Functional evaluation of a novel kisspeptin analogue on the reproduction of female goldfish

Abstract

Kisspeptin (kp) is a key regulator of reproduction, which stimulates sexual maturation and gametogenesis in mammals, amphibians, and teleosts. In the present study, to enhance the biological activity of kp10, a novel analog (referred to as M-kp10) was designed based on the endogenous goldfish variant, in which phenylalanine 6 was substituted by tryptophan and the N-terminus was acetylated. Compared with the native kp-10 and salmon gonadotropin-releasing hormone (GnRH3), the effect of M-kp10 on sexual hormones and reproductive indices as well as the expression of kiss1, cyp19a1, and kiss1ra genes in goldfish (Carassius auratus) was investigated. In practice, peptides were synthesized based on the standard Fmoc-solid-phase peptide synthesis and purified by employing RP-HPLC, followed by approving their structure using ESI-MS. The results showed that M-kp10 increased significantly 17,20β-DHP, LH, FSH and E2 as well as fecundity, hatching and fertilization percentages than the other peptides. Histological studies revealed that M-kp10 led to the faster growth of ovarian follicles compared to the kp-10 and GnRH3. The genes of cyp19a1, kiss1ra, and kiss1 were remarkably more expressed after treatment with M-kp10. In conclusion, the results indicated the superiority of M-kp10 over kp-10 in inducing sexual maturation and accelerating the percentage of fecundity, suggesting that M-kp10 could be a promising candidate for application in the artificial breeding of fish.

Figure 1

Variations of the luteinizing hormone (LH), the follicle stimulating hormone (FSH) and the 17β estradiol (E2) in the female goldfish treated with kp-10, M-kp10 and GnRH3, 6 h post-injection. The different letters above columns are showing significant differences based on one-way ANOVA and Tukey’s post hoc test.

Figure 2

Variations of 17α-20β-Hydroxy-4-peregnen-3-one (DHP), 11-keto testosterone (11KT), lipoprotein lipase (LPL) and cortisol in the female goldfish treated with kp-10, M-kp10 and GnRH3, 6 h post-injection. The different letters above columns are showing significant differences based on one-way ANOVA and Tukey’s post hoc test.

Figure 3

Changes in the expressions of kiss1 gene in the ovarian and hypothalamus tissues of female goldfish based on the RT-PCR (The actb is considered as the control). The different letters above columns are showing significant differences based on one-way ANOVA and Tukey’s post hoc test.

Figure 4

Changes in the expressions of cyp19a1 gene in the ovarian and hypothalamus tissues of female goldfish based on the RT-PCR (The actb is considered as the control). The different letters above columns are showing significant differences based on one-way ANOVA and Tukey’s post hoc test.

Figure 5

Changes in the expressions of kiss1ra gene in the ovarian and hypothalamus tissues of female goldfish based on the RT-PCR (The actb is considered as the control). The different letters above columns are showing significant differences based on one-way ANOVA and Tukey’s post hoc test.

Figure 6

Photomicrographs of H&E-stained ovary in the female goldfish. (A) Negative control, (B) Domperidone control, (C) kp-10, (D) M-kp10, (E) GnRH3 (250 μg/ml), and (F) GnRH3 (100 μg/ml) and maturation percentage of oocytes (G). Abbreviations: PVO, pre-vitellogenic oocyte; EVO, early-vitellogenic oocyte; VO, vitellogenic oocyte; LVO, late-vitellogenic oocyte; PMO, pre-mature oocyte; MO, mature oocyte. Scale bar = 500 µm.

Figure 7

Relative fecundity, percentage of fertilization and hatching in the female goldfish treated with kp-10, M-kp10 and GnRH3. The different letters above columns are showing significant differences based on one-way ANOVA and Tukey’s post hoc test.

Figure 8

Sequence and structure of peptides. Sequences of (a) native kp-10, (b) M-kp10 in which Phe 6 is substituted with Trp and the N-terminus is acetylated, and (c) GnRH3, and structure of (d) kp-10, (e) M-kp10, and (f) GnRH3.