Quantification of rainbow trout (Oncorhynchus mykiss) estrogen receptor-alpha messenger RNA and its expression in the ovary during the reproductive cycle

Abstract

This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-(ER ) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers based on the known ER DNA sequence in this species, cDNA sequences representing most of the protein coding region were obtained from ovary poly A(+) RNA. Using these DNA sequences as probes in Northern blot hybridizations confirmed that a single transcript of 4·2 kilobases in poly A(+) RNA could be detected in liver and ovary RNA. For the quantitative RT-PCR assay an internal standard RNA molecule was produced to control for inherent inter-tube differences in amplification efficiency and permit accurate quantification of ER mRNAs. The quantitative RT-PCR assay proved to be highly specific for ER mRNA with a detection limit of 6·9 fg, which corresponds to 273 fg ER mRNA/µg total RNA. The quantitative RT-PCR assay was used to measure the levels of ER mRNA in ovaries of rainbow trout at different stages of reproductive development. Ovarian ER mRNA expression was found during two distinct periods of reproductive development, in pre-vitellogenic ovaries of fish with ovarian follicle diameters (OFDs) c100 µm and in mid-vitellogenic ovaries with OFDs >1000 µm. ER mRNA could not be detected in the ovaries of fish with OFDs >100 µm but c1000 µm. The highest levels of ER mRNA were found in late vitellogenic ovaries of fish with OFDs >2000 µm.

Distinct expression of three estrogen receptors in response to bisphenol A and nonylphenol in male Nile tilapias (Oreochromis niloticus)

“…As gonadal development progressed, the expression levels of all three ntERs declined. Similar observations have been reported in rainbow trout follicles where ERa mRNA was detectable in very immature follicles and was rapidly down-regulated as oocyte development progressed (Nagler et al 2000). Future studies on the cellspecific expression pattern of each ER are required for better elucidation of the molecular mechanism of estrogen receptor-mediated effects on male reproduction.…”

Sex Differences: A Resultant of an Evolutionary Pressure?

“…In higher phyla estrogens and their receptors (ER) acquired an indispensable role in the reproductive organs, where controlled the mechanisms leading to the maturation of the egg, and in the liver, where became essential for vitellogenin synthesis (Della Torre et al, 2014;Flouriot et al, 1997;Flouriot et al, 1996;Hayward et al, 1982;May et al, 1981;Nagler et al, 2000;Pakdel et al, 1991;Takase and Iguchi, 2007). Supporting this view, from anguilliformes to mammals, ER expression or activity is highest in ovaries and liver (Ciana et al, 2003;Griffin et al, 1999;Nagler et al, 2000;Todo et al, 1996). In addition, studies in female mouse showed a specific synchrony of ER transcriptional activity in liver and reproductive tissues (Ciana et al, 2003;Della Torre et al, 2011a) with a rhythm imposed by the reproductive cycle.…”

Relationships between the transcriptome and physiological indicators of reproduction in female rainbow trout over an annual cycle

“…The mRNAs for the four ER isoforms (ERa1, ERa2, ERb1, ERb2) were quantified using primers and protocols for quantitative reverse transcription polymerase chain reaction originally described in Nagler et al [21]. Enhanced green fluorescent protein complementary RNA generated by in vitro transcription was used as an internal standard to account for amplification differences between samples, because an endogenous gene could not be found that consistently amplified in samples of both the liver and the ovary.…”

“…Enhanced green fluorescent protein complementary RNA generated by in vitro transcription was used as an internal standard to account for amplification differences between samples, because an endogenous gene could not be found that consistently amplified in samples of both the liver and the ovary. Generation of the enhanced green fluorescent protein plasmid and application of the in vitro transcription protocol were as described in Nagler et al [21]. Messenger RNA levels for all target genes and the enhanced green fluorescent protein internal standard were determined using absolute quantities calculated from standard curves as described in Nagler et al [21].…”

“…Generation of the enhanced green fluorescent protein plasmid and application of the in vitro transcription protocol were as described in Nagler et al [21]. Messenger RNA levels for all target genes and the enhanced green fluorescent protein internal standard were determined using absolute quantities calculated from standard curves as described in Nagler et al [21]. Each ER isoform was measured in three independent quantitative reverse transcription polymerase chain reaction (i.e., three technical replicates), and an average of these values was used to represent the level of that isoform for each individual.…”