Steroids may exert their action by two distinct ways 1- a direct control of gene transcription involving classic nuclear receptors [
1]; 2- a rapid non-nuclear action, independent of direct regulation of gene transcription [
2,
3]. This second type of steroid action has been proposed to be involved in steroid-induced oocyte maturation in lower vertebrates. Thus, in amphibians, it has been assumed for a long time, that the actinomycin D-insensitive progesterone-induced oocyte maturation [
4] was mediated by a membrane steroid receptor rather than a nuclear receptor. The recent cloning and sequencing of a gene coding for a membranous progesterone receptor [
5] in African clawed frog (
Xenopus laevis) brought evidence for the existence of both intracellular and membrane progestin receptors [
6].
In fishes, 2 distinct progestins, 17,20β-dihydroxy-4pregnen-3one (17,20βP) and 17,20β,21-trihydroxy-4pregnen-3one (17,20β,21P), have been identified as the natural Maturation-inducing steroids (MIS), depending on the species [
7‐
9]. High affinity binding for 17,20βP, the MIS in salmonids, has been found in ovarian plasma membranes of rainbow trout (
Oncorhynchus mykiss) [
10], brook trout (
Salvelinus fontinalis) and Artic char (
Salvelinus alpinus) [
11,
12]. This specific 17,20βP binding was high in non-matured post-vitellogenic rainbow trout and medaka (
Orizias latipes) oocytes, but low in matured oocytes [
13]. On the other hand, both nuclear and membrane progestin receptors were fully characterized in the spotted seatrout (
Cynoscion nebulosus), the membrane receptor being more specific for 17,20β,21P, which is the MIS in this species, than the nuclear receptor which exhibited a higher affinity for 17,20βP [
14,
15]. Furthermore, membrane receptor capacity of the spotted seatrout ovary has been shown to be correlated with oocyte responsiveness to 17,20β,21P for maturation, assessed by the occurrence of germinal vesicles breakdown (GVBD) [
16]. In addition, the cDNA encoding for a putative membrane progestin receptor (mPR) was recently cloned in spotted seatrout [
5]. It belongs to a novel family of putative membrane receptors genes present in various vertebrates and displaying three subtypes (α, β and γ) [
17]. To date, the 3 forms have been identified in human, mouse, catfish (
Ictalurus punctatus) and fugu (
Takifugu rubripes), while 2 forms, α and β, have been reported in pig and zebrafish (
Danio rerio) and only 1 form in African clawed frog (β), goldfish (
Carassius auratus) (α) and spotted seatrout (α) [
5,
18,
19]. The recombinant protein produced in an
E. coli expression system using the spotted seatrout mPRα cDNA sequence exhibited a specific and saturable progesterone binding. Steroid competition studies showed that binding was highly specific for progesterone and 17-hydroxyprogesterone. However, no displacement of tritiated progesterone was observed with 17,20β,21P, the natural spotted seatrout MIS, which has been hypothesized to be due to a lack of adequate post-translational modifications in the
E. coli expression system [
5]. Microinjection of mPRα antisense nucleotide in oocytes resulted in an almost complete inhibition of 17,20βP-induced oocyte maturation in zebrafish [
5] while a partial inhibition was observed in goldfish [
19]. Similar experiments conducted in zebrafish oocytes using the mPRβ subtype resulted in an almost complete inhibition of oocyte maturation suggesting that mPRβ could also participate in the control of oocyte maturation in zebrafish [
20]. Indeed, within the same experiment, the maximum inhibition was observed using the mPRβ subtype [
20]. Finally, mRNA tissue distribution of the 3 subtypes have been studied in catfish and mPRβ showed a predominant expression in the ovary [
18]. Apart from oocyte maturation, mPRα protein has been detected by Western blotting in Atlantic croaker (
Micropogonias undulatus) sperm, a species in which a 17,20β,21P action on spermatozoa motility was suggested [
21].
Some years ago, a totally distinct progesterone binding protein was isolated from porcine liver microsomal membranes and partially sequenced [
22]. This protein, originally termed membrane progesterone receptor, is now termed progesterone membrane receptor component 1 (PGMRC1) [
23]. Porcine PGMRC1 cDNA was subsequently cloned [
24] as well as the human [
25] and rat [
26] orthologs. In addition, a related sequence, PGMRC2, belonging to the same family was also reported in humans [
25,
27]. Human PGMRC1 mRNA has been detected in a wide variety of tissues (Table
1) and has a predominant expression in liver and kidney [
25]. In addition, PGMRC1 and PGMRC2 mRNAs have been found in human spermatozoa [
27]. PGMRC1 and PGMRC2 cDNAs were also generated from several species subjected to massive cDNA sequencing programs, including mouse, chicken, African clawed frog and zebrafish, and the corresponding sequences deposited in GenBank (Table
1). Antibodies raised against porcine PGMRC1 can mitigate the progesterone induced acrosome reaction [
28,
29]. PGMRC1 protein was also detected in human spermatozoa [
27] and an antibody raised against PGMRC1 could suppress the rapid progesterone-initiated Ca
2+ flux observed in sperm. Together, this suggests that PGMRC1, if present in the oocyte, is a candidate for mediating progestin-induced oocyte maturation in fish. However, besides sequences originating from genome sequencing programs, PGMRC1 was never characterized in any fish species. Furthermore, to our knowledge, PGMRC1 protein or mRNA expression was never reported in the oocyte of any vertebrate species (Table
1).
Table 1
Sequence accession number, cDNA source or expression data and sequence identity with rainbow trout (rt) PGMRC1 of vertebrate PGMRC1 and PGMRC2 proteins. The tissues exhibiting a predominant expression are underlined.
PGMRC1 | NP_006658 | human | heart, brain, placenta, lung, liver, skin, kidney, pancreas | 72 % |
| NP_999076 | porcine | brain, liver, lung, kidney, spermatozoa | 71% |
| O55022 | mouse | liver | 71% |
| NP_068534 | rat | brain, liver | 68% |
| CAG31527 | chicken | 2-week embryo | 67% |
| AAH76926 |
Xenopus tropicalis
| whole body (male) | 74% |
| AAH72727 |
Xenopus laevis
| embryo | 75% |
| CAF97306 |
Tetraodon nigroviridis
| - | 77% |
| AAH85558 | zebrafish | liver | 89% |
PGMRC2 | AAH92478 | human | heart, brain, placenta, lung, liver, skin, kidney, pancreas | 60% |
| AAH44759 | mouse | mammary tumor | 59% |
| NP_001008375 | rat | - | 59% |
| XP_420466 | chicken | - | 59% |
| AAH64268 |
Xenopus tropicalis
| embryo (tailbud) | 62% |
| AAH81155 |
Xenopus laevis
| brain | 60% |
| CAG03786 |
Tetraodon nigroviridis
| - | 63% |
| AAH53415 | zebrafish | whole body | 60% |
Therefore, the present study aimed at 1- characterizing the PGMRC1 cDNA sequence in rainbow trout, 2- studying the rainbow trout PGMRC1 mRNA tissue distribution with special interest for any oocyte mRNA expression and, 3- if present in the oocyte, characterizing PGMRC1 mRNA expression profiles during oogenesis. In addition, in order to evaluate PGMRC1 eligibility as a possible participant in the progestin-induced oocyte maturation, we aimed at performing a similar analysis for the so far unknown rainbow trout mPRβ subtype that has recently been implicated in the control of meiosis resumption in zebrafish, a species exhibiting the same maturation-inducing steroid (17,20βP) than rainbow trout. Among mPR subtypes, mPRβ was chosen because it has a predominant expression in catfish ovary [
18] and exhibits the maximum oocyte maturation inhibition in antisense oligonucleotide studies [
20]. In addition, mPRβ was the only mPR subtype that could be identified in African clawed frog [
5].