J. Tentler

Coupled reverse transcription-polymerase chain reaction (RT-PCR) technique is comparative, quantitative, and rapid: uses in alcohol research involving low abundance mRNA species such as hypothalamic LHRH and GRF.

The measurement of alterations in low abundance mRNAs such as the hypothalamic hormones luteinizing hormone-releasing hormone (LHRH) and growth hormone-releasing hormone (GHRH or GRF) from individual hypothalamic tissues in rats has previously been difficult and usually required either isolation of poly(A) mRNA or the pooling of numerous animals to obtain a reasonable signal on Northern blots. Although more sensitive detection methods exist, such as the use of RNA probes or solution hybridization (RNase protection), we have found the most reliable, sensitive, rapid, and accurate method is the reverse transcription-polymerase chain reaction (RT-PCR) using histone H3.3 as an internal control for both steps of this procedure. H3.3 is a cell-cycle independent and constitutively expressed gene in all tissues. We have developed an RT-PCR assay for LHRH and GRF mRNA quantitation and comparative analysis for hypothalamic and extrahypothalamic brain tissues and present the use of RT-PCR for LHRH quantitation in ethanol (EtOH) studies.

In vivo effects of acute EtOH on rat α and β luteinizing hormone gene expression

The suppressive effects of ethanol (EtOH) on the male rodent reproductive axis, especially on serum luteinizing hormone (LH) levels, are well known. In this study we examined, in coordinate fashion, the effects of EtOH on LH secretion and on steady-state levels of the mRNA for the two genes that direct LH synthesis, namely α- and β-LH. A single intraperitoneal (IP) injection of EtOHgiven to castrated male rats produced a statistically significant fall in serum LH levels at 1.5 (p<0.05) and 3 hours (p<0.01) after injection compared to saline-injected controls

Further characterization of the impact of ethanol on βLH: alterations in polyribosome association of βLH mRNA

We have previously reported a decrease in Luteinizing Hormone (LH) levels in serum afterin vivo acute ethanol exposure in male rats. Accompanying these changes, a rapid and marked decrease of β-LH mRNA was observed. A similar decrease was not detected in the common α-subunit or β-FSH mRNA. The studies presented here examined the possible mechanisms of decreasing β-LH mRNA by using S1 nuclease protection assay to evaluate the effect of acute ethanol exposure on the levels of β-LH heteronuclear RNA (hnRNA). There was no significant difference detected in the level of β-LH hnRNA after ethanol exposure. Polysome distribution analysis was used to evaluate the association and disassociation of β-LH mRNA with polyribosomes since non-polyribosome associated mRNA may be more vulnerable to degradation by RNAases. The results indicated a decrease in the association of the β-LH mRNA with polysomes following acute ethanol exposure. This decrease in polyribosome association would increase the exposure of the β-LH transcript making it more susceptible to RNases. We conclude that the decrease in steady-state β-LH mRNA levels after ethanol exposure occurs because of increasing degradation of the transcript rendered vulnerable by displacement from polysomes and not through a decreased transcriptional rate.

Failure of ethanol to induce changes in gonadotropin gene expression in selectively bred ethanol-sensitive rats

The recent availability of genetically altered rat lines differing in sensitivity to ethanol (EtOH) has allowed deeper investigation into the mechanisms of EtOH-induced cellular toxicity in several systems. Since the male central reproductive axis has been demonstrated to be exquisitely sensitive to EtOH, studies were undertaken to determine if the gonadotropin suppression reported earlier could be duplicated in one of these selected rat lines. Castrated high alcohol sensitivity (HAS), low alcohol sensitivity (LAS) and control alcohol sensitivity (CAS) rats were given EtOH or saline acutely. Castrated non-selectively bred Sprague Dawley rats were treated similarly and used as an additional control. At sacrifice, serum and pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were obtained and the mRNA levels for both gonadotropins assessed. In the selectivity bred animal there was essentially no change in serum or pituitary LH or FSH levels between EtOH and saline treated animals. The mRNA levels for both LH and FSH similarly were unaffected by EtOH, in striking contrast to the non-selectively bred Sprague Dawley rats where serum LH, FSH and beta-LH mRNA levels are markedly suppressed after EtOH exposure. The selectively bred lines of rats genetically manipulated for high or low EtOH sensitivity, as well as their non-selected controls, appeared to have a hypothalamic-pituitary reproductive unit that is resistant to EtOH. This is in contrast to Sprague-Dawley rats, where suppression of this axis previously has been consistently demonstrated.

Cross-Reaction of Albumin with Polyclonal Lh Antibody on Western Blots

When pituitary tissue was subjected to Western blot analysis utilizing polyclonal antibody NIDDK-rLH-S-10, bands at 17 and 19 Kd representing LH subunits were identified. In addition, a high molecular weight 66 Kd band was seen. Surprisingly this high molecular weight band was also seen in rat cerebral cortex, brain stem, hypothalamus, spinal cord, lung, liver, pancreas, spleen, kidney, testis, and serum. Antibody preabsorbed with iodination grade rat LH antigen no longer recognized the 17 and 19 Kd bands in pituitary, but recognized the 66 Kd bands in pituitary and the other tissues examined. Since 66 Kd is the molecular weight of albumin, we found that antisera to rat albumin recognized this same high molecular weight band in the tissues examined. Preabsorption of LH antibody with albumin reduced the ability of that antibody to recognize this 66 Kd. A monoclonal antibody to bovine LH beta-subunit recognized only the LH protein in anterior pituitary, but no high molecular weight band in either pituitary or the other tissues studied. Finally, 10, 100, and 1000 micrograms of rat albumin caused no substantial interference under conditions of RIA. We conclude that the polyclonal antibody, provided by the NIH, is excellent for conditions of RIA, but caution must be exercised when it is used for Western analysis where some lots of this antibody may recognize other unrelated proteins.