma testosterone (T) and estradiol (E2) levels in male and female fish of reproductively active spawning and quiescent phases were correlated with lep and lepr expression in their immune organs. Splenic lep and lepr showed a negative correlation with T in both male and female, while with E2 there was a positive correlation in male and negative in female C. punctata. In head kidney, a contrasting correlation was observed as compared to spleen. To validate the implication of sex steroids in regulating leptin system in immune organs, in vivo and in vitro experiments were performed with DHT and E2. Upon administration, lep and lepr expression in tissues of either sex was downregulated. In addition, in vitro results with either of the sex steroids exemplified their direct involvement. Overall, this study, for the first time, reports correlation between sex steroids and transcript expression of leptin system in immune organs of a seasonally breeding vertebrate.

expression of lep and lepr during different reproductive phases in both, male and female C. punctata, exhibited marked variation depending on their reproductive state (one-way analysis of variance, ANOVA, p < 0.0001; Fig. 1a,b). In male, lowest lep transcripts level was observed during preparatory and spawning phases. Compared to these reproductively active phases, lep expression was considerably (p < 0.05) higher in reproductively quiescent phases, i.e., postspawning phase and resting phase. An appreciable (p < 0.05) increase in its expression observed during postspawning phase further increased significantly (p < 0.05) in resting phase (Fig. 1a). The lep expression in female was largely comparable to male, considerably (p < 0.05) lower during reproductively active than quiescent phases (Fig. 1a). In case of phase-dependent expression of lepr, unlike lep, minimal expression was recorded during postspawning phase in both the sexes (Fig. 1b). Nonetheless, maximum lepr transcript level was observed during resting phase in male as well as female. After resting phase, a sharp (p < 0.05) decline in lepr expression was detected during preparatory phase. In spawning phase, an appreciable (p < 0.05) increase in lepr level was observed compared to preparatory phase, though the level was significantly (p < 0.05) lower than resting phase in both the sexes.
Head kidney. Although expression of lep and lepr in head kidney of male and female C. punctata showed significant variation depending on reproductive phases (one-way ANOVA, p < 0.0001; Fig. 1c,d), their expression pattern was different with that of spleen. The lowest expression of lep was observed during resting phase in both male and female C. punctata (Fig. 1c). Thereafter, lep expression highly (p < 0.05) increased during preparatory phase in both the sexes. Although level of lep declined considerably (p < 0.05) during spawning phase in male as well as female, it remained unaltered until postspawning phase in male while significantly (p < 0.05) increased in female. Regarding reproductive phase-dependent expression of lepr (Fig. 1d), the expression pattern in both the sexes was found to be quite similar, with maximum transcript levels during spawning phase and minimum during preparatory and postspawning phases. In resting phase, lepr expression was considerably (p < 0.05) higher than preparatory and postspawning phases but significantly (p < 0.05) lower than spawning phase.
Sexual dimorphism in expression of lep and lepr in lymphoid organs. Sex-related differential expression of lep and lepr in spleen and head kidney during spawning and resting phases showed marked (p < 0.02) difference in their transcripts level in both the lymphoid organs only during reproductively active spawning phase (Fig. 2). During active phase, expression of lep was 2.5 to 3-fold higher in spleen (p < 0.0001) and head kidney (p = 0.0052) of male than that in respective tissues of female C. punctata (male vs female lymphoid tissue, unpaired t-test, Fig. 2a,b). Regarding sexually dimorphic expression of lepr, contrasting results were observed between primary and secondary lymphoid organs. In case of spleen, transcript level of lepr was considerably (~0.4 fold, p = 0.0115) higher in female than male while in head kidney it was 2.5-fold (p = 0.0012) higher Relative fold change was calculated considering resting phase as reference. One-way analysis of variance (ANOVA, p < 0.0001, N = 8 for each tissue of either sex in each reproductive phase) followed by Student's Newman Keuls (SNK) post-hoc test (p < 0.05) was used to determine the significant difference between the groups. Different superscripts (alphabets for male and numerals for female) above error bars indicate significant differences between the groups. The data is represented as mean fold change ± standard error of mean (SEM).
correlation analyses. Correlation of lep and lepr expression with sex steroids. The plasma levels of sex steroids, T and E 2 , in male and female C. punctata during reproductively active spawning and quiescent resting phases correlated with expression of lep and lepr in spleen and head kidney showed differential results depending on sex, specific sex steroid and type of lymphoid tissue (Fig. 3). In male, a strong positive correlation was observed between level of plasma E 2 and expression of splenic lep (r = 0.8295, p = 0.0002) as well as lepr (r = 0.7761, p = 0.0011) (Fig. 3c,d) while an insignificant negative correlation was seen between plasma T and splenic leptin system (lep: r = −0.3824, p = 0.2754; lepr: r = −0.697, p = 0.051; Fig. 3a,b). Contrary to spleen, a marked negative correlation between plasma E 2 levels and lep (r = −0.8285, p = 0.0003) / lepr (r = −0.6929, p = 0.006) expression Correlation between expression of lep and its receptor (lepr). No significant correlation was observed between relative expression of lep and lepr in any of the immune organs of either male or female C. punctata during different reproductive phases (Supplementary Table S3).

Role of sex steroids in regulation of lep and lepr expression in lymphoid organs.
In vivo experiment. Effect of dihydrotestosterone (DHT) on lep and lepr expression in male fish. The male C. punctata receiving varying doses of DHT (9, 45 and 90 ng per day/fish for 3 days) during resting phase exhibited dose-and tissue-related differential effect of non-aromatizable androgen on lep and lepr expression in spleen and head kidney (ANOVA, p < 0.01, Fig. 4a,b). Compared to splenic lep expression in vehicle-injected control, a marked (p < 0.05) decrease in its expression was observed after treatment with low dose (9 ng/fish/day) of DHT. The DHT-induced down-regulation became severely pronounced with an increase of its dose to 45 ng/fish/day (9 ng vs 45 ng, p < 0.05; Fig. 4a). However, the highest dose of DHT (90 ng/fish/day) was seen ineffective in influencing splenic lep expression. In case of head kidney, all the doses of DHT were effective in significantly (p < 0.05) reducing lep expression, though maximal inhibition was observed at the moderate dose of 45 ng/fish/day (Fig. 4b).
Unlike lep that was inhibited by DHT in both the lymphoid organs, expression of lepr after DHT treatment showed contradictory results, a marked (p < 0.05) decline in spleen while a robust (p < 0.05) increase in head kidney. However, only the moderate dose of DHT (45 ng/fish/day) was found to be effective in modulating lepr expression in spleen whereas the lowest dose (9 ng/fish/day) in case of head kidney (Fig. 4). . Sex-related differential expression of (a,b) lep and (c,d) lepr in lymphoid organs during reproductively quiescent resting phase (RP) and active spawning phase (SP) of C. punctata. The relative fold change was calculated considering female as reference. Student's unpaired t-test was employed and graphs are represented as mean fold change ± SEM (N = 8). Asterisk '*' above error bars indicates significant (p < 0.02) difference in gene expression in a lymphoid tissue of male and female during same reproductive phase.
Reproductive phase E 2 level (mean ± SEM in pg/ml) T level (mean ± SEM in pg/ml) www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of 17β-estradiol (E 2 ) on lep and lepr expression in female fish. Treatment of female C. punctata with different doses of E 2 during resting phase resulted in a marked (one-way ANOVA, p = 0.0035) decrease of lep expression in spleen at all the doses (50, 250 or 500 ng E 2 /fish/day for 3 days), though a significant (p < 0.05) decline in splenic lepr was observed only at the dose of 250 ng/fish/day when compared to that of vehicle-injected female control (Fig. 4c). Unlike spleen, in head kidney E 2 treatment failed to affect lep expression (p < 0.05) at any of its dose while considerably (one-way ANOVA, p = 0.0012) increased lepr expression at the doses of 50 and 250 ng/fish/day (Fig. 4d).    www.nature.com/scientificreports www.nature.com/scientificreports/ Criss-cross experiment: effect of DHT in female and E 2 in male fish. The administration of DHT in female fish of resting phase led to a significant (p < 0.0001) decrease of lep as well as lepr expression in both the immune organs, spleen and head kidney when compared to vehicle-injected female control, with an exception at the dose of 90 ng/fish/day for lepr in head kidney where no marked alteration in its expression was observed (Fig. 5a,b). Like inhibitory effect of the DHT in female, E 2 administration in male caused a marked (p < 0.01) decline in lep and lepr expression in spleen as well as head kidney when compared to their expression in respective immune organ of vehicle-injected male fish of resting phase (Fig. 5c,d).
In vitro experiment. Effect of DHT on male lymphoid organs. The pieces of spleen incubated with different concentrations of DHT (p < 0.0001) showed concentration-related dual effects on lep expression, stimulatory (p < 0.05) at the lowest concentration while inhibitory (p < 0.05) at the highest concentration when compared to that incubated in medium alone (control). However, on splenic lepr expression, DHT had marked (p < 0.0001) inhibitory effect at all the concentrations (Fig. 6a). In case of head kidney, expression of both, lep and lepr, considerably (p < 0.0001) decreased after incubation with varying concentrations of DHT, except 34.4 µM for lepr (Fig. 6b).
Effect of E 2 on female lymphoid organs. The expression of lep and lepr in spleen and head kidney incubated with varying concentration (0.36, 3.67 and 36.7 µM) of E 2 exhibited differential results (Fig. 6c,d). In spleen, E 2 significantly (p < 0.05) inhibited the expression of lepr at all the concentrations while it appreciably (p < 0.05) augmented the expression of lep at the lowest concentration and failed to alter at the subsequent higher concentrations (Fig. 6c). In contrast to spleen, in head kidney E 2 markedly (p < 0.05) reduced lep expression at all the concentrations though failed (p = 0.2039) to affect lepr expression at any of its concentration (Fig. 6d).
Criss www.nature.com/scientificreports www.nature.com/scientificreports/ appreciable (p < 0.05) increase in splenic lep and lepr expression only at the lowest concentration (Fig. 7a) as their expression at subsequent higher concentrations were comparable to that incubated in medium alone (control). Unlike spleen, DHT significantly inhibited the expression of both, lep (p < 0.0001) and lepr (p = 0.0013), in head kidney (Fig. 7b). In case of male, pieces of lymphoid organs incubated with varying concentrations of E 2 exhibited dual effects (p < 0.0001) on splenic lep expression, stimulatory at lower concentration (0.36 µM; p < 0.05) while inhibitory at higher concentrations (3.67 and 36.7 µM; p < 0.05). However, expression of splenic lepr decreased considerably (p < 0.0001) at all the concentrations of E 2 when compared to control (Fig. 7c). With regard to expression of leptin system in head kidney, an inconsistent effect of E 2 was observed on lep expression, inhibitory (p < 0.05) at 0.36 and 36.7 µM concentrations while no effect at 3.67 µM. On lepr, an appreciable (p < 0.05) increase was recorded at the highest concentration of E 2 (Fig. 7d).

Discussion
The present study aimed to investigate the reproductive phase-dependent and sex-related variations in expression of leptin and leptin receptor in primary and secondary lymphoid organs, head kidney and spleen, respectively, of an adult Channa punctata. The sexually dimorphic expression of lep and lepr during reproductively active but not during the quiescent phase, led the authors to hypothesize the involvement of sex steroids in modulating transcripts level of leptin and its receptor. Therefore, in vivo and in vitro experiments were undertaken to elucidate the role of dihydrotestosterone and 17β-estradiol on the expression of lep and lepr in spleen as well as head kidney of adult male and female C. punctata.
The expression of leptin and its receptor has been demonstrated in lymphoid organs of a few fishes 10,11,32,33 . Likewise, an intense expression of lep and lepr was observed in spleen and head kidney of C. punctata though the pattern varied from secondary to primary lymphoid organ. Recent studies in fishes have also shown marked difference between spleen and head kidney with respect to expression pattern of immune-related genes and their respective proteins in response to bacterial infection [34][35][36] . These differences have been attributed to differential role of primary and secondary lymphoid organs as teleostean spleen is proposed to be majorly involved in cellular responses while head kidney in humoral responses 34 . Another possibility could be due to differences in mRNA turnover of lep and lepr between two lymphoid organs as mRNA turnover has been suggested to be dependent on RNA binding proteins (RBPs) and expression of these RBPs is reported to vary from tissue to tissue in humans 37 . Nonetheless, expression of lep and lepr in spleen and head kidney point towards the localized role of leptin system in fish immune organs. Till date, only two studies have been conducted in fishes highlighting the direct implication of leptin in immune responses. Leptin knockdown in zebrafish caused an increase in bacterial load due to marked reduction in ability to fight pathogens 38 . Another study reports decreased superoxide production from blood leucocytes of trout when incubated with homologous leptin under in vitro condition 39 . Moreover, in mammals, role of leptin in regulating immune functions is well demonstrated where it is shown to modulate both the arms of immunity, innate as well as adaptive 5 . Taken together, presumptive immunomodulatory role of leptin is speculated in C. punctata.
Interestingly, lep and lepr expression during different reproductive phases has not been estimated so far in lymphoid organs of fishes or any other seasonally breeding vertebrate despite the fact that immune defence process varies depending on season 14,15 . The current study for the first time reports reproductive phase-dependent variation in lep and lepr expression in lymphoid organs of vertebrates. In spleen of male C. punctata, expression of lep remained considerably low during preparatory and spawning phases while high in resting and postspawning phases. The reproductive phase-dependent pattern of splenic lep expression in female C. punctata was largely comparable to that of male. Also, male and female C. punctata exhibited similar expression pattern for splenic lepr, being high in resting and low in preparatory and postspawning phases. In case of male head kidney, level of lep remained low during resting, spawning and postspawning phases while substantially high during preparatory phase. The pattern of lep expression in head kidney of female during different reproductive phases was similar to that of male, except postspawning phase. Regarding head kidney lepr, the expression pattern in both the sexes was found to be identical along the reproductive phases. These observations in the present study led to speculate a correlation between reproductive phase-dependent variations in expression of lep and lepr in lymphoid organs and relative concentration of androgen and estrogen in male and female C. punctata. In male C. punctata, a peak of T and 11-ketotestosterone was attained during preparatory phase while E 2 in resting phase 40 . Both T and E 2 remained high during preparatory phase of female C. punctata. Further, our assumption gets support from studies in mammals where male and female sex steroids are shown to have opposite effects on leptin production from adipose tissue 17,18,41 . As far as interrelation between ligand and its receptor expression is concerned, lep did not correspond to lepr in both male and female C. punctata depending on reproductive phase and lymphoid tissue. Similar to the findings of present study, expression pattern of lep has been reported to be opposite to lepr in liver of Oryzias latipes 8 , Salmo salar 42 , Pelteobagrus fulvidraco 11 , Epinephelus coioides 33 and Oreochromis niloticus 43 . Although possible explanation for opposite expression pattern of lep and lepr has not been proposed in these studies, one could speculate the implication of an unknown receptor-mediated mechanism as suggested by Farooqi and colleagues 44 . They observed that leptin-deficient patients show more severe phenotypes as compared to leptin receptor-deficient ones. Moreover, difference between half-life of ligand and its receptor mRNA and/or alteration in binding affinity of leptin receptor depending on reproductive phase could be taken into account for reverse pattern between ligand and its receptor during postspawning phase in C. punctata. The sexual dimorphism in plasma levels of leptin [17][18][19]45,46 and its soluble receptor 46 is well demonstrated in mammals. Regarding their sex-related expression in tissues, studies are largely confined to adipocytes 19,45 , skeletal muscle 46 and hypothalamus 22 . The levels of leptin and its receptor in these studies are reported to be higher in females than males. However, no attempt has been made to investigate the sexually dimorphic expression of LEP and LEPR in immune organs of mammals. In fishes, a handful of studies are available on sex-related dimorphism in levels of leptin and its receptor, and the results are inconsistent [23][24][25][26] . The plasma levels of leptin in Lota lota is shown to be high in female as compared to male before, during and after spawning season 23 while no sex-related difference in plasma leptin levels is reported in Cyprinus carpio and Capoeta trutta 24 . It is to be noted that the reproductive phase during which plasma leptin was assayed in C. carpio and C. trutta is not highlighted in the report. The efforts have also been made to examine sex-related differential expression of lep and lepr in various tissues (brain, liver, hypothalamus, pituitary and gonads) during different stages of germ cell development in male and female Megalobrama amblycephala 25 . The results varied depending on tissues and stages of spermatogenesis/ folliculogenesis. Similarly, tissue-wise sex-related marked difference in lep expression is demonstrated in several tissues (brain, liver, gills, intestine, kidney, heart, muscle and spleen) but adipocytes in sexually immature, and also in gonads of sexually mature Tanichthys albonubes 26 . This is the only study in fishes that reports sexual dimorphism in expression of lep in a lymphoid organ in which leptin transcript level is shown to be high in male than female 26 . However, sex-related differential expression of lepr has not been explored so far in primary or secondary lymphoid organs of fishes. In the current study, sexually dimorphic expression of lep and lepr was observed in spleen as well as head kidney of C. punctata during reproductively active phase when level of sex steroids remain high and not in quiescent phase when their levels remain basal. As observed in immature T. albonubes 26 , splenic lep was high in male than female C. punctata. A similar pattern of lep expression was also seen in head kidney. However, a contrasting expression pattern of its receptor was observed between primary and secondary lymphoid organs of C. punctata as level of lepr was recorded higher in spleen of female while head kidney of male. Taken together, we speculated the involvement of sex steroids in sexually dimorphic expression of lep and lepr in immune organs of C. punctata.
In the present study, role of sex steroids in control of leptin and its receptor expression in immune organs of C. punctata was deduced based on observations during different reproductive phases in both the sexes and also between opposite sexes in the same reproductive phase. This inference was backed by correlation analysis between levels of plasma sex steroids and lep as well as lepr expression in immune organs during reproductively active and quiescent phases in male and female C. punctata. Although efforts have not been made to examine an interrelationship between levels of plasma sex steroids and leptin system in immune organs of seasonally breeding vertebrates, studies in mammals have suggested that plasma leptin is negatively associated with androgens in males 47,48 while positively with estradiol in females 49 . In male C. punctata, a positive correlation between levels of plasma T and lep and lepr was observed in primary lymphoid organ, i.e., head kidney while negative in case of secondary lymphoid organ, i.e., spleen. The relationship with E 2 in male immune organs was contradictory to T, negative with lep and lepr in head kidney while positive with that in spleen. Interestingly, in female C. punctata, lep and lepr expression in spleen exhibited negative correlation with both T and E 2 and vice versa in head kidney. This implies that lep and lepr expression in immune organs depends on sex, prevalence of sex steroids and type of lymphoid organ.
The inference drawn from correlation analyses gets support from our in vivo study, including criss-cross experiments in which male and female C. punctata received E 2 and DHT, respectively. Sex steroids, depending on dose, had either inhibitory or no effect on lep and lepr expression in primary as well as secondary lymphoid organs of both the sexes, except on lepr in head kidney where DHT had marked stimulatory effect in male and E 2 in female. Since the effect of sex steroids on lep and lepr expression remain unexplored in immune organs of vertebrates, we analysed our results in light of observations in other tissues of fishes 26,28,29 and mammals 41,50-54 . Our findings on lep and lepr expression in immune organs of C. punctata are largely contrasting to a recent study in immature male Salmo salar in which testosterone is reported to increase lep expression in liver and pituitary while plasma level of leptin has been shown to remain unchanged in androgen-treated fish 28 . In another study, estradiol has been shown to upregulate hepatic lep expression in a dose-and time-dependent manner in immature female as well as male T. albonubes 26 . Regarding leptin receptor, testosterone is reported to stimulate the transcription Scientific RepoRtS | (2020) 10:999 | https://doi.org/10.1038/s41598-020-57922-x www.nature.com/scientificreports www.nature.com/scientificreports/ in pituitary but not in testis of immature male S. salar. However, 11-ketoandrostenedione had no effect on the expression of both, lep and its receptor, in any of the tissues 28 . In mammals, male and female sex steroids have contrasting effects on expression and production of leptin from adipose tissue, inhibitory effect of androgen in male as well as female 17,18 while stimulatory effect of estrogen in female 41 . With regard to sex steroid-induced modulation of LEPR expression, studies are meagre and restricted to estrogen only. Estradiol is reported to stimulate hypothalamic LEPR expression in rat 50 but not in heifers 51 . Also, effect of E 2 on the receptor expression is shown to vary with tissues in heifers as it has been inhibitory in uterine endometrium and mammary adipose tissue while ineffective in liver, muscle and subcutaneous adipose tissue 51 . Based on these studies and our observations in C. punctata, it is evident that role of sex steroids in modulation of leptin and its receptor expression vary with sex, tissue and species.
In addition, in vitro experiments with DHT and E 2 in the present study exemplifies direct implication of sex steroids in regulation of leptin and its receptor expression in lymphoid organs of male and female C. punctata. Although no such in vitro experiments are conducted with immune tissues of fishes or other vertebrates, culture of hepatocytes with androgen and estrogen, separately, has shown upregulation of lep expression in immature/ adult male and immature female S. salar 27 . In contrast, male as well as female sex steroids have been demonstrated to downregulate the expression of lep and its receptor in hepatocytes of immature female Cynoglossus semilaevis 31 . Apart from fishes, direct role of sex steroids has been explored also in mammals where androgens and estrogen are shown to have differential effects, inhibitory, stimulatory or no effect, on leptin production from adipocytes 18,[52][53][54] . Surprisingly, in vitro study to demonstrate the effect of sex steroids on expression of leptin receptor is missing in mammals. In the current in vitro study, DHT and E 2 downregulated the transcription of leptin and its receptor in spleen and head kidney of C. punctata, except DHT on lep and lepr in female spleen and E 2 on lep in female spleen and lepr in male head kidney where upregulation in transcription was observed. Also, dual effects of sex steroids depending on concentration were observed on lep expression in male spleen. This largely ratify the in vivo observations that have evidenced variable role of sex steroids depending on dose/concentration, type of lymphoid organs and sex of fish, in modulating the leptin system.

conclusion
The expression of lep and lepr in immune organs of C. punctata preempts the direct role of leptin in regulation of immune system in fishes. Also, seasonal variation in expression of lep and lepr in primary as well as secondary lymphoid organs of C. punctata point towards an axis operating between reproduction-leptin-immunity in fishes. A marked sexual dimorphism in splenic and head kidney lep and lepr expression during reproductively active phase only suggested the involvement of sex steroids in regulating leptin system of lymphoid organs. This hypothesis was validated by in vivo and in vitro experiments with sex steroids. In view of this, sex steroids emerge as important modulator of leptin system in lymphoid organs of fishes.

Materials and Methods
Reagents and culture medium. The culture medium RPMI 1640 was supplemented with 0.1 mg/ml streptomycin sulphate and 40 µg/ml gentamycin sulphate. Stock solution of sex steroids, 5α-dihydrotestosterone (DHT) and 17β-estradiol 3-benzoate (E 2 ), were made in ethanol (1 mg/ml). Their further dilutions ranging from 10 µg/ml to 100 ng/ml were prepared in phosphate buffered saline (PBS, pH 7.4) containing above said concentrations of streptomycin and gentamycin. Finally, working concentrations of sex steroids were made in culture medium. The media of control groups contained the maximum concentration of ethanol (0.001%). Estradiol and testosterone enzyme-linked immunosorbent assay (EIA) kits were purchased from Cayman Chemical.
Animals and ethics statement. Fishes (90-120 g) were procured from wild population (National Capital Region of Delhi) and acclimated to laboratory conditions for a week at 12 L:12 D prior to experiments. They were fed ad libitum with minced chicken liver. An overdose of 2-phenoxyethanol (5 ml/L water) was used to sacrifice the fish. Our protocol has been approved by the Institutional Animal Ethics Committee (DU/ZOOL/ IAEC-R/2017/06), Department of Zoology, University of Delhi. All experiments were carried out following relevant guidelines and regulations of IAEC.

Differential expression of lep and lepr.
Reproductive phase-dependent. On the basis of histological observations of gonads, reproductive cycle has been broadly delineated into resting, preparatory, spawning and postspawning phases in male 40 and female 55 Channa punctata. Fishes were procured during the peak of each reproductive phase to examine differential expression of leptin (lep) and leptin receptor (lepr) in primary and secondary lymphoid organ, head kidney and spleen, respectively. In each reproductive phase, spleen and head kidney from male and female fish (N = 8 for each sex) were dissected out, quickly frozen in liquid nitrogen and stored at −80 °C until RNA extraction.

Sex-dependent.
To examine sex-related differential expression of lep and lepr in lymphoid organs, data of contrasting reproductive phases, spawning (active) and resting (quiescent), were selected. The expression of lep and lepr in spleen or head kidney of female during resting or spawning phase was compared with respective gene expression in the same lymphoid organ of male of that particular phase. Experiment: Effect of sex steroids on lep and lepr expression in lymphoid organs. To examine the role of sex steroids in regulation of lep and lepr expression in spleen and head kidney, in vivo and in vitro experiments were performed with sex steroids during resting phase when plasma T and E 2 remain at basal level in male and female C. punctata, respectively.
In vivo experiment. The range of doses for DHT and E 2 was determined based on plasma levels of T and E 2 in adult male and female C. punctata, respectively, during different reproductive phases. Each male fish of group I, II and III received 9, 45 and 90 ng of DHT/injection/day, respectively. Likewise, three groups of female fish were made to receive different doses of E 2 (50 ng, 250 ng and 500 ng per injection/day/fish).
Given the facts that female sex steroid E 2 plays critical role in regulation of male reproduction 40,56 and vice versa for testosterone in female fish 57,58 , a criss-cross experiment was designed where male fish received different doses of E 2 (250 and 500 ng/injection/fish/day) while females were administered 45 and 90 ng of DHT/injection/ fish/day. For controls, fishes of both the sexes were injected with comparable volume of vehicle (100 µl of 0.6% saline/injection/fish/day).
Fish of all the groups (N = 8 for each experimental group of male or female) received injections for three consecutive days. They were sacrificed after 18 h of the last injection. Their spleens and both side head kidneys were dissected out and used for gene expression analysis.
In vitro experiment. The experiment was designed to determine the direct role of sex steroids in regulation of lep and lepr expression in immune organs. Spleens and both side head kidneys from six adult male and same number of female C. punctata were dissected out, pooled sex-wise, washed and chopped into small pieces. Prior to incubation with sex steroids, tissue (10-15 mg/well) from spleen or head kidney was cultured in medium for 2 h in a 24-well culture plate. Thereafter, spent media was aspirated out and fresh media containing different concentrations of E 2 (0.36 µM, 3.67 µM, 36.7 µM) or DHT (0.68 µM, 3.44 µM, 34.4 µM) were added to each well of culture plate. For controls, tissues were incubated in medium alone. The culture was run in hexaplicate. After12 h of incubation, tissues were collected, zap frozen in liquid nitrogen and stored at −80 °C until RNA extraction.
Relative gene expression using quantitative real-time PCR. Total RNA was extracted from spleen and head kidney using TRI reagent following manufacturer's instructions. RNA samples having optical density ratio (A 260/280 ) between 1.8 to 2.0 and optimal integrity were processed for cDNA preparation. In brief, one microgram RNA was incubated with 10U DNase I at 37 °C for 30 min. Thereafter, DNase was heat-inactivated at 65 °C for 10 min in the presence of EDTA (50 mM, pH 8.0). Further, random hexamer primer was used to reverse transcribe the DNase-treated RNA. The cDNA thus prepared was validated by reverse transcription Polymerase Chain Reaction (RT-PCR) using a housekeeping gene 18S ribosomal RNA (18S rRNA). Primers specific for leptin and leptin receptor (Supplementary Table S1) were designed using testicular transcriptome data of C. punctata (NCBI bioproject accession no. PRJNA304088). The obtained sequences (accession number: lep-MK039679, lepr-MK039680) were used to design the primers for quantitative PCR (qPCR, Supplementary Table S2). The percentage efficiency of qPCR primers is also listed in Supplementary Table S2. In parallel, as a reference gene, specific primers of 18S rRNA were used for qPCR. The reaction was carried out using power SYBR Green PCR Master Mix following manufacturer's protocol. All the samples were run in duplicate and no template control were run with each reaction. estimation of plasma sex steroids. For estimation of E 2 and T during reproductively active spawning and quiescent resting phases in female fish (N = 6 for each reproductive phase), blood was collected and centrifuged at 2300 g for 10 min. Plasma was extracted out and stored at −80 °C until steroid estimation. To extract the total steroid, 1 ml diethyl ether was added to 200 µl plasma sample, vortexed for 5 min, kept at room temperature for 15 min and ether phase was separated out. This procedure was repeated thrice to maximally extract total steroid from the same aliquot of a plasma sample. The collected ether phases were pooled, evaporated at room temperature, dried and kept at −20 °C until assayed for E 2 and T. Each sample was reconstituted in 200 µl of assay buffer and loaded in duplicates for estimation of E 2 and T using respective EIA kit. As per manufacturer's protocol, minimum detection limit of E 2 and T was 19 and 6 pg/ml, respectively. The accuracy of kit was verified by percentage recovery and linearity of detection using serial dilutions of sample. Our earlier report on plasma level of sex steroids in male C. punctata 53 , showing high level of T and low level of E 2 during spawning than resting phase, was used in the present study for correlation analysis. correlation analyses. A correlation between expression of lep and lepr in lymphoid organs and plasma level of sex steroids was examined in male as well as female C. punctata during reproductively active and quiescent phases, spawning and resting, respectively. To understand immune organ-specific reproductive phase-dependent interrelation between expression of ligand and its receptor, a correlation analysis was also carried out between expression of lep and lepr in each lymphoid organ of male as well as female C. punctata along their reproductive phases.
Statistical analysis. After normalization with 18S rRNA expression, relative fold change was calculated following an optimized method 59 . In case of lep expression in head kidney of male and female, fold change values were log-transformed to meet normality and heterogeneity of variance. One-way analysis of variance followed by Student-Newman-Keuls (SNK) multiple range test was employed to analyse expression of lep and lepr in each lymphoid organ of either sex depending on reproductive phases or after treatment (in vivo/in vitro) with sex steroids. Student's unpaired t-test was applied for sex-related differential expression (male vs female, p < 0.05). Correlation analyses were carried out using Pearson's correlation test to examine interrelation between levels of plasma sex steroids and expression of lep and lepr in spleen and head kidney during reproductively active and quiescent phases in male as well as female C. punctata. Also, a correlation between lep and lepr expression was analysed (Spearman's correlation test at 95% confidence interval) in each lymphoid tissue of male as well as female along their reproductive phases. The statistical analyses were carried out using GraphPad Prism5 software.