PLASMA CONCENTRATIONS OF LUTEINIZING HORMONE, FOLLICLE STIMULATING HORMONE AND PROLACTIN IN OVARIECTOMIZED, HYSTERECTOMIZED AND INTACT SWINE

In order to critically evaluate the temporal patterns of plasma luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL); blood samples were collected daily at 0600, 1200, 1800 and 2400 hrs from intact, sham, ovariectomized and hysterectomized Yorkshire sows via indwelling vena caval cannulae. The maximum preovulatory plasma LH concentration in the intact and sham operated swine (mean ±l SD, 7.06±2.40 ng/ml) was reached between 2400 hrs on the first day of estrus (day 0) and 0600 hrs on day 1. Preovulatory LH peaks averaged 28.75±4.76 hrs duration from initial rise from, and return to, a baseline concentration of 1.19±.25 ng/ml plasma. In addition to the preovulatory LH peak, an average of 8.75 luteal phase peaks was observed during each estrous cycle . Approximately 69.5% ·of the luteal phase peaks had an average duration of 12.65±1.79 hrs, an average maximum plasma concentration of 2.29±.55 ng/ml and occurred during days 2 to 17. The remaining luteal phase peaks reached a maximum concentration of 2.86±1.04 ng/ml and occurred during days 2 to 13. The average duration of these luteal phase peaks was 18.61± 3.49 hrs. The largest and most consistent plasma FSH peak in the intact and sham operated swine occurred between days 2 and 3. This postovulatory peak had a duration of 58.17±16.25 hrs and reached an average maximum concentration of 13.14±9.42 ng/ml before returning to a baseline of 4.76± 1.56 ng/ml plasma. The initial rise of the postovulatory FSH peak coincided with the initial rise of the preovulatory LH peak, but did not reach maximum concentration until 2.66±.45 days after the preovulatory LH peak had reached maximum concentration. In some animals, a luteal phase FSH peak occurred between days 8 to 14. The luteal phase FSH peaks had a duration of 18.09±5.36 hrs and an average maximum concentration of 9.76± 3.78 ng/ml plasma. An average of 2.58±1.08 plasma PRL peaks was observed between day 0 and day 2 of the estrous cycle in the intact and sham operated swine. These peaks averaged 20.54±4.02 hrs in duration and reached maximum concentrations of 11.49±2.97 ng/ml before returning to a baseline of 6.54± 1.29 ng/ml plasma. They reached their maximum concentration approximately 9.12±9.84 hrs after the maximum concentration of the preovulatory LH peak had been reached. Larger prolactin peaks occurred during the follicular phase of the estrous cycle. An average of 3.09±1.38 peaks occurred between days 14 and 19.· These peaks had an average duration of 18.98±3.30 hrs and reached a maximum concentration of 14.28±4.27 ng/ml plasma. Shortly after removal of the ovaries (6 to 8 hrs), baseline LH (2.2± .14 ng/ml) and FSH (12.20±2.90 ng/ml) plasma concentrations in the ovariectomized swine were significantly greater (P<.05) than those found in the intact and sham operated animals, but baseline prolactin concentrations (6.70±2.10 ng/ml) were not significantly different (P>.05) from the controls. The temporal patterns of maximum plasma concentrations associated with LH (3.47±.21 ng/ml), FSH (21.43±.82 ng/ml) and PRL (9.32±2.00 ng/ml) peaks were not similar among the ovariectomized animals. Baseline LH (1.12±.34 ng/ml), FSH (5.30±2.09 ng/ml) and PRL (6.05± 1.89 ng/ml) concentrations in the hysterectomized swine were not significantly different (P>.05) from the baseline concentrations in the intact and sham operated animals. Numerous plasma LH peaks (14.30±4.59) were observed during the 20-day collection period. These peaks averaged 19.50 ±1.49 hrs in duration and reached maximum concentrations of 2.63±.23 ng/ ml before returning to baseline. Plasma FSH and PRL peaks averaged 21.00 ±4.51 and 17,69±3.50 hrs in duration and reached maximum concentrations of 8.12±1.95 and 10.18±2.95 ng/ml before returning to baseline. Mean plasma LH concentrations in the intact, sham, ovariectomized and hysterectomized swine were significantly greater at the 2400 hr collections than at the 1800 hr collections. A critical assessment of plasma hormone concentrations is an essential prerequisite to a basic understanding of the physiological mechanisms governing the reproductive process. The descriptive data provided by this study will enable future investigators to design critical experiments to evaluate the physiological importance of these hormone fluctuations, thereby broadening our basic understanding of the mechanisms which govern the reproductive cycle

DNAzol®: A Reagent for the Rapid Isolation of Genomic DNA

In this report, we present DNAzol®, a patent-pending DNA isolation reagent containing guanidine thiocyanate and a detergent mixture. It is a complete, nontoxic and ready-to-use reagent for the isolation of genomic DNA from various biological sources. In the DNAzol protocol, a biological sample is homogenized (or lysed) in DNAzol, and the DNA is precipitated with ethanol, washed and dissolved in 8 mM NaOH. Following pH adjustment, the DNA can be used immediately for analysis or stored at 4°C. The entire isolation can be completed in 20–30 min, and a wide range of DNA molecules can be isolated including genomic DNA and DNA fragments down to 0.1 kb in length. If necessary, samples can be stored in DNAzol at room temperature for extended periods of time. The isolated DNA is ready for PCR, Southern blotting and other molecular biology applications without any additional purification

Inter-Individual Differences in RNA Levels in Human Peripheral Blood

<div><p>Relatively little is known about the range of RNA levels in human blood. This report provides assessment of peripheral blood RNA level and its inter-individual differences in a group of 35 healthy humans consisting of 25 females and 10 males ranging in age from 50 to 89 years. In this group, the average total RNA level was 14.59 μg/ml of blood, with no statistically significant difference between females and males. The individual RNA level ranged from 6.7 to 22.7 μg/ml of blood. In healthy subjects, the repeated sampling of an individual’s blood showed that RNA level, whether high or low, was stable. The inter-individual differences in RNA level in blood can be attributed to both, differences in cell number and the amount of RNA per cell. The 3.4-fold range of inter-individual differences in total RNA levels, documented herein, should be taken into account when evaluating the results of quantitative RT-PCR and/or RNA sequencing studies of human blood. Based on the presented results, a comprehensive assessment of gene expression in blood should involve determination of both the amount of mRNA per unit of total RNA (U / ng RNA) and the amount of mRNA per unit of blood (U / ml blood) to assure a thorough interpretation of physiological or pathological relevance of study results.</p></div

Robust regression analysis of total RNA vs the product of blood DNA level (μg DNA / ml blood) and cellular RNA content (pg RNA/ cell).

<p>The blue dashed line represents the 95% prediction interval for the group of 35 samples. An adjusted R-square value for this regression analysis is 0.947.</p

Bioanalyzer profiles of the large RNA fractions isolated from donors with the lowest and highest blood RNA level.

<p>The large RNA fractions (200 ng) from the low RNA level sample 180 (6.7 μg RNA/ml blood) and the high RNA level sample 162 (22.7 μg RNA/ml blood) were separated using the Bioanalyzer RNA Nano 6000 Kit. Shown are positions of: the (18S) and (28S) ribosomal RNA; the 600 nt globin region (G); and Bioanalyzer marker (M).</p

Characteristics of RNA isolated from healthy donors.

<p>a) Proportion of the large and small RNA fractions in relation to increasing quantities of total RNA in human peripheral blood. Samples of RNA from the 35 individuals are sequentially ranked in accord with the increasing amount of total RNA in the sample. For each sample, the amounts of the large RNA fraction (inverted triangle) and small RNA fraction (square) are depicted. b) The large RNA and small RNA fractions are expressed as a percentage of the total RNA in the sample. c) Bioanalyzer RIN values of the large RNA fractions from 35 samples.</p

The total RNA level in blood collected over a period of 30 to 270 days from female (F) and male (M) donors.

<p>Total RNA was extracted from six blood donors using RNAzol BD as described in the Methods section. The calculated within-individual coefficient of variation of blood RNA level for the six donors was 9.2, 4.8, 6.2, 1.1, 7.6 and 6.9, respectively.</p