Background
Polyunsaturated fatty acids (PUFA) have long been implicated in various aspects of vertebrate reproduction. Both the
n-6 and
n- 3 PUFA influence reproductive processes through a variety of mechanisms, which includes provision of precursors for prostaglandin synthesis, inducement of steroidogenesis and regulation of transcription factors involved in reproductive process [
1]. Among the PUFA, eicosapentaenoic acid (EPA, C20:5
n-3), docosahexaenoic acid (DHA, C22:6
n-3) and arachidonic acid (ARA, C20:4
n-6), also collectively known as highly unsaturated fatty acids (HUFA) have been shown to play pivotal role in regulation of oocyte maturation and ovulation [
2,
3]. In aquaculture, supplement of HUFA in broodstock diet is essential to increase probability of spawning success [
4,
5].
In vitro studies using teleost follicles have reported the stimulation of maturation and ovulation by ARA [
6,
7]. Besides the provision of adequate dietary ARA, EPA and DHA levels respectively, studies have also demonstrated the importance of having a balanced dietary ratio of these three HUFA for better reproductive performance [
4,
5]. Studies have shown that several marine fish species selectively transfer HUFA from muscle reserves to oocytes as preparation of long spawning season [
8,
9]. In addition, comparative analysis of fatty acid composition in ovary of wild and captive farmed fish have proposed inferior or imbalanced ratio of HUFA as the main reason for poor spawning performance of farmed broodstock [
3,
10].
Freshwater fish species possess the capacity to synthesize EPA and DHA from linolenic acid (LNA, 18:3
n-3) and ARA from linoleic acid (LA, 18:2
n-6) respectively, through two separate pathways involving desaturation and elongation of their respective precursors [
11,
12]. The extent to which animals, including fish, can convert LNA and LA, to HUFA differs according to species and depend on the activities of the desaturase and elongase enzymes. In most freshwater fish, EPA is synthesized from LNA by desaturation at the Δ6 position, followed by a 2-carbon elongation, and a further desaturation at the Δ5 position. Subsequently, synthesis of DHA from EPA is believed to proceed via a C24 intermediate, requiring two successive elongations to 22:5
n-3 and 24:5
n-3, followed by desaturation at the Δ6 position, and lastly a chain shortening process to produce DHA [
13]. Production of ARA involves desaturation at Δ6 position of LA followed by a 2-carbon elongation process and lastly a desaturation at the Δ5 position [
13]. We have previously reported the presence of desaturase and elongase mRNA in oocytes of two freshwater fish species, swordtail and zebrafish [
14,
15]. Despite the known importance of PUFA in vertebrate reproduction and the proven selective accumulation of unsaturated fatty acids in oocyte and fertilized eggs, very little is known about the existence, role and regulation of the HUFA biosynthesis system in ovary [
16].
In addition to its value as a model organism in developmental biology, the zebrafish is emerging as a useful model to understand oogenesis and folliculogenesis [
17]. The female zebrafish is a prolific spawner with the oocytes easily differentiated to 4–5 distinct follicle stages. In addition, gonadotropin and steroid induced
in vitro maturation of zebrafish oocytes have also been developed [
18,
19]. Accordingly, transcriptome and proteome analysis have been carried out to map vital molecular pathways responsible for both maturation and ovulation processes in zebrafish [
20,
21]. Based on the importance of HUFA in reproduction, we reason that localized desaturation and elongation activities in oocyte could potentially be a source of HUFA for maturation and ovulation processes. As a prerequisite to this hypothesis, we investigated the mRNA levels of desaturase and elongase and unsaturated fatty acids composition in different zebrafish follicle stages. Collectively, the mRNA expression pattern and fatty acid composition indicate the synthesis of ARA and DHA in zebrafish follicles as they enter into maturation and ovulation stages.
Discussion
Oocyte fatty acid composition is species-specific in terms of its apparent abundance and utilization. Among the fatty acids, EPA, DHA and ARA, have been implicated in many key aspects of reproduction such as precursors for prostaglandin synthesis, steroidogenesis and regulation of essential reproductive related transcription factors [
1]. In vertebrates, depending on species, HUFA are either generated by
de novo synthesis or are absorbed from the diet. Freshwater fish species possess the ability to synthesize EPA, DHA and ARA from their respective precursors via 2 separate pathways involving desaturation and elongation processes. In contrast, this ability is limited or non-present in marine species, which also meant that the supply of EPA, DHA and ARA in these species comes exclusively from dietary intake. Despite the known importance of unsaturated fatty acids in oocytes, very little is understood on the presence, role and regulation of their synthesis in oocytes. In contrast, this subject has been well investigated in the mammalian testis [
26]. As a prerequisite to address this issue, we showed here the EPA, ARA and DHA composition of different zebrafish follicle stages and mRNA expression pattern of desaturase and elongase, 2 enzymes involved in the HUFA biosynthesis pathways.
In this present study, histone H2A was used as a housekeeping gene for validation of desaturase and elongase expression in different oocyte stages. Expression of commonly used housekeeping genes such as beta-actin, acidic ribosomal phosphoprotein, elongation factor-1 alpha and glyceraldehyde 3-phosphate dehydrogenase have been reported to show decreasing pattern as follicles matured, making them unsuitable for the traditional normalization method for gene expression determination [
27,
28]. A proposed solution to this decreasing pattern was to create an equivalent average for each follicular stage prior to normalization of gene of interest [
27,
29‐
31]. Recently histone H2A was also used as reference gene in a study on gene expression in trout oocyte stages [
32]. Our results showed the consistency in expression of zebrafish histone H2A in the 5 follicle stages analyzed, confirming its suitability as a reference gene.
There was an increase in ARA and DHA levels in matured follicles. The elevated levels of ARA is in agreement with a study detailing effect of maturation process on lipid profiles of
Bufo arenarum oocyte, which reported significant increase in ARA in phosphatidylserine[
33]. ARA is the precursor for eicosanoids, a group of prostaglandins known to have pivotal roles in oocyte maturation and ovulation [
34‐
36]. The finding showing fortification of matured zebrafish oocytes with ARA strongly indicate the capacity of oocytes to synthesize eicosanoids for follicular maturation and ovulation. In oocytes of pig, cattle and sheep, ARA, together with LA was reported to be the most abundant polyunsaturated fatty acid [
7]. ARA has also been implicated to play essential role in steroidogenesis[
37]. In addition, the trend of increasing ARA level coupled with decreasing EPA level in maturing zebrafish oocytes could be due to the need to increase the ratio of ARA:EPA as EPA may inhibit ARA-based prostaglandin synthesis due to increased competition between EPA with ARA for binding to prostaglandin H synthase [
5,
38]. In fish farming, low ARA:EPA ratio in ovaries and eggs have often been cited as a reason for poor performance in captive broodstock [
3,
10]. Studies in warm and cold water fish species have shown that DHA, together with EPA inhibit gonadotropin-stimulated steroidogenesis, implying the importance of these two HUFAs as regulator of maturation in fish ovary [
39].
Semi-quantitative RT-PCR analysis of elongase gene in different zebrafish oocyte stages revealed high levels of mRNA during the initial PV stage, followed by a reduction in expression during the EV and LV stages. There was a slight increase in expression during maturation. To our knowledge, this is the first report on differential mRNA expression of a long chain fatty acid elongase in different oocyte stages. Although actual enzymatic activities of both genes were not measured here, studies on several fish species have shown that expression of desaturase and elongase mRNA correlates with their enzymatic activities [
14,
15,
40]. The primers utilized for elongase amplification in this study yielded a partial cDNA sequence, which was 100% identical to a zebrafish
elovl family member 5. Elsewhere, functional characterization of zebrafish
elovl5 gene using recombinant
Saccharomyces cerevisiae showed that this enzyme display broad substrate specificities, with ability to elongate monounsaturated fatty acids and the C18, C20 and C22 PUFA [
41]. In addition, the zebrafish
elovl5 is able to perform all the elongation steps necessary to produce DHA and ARA from LA and LNA respectively [
41]. In zebrafish,
elovl5 was also reported to have the capability to elongate the C20 and C22 fatty acids to tetracosapentaenoic acid (C24:5
n-3). This is important as study in rat liver have shown that C24:5
n-3 can be further processed to the physiologically important DHA [
42]. Elsewhere, a mouse ovulatory-selective cDNA library reported increased expression of elongase, which indicates its potential role during ovulation processes [
43]. Recently, it has been shown the mouse
elovl6 is regulated by the sterol regulatory element binding proteins (SREBP) [
44]. Elsewhere, SREBP have also been implicated in the development of avian follicles [
45]. Similarly, the lipid x receptor (LXR), another regulator of elongase is also vital for oocyte maturation, as LXR-deficient mice showed reduced fertility and inability of oocytes to resume meiosis [
46]. Therefore, a possible role for SREBP and LXR in follicle maturation could be regulation of elongase to synthesize essential unsaturated fatty acids, although further experiments will be required to confirm this.
There was also an increase in zebrafish putative delta-6 fatty acyl desaturase (
fadsd6) mRNA levels in matured zebrafish follicles. This gene has been characterized and is reported to encode for a desaturase enzyme with bifunctional activities, capable of both Δ5 and Δ6 activities, with slight preference towards
n-3 substrates and towards the Δ6 desaturation [
47]. In rat, characterization of expression of stearoyl-coenzyme A desaturase, the rate limiting enzyme in the biosynthesis of monounsaturated fatty acids from saturated fatty acids seems to suggest the importance of this enzyme in production of unsaturated fatty acids to enable follicular maturation [
48]. The decrease in expression of desaturase and elongase in ovulation stage could be due to a negative feedback loop from the high levels of EPA, DHA and ARA in the maturation stage. In human, high levels of PUFA have been shown to negatively regulate desaturase mRNA expression[
49].
Although we do not rule out the possibility of more isoforms of fatty acid desaturase or elongase genes in zebrafish ovaries, it is conceivable that both the genes investigated here are capable of producing all three HUFA from the C18 fatty acids in ovary. Functional studies on both these genes have shown activities encompassing the whole pathway of production of EPA, DHA and ARA from LA and LNA respectively [
41,
47]. This is in contrast with other teleost species such as salmon, which possess specific desaturase enzymes having either only Δ5 or Δ6 activity respectively [
50]. In addition, low desaturase and elongase activities have been cited as main reason for limited HUFA biosynthesis capabilities in marine species like cod and turbot [
51,
52]. Thus, it will be important to actually determine the origins of the HUFAs in oocyte, as these fatty acids are also transported from reserves in muscles as compared to those presumably synthesized in oocytes.
Future experiments will be required to further support the suggestion of localized HUFA biosynthesis activities within the oocytes. In addition to measurement of actual enzymatic activities of both desaturase and elongase, it is also possible to elucidate their function by using a morpholino knockdown technique to block the translation process of these two genes in the oocyte. This technique has been used to confirm the function of candidate genes essential for oocyte maturation in zebrafish follicles [
53,
54]. Since the zebrafish oocyte fatty acid composition can be manipulated by dietary fatty acid intake, it is possible to combine the morpholino method with different dietary fatty acid treatment, which will provide insight on the importance of desaturase and elongase under different condition of dietary fatty acid intake.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SDI, SHT and HKK carried out the RNA extraction, PCR and data interpretation. AJR and YLE assisted in separation and identification of oocyte stages. Design of primers was done by MKK. AC conceived the study, designed the experiments and structured the manuscript. All authors proofread and approved the final submitted manuscript.