Development of real-time PCR assays in the study of gonadotropin subunits, follistatin and prolactin genes expression in the porcine anterior pituitary during the preovulatory period.
OBJECTIVE
Neural control of the anterior pituitary function consists of the interplay of neuropeptides action, gonadal steroid hormones and many other factors. The physiological effect of this regulatory action is the release and synthesis of protein hormones in the precise time and quantity. The main factor responsible for the gonadotropins release and synthesis is the gonadotropin-releasing hormone (GnRH). We must still study the modulation of the synthesis of the gonadotropins subunits - LHbeta, FSHbeta and alpha subunit by different forms of GnRH and by its analogs, in order to better understand the regulation of gonadotropin release and synthesis. THE AIM of this study was to develop real-time PCR assays of five candidate reference genes for normalization purposes in order to quantify target transcripts in anterior pituitary cells during the preovulatory period. Moreover, we focused on the influence of GnRH receptor antagonist (antide) treatment on mRNA expression levels of GPalpha, LHbeta, FSHbeta, FST(follistatin) and PRL(prolactin) genes in these cells.


METHODS
Anterior pituitary cells were obtained from pituitary glands of four mature pigs at the preovulatory phase. Cells were incubated with or without antide and relative mRNA level of target genes was measured using the Applied Biosystems 7500 Real Time System. For an exact comparison of mRNA quantity, the stability of five reference genes, ACTB, B2M, GAPDH, RPL1, and TOP2B was evaluated to choose the most appropriate reference gene for qRT-PCR normalization in the pituitary cells. Expression stability of reference genes was calculated using the geNorm application. The developed method of PCR assay was applied to study gene expression in pig pituitary cells in short culture.


RESULTS
The most stably expressed genes in the pituitary cells were GAPDH and TOP2B. The expression of ACTB, B2M and RPL1 appeared to be highly unstable. After normalization to the GAPDH/TOP2B, results showed that the mRNA expression of the FSHbeta gene was highest in comparison with LHbeta, GPalpha, FST and PRL genes (p<0.005). Pre-treatment of cells by the antide resulted in lower mRNA expression of these genes, while FSHbeta mRNA had a significantly lower expression (p<0.05) in comparison with control.


CONCLUSIONS
Real-time PCR analysis of the expression of LHbeta, FSHbeta, alpha subunit, follistatin and prolactin genes in porcine anterior pituitary cells during the preovulatory period is suitable for the study of modulatory action of metal complexes with GnRH on the expression of these genes.
Lisowski
Pawel
P
Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, 05-552 Wólka Kosowska, Poland.
Robakowska-Hyzorek
Dagmara
D
Blitek
Agnieszka
A
Kaczmarek
Monika M
MM
Gajewska
Alina
A
Kochman
Helena
H
Zwierzchowski
Lech
L
Ziecik
Adam J
AJ
Kochman
Kazimierz
K
D000704:Analysis of Variance; D000818:Animals; D030762:Estrous Cycle / Q000378:metabolism*; D005260:Female; D037201:Follicle Stimulating Hormone, beta Subunit / Q000032:analysis* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D038681:Follistatin / Q000032:analysis* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D007987:Gonadotropin-Releasing Hormone / Q000037:antagonists & inhibitors; D006727:Hormone Antagonists / Q000494:pharmacology; D037101:Luteinizing Hormone, beta Subunit / Q000032:analysis* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010060:Ovulation / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011388:Prolactin / Q000032:analysis* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011966:Receptors, LHRH / Q000037:antagonists & inhibitors / Q000378:metabolism; D012015:Reference Standards; D018709:Statistics, Nonparametric; D013552:Swine
D037201:Follicle Stimulating Hormone, beta Subunit; D038681:Follistatin; D006727:Hormone Antagonists; D037101:Luteinizing Hormone, beta Subunit; D009842:Oligopeptides; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D011388:Prolactin; C057022:iturelix