The Androgenic Regulation of Abundant mRNA in Rat Ventral Prostate

Abstract

The most abundant poly(A)-containing RNA accounting for approximately half the total is regulated by testosterone in rat ventral prostate. A complementary DNA probe to these sequences was isolated by hybridizing the total cellular poly(A)-containing RNA with its complementary DNA to an rotxi, value of 2 x mol I-' s and separating the abundant cDNA from the nonabundant cDNA by hydroxyapatite chromatography. The abundant cDNA, complementary to about three different poly(A)-containing RNA sequences comprising 45 % of the total poly(A)-containing RNA, has been used to investigate the effect of androgens on their metabolism.Following castration there is a progressive decrease in the abundant sequences from 45% of the total in normal animals to less than 0.03% after 14 days; testosterone administration in vivo results in their regeneration. The sequences were not detected in seminal vesicle, liver, heart or spleen but represented about 0.01 % of the poly(A)-containing RNA of kidney.Translation of the poly(A)-containing RNA in vitro in a cell-free system derived from wheat germ resulted in the synthesis of four major proteins which could be separated by polyacrylamide gel electrophoresis. Their synthesis, as measured by [35S]methionine incorporation, accounted for 40 -50 % of the total incorporation and declined after castration but was restored by testosterone treatment. We conclude that a class of abundant poly(A)-containing RNA which codes for four major proteins is regulated by testosterone in rat ventral prostate.The regulation of protein synthesis by steroids is thought to be due to hormonal effects primarily on specific mRNA concentration [l -31. The rat ventral prostate is dependent for its growth and maintenance on androgenic steroids and has been used extensively to study the molecular mechanism of androgen action [4 -71. Much is known about the metabolism of testosterone and its interaction with a cytoplasmic receptor [8 -111 but studies of subsequent events have been hindered by the failure to detect suitable protein markers induced by the hormone.Recently we have approached this problem by examining the effects of androgens on the complexity of prostatic poly(A)-containing RNA [ 121. We demonstrated that prostatic RNA comprised three distinct classes of RNA of different relative abundance and testosterone appeared to have a marked effect on the most abundant class. In order to study the hormonal regulation of this class in more detail we have isolated a cDNA probe specific for the most abundant species of poly(A)-containing RNA. Hybridization of total rH]cDNA with total poly(A)-containing RNA to an rot,,2 value of 2 x mol 1-' s results in the hybridization of the most abundant cDNA whereas the moderate and least abundant species of cDNA will be largely unhybridized. The hybridized and unhybridized cDNA can then be separated by hydroxyapatite chromatography. The abundant cDNA was subsequently used as a probe to investigate the androgenic regulation of the abundant poly(A)-containing RNA sequences.In additio...

Androgenic Regulation of Messenger RNA Sequence Complexity in Accessory Sexual Tissues of the Male Rat Studied with Fractionated Complementary DNA

“…This is in distinct contrast to the situation in the ventral prostate, another androgen-dependent tissue of the male reproductive tract. Here, too, testosterone controls synthesis of major secretory proteins coded for by highly abundant mRNA [4,5] but markedly differential effects of testosterone are seen. Thus the highly abundant mRNAs constitute 45% of total prostatic mRNA in normal rats but only 1 % one week after castration and less than 0.03% after two weeks [4].…”

“…This difference in response to androgenic steroids is confirmed by studies on the effects of testosterone on the sequence complexity of mRNA from the two target tissues. Again in common with many other steroid-responsive systems [2], the most abundant prostatic mRNA sequences are grossly reduced in concentration within the mRNA population following castration [3,4]. In the seminal vesicle on the other hand, castration has little or no effect on the total mRNA sequence complexity nor do there appear to be any major changes in the relative concentrations of mRNA species within the mRNA as a whole 191.…”

“…R N A was used at 300 pg/ml (0) and 1000 pg/ml (0). The broken line indicates the homologous reaction using poly(A)-rich RNA from prostates of normal rats [4] have been unable to obtain satisfactory data above r o t l / 2 z lo3 mol 1-l s, so we are uncertain of the final extent of hybridisation and the rOt1p. Consequently we cannot say whether all the moderate and abundant sequences are present in prostatic RNA nor can we determine their precise concentrations.…”

“…Similar calculations suggest that the moderate seminal vesicle sequences account for less than 0.004% of prostatic mRNA, equivalent to less than 12 copies/cell. By the same methods as used here, we have previously isolated cDNA complementary to abundant prostatic mRNA sequences coding for major prostatic secretory proteins [4]. We have used this cDNA to examine seminal vesicle mRNA for sequences homologous with this abundant prostatic mRNA.…”

“…Thus after castration the concentrations of abundant mRNAs coding for the major proteins are greatly decreased within the mRNA population but are restored to normal by testosterone in vivo [3,4]. Consequently the proportion of total prostatic protein synthesis devoted to these proteins shows wide variation with androgen status [5].…”