Regulation of pituitary alpha-subunit, beta-luteinizing hormone and prolactin messenger ribonucleic acid by gonadotropin-releasing hormone and estradiol in hypogonadal mice.

We have investigated the effects of three different GnRH injection regimens and the effects of estradiol benzoate (EB) on expression of the common alpha-subunit, beta-LH, and PRL genes in male and female hpg mice. GnRH was injected once daily (100 ng), every 2 h (100 ng) or every 30 min (25 ng), and EB (10 micrograms) was injected once daily. The effects of continuous exposure to the superactive agonist D-Trp6-GnRH released from microcapsules were also studied. Northern blot analysis showed that administration of GnRH increased alpha-subunit mRNA levels 2- to 10-fold in male and female hpg but not normal mice and had no significant effect on beta-LH or beta-TSH mRNA levels. The greatest increase in alpha-mRNA occurred when 100 ng GnRH were injected every 2 h and could be detected within 6 h of the first GnRH injection. More frequent injections (25 ng every 30 min) were less effective in increasing alpha-mRNA, as was prolonged exposure to the D-Trp6-GnRH superagonist. The increase in alpha-mRNA was associated with an increase in pituitary FSH content of similar magnitude. Continuous exposure of the pituitary gland to D-Trp6-GnRH (approximately 1500 ng/day) resulted in a smaller (2-fold) increase in alpha-mRNA and pituitary FSH content, suggesting that desensitization had occurred. EB had little effect on beta-LH mRNA and did not alter alpha-mRNA levels or affect the increase in alpha-mRNA caused by GnRH. Injection of GnRH every 2 h increased pituitary PRL mRNA levels in female but not male hpg mice, probably due to an indirect effect resulting from increased estrogen secretion. We conclude that GnRH administration to hpg mice significantly increases alpha-subunit but not beta-LH mRNA levels and that maximal effects occur with 100 ng GnRH injections every 2 h. Although EB does have direct effects upon pituitary gonadotropin content in hpg mice, the absence of significant changes in alpha- and beta-LH mRNA suggests that these effects may be largely posttranscriptional.

Prolactin messenger ribonucleic acid levels in the normal and hypogonadal mouse pituitary gland.

The effects of estrogen on the synthesis of pituitary PRL mRNA and PRL and the release of PRL into plasma were investigated in male and female normal and hypogonadal (hpg) mice. Because the hpg mouse is totally deficient in hypothalamic GnRH, it provides an excellent model for investigating the effects of estrogen and GnRH on PRL synthesis and release in the presence of a totally inactive gonadotropin-gonadal system. Estrogen stimulated the synthesis and release of pituitary PRL in both sexes of normal and hpg mice, and this marked increase in PRL synthesis correlated with an equally dramatic increase in pituitary PRL mRNA levels. Administration of GnRH alone produced a slight but significant increase in pituitary PRL content, which is consistent with an action of GnRH on prolactotropes, either directly or by way of a paracrine action involving gonadotropes.

Molecular evolution of the family Camelidae: a mitochondrial DNA study.

We report the first molecular evolutionary analysis of the family Camelidae by analysing the full DNA sequence of the mitochondrial cytochrome b gene. Estimates for the time of divergence of the Old World (Camelini) and New World (Lamini) tribes obtained from sequence data are in agreement with those derived from the fossil record. The DNA sequence data were also used to test current hypotheses concerning the ancestors of the domesticated llama and alpaca. The results show that hybridization has occurred in the ancestry of both domesticated camelids, obscuring the origin of the domestic species.

Genetic analysis reveals the wild ancestors of the llama and the alpaca.

The origins of South America's domestic alpaca and llama remain controversial due to hybridization, near extirpation during the Spanish conquest and difficulties in archaeological interpretation. Traditionally, the ancestry of both forms is attributed to the guanaco, while the vicuña is assumed never to have been domesticated. Recent research has, however, linked the alpaca to the vicuña, dating domestication to 6000-7000 years before present in the Peruvian Andes. Here, we examine in detail the genetic relationships between the South American camelids in order to determine the origins of the domestic forms, using mitochondrial (mt) and microsatellite DNA. MtDNA analysis places 80% of llama and alpaca sequences in the guanaco lineage, with those possessing vicuña mtDNA being nearly all alpaca or alpaca-vicuña hybrids. We also examined four microsatellites in wild known-provenance vicuña and guanaco, including two loci with non-overlapping allele size ranges in the wild species. In contrast to the mtDNA, these markers show high genetic similarity between alpaca and vicuña, and between llama and guanaco, although bidirectional hybridization is also revealed. Finally, combined marker analysis on a subset of samples confirms the microsatellite interpretation and suggests that the alpaca is descended from the vicuña, and should be reclassified as Vicugna pacos. This result has major implications for the future management of wild and domestic camelids in South America.

Synthesis of specific brain proteins is influenced by testosterone at mRNA level in the neonatal rat.

Sex differences in brain protein synthesis were investigated by analyzing the in vitro translation products of poly (A)+ mRNA isolated from the hypothalamus-preoptic area of male, female and androgenized female (injected with testosterone propionate at day 4) rats at different stages of development. Poly (A)+-enriched mRNA, prepared by oligo (dT)-chromatography of total RNA extracted by guanidine thiocyanate, was translated in either a rabbit reticulocyte lysate or cell-free wheat germ system supplemented with either [35S]methionine or a [3H]amino acid mix. The translation products were analyzed by one- and two-dimensional SDS-PAGE. Several developmental and androgen-induced changes were found in the translation products of specific mRNA species, the most important of which was a sex difference (higher in the male) in the apparent amount of mRNA coding for proteins with the electrophoretic characteristics of actin and tubulin. These developmental and sex differences in the apparent amounts of specific mRNA species in brain may be the cause or result of functional and structural changes such as neurite growth and synapse formation.

The short-term effects of testosterone on brain protein synthesis in 4-day-old rats: an electrophoretic study of proteins following intraventricular injection of [35S]methionine.

The short-term effects of administering testosterone propionate (TP) to 4-day female rats (androgenized females) on the synthesis of specific brain proteins were investigated. At various times after the intraperitoneal injection of TP or arachis oil, [35S]methionine was injected into the third ventricle and 1 h later proteins were extracted from the hypothalamus-preoptic area (H-POA) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Changes in synthesis of two 13,000-14,000 mol. wt. proteins in the 4-day rat brain were detected, and these changes appeared to be specific for the H-POA. In addition, silver staining revealed a 50,000 mol. wt. protein which was present in the male and androgenized female H-POA but not in the oil-treated female H-POA. An incidental, but potentially important finding, was the synthesis of a 77,600 mol. wt. protein in the H-POA of one animal which had several seizures during the experiment. These results show that changes in the synthesis of specific proteins occur in the H-POA of neonatal female rats within 8 h of TP administration and support the hypothesis that hormone-induced changes in brain protein synthesis may be involved in sexual differentiation of the brain.

Brain protein changes during development and sexual differentiation in the rat.

Subcellular fractions were prepared from the hypothalamus-preoptic area and the 'remainder of the brain' of intact male and female rats at 0, 8, 25 and 72 days of age. Proteins associated with each fraction were subjected to SDS-PAGE chromatography and stained with Coomassie Brilliant Blue. Developmental changes were found to occur in proteins associated with the soluble (14,600, 15,000, 29,900, 38,900 and 49,000 mol. wt), nuclear (40,000-50,000 and 13,800-16,000 mol. wt.), mitochondrial-lysosomal (49,000-52,000 mol. wt.) and microsomal (14,400, 20,000, 50,100, 56,900 and 130,000 mol. wt.) fractions. In addition, soluble proteins were greater in males than in females at days 0 (53,000-56,000 mol. wt.; probably tubulin) and 25 (14,600 and 15,000 mol. wt.). These changes in brain proteins probably reflect important structural and functional changes that occur during maturation and sexual differentiation of the brain.

Changes in catecholamine concentrations in the hypothalamus and region of the midbrain raphe in male and female rats during postnatal development.

To investigate possible sex differences in central catecholaminergic systems, the concentrations of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by high-performance liquid chromatography with electrochemical detection in the hypothalamus-preoptic area (H-POA) and midbrain raphe (MR) region of male and female rats throughout postnatal development. Other than a small but significant sex difference in DA concentration in the MR at 36 h (higher in females), the results showed that there were no sex differences in the concentrations of DA or DOPAC at any age in either brain region. The developmental profiles of the concentrations of DA and DOPAC showed that DAergic systems in the H-POA are immature at birth, with concentrations increasing steadily until 80 days of age, but in the MR concentrations reach a maximum at 20 days of age; a situation which is perhaps a reflection of differing monoamine metabolism in the two areas.

Lumbar muscle electromyographic dynamic topography during flexion-extension.

The objective of this study is to introduce dynamic topography of surface electromyography (SEMG) to visualize lumbar muscle myoelectric activity and provides a new view to analyze muscle activity in vivo. A total of 20 healthy male subjects and 15 males LBP were enrolled. An electrode-array was applied to the lumbar region to collect SEMG. The root mean square (RMS) value was calculated for each channel, and then a 160x120 matrix was constructed using a linear cubic spline interpolation of each scan to create a 2-D color topographic image. Along a definite interval of action, a series of RMS topography matrices was concatenated as a function of position and time, to form a dynamic topographical video of lumbar muscle activity. Relative area (RA), relative width (RW), relative height (RH) and Width-to-Height Ratio (W/H) were chosen as the four quantitative parameters in measuring topographic features. Normal RMS dynamic topography was found to have a consistent, symmetric pattern with a high intensity area in the paraspinal area. LBP patients had a different RMS dynamic topography, with an asymmetric, broad, or disorganized distribution. Quantitative SEMG features were found significantly different between normal control and LBP. After physiotherapy rehabilitation, the dynamic topography images of LBP tended towards the normal pattern. There are obvious differences in lumbar muscle coordination between healthy subjects and LBP patients. The dynamic topography allows the continuous visualization of the distribution of surface EMG signals and the coordination of muscular contractions.

A study of the teaching of nursing research using the project method to post-basic registered nurses on ENB course 100 (general intensive care nursing for RGN).

The study examined how a project based on the research process was included in ENB Course 100 and how effective this method was in teaching course members about nursing research. The data was collected by a postal questionnaire to course teachers and by pre- and post-project questionnaires to 12 course members taught by the investigator. Most course teachers include a project in their programme and felt this met the research objective required by the ENB. Advantages of the method described were improved problem-solving skills, awareness and understanding of research and implications arising for clinical practice. Disadvantages include limitations of time, quality, ethical considerations and selecting suitable topic areas. The course members had received little instruction on nursing research prior to the course, but their attitudes to it were positive. Following the project, they described the practical experience of data collection and analysis as the most enjoyable aspects, and the least enjoyable as interpretation and writing up of results. Attitudes to nursing research had improved or remained positive from the experience of the project method. It was recommended that the project be considered as a method of teaching nursing research in this post-basic course.

Indoleamines in the hypothalamus and area of the midbrain raphe nuclei of male and female rats throughout postnatal development.

We have investigated the changes in the concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus-preoptic area (HPOA) and midbrain raphe (MR) region of male and female rats throughout development. No sex differences were found in the concentrations of the indoleamines in either of the areas investigated at any stage of development. The 5-HIAA/5-HT molar ratio was maximal at 4 days of age in both the HPOA and MR. A small but significant sex difference in the 5-HIAA/5-HT ratio occurred in the HPOA (higher in males) but not in the MR at 40 days of age, and the ratio was significantly greater in 80-day-old female rats in the HPOA but not MR. Studies using NSD 1015 to block the conversion of 5-hydroxytryptophan to 5-HT showed that a higher rate of 5-HT synthesis occurred in the HPOA of 80-day-old rats compared with the neonatal HPOA and that in 80-day-old rats the rate of 5-HT synthesis was significantly higher in females. These results show that endogenous steroids do not affect the activity of serotoninergic neurons in the HPOA or MR either during or immediately after the critical period of sexual differentiation. In addition, the results from the NSD 1015 experiments show that the high 5-HIAA/5-HT ratio found in neonates does not involve a higher rate of 5-HT synthesis than that in adult rats.

Worldwide patterns of mitochondrial DNA differentiation in the harbor seal (Phoca vitulina).

The harbor seal (Phoca vitulina) has one of the broadest geographic distributions of any pinniped, stretching from the east Baltic, west across the Atlantic and Pacific Oceans to southern Japan. Although individuals may travel several hundred kilometers on annual feeding migrations, harbor seals are generally believed to be philopatric, returning to the same areas each year to breed. Consequently, seals from different areas are likely to be genetically differentiated, with levels of genetic divergence increasing with distance. Differentiation may also be caused by long-standing topographic barriers such as the polar sea ice. We analyzed samples of 227 harbor seals from 24 localities and defined 34 genotypes based on 435 bp of control region sequence. Phylogenetic analysis and analysis of molecular variance showed that populations in the Atlantic and Pacific Oceans and east and west coast populations of these oceans are significantly differentiated. Within these four regions, populations that are geographically farthest apart generally are the most differentiated and often do not share genotypes or differ in genotype frequency. The average corrected sequence divergence between populations in the Atlantic and Pacific Oceans is 3.28% +/- 0.38% and those among populations within each of these oceans are 0.75% +/- 0.69% and 1.19% +/- 0.65%, respectively. Our results suggest that harbor seals are regionally philopatric, on the scale of several hundred kilometers. However, genetic discontinuities may exist, even between neighboring populations such as those on the Scottish and east English coasts or the east and west Baltic. The mitochondrial data are consistent with an ancient isolation of populations in both oceans, due to the development of polar sea ice. In the Atlantic and Pacific, populations appear to have been colonized from west to east with the European populations showing the most recent common ancestry. We suggest the recent ancestry of European seal populations may reflect recolonization from Ice Age refugia after the last glaciation.