Luteinizing and follicle-stimulating hormones. Cell-free translations of messenger RNAs coding for subunit precursors.

J.E Godine,W.W Chin,J.F Habener

doi:10.1016/s0021-9258(18)43570-3

Abstract

Polyadenylated RNA prepared from pituitary glands of normal and castrate rats was translated in membrane-free heterologous cell-free systems derived from wheat germ and rabbit reticulocytes. Sodium dodecyl sufate (SDS)-polyacrylamide gel electrophoresis and autoradiography demonstrated a major [35S]methionine-labeled translation product of castrate pituitary mRNA with apparent Mr = 14,000. This Mr = 14,000 product was specifically immunoprecipitated by anti-sera to the alpha subunit of rat and bovine luteinizing hormone (LH alpha) and was much reduced in the translation products of normal pituitary RNA. Additional minor translation products of normal pituitary RNA. Additional minor translation products with apparent Mr = 15,000 and 16,000 were identified by immunoprecipitation with antisera to rat LH beta and to the beta subunit of rat follicle-stimulating hormone (FSH beta), respectively. The immunological identity of the alpha, LH beta, and FSH beta precursors was confirmed by successful competition of these immunoprecipitation reactions by unlabeled homologous LH alpha, LH beta, and FSH, respectively. These results indicate that the gonadotropin subunits are synthesized as precursors from separate mRNAs. This system is responsive to endocrine manipulations and is appropriate for studies of the regulation of gonadotropin biosynthesis.

Highlights

PolyadenylatedRNA prepared from pituitary glands TSH in mouse thyrotrope tumors [5,6,7], and of LH in steer ofnormaland castrate rats was translatedinmem- pituitary glands [8]are synthesized from separatemRNAs
LHa, LHB, and FSH,respectively.These results indicate Sprague-Dawley rats were individually homogenized in 0.5 ml of 0.01 that the gonadotropin subunits are synthesizedas pre- M PO,0.15 M NaCl buffer, pH 7.6, in a glass homogenizer using a cursors from separate mRNAs
These studies demonstrate that polyadenylated RNA extracted from pituitary glands of ovariectomized rats directs the synthesis of a major product with apparent M,= 14,000in two cell-free translation systems

Summary

RESULTS

Unlabeled FSH was used for Pituitary content of LH and FSH". Pituitary contentof LH and FSH (micrograms of NIAMDD standard RP-1 per pituitary) for normal and castrate male and female. Sprague-Dawley rats weighing 200 to 250 gr. Castration was performed four weeks prior to killing animals. Normal Castrate Normal Castrate ment of pre-a subunipt roduction directedby pituitary mRNA. 4 7 f 8 " 5 2 2 f 175 1 3 2 f 11 711 f 9 1 in cell-free systems. 6 0 f 1 7 1 3 3 f 1 0 for the LHB and FSHP precursors under theconditions used " Values represent mean f standarddeviation of triplicate or for the cell-free translations. The M , = 15,000 and quadruplicate preparations

LH and FSH Subunit Precursors

DISCUSSION