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Evolution of 5S rDNA units and their chromosomal localization in Allium cepa and Allium schoenoprasum revealed by microdissection and FISH.

Allium cepa and Allium schoenoprasum each possess 5S rDNA units of two different sizes. The evolution of the two repeat units and their chromosomal localization were investigated. A. cepa has 5S rDNA loci in the proximal and distal regions of the short arm of chromosome 7. When the proximal and distal segments of the short arm of chromosome 7 were microdissected separately, and used as templates for PCR, the short and long 5S rDNA fragments were amplified predominantly from the proximal and distal segments, respectively. The nucleotide sequence of the long 5S rDNA unit resulted from partial duplication of a non-transcribed spacer (NTS) and the insertion of a unique sequence. FISH using a probe consisting of the unique sequence demonstrated that the long unit was distally localized. In A. cepa, the long 5S rDNA unit is only present distally and the short unit is predominantly located proximally on the short arm of chromosome 7. In A. schoenoprasum, the NTSs of the two different-sized 5S rDNAs had quite different sequences. The two 5S rDNA loci were localized very close together in the interstitial region of chromosome 6. FISH, using long and short 5S rDNA unit probes with a competitor of a 120-bp sequence of the 5S rRNA gene, indicated that the long 5S rDNA unit was localized proximally and the short unit distally. Although the NTSs of the 5S rDNA of A. cepa and A. schoenoprasum had quite different nucleotide sequences, the long 5S rDNA units of A. cepa and A. schoenoprasum share a common 75-bp sequence. This sequence might act in the formation of the long 5S rDNA unit in Allium species.

Journal Article↗

Improved survival of patients with papillary thyroid cancer after surgical microdissection.

A total of 195 patients had surgery for papillary thyroid cancer. The mean age at operation was 50 years. A microdissection technique was used for total thyroidectomy and lymph node clearance. Postoperative radioiodine tests showed no uptake or an uptake close to the background activity in 77% of the examined patients. By counting the lymph nodes removed at surgery we were able to check on the quality of the lymph node dissection. Men had a higher incidence (70%) of lymph node metastases than women (45%). Only 4% of the patients had radioiodine ablation of the thyroid remnant. The median follow-up time was 13 years. None of the patients below 45 years of age at surgery died of thyroid cancer. In the older age group eight patients died of thyroid cancer at a mean age of 75 years. Five of those who died of a thyroid carcinoma had distant metastases at diagnosis. Among patients with resectable disease, three (1.6%) died of thyroid cancer, all of whom had lived for more than 17 years after surgery. Hence longer follow-up is needed before we know the final mortality in our series. The results suggest that surgical technique and strategy can positively influence the survival of patients with papillary thyroid cancer.

Adolescent↗

The fetal gallbladder: morphology and morphometry by microdissection.

Most studies of the fetal gallbladder have been performed using ultrasonography. The identification of the fetal gallbladder and the presence of gallstones have been determined this way. The purpose of this study was to investigate the morphology and morphometry of the actual fetal gallbladder by microdissection and to examine its internal features and content. Eighty-nine formalin-embalmed fetuses of CR age 35 mm to 342 mm, i.e. 10 weeks to 36 weeks gestational age (GA), were studied by hepatic evisceration. The fetal gallbladder was examined in-situ in its bed, in relation to the umbilical vein, the anterior hepatic margin and its protrusion below the inferior hepatic surface of the liver. After excision, the form, length and diameters of the gallbladder were determined and its internal surface, lumen and content also examined. The mean length of the fetal gallbladder for the gestational ages studied ranged from 2.21 mm (10 weeks GA) to 281.6 mm (32 weeks GA); the mean fundal diameter ranged from 0.4 mm (one specimen only) to 9.42.4 mm for the same period, while the infundibular width ranged from 0.41 mm (one specimen only) to 9.01.6 mm, and the antero-posterior diameter at the fundus ranged from 0.90.3 mm to 9.03.3 mm for the same period. The parameters of the gallbladder for the period examined showed a curvilinear increase in size and were consistent with the ultrasonographic studies. The distance of the fetal gallbladder from the umbilical vein was variable and, as the gallbladder lengthened, the fundus encroached the anterior hepatic margin towards 34 weeks. A descent of the gallbladder from an "intra-hepatic" position early in fetal life to a sub-hepatic position later was clearly evident. The gallbladder wall was thick in early fetal life (10-13 weeks GA) and contained crumbly debris. Bile staining occurred at 14 weeks gestational age and the mucosa took on the normal appearance with the bile having an adult colour and consistency at 20 weeks GA.

Dissection↗

A new gene (rmSTG) specific for taste buds is found by laser capture microdissection.

Getting pure populations of taste buds suitable for molecular analysis has hampered the characterization of genes specifically expressed in taste cells. To solve this problem, we prepared specific cDNA libraries from small numbers of taste cells and surrounding epithelium isolated by laser capture microdissection (LCM) and report the discovery of a rhesus monkey novel gene (rmSTG) expressed specifically in taste cells, as found by differential screening of the cDNA libraries and RNA in situ hybridization. RNA in situ hybridization shows the preferential expression of this gene in taste buds from circumvallate, foliate, and fungiform papillae of the tongue. RT-PCR and Northern analysis of RNA from different non-taste organs showed no expression, pointing to a very specialized function of the protein in taste cells. Analysis of extended cDNAs and genomic DNA showed two exons and one intron. Northern analysis of circumvallate papillae showed a transcript of 1.3 kb as established in the gene model. BLAST search analysis showed that the human homolog is localized in the recently completely sequenced HLA class I region of Chromosome 6p21 and is sublocalized to the main susceptibility region for psoriasis vulgaris. The predicted gene encodes a protein of 314 amino acids with an N-terminal signal peptide and cleavage site, suggesting a membrane-bound or secreted protein with an extracellular role in taste cell physiology. The monkey, human, and mouse STG proteins contain potential O-glycosylation sites and tandem repeats inside a region showing approximately 50% similarity with prion proteins.

Amino Acid Sequence↗

Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogastermutant Suppressor of Underreplication.

In the Suppressor of Underreplication( SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of beta-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [ su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes.

Animals↗

Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb. & Zucc.

Japanese red pine, Pinus densiflora, has 2n=24 chromosomes, of which most carry chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) bands at their centromere-proximal regions. It was proposed that these regions contain highly repetitive DNA. The DNA localized in the proximal fluorescent bands was isolated and characterized. In P. densiflora, centromeric and neighboring segments of the somatic chromosomes were dissected with a manual micromanipulator. The centromeric DNA was amplified from the DNA contained in dissected centromeric segments by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and a cloned DNA library was constructed. Thirty-one clones carrying highly repetitive DNA were selected by colony hybridization using Cot-1 DNA from this species as a probe, and their chromosomal localization was determined by fluorescent in situ hybridization (FISH). Clone PDCD501 was localized to the proximal CMA band of 20 chromosomes. This clone contained tandem repeats, comprising a 27 bp repeat unit, which was sufficient to provide the proximal FISH signal, with a 52.3% GC content. The repetitive sequence was named PCSR (proximal CMA band-specific repeat). Clone PDCD159 was 1700 bp in length, with a 61.7% AT content, and produced FISH signals at the proximal DAPI band of the remaining four chromosomes. Four clones hybridized strongly to the secondary constriction and gave weak signals at the centromeric region of several chromosomes. Clone PDCD537, one of the four clones, was homologous to the 26S rRNA gene. A PCR experiment using microdissected centromeric regions suggested that the centromeric region contains 18S and 26S rDNA. Another 24 clones hybridized to whole chromosome arms, with varying intensities and might represent dispersed repetitive DNA.

Base Sequence↗

Novel tissue preparation method and cell-specific marker for laser microdissection of Arabidopsis mature leaf.

Laser microdissection (LMD) is a powerful tool to isolate pure cell populations from heterogeneous tissues. This system has been successfully used for animal research; however, the reports of its application to plant tissues remain limited. One of the challenges of LMD for plant material is the tissue preparation. Although cryosectioning is commonly used for animal tissues, this is not a desirable method for fragile plant material with large central vacuoles. While paraffin preparation provides high histological quality and stability, the procedure is highly time consuming and may result in degradation of molecules of interest. In addition, conventional fixation and paraffin preparation methods do not preserve the structural integrity of very delicate plant tissues such as mature Arabidopsis thaliana leaves. Here, we used the rapid microwave paraffin preparation method with no fixative for preparation of Arabidopsis leaf tissue for LMD. This method resulted in Arabidopsis leaf sections with excellent preservation of leaf internal structure as evidenced by well-defined vascular bundles, phloem, and chloroplasts, and expanded and rounded epidermal cells. RNA extracted from leaf epidermal and mesophyll cells was of sufficient yield and specificity to use in downstream applications such as microarray analysis of the amplified mRNA. We employed the mesophyll cell-specific molecular marker, chloroplastic carbonic anhydrase, and developed an epidermal cell-specific marker, the very-long-chain fatty acid-condensing enzyme, CUT1, to assess specificity of harvested Arabidopsis leaf cell types by reverse transcription polymerase chain reaction. The described method is also likely to be superior for the preparation of other fragile botanical tissue for LMD and downstream applications.

Arabidopsis↗

Detection of gene expression of vascular endothelial growth factor and flk-1 in the renal glomeruli of the normal rat kidney using the laser microdissection system.

The recent development of the laser microdissection (LMD) technique enables one to target particular tissues or cells for gene or protein analyses. The purpose of this study was to detect local mRNA expression of vascular endothelial growth factor (VEGF) and its receptor, flk-1, in the glomeruli of normal rat kidneys using the LMD system. Frozen sections of the kidney of 8-week-old male Wistar rats were made. The glomeruli were dissected from the frozen sections with the LMD system, and total RNA was extracted from 200 glomeruli in each kidney. Reverse-transcription polymerase chain reaction (RT-PCR) revealed the local mRNA expression of three isoforms of VEGF, flk-1 and GAPDH in the glomeruli. Moreover, the real-time PCR was performed to evaluate the experimental condition for quantification of VEGF and flk-1 mRNA expression using this system, and the results showed that at least 10 glomeruli might be needed for quantifying local VEGF mRNA expression. However, cDNA from 200 glomeruli was not enough for quantitative evaluation of flk-1 mRNA with this system. These results demonstrate the reproducibility of the analysis of mRNA expression in the renal glomeruli using the LMD system and also suggest that the application of the LMD technique will provide information to further our understanding of the mechanisms involved in kidney diseases.

Angiogenesis Inducing Agents↗

Differential somatic CAG repeat instability in variable brain cell lineage in dentatorubral pallidoluysian atrophy (DRPLA): a laser-captured microdissection (LCM)-based analysis.

Employing a laser-captured microdissection (LCM), we have investigated the somatic instability of CAG repeats in the variable brain cell lineage in three patients with dentatorubral pallidoluysian atrophy (DRPLA). LCM enables the isolation of single lineage brain cells for subsequent molecular analysis. We have found that CAG repeat size and the range of CAG repeats in the cerebellar granular cells is smaller than those in cerebellar glial cells. Similarly, those in the cerebral neuronal cells are significantly shorter than those in cerebral glial cells. These data directly indicate that the CAG repeat is relatively more stable in neuronal cells than in glial cells. Furthermore, cerebellar granular cells show significantly smaller main CAG repeat size and CAG repeat range than either Purkinje cells or cerebral neuronal cells, suggesting that somatic instability in the CAG repeat is markedly variable even among the different types of neuronal populations. The cell-specific CAG repeat instability may thus be more complex than has previously been considered. LCM is a powerful tool for elucidating the mechanism of the triplet repeat instability of each cell type.

Adult↗

Localization of Aa-nat mRNA in the rat retina by fluorescence in situ hybridization and laser capture microdissection.

Arylalkylamine N-acetyltransferase (AA-NAT) is the key regulatory enzyme in the melatonin biosynthetic pathway. Previous investigations have reported that Aa-nat mRNA in rat is only detected in a sub-population of photoreceptor cells that resemble cones in shape and size. In the present study, we investigated Aa-nat expression in the rat retina by using in situ hybridization and laser capture microdissection combined with the reverse transcription/polymerase chain reaction technique. Our results demonstrate that, contrary to previous reports, Aa-nat transcripts are present not only in the photoreceptor cells, but also in the inner nuclear layer and in the ganglion cell layer. However, the rhythmic expression of Aa-nat mRNA was observed only in photoreceptor cells.

Animals↗

Cardiac myocytes and dendritic cells harbor human immunodeficiency virus in infected patients with and without cardiac dysfunction: detection by multiplex, nested, polymerase chain reaction in individually microdissected cells from right ventricular endomyocardial biopsy tissue.

Two hundred fifteen patients infected with human immunodeficiency virus (HIV) participated in a prospective longitudinal study of HIV-related heart disease. Evaluation included signal-averaged electrocardiography and echocardiography. Fifteen patients underwent endomyocardial biopsy, 5 had cardiovascular symptoms and 10 did not. Cardiac myocytes or dendritic cells were prepared by individual cell microdissection to sort them from other cell types such as interstitial cells or circulating blood elements. HIV proviral sequences were amplified in samples of 15 to 20 cells of each type by multiplex, nested, polymerase chain reaction and hybridized to 32P-labeled probes specific for regions within the gag and pol genes of HIV-1. The results showed the presence of HIV sequences in myocytes of 2 of 5 patients with cardiac symptoms and in 6 of 10 without. Thus, symptomatic HIV cardiomyopathy did not appear to be a direct consequence of the virus on myocardial cells. In dendritic cells, HIV sequences were detected in 5 of 5 patients with cardiac symptoms and in 8 of 10 with apparently normal ventricular function. Furthermore, dendritic cells were somewhat more numerous in the myocardium of symptomatic than asymptomatic patients. Our studies are the first to directly detect the HIV genome in purified cardiac myocytes from patients with and without cardiac dysfunction. Our findings do not support a direct role of the virus in myocardial dysfunction. However, the results do suggest that the interstitial dendritic cells may be involved in some manner in the development of cardiac dysfunction observed in HIV-infected patients.

Adult↗

Effects of castration and adrenalectomy on in vitro rates of tryptophan hydroxylation and levels of serotonin in microdissected brain nuclei of adult male rats.

Rates of 5-hydroxytryptophan (5-HTP) synthesis and levels of serotonin (5-HT) were measured in microdissected brain nuclei following castration or adrenalectomy of adult male rats. Fourteen days following gonadectomy, 5-HTP synthesis decreased in the nucleus raphe dorsalis (DR) and nucleus centralis superior (NCS), while levels of 5-HT were unchanged in the 7 brain nuclei examined. Administration of testosterone to castrated rats not only did not reverse the castration-induced decrease in 5-HTP synthesis in the DR and NCS, but also decreased 5-HT synthesis in the nucleus amygdaloideus centralis (AGC) and the nucleus septalis lateralis (LS). Following administration of testosterone, 5-HT levels were unchanged. 10 days following bilateral adrenalectomy, 5-HTP synthesis increased in the NCS and the median eminence. Levels of 5-HT increased only in the median eminence. The increased 5-HTP synthesis and 5-HT levels following adrenalectomy were not reversed by corticosterone administration. In addition, these selective changes in 5-HT metabolism did not result from hormonal effects on the availability of tryptophan to the brain. We conclude that there are subsets of serotonergic neurons in rat central nervous system which respond uniquely to removal of the gonads and adrenals. Furthermore, the dissociation between serum and brain tryptophan concentrations and changes in rates of 5-HTP synthesis argue against tryptophan availability as being a primary determinant of 5-HT biosynthesis and for a direct endocrine central nervous system interaction with serotonergic neurons.

5-Hydroxytryptophan↗

Age-related alterations in dopamine and norepinephrine activity within microdissected brain regions of ovariectomized Long Evans rats.

The ability of several stimuli which augment central catecholamine (CA) neuronal activity to reinitiate estrous cycles in old constant estrous (CE) rats suggests CA neuronal function is impaired with advanced age. We examined the effects of age on dopamine (DA) and norepinephrine (NE) levels and turnover rates within microdissected brain regions of previously normally cycling young (3-4 months old) and middle-aged (10 months old) and CE old (20-22 months old) Long Evans 2 weeks after ovariectomy. Steady-state DA concentrations were significantly decreased in old compared to young rats in the nucleus accumbens (34%), anterior hypothalamic nucleus (54%, NHA ), neurointermediate pituitary lobe (51%, NIL) and median eminence (74%, ME). The rate constant of DA loss, an estimate of neuronal activity, decreased in old versus young rats only in the preoptic area suprachiasmatica (60%, POAs ) and NHA (60%) and was unchanged or augmented in the 7 other regions. In contrast, a decline in DA turnover rate of 29-67% was observed in 6 of 9 regions in middle-aged rats and 45-81% in 5 of 9 regions in old rats. Steady-state NE concentrations similarly were significantly decreased in old versus young rats in the POAs (54%), medial forebrain bundle (44%), nucleus suprachiasmatica (49%) and ME (59%). The rate constant of NE loss progressively decreased with increasing age only in the POAs and was unchanged or augmented in other regions. Turnover rate of NE was decreased from 21 to 98% in 4 of 8 regions from old animals. A strong positive correlation was noted between the rate constant of NE (but not DA) loss measured in young rats and the magnitude of the age-related depletion in NE concentrations within specific brain regions. Collectively these data indicate that with increasing age: CA neuronal function is differentially altered in nuclei located along the preoptico-tuberal pathway; substantial declines in both DA and NE concentrations are the primary contributor to the reduced amine turnover noted in several of these regions; and the observed age-related alterations in CA turnover may contribute to impaired LH response and the persistent hyperprolactinemia in old CE rats.

Aging↗

Development of methionine-enkephalin in microdissected areas of the rabbit brain.

Microdissected areas of the rabbit brain were isolated at prenatal day E-29, postnatal days P-3, 7, 14, 21, 2 months and adults. Methionine-enkephalin (ME) was assayed by RIA and ME concentration [ME] was expressed relative to the protein content of the extracted brain tissues. In brain nuclei with important roles in respiratory control [ME] was higher in prenatal and early postnatal life than in adults. In contrast, the prenatal and early postnatal [ME] levels in other nuclei were lower than or equal to adult values. These data suggest an important and changing role for ME in respiratory control throughout development. Early high [ME] levels within brainstem respiratory control nuclei may contribute to the newborn's increased susceptibility to respiratory depression.

Age Factors↗

Distribution of androgen receptor in microdissected brain areas of the female baboon (Papio cynocephalus).

We measured androgen receptors in the brain and pituitary of 4 female baboons (Papio cynocephalus) by the in vitro binding of methyltrienolone (R1881) to cytosols from 17 brain subregions as well as anterior and posterior pituitaries. High levels of AR were detected in anterior (22.1 +/- 7.1 (S.E.M.) fmol/mg protein) and posterior pituitary (12.6 +/- 3.3 fmol/mg protein). In brain tissue, the highest androgen receptor levels were found in the infundibular nucleus/median eminence (9.4 +/- 2.3 fmol/mg protein), ventromedial nucleus (6.3 +/- 1.7 fmol/mg protein) and periventricular area (4.9 +/- 1.3 fmol/mg protein). Saturation analysis of anterior pituitary and brain tissue (pool of hypothalamic, preoptic area, amygdala and septum remaining after microdissection of brain nuclei) showed that [3H]R1881 binds to the androgen receptor with high specificity and affinity (Kd = 1.25 x 10(-10) M, 0.45 x 10(-10) M, in anterior pituitary and HPA cytosol, respectively). Serum testosterone levels were low in all animals (0.59 +/- 0.26 ng/ml). With these data we described the quantitative distribution of androgen receptor in the pituitary and in specific brain nuclei in a species of nonhuman primate. The distribution is similar in many respects to that described in the male rat and the data suggest a conservation of androgen receptor distribution across species.

Animals↗

Sex differences in cytosolic progestin receptors in microdissected regions of the hypothalamus/preoptic area of guinea pigs.

Cytosolic progestin receptors (CPRs) were measured in microdissected nuclei of the hypothalamus and preoptic area of male and female guinea pigs. Adult gonadectomized animals were given 3 daily injections of 20 micrograms/day estradiol benzoate (EB) or oil vehicle. 24 h later, animals were sacrificed and cytosolic progestin receptors were measured using the synthetic progestin 3H-R5020. CPR levels did not differ significantly between oil treated males and oil treated females in any brain areas examined. With EB treatment, males showed significant increases in CPRs in most of the brain areas in which females showed increases, i.e. in the medial preoptic area, the periventricular part of the preoptic area, the periventricular part of the anterior hypothalamus, the ventromedial nucleus of the hypothalamus, the periventricular part of the medial hypothalamus and the arcuate-median eminence. However, EB treated males showed significantly lower CPR levels than EB treated females in both the periventricular part of the preoptic area and the periventricular part of the medial hypothalamus.

Animals↗

Aging in male rats modifies castration and testosterone-induced neuropeptide Y response in various microdissected brain nuclei.

Neuropeptide Y (NPY) is localized in several hypothalamic sites which are implicated in the control of hypothalamic luteinizing hormone-releasing hormone (LHRH) and pituitary luteinizing hormone (LH) release. We have observed previously that in young rats castration decreases and testosterone (T) replacement restores NPY levels in selected hypothalamic sites. However, in aged male rats, NPY levels were decreased in all hypothalamic sites studied. Since testicular function is diminished in aged rats, we reasoned that decreased T feedback may be responsible for the reduction of NPY in the hypothalamus. Therefore, we compared the effects of castration and T-replacement on NPY levels in microdissected hypothalamic sites of 2.5-month- (young) and 15-month-old (aged) male rats. Serum LH and T levels were markedly reduced in aged as compared to those observed in young rats. In association with the decreased hormone levels, NPY levels were significantly reduced in each of the 7 hypothalamic sites of aged as compared to young rats. Further, in young rats, castration reduced and T-replacement prevented the castration-induced depletion in only 3 sites, viz. the ventromedial hypothalamic nucleus (VMN), arcuate nucleus (ARC) and median eminence (ME). In contrast, castration in aged rats reduced NPY levels not only in the VMN as in young rats, but also in the medial preoptic area (MPOA) and dorsomedial nucleus (DMN). However, the marked reduction in the ME and ARC NPY levels of young rats following castration was not observed in the ME and ARC of aged rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

Prazosin treatment does not affect progestin receptor induction in microdissected regions of the rat hypothalamus.

Sex differences in estrogen-induced progestin receptors have been described in specific regions of the rat brain. To determine if alpha 1-neurotransmission plays a role in the expression of the sex differences in progestin receptor induction, the effects of the alpha 1-antagonist, prazosin, on progestin binding in microdissected regions of the rat brain was determined. Adrenalectomized/gonadectomized male or female rats were administered various doses of estradiol benzoate (EB) in combination with prazosin. With all treatment paradigms, and in both sexes, no significant effect of prazosin treatment on progestin receptor levels was observed. These results are consistent with the idea that sex differences in the estrogen-induction of progestin receptors in the rat hypothalamus are not due to sex differences in the alpha 1-adrenergic regulation of progestin receptor synthesis.

Adrenergic Fibers↗