Exploring the Fanconi Anemia Gene Expression and Regulation by MicroRNAs in Gilthead Seabream (Sparus aurata) at Different Gonadal Development Stages

Abstract

Fanconi anaemia (FA) is a rare autosomal recessive disease in humans that is distributed worldwide. Fanconi anemia complementation (Fanc) proteins are essential for the appropriate functioning of the FA DNA repair pathway. They are also linked to a number of other biological processes, including oxygen metabolism, cell cycle regulation, haematopoiesis and apoptosis. So far, little research has been conducted on teleosts, but evidence shows that Fanc proteins play a significant role in immune response and sex reversal. For the examination of the expression of three fanc genes (fancc, fancl, and fancd2), as well as the potential regulation of these genes by microRNAs (miRNAs) in gonadal tissues at different stages of development, the present study has selected the gilthead seabream (Sparus aurata), a significant aquaculture species that exhibits protandrous hermaphroditism. The obtained data suggested the role of fancl and fancd2 in the maturation of female gonads and the miRNAs miR-210, miR-217 and miR-10926 have been identified as putative regulators of fancd2, fancc and fancl, respectively. Overall, the data indicated the potential use of fancl and fancd2 genes as sex biomarkers in conjunction with their respective regulation by miRNAs. To the best of our knowledge, this is the first study demonstrating the importance of fanc genes, along with putative regulatory miRNAs, in the reproduction of an important marine aquaculture species.

Keywords: Expression; Fanconi anemia genes; Gonadal stages; Hermaphrodite; Teleost; miRNA.

Fig. 1

Histological sections of ovaries and testes of 6-year-old gilthead seabream during the reproductive period (February). a, b and c. Group F ovaries collected from mature females with vitellogenic oocytes (Vg); d, e and f. Group fM ovaries (inactive and immature ovarian part of male fish), consisting exclusively of primary oocytes (po); g, h and i. Group M testes (active and mature testicular part of male fish) containing spermatocytes (sc) and spermatozoa (sz). The scale bars of each row correspond to the three photographs of the same row (200 μm for photographs a-f and 100 μm for photographs g-i)

Fig. 2

Total RNA profiles in DNAnalyzer from mature female gonads (group F, a), inactive and immature ovarian part of male fish (fM, b) and active and mature testicular part of male fish (M, c) of 6-year-old gilthead seabream collected during the reproductive season (February). The numbers shown at the top right of each graph represent the 5S/18S index

Fig. 3

Differential expression values of fancl, fancc and fancd2 genes among M, F and fM gonads of the gilthead seabream during the reproductive season determined by qPCR. Different letters indicate statistically significant differences in the expression of fanc genes among the three gonadal types (p < 0.05). Values are presented as mean ± SEM (n = 3)

Fig. 4

a Read length distribution of small RNA sequences from 6-year-old gilthead seabream gonads during the reproductive season. Red: mature ovary (F), purple: inactive and immature ovarian part of male fish (fM), blue: active and mature testicular part of male fish (M). b PCA analysis of sncRNAs of mature female gonads (F-1, F-2, and F-3), inactive and immature ovarian part of male fish (fM-1, fM-2 and fM-3) and active and mature testicular part of male fish (M-1, M-2 and M-3) of 6-year-old seabream. The first principal component (PC1) is expected to discriminate samples from different biological conditions. The first two components of the PCA are presented, with the percentages of variance associated with each axis

Fig. 5

Hierarchical clustering of the differentially expressed miRNAs among mature and active male parts of the male gonads (M), the mature female gonads (F) and the immature and inactive ovarian parts of male gonads (fM) of sharpsnout seabream (padj < 0.005) collected during the reproductive season. Individual gonadal samples of each group (M F, and fM) are indicated at the bottom of each column. Each row represents the expression of one miRNA and blue and yellow represent low and high abundance, respectively. The expression of the fanc genes, being putatively targeted by miR-210, miR-10926 and miR-217 are shown on the right

Fig. 6

Putative hybidization of (a) miR-210 with the 3’ untranslated region (3’UTR) of fancd2, (b) miR-10926 with the 3’ UTR of fancl (c) miR-217 with the 3’ UTR of fancc, applying RNAhybrid analysis, with minimum freedom energy (mfe) of < −20 and with complete seed region complementarity

Fig. 7

Total number of reads of the ten most abundant differentially expressed miRNAs in the three different gilthead seabream gonad comparisons, between F and M gonads (a), between fM and M gonads (b) and between F and fM gonads (c) during the reproductive season. F represents mature female gonads, M represents the mature and active male parts of male gonads and fM represents the immature and inactive parts of male gonads