Evidence of gonadotropin-releasing hormone mRNA in the rat anterior pituitary.
We have previously reported the presence and the persistence of immunoreactive GnRH cells in the rat anterior pituitary in the absence of exogenous GnRH. To substantiate the hypothesis of its endogenous production, we have investigated the presence of GnRH mRNA in rat anterior pituitary tissue using a reverse transcription-polymerase chain reaction (RT-PCR) amplification protocol. Total RNA from rat anterior pituitary, hypothalamus (positive control), and muscle (negative control) was reverse transcribed to the first strand of cDNA and amplified by PCR using a set of three exon-specific primers for a target sequence of the GnRH gene, i.e. two external 5' and 3' primers and one internal 3' primer. UV light analysis of the size-fractionated RT-PCR products showed two bands of the predicted sizes [511 and 397 base pairs (bp)] hybridizing with GnRH probes in Southern analysis only in hypothalamus and in anterior pituitary but not in muscle. These two bands were far more intense in the hypothalamus than in the anterior pituitary. Restriction enzyme analysis evidenced that the two RT-PCR products included the HindIII endonuclease site of cleavage present in the GnRH cDNA sequence spanned by the primers, yielding two digested products of the anticipated sizes (86 and 425 bp for the 511-bp fragment, and 86 and 311 bp for the 397-bp fragment). The complementary sequences for the GnRH probes were conserved in the 425-bp and 311-bp fragments. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target GnRH sequences in the anterior pituitary as well as in the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)
Pagesy
P
P
INSERM U. 223, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
Li
J Y
JY
Berthet
M
M
Peillon
F
F
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010903:Pituitary Gland, Anterior / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase