Abstract
Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17β-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.
Highlights
The pituitary gland is a key organ integrating both intrinsic and extrinsic stimuli to regulate vertebrate physiology, including for example reproduction, growth or stress [1, 2]
Pituitary fshb expression levels were at least 10-fold higher than lhb, and at least 100-fold higher than gnrhr2bba levels in immature fish of both photoperiod regimes (Figures 1B, E). fshb, lhb and gnrhr2bba transcripts all increased in the maturing fish exposed to stimulatory conditions (Figure 1E), and all transcripts showed significant correlations with the gonado-somatic index (GSI) values (Supplementary Figures 2B, E, H)
A significant increase in plasma 11-KT was observed in maturing fish (Figure 1F), and its levels correlated to GSI and fshb expression values (Supplementary Figure 3)
Summary
The pituitary gland is a key organ integrating both intrinsic and extrinsic stimuli to regulate vertebrate physiology, including for example reproduction, growth or stress [1, 2]. The anterior pituitary (pars distalis of the adenohypophysis) contains various cell types, together producing a range of peptide hormones Among those cell types, gonadotropes produce the key regulators of reproduction, the gonadotropins folliclestimulating hormone (FSH) and luteinizing hormone (LH) [3], which are produced by two distinct gonadotrope cell types in teleost fish [4]. Androgenic and estrogenic sex steroids exert complex feedback effects on the teleost pituitary, depending among others on gender, stage of maturation, or species examined [as reviewed by Fontaine et al [13]]. In both Atlantic salmon (Salmo salar) [14] and coho salmon (Oncorhynchus kisutch) [11], in vivo treatment with testosterone (T) −and with 17b-estradiol (E2) in the latter species− stimulated the pituitary expression of lhb but not of fshb. Further studies using gonad-intact and gonadectomized Atlantic salmon parr showed that plasma Fsh levels decreased after implanting aromatizable and nonaromatizable androgens [15], demonstrating an important role of androgens in mediating negative feedback on Fsh release
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