Testis development in the Japanese eel is affected by photic signals through melatonin secretion

Abstract

Objective: According to reported spawning characteristics of Japanese eel, Anguilla japonica, which exhibit spawning and migration patterns that are synchronized with lunar cycles and photoperiod, we hypothesized that a close association exists between specific photic signals (daylight, daylength, and moonlight) and endocrinological regulation. Given the photic control in melatonin secretion, this hypothesis was tested by investigating whether melatonin signals act as mediators relaying photic signals during testis development in the eel.

Methods: We examined changes in melatonin-secretion patterns using time-resolved fluorescence immunoassays in sexually immature and mature male Japanese eels under the condition of a new moon (NM) and a full moon (FM).

Results: The eye and plasma melatonin levels exhibited a nocturnal pattern under a 12-h light: dark cycle (12L12D) or under constant darkness (DD), but not with constant light (LL). Eye melatonin levels were similar under the 12L12D and short-day (9L15D) conditions. In the long-day condition (15L9D), secreted plasma melatonin levels were stable, whereas short-day melatonin secretion began when darkness commenced. Sexual maturation began at 8 weeks following intraperitoneal injection of human chorionic gonadotropin (hCG), and NM exposure led to significantly higher eye and plasma melatonin levels compared with those detected under FM exposure.

Keywords: Eel migration; Japanese eel; Melatonin; Moonlight; Photic signal; Photoperiod.

Figure 1. Experimental design for determining melatonin levels in ocular and plasma according to exposure to an NM or an FM.

The solid and open squares represent the NM and FM groups, respectively. Following acclimation to fresh water for 1 week, the eels were gradually increased to a salinity approximating that of seawater for 1 week. After acclimation to seawater, the fish were transferred to two separate outdoor tanks for inducing sexual maturation. The fish were weighed and intraperitoneally injected with hCG (hCG+ group) or 0.6% saline (hCG–  group) once a week on the same day (totally eight times) until the sampling date (NM, November 22, 2014; FM, December 06, 2014).

Figure 2. Variations of daily and circadian eye and plasma melatonin rhythms after 3 days of rearing under the 12L12D (eye; A, plasma; D), DD (eye; B, plasma; E), and LL (eye; C, plasma; F) conditions.

The values shown for the melatonin levels represent the means ± standard errors of the mean (SEMs) (n = 5–6 fish per time point), where duplicate determinations were performed for each sampling time. The open and solid bars of each graph represent the scotophase and photophase, respectively. Significant differences between the means at each sampling time are indicated by different letters (one-way ANOVA, A; F = 12.22, df = 33, B; F = 6.41, df = 33, C; F = 2.456, df = 33, D; F = 5.345, df = 30, E; F = 4.583, df = 31, F; F = 2.702, df = 32, P <  0.05).

Figure 3. Photoperiodic changes of eye (A) and plasma (B) melatonin levels under a short-day condition (SD, 9L15D) and a long-day condition (LD, 15L9D).

The values of the melatonin levels shown represent the means ± SEM (n = 5 per time point) of duplicate determinations for each sampling time. The solid and open circles indicate the SD and LD conditions, respectively. The open and solid bars at the top of each graph represent the scotophase and photophase, respectively. The asterisk indicates statistically different levels of melatonin observed between same sampling points (unpaired t-test, P < 0.05).

Figure 4. Histological observation of the testis development.

Light-micrographs images of the testes from the groups acclimated to FW (A) and SW (B). After an 8 weeks treatment with hCG or saline, histological observations were performed to examine the testes from the FM (hCG–; C, hCG+; E) and NM (hCG–; D, hCG+; F) groups. The testes were sectioned after paraffin embedding and stained with hematoxylin and eosin.

Figure 5. Changes of EI and GSI values.

The black and yellow circles represent data from the NM and FM groups, respectively. The GSI (A; F = 69.04, df = 45) and EI (B; F = 130.6, df = 45) values shown were determined after FW and SW acclimation, for Japanese eels in the NM and FM groups. In all graphs, the significance levels are as follows: ^∗P < 0.05; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001. The error bars represent the SEMs (n = 5–9 fish/treatment).

Figure 6. Changes in eye and plasma melatonin levels between Japanese eels exposed to an NM or FM, according to testis development.

The black and yellow circles represent eels from the NM and FM groups, respectively. The levels of eye (A; F = 14.93, df = 42) and plasma melatonin (B; F = 16.43, df = 43) in hCG+ group after NM exposure were significantly higher than those in the other groups. In all graphs, the significance levels are as follows: ^∗∗P < 0.01; ^∗∗∗∗P < 0.0001. The error bars represent the SEM (n = 5–9 fish/treatment).