Testicular Steroidogenesis and Locomotor Activity Are Regulated by Gonadotropin-Inhibitory Hormone in Male European Sea Bass

Abstract

Gonadotropin-inhibitory hormone (GnIH) is a neurohormone that suppresses reproduction by acting at both the brain and pituitary levels. In addition to the brain, GnIH may also be produced in gonads and can regulate steroidogenesis and gametogenesis. However, the function of GnIH in gonadal physiology has received little attention in fish. The main objective of this study was to evaluate the effects of peripheral sbGnih-1 and sbGnih-2 implants on gonadal development and steroidogenesis during the reproductive cycle of male sea bass (Dicentrarchus labrax). Both Gnihs decreased testosterone (T) and 11-ketotestosterone (11-KT) plasma levels in November and December (early- and mid-spermatogenesis) but did not affect plasma levels of the progestin 17,20β-dihydroxy-4-pregnen-3-one (DHP). In February (spermiation), fish treated with sbGnih-1 and sbGnih-2 exhibited testicles with abundant type A spermatogonia and partial spermatogenesis. In addition, we determined the effects of peripheral Gnih implants on plasma follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) levels, as well as on brain and pituitary expression of the main reproductive hormone genes and their receptors during the spermiation period (February). Treatment with sbGnih-2 increased brain gnrh2, gnih, kiss1r and gnihr transcript levels. Whereas, both Gnihs decreased lhbeta expression and plasma Lh levels, and sbGnih-1 reduced plasmatic Fsh. Finally, through behavioral recording we showed that Gnih implanted animals exhibited a significant increase in diurnal activity from late spermatogenic to early spermiogenic stages. Our results indicate that Gnih may regulate the reproductive axis of sea bass acting not only on brain and pituitary hormones but also on gonadal physiology and behavior.

Fig 1. In vivo effect of sbGnih-1 and sbGnih-2 in male sea bass steroid plasma levels.

Plasma levels of testosterone (T)(A, B, C), 11-ketotestosterone (11-KT)(D, E, F) and 17α,20β-dihydroxy-4-pregnen-3-one (DHP)(G, H, I) were analyzed in sbGnih-1- (B, E, H) and sbGnih-2- (C, F, I) treated animals throughout the reproductive cycle. Vehicle implanted animals represent the control group (A, D, G). Values are expressed as mean ± SEM (n = 12–14). Different lower case letters indicate significant differences throughout the reproductive cycle within each condition (ANOVA, Student-Newman Keuls test, P<0.05). Asterisks (*) denote significant differences between Gnih-treated animals versus controls in a particular month (P<0.05).

Fig 2. Histological analysis of testicles of European sea bass at end of the experiment (February, spermiation).

Full spermiating control fish (A) with lobules mostly filled with sperm (inset). Gnih-1 (B) and Gnih-2 (C) treated fish testicles exhibiting only isolated clusters of sperm and abundant type A spermatogonia (SgA, insets). Bars 50 μm, 100 μm (inset in A) and 10 μm (insets in B and C).

Fig 3. Effect of in vivo implants of sbGnih-1 and sbGnih-2 on brain expression of reproductive hormone genes.

Data show transcript levels of gnrh1 (A), gnrh2 (B), gnrh3 (C), gnrhr-II-2a (D), sbgnih (E) and sbgnihr (F) in male sea bass specimens at the spermiation stage (February). Vehicle implanted animals represent the control group. Values are expressed as mean ± SEM (n = 12–14). Different lower case letters indicate significant differences between treatments (ANOVA, Student-Newman Keuls test, P<0.05).

Fig 4. Effect of in vivo implants of sbGnih-1 and sbGnih-2 on brain expression of reproductive hormone genes.

Data show transcript levels of kiss1 (A), kiss1r (B), kiss2 (C) and kiss2r (D) in male sea bass specimens at the spermiation stage (February). Vehicle implanted animals represent the control group. Values are expressed as mean ± SEM (n = 12–14). Different lower case letters indicate significant differences between treatments (ANOVA, Student-Newman Keuls test, P<0.05).

Fig 5. Effect of in vivo implants of sbGnih-1 and sbGnih-2 on pituitary expression of reproductive hormone genes.

Data show transcript levels of fshβ (A), lhβ (B) and gnrhr-II-1a (C) in male sea bass specimens at the spermiation stage (February). Vehicle implanted animals represent the control group. Values are expressed as mean ± SEM (n = 12–14). Different lower case letters indicate significant differences between treatments (ANOVA, Student-Newman Keuls test, P<0.05).

Fig 6. Effect of in vivo implants of sbGnih-1 and sbGnih-2 on plasma levels of Fsh and Lh.

A, plasma levels of Fsh and B, plasma levels of Lh in male sea bass specimens at the spermiation stage (February). Vehicle implanted animals represent the control group. Values are expressed as mean ± SEM (n = 12–14). Different lower case letters indicate significant differences between treatments (ANOVA, Student-Newman Keuls test, P<0.05).

Fig 7. Effect of sbGnih-1 and sbGnih-2 implants on locomotor activity of male sea bass.

Average diel profiles of locomotor activity (mean waveforms) of controls (A), sbGnih-1- (B) and sbGnih-2- (C) implanted fish from October to February. Each point in the mean waveform was calculated as mean ± SEM from the 10-min binned data over the days of the respective month, indicated at the top right of the mean waveform. The grey area in the waveform represents the mean values and the continuous line the SEM. Horizontal bars above graphs indicate day-time (open bars) and night-time (solid bars).

Fig 8. Effect of sbGnih-1- and sbGnih-2-implants on average monthly percentage of diurnal locomotor activity in male sea bass.

Values are expressed as mean ± SEM from October to February. Different lower case letters indicate significant differences between treatments (Kruskal-Wallis ANOVA on ranks followed by Bonferroni’s test, P<0.0001).