Comparative transcriptomic analysis of follicle-enclosed oocyte maturational and developmental competence acquisition in two non-mammalian vertebrates

Abstract

Background: In vertebrates, late oogenesis is a key period during which the oocyte acquires its ability to resume meiosis (i.e. maturational competence) and to develop, once fertilized, into a normal embryo (i.e. developmental competence). However, the molecular mechanisms involved in these key biological processes are far from being fully understood. In order to identify key mechanisms conserved among teleosts and amphibians, we performed a comparative analysis using ovarian tissue sampled at successive steps of the maturational competence acquisition process in the rainbow trout (Oncorhynchus mykiss) and in the clawed toad (Xenopus laevis). Our study aimed at identifying common differentially expressed genes during late oogenesis in both species. Using an existing transcriptomic analysis that had previously been carried out in rainbow trout, candidate genes were selected for subsequent quantitative PCR-based comparative analysis.

Results: Among the 1200 differentially expressed clones in rainbow trout, twenty-six candidate genes were selected for further analysis by real-time PCR in both species during late oogenesis. Among these genes, eight had similar expression profiles in trout and Xenopus. Six genes were down-regulated during oocyte maturation (cyp19a1, cyp17a1, tescalcin, tfr1, cmah, hsd11b3) while two genes exhibited an opposite pattern (apoc1, star). In order to document possibly conserved molecular mechanisms, four genes (star, cyp19a1, cyp17a1 and hsd11b3) were further studied due to their known or suspected role in steroidogenesis after characterization of the orthology relationships between rainbow trout and Xenopus genes. Apoc1 was also selected for further analysis because of its reported function in cholesterol transport, which may modulate steroidogenesis by regulating cholesterol bioavailability in the steroidogenic cells.

Conclusions: We have successfully identified orthologous genes exhibiting conserved expression profiles in the ovarian follicle during late oogenesis in both trout and Xenopus. While some identified genes were previously uncharacterized during Xenopus late oogenesis, the nature of these genes has pointed out molecular mechanisms possibly conserved in amphibians and teleosts. It should also be stressed that in addition to the already suspected importance of steroidogenesis in maturational competence acquisition, our approach has shed light on other regulatory pathways which may be involved in maturational and developmental competence acquisitions that will require further studies.

Figure 1

Gene expression profiling during rainbow trout late oogenesis Supervised average linkage clustering analysis of 1200 clones in rainbow trout ovary during late vitellogenesis (LV, n = 3), post-vitellogenesis (PV, n = 4) and during maturation (Mat., n = 6). Each row represents a gene and each column an ovarian RNA sample. The 13 samples are supervised according to the natural time-course of oogenesis. Data were log2-transformed and median-centered prior to the clustering analysis. For each gene the expression level within sample set is indicated using a color density scale. Red and green are used for over- and under-expression respectively, while black is used for median expression.

Figure 2

Expression of 26 genes assessed by real-time PCR in Oncorhynchus mykiss and Xenopus laevis ovarian samples. QPCR analysis of the 26 candidate genes in rainbow trout (A) ovary during late vitellogenesis (LV, n = 6), post-vitellogenesis (PV, n = 14) and during maturation (Mat., n = 8), and in Xenopus (B) ovarian follicles at stage IV, stage VI and after oocyte maturation (metaphase-II arrested oocytes) from six adult females. Data were normalized to the abundance of 18S, log2-transformed, and median-centered prior to the clustering analysis. The expression data sets have been supervised according to oogenesis stage. Genes with similar expression profiles in both species are bolded. The dendrograms on the left represent correlation distances between the profiles of studied trout genes. For each gene the expression level within sample set is indicated using a color density scale. Red and green are used for over- and under-expression respectively, while black is used for median expression (grey boxes, not determined).

Figure 3

Cyp17a1 amino acid sequence alignments among vertebrates. Amino acid sequence alignments between human CYP17A1 (ENSG00000148795, H. sapiens), mouse Cyp17a1 (NP_031835.3, M. musculus), chicken CYP17A1 (ENSGALG00000008121 peptide ENSGALP00000032532, G. gallus), clawed toad Cyp17a1 (AAG42003, X. laevis), zebrafish Cyp17a1 (AAI62669.1, D. rerio) and rainbow trout Cyp17a1 (NP_001118219.1, O. mykiss). Multiple amino acid sequence alignments were constructed using ClustalW software. The conserved domains previously identified are indicated: domain I (heme-binding domain [22]); domain II (putative steroid-binding domain [23]); domain III (CYP17 specific domain [21]). The amino acids that have been evidenced as essential for human CYP17 activity are indicated with asterisks (serine 106, aspartic acid 487, serine 488 and phenylalanine 489) [24,25].

Figure 4

hsd11b3 amino acid sequence alignments among vertebrates. Amino acid sequence alignments between human HSD11B3 (ENSP00000340436, H. sapiens), chicken HSD11B3 (NP_001001201.1, G. gallus), clawed toad hsd11b3 (BC106472, X. laevis), zebrafish hsd11b3 (ENSDARG00000004562, D. rerio) and rainbow trout hsd11b3 (CA348069, O. mykiss). Multiple amino acid sequence alignments were constructed using ClustalW software. The superfamily Rossmann-fold NAD(P)H/NAD(P)(+) binding domain is underlined.

Figure 5

apoC1 amino acid sequence alignments among vertebrates. Amino acid sequence alignments between human APOC-I (NP_001636.1, H. sapiens), mouse APOC-I (NP_031495.1, M. musculus), rat APOC-I (NP-036956.1, R. norvegicus), the clawed toad apoC1 (CF547196, X. laevis), tetraodon apoC1 (CAF95503, T. nigroviridis), the gilthead seabream apoc1 (AAT45249.1, S. auratus) and the deduced amino acid sequence of rainbow trout apoC1 (CA353171, O. mykiss). Multiple amino acid sequence alignments were constructed using ClustalW software. The signal peptide is indicated.

Figure 6

star expression profiles during late oogenesis and tissue expression profiles. Expression profiles of star in rainbow trout ovary sampled from females during late vitellogenesis (LV, n = 6), post-vitellogenesis (PV, n = 14) and during maturation (Mat., n = 8) (A), in Xenopus laevis ovarian follicles sampled from six females, at stage IV, stage VI and after in vitro maturation (st VI MII) (B). Expression of star mRNA in rainbow trout tissues: brain (Br), heart (He), stomach (St), liver (Li), intestine (In), muscle (Mu), skin (Sk), post-vitellogenic ovary (Ov), and testis (Te) (C) and in Xenopus laevis tissues: brain (Br), heart (He), stomach (St), liver (Li), intestine (In), muscle (Mu), skin (Sk), ovary (Ov), testis (Te). Data were normalized to the abundance of 18S. Mean and SEM are shown. Bars sharing the same letter(s) are not significantly different (p > 0.05). In tissue, expression levels which are not significantly different from background signal are indicated with #.

Figure 7

aromatase expression profiles during late oogenesis and tissue expression profiles. Expression profiles of aromatase (cyp19a1a for trout and p450arom-A for Xenopus) during late oogenesis and in various tissues. Experiments were conducted in parallel for all genes. See fig. 6 legend for details.

Figure 8

cyp17a1 expression profiles during late oogenesis and tissue expression profiles. Expression profiles of cyp17a1 during late oogenesis and in various tissues in rainbow trout and Xenopus. Experiments were conducted in parallel for all genes. See fig. 6 legend for details.

Figure 9

hsd11b3 expression profiles during late oogenesis and tissue expression profiles. Expression profiles of hsd11b3 for trout and Xenopus. Experiments were conducted in parallel for all genes. See fig. 6 legend for details.

Figure 10

apoC1 expression profiles during late oogenesis and tissue expression profiles. Expression profiles of apoC1 for trout and Xenopus. Experiments were conducted in parallel for all genes. See fig. 6 legend for details.