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

Abstract

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 (E 2) 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 e 2 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 e 2. 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. Leptin encoded by obese gene Lep also known as Ob is a 16 kDa non-glycosylated protein belonging to class I cytokine family and is primarily produced from adipose tissue in mammals 1. It is reported to act through long form of membrane-bound leptin receptor (Lepr) 2. Besides acting as a central link between feeding, adiposity and energy homeostasis 3 , leptin regulates several other physiological functions in mammals including reproduction 4 and immunity 5. The orthologs of Lep and Lepr have been identified in several teleosts 6-10 in which liver, and not the adipocytes, is reported to be the major leptin-expressing organ. Regarding physiological significance, hepatic lep is suggested to be majorly involved in orchestration of energy trade-off rather than regulating food intake in fishes 9,11-13. In addition to liver, expression of lep is shown in other tissues, including immune organs 6,10,11. Since immune defence varies with state of reproductive activity in seasonally breeding vertebrates 14,15 , an effort needs to be made to examine correlation between leptin, immunity and reproduction. In addition, sexual dimorphism is unveiled in levels of leptin in blood plasma 16,17 and adipose tissue 18,19 in mammals, being significantly higher in females than males. Interestingly, these sex-related differences in leptin protein and mRNA levels exist regardless of the amount of body fat 16. This led to hypothesize the involvement of sex steroids in modulating leptin expression 20. With regard to sexually dimorphic expression of leptin receptor, reports in mammals are limited and present contradictory results 21,22. In teleosts, studies on sex-related variation in plasma levels of leptin 23,24 or expression of lep and lepr have been me...

Reproductive phase–dependent and sexually dimorphic expression of leptin and its receptor in different parts of brain of spotted snakehead Channa punctata

“…The levels of lep and lepr were higher in middle and posterior parts of the male brain than that of the female brain during reproductively active as well as quiescent phases. A minimal level of testosterone/11‐ketotestosterone in male and 17β‐estradiol in female during resting phase and a maximal level of respective sex steroids during active phase of C. punctata (Bakshi & Rai, 2020; Basak et al ., 2016) indicate that factors other than sex steroids might be playing a role in sexually dimorphic lep and lepr expression in these regions of brain. A similar dimorphic expression pattern of lep has been reported in hypothalamus of M .…”

“…Previous findings of the authors demonstrate high plasma level of testosterone (T) in male and 17β-estradiol (E 2 ) in female spotted snakehead C. punctata during the reproductively active spawning phase. The levels of T and E 2 have been found to significantly decrease during the reproductively quiescent resting phase in male and female fish, respectively (Bakshi & Rai, 2020;Basak et al, 2016). On this basis, the data of these contrasting reproductive phases were selected to scrutinise sex-wise variation in the expression of lep/lepr in anterior, middle and posterior parts of the brain (male vs. female).…”

“… TABLE S1. Primer sequences (5′➔3′) used for quantitative real‐time PCR (qPCR) of lep and lepr in C. punctata (Bakshi & Rai, 2020) …”

In silico analyses of leptin and leptin receptor of spotted snakehead Channa punctata

“…Thermocycling conditions adopted for PCR are enlisted as: preliminary denaturation (94 ο C for 1 min), and thereafter 30 cycles of each, denaturation (94 ο C for 30 sec/ cycle), annealing (68.3 ο C for 30 sec/ cycle) and extension (72 ο C for 3 min/ cycle). For validation of cDNA, qPCR primers of lepr [ 30 ] were used as positive control. The amplified product was resolved by electrophoresis on 1% agarose gel containing 0.05% ethidium bromide and observed under ultraviolet illumination.…”