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Biomedical subjects

S O Mack

Publications and source records attributed to S O Mack.

11 recordsLinked to original sources
PubMed2006

Stimulation of the hypothalamic paraventricular nucleus modulates cardiorespiratory responses via oxytocinergic innervation of neurons in pre-Botzinger complex.

Previously we reported that oxytocin (OT)-containing neurons of the hypothalamic paraventricular nucleus (PVN) project to the pre-Bötzinger complex (pre-BötC) region and phrenic motoneurons innervating the diaphragm (D). The aim of these studies was to determine pathways involved in PVN stimulation-induced changes in upper airway and chest wall pumping muscle activity. In addition, we determined the role of OT-containing neurons in the PVN in mediating increased respiratory output elicited by PVN stimulation. Neuroanatomical experiments, using pseudorabies virus (PRV) as a transneuronal tracer in C8 spinalectomized animals showed that PVN neurons project to hypoglossal motoneurons innervating the genioglossus (GG) muscle. Furthermore, microinjection of the PVN with bicuculline, a GABA(A) receptor antagonist, significantly increased (P < 0.05) peak electromyographic activity of GG (GG(EMG)) and of D(EMG), frequency discharge, and arterial blood pressure (BP) and heart rate. Prior injection of OT antagonist [d-(CH(2))(5),Tyr(Me)(2),Orn(8)]-vasotocin intracisternally or blockade of OT receptors in the pre-BötC region with OT antagonist l-368,899, diminished GG(EMG) and D(EMG) responses and blunted the increase in BP and heart rate to PVN stimulation. These data show that PVN stimulation affects central regulatory mechanisms via the pre-BötC region controlling both respiratory and cardiovascular functions. The parallel changes induced by PVN stimulation were mediated mainly through an OT-OT receptor signaling pathway.

Animals↗
PubMed2002

Paraventricular oxytocin neurons are involved in neural modulation of breathing.

In this study, we determined the projections of oxytocin-containing neurons of the paraventricular nucleus (PVN) to phrenic nuclei and to the rostral ventrolateral medullary (RVLM) region, which is known to be involved in respiratory rhythm generation. Studies were also designed to determine oxytocin-receptor expression within the RVLM and the physiological effects of their activation on respiratory drive and arterial blood pressure. Oxytocin immunohistochemistry combined with cholera toxin B, a retrograde tracer, showed that a subpopulation of oxytocin-containing parvocellular neurons in the dorsal and medial ventral regions of the PVN projects to phrenic nuclei. Similarly, a subpopulation of pseudorabies virus-labeled neurons in the PVN coexpressed oxytocin after injection of pseudorabies virus, a transynaptic retrograde marker, into the costal region of the diaphragm. A subpopulation of oxytocin expressing neurons was also found to project to the RVLM. Activation of this site by microinjection of oxytocin into the RVLM (0.2 nmol/200 nl) significantly increased diaphragm electromyographic activity and frequency discharge (P < 0.05). In addition, oxytocin increased blood pressure and heart rate (P < 0.05). These data indicate that oxytocin participates in the regulation of respiratory and cardiovascular activity, partly via projections to the RVLM and phrenic nuclei.

Animals↗
PubMed2001

Monoaminergic neurons, chemosensation and arousal.

In recent years, immense progress has been made in understanding central chemosensitivity at the cellular and functional levels. Combining molecular biological techniques (early gene expression as an index of cell activation) with neurotransmitter immunohistochemistry, new information has been generated related to neurochemical coding in chemosensory cells. We found that CO(2) exposure leads to activation of discrete cell groups along the neuraxis, including subsets of cells belonging to monoaminergic cells, noradrenaline-, serotonin-, and histamine-containing neurons. In part, they may play a modulatory role in the respiratory response to hypercapnia that could be related to their behavioral state control function. Activation of monoaminergic neurons by an increase in CO(2)/H(+) could facilitate respiratory related motor discharge, particularly activity of upper airway dilating muscles. In addition, these neurons coordinate sympathetic and parasympathetic tone to visceral organs, and participate in adjustments of blood flow with the level of motor activity. Any deficit in CO(2) chemosensitivity of a network composed of inter-related monoaminergic nuclei might lead to disfacilitation of motor outputs and to failure of neuroendocrine and homeostatic responses to life-threatening challenges (e.g. asphyxia) during sleep.

Animals↗
PubMed2000

Absence of correlation between in situ expression of cytochrome P450 17alpha hydroxylase/lyase and 3beta-hydroxysteroid dehydrogenase/(Delta5-4) isomerase messenger ribonucleic acids and steroidogenesis during pubertal development in the rat testis.

Changes in expression of Leydig cell 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase/C17-20 lyase (P450(17alpha)) messenger RNA (mRNA) during pubertal development have not been well characterized in the rat. In the present study, expression of 3betaHSD and P450(17alpha) were determined in frozen sections of testes of immature (days 21 and 28), pubertal (days 45 and 60) and adult (day 90) rats by in situ hybridization using digoxigenin-labeled riboprobes and quantified densitometrically. Measures of steroidogenesis in this study, 3betaHSD and P450(17alpha) enzyme activities per testis and plasma testosterone concentration, increased during pubertal development, peaking at 45-60 days of age. Expression of 3betaHSD protein, a marker for Leydig cell function, was abundantly immunolocalized to the interstitial compartment of the testis. Quantified densitometrically, the amount of 3betaHSD protein did not vary significantly during pubertal development. Transcripts of 3betaHSD and P450(17alpha) were expressed abundantly by clusters of immature Leydig cells in immature animals. However, in contrast to measures of steroidogenesis during pubertal development, mRNA of 3betaHSD and P450(17alpha) decreased to undetectable levels at the age of 45 and 60 days, respectively. The decline in mRNA of 3betaHSD and P450(17alpha) was confirmed by Northern analysis. Expression of 3betaHSD and P450(17alpha) transcripts rebounded in the adult at 90 days and were comparable to levels of expression observed in immature animals. These results show that during pubertal development the steady-state accumulation of mRNA of 3betaHSD and P450(17alpha) are not correlated with accumulation of 3betaHSD protein, enzyme activities of 3betaHSD and P450(17alpha), or testosterone secretion. Possible explanations of the depletion of transcripts during pubertal development include: specific inhibition of transcription, increased mRNA instability, or high translational activity.

Androgens↗
PubMed2000

In situ analysis of the changes in expression of ovarian inhibin subunit mRNAs during follicle recruitment after ovulation in pigs.

In situ hybridization was used on frozen tissue sections with digoxigenin-labelled antisense riboprobes to inhibin/activin alpha and beta(A) subunits to determine whether inhibin/activin subunit mRNA expression was associated with development of growing, steroidogenically active follicles during follicle recruitment after ovulation. Cell proliferation-associated nuclear antigen Ki-67 protein and cytochrome P450 aromatase expression in granulosa cells were determined immunohistochemically and used as markers for granulosa cell proliferation and steroidogenesis, respectively, on days 3, 5 and 7 after the onset of oestrus. The amounts of inhibin/activin alpha and beta(A) subunit mRNA and P450 aromatase protein were greater (102, 93, and 238%, respectively; P < 0.05) in medium than in small non-atretic follicles and were positively correlated with Ki-67 and with each other. Inhibin/activin alpha and beta(A) mRNA, P450 aromatase, and Ki-67 in granulosa cells were reduced by 66-83% (P < 0.001) in atretic follicles compared with non-atretic follicles. In addition, inhibin/activin alpha and beta(A) mRNA and P450 aromatase in small (1-2 mm) non-atretic follicles decreased (P < 0.05) between day 3 and day 7 independently of morphological or biochemical signs of atresia. The pattern of inhibin/activin subunit mRNA expression supports the notion that activin and inhibin have roles in growth and steroidogenesis in follicle recruitment during the early luteal phase of the oestrous cycle.

Analysis of Variance↗
PubMed1991

3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase expression in rat and characterization of the testis isoform.

The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3 beta-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3 beta-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3 beta-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 microM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3 beta-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3 beta-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3 beta-HSD content, a 3 beta-HSD is also expressed in liver in a sex-specific manner.

Amino Acid Sequence↗
PubMed1990

Expression of cytochrome P-450(17) alpha, 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase, and steroid 5 alpha-reductase in rat H540 Leydig tumor cells.

The rat H540 Leydig tumor cell is established as a model for acute lutropin action on the initial step of steroidogenesis, namely the conversion of cholesterol to pregnenolone. Herein, we demonstrate that H540 cells express high levels of three steroid-metabolizing enzymes which are involved in the further processing of pregnenolone in the endoplasmic reticulum of the steroidogenic cell. In particular, in addition to expressing 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) and 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta-HSD), H540 cells also showed high levels of steroid 5 alpha-reductase mRNA and activity. The H540 cells therefore exhibit similarity to Leydig cells from sexually immature animals which also demonstrate high 5 alpha-reductase activity. Thus, after 3 beta-HSD-catalyzed formation from pregnenolone, progesterone was efficiently converted to 5 alpha-pregnan-3,20-dione (5 alpha-dihydroprogesterone) and subsequent metabolism to the corresponding 17 alpha-hydroxylated derivative and 5 alpha-androstan-3,17-dione in a reaction catalyzed by P-450(17) alpha. H540 cells have apparently very low 17-ketosteroid reductase activity and, therefore, a principal end-product of the steroidogenic pathway in these cells was 5 alpha-androstan-3,17-dione. H540 cells maintained in primary culture under serum-free conditions accumulated demonstrable levels of mRNA species for P-540 17 alpha (1.7 kb), 3 beta-HSD (1.6 kb) and 5 alpha-reductase (2.7 kb). This finding suggests that the H540 tumor cell model will not only be of utility in the study of acute lutropin action but also in the elucidation of mechanisms involved in the regulation of expression of various families of microsomal steroid-metabolizing enzymes.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1989

Human sperm capacitation in gelatin-fortified medium.

Gelatin was shown to be an effective substitute for serum albumin in human sperm capacitation. No protein-dependent differences were seen with regard to long-term retention of sperm motility, including hyperactivation, in acrosomal status or in the acquisition of fertility potential. Gelatin may serve as a cost-effective substitute for serum albumin, which carries minimal risk of infectious disease transmission.

Culture Media↗
PubMed1989

Effect of measurement conditions on quantification of hyperactivated human sperm subpopulations by digital image analysis.

The effect of different measurement conditions on the quantification of hyperactivated (HA) motility in human sperm by digital image analysis (DIA) was determined with respect to chamber depth, temperature, sperm concentration, time in the analysis chamber, culture medium, and type as well as concentration of extracellular protein components. In whole semen, the level of HA (less than 1%) as well as the values for all other motility parameters (curvilinear and straight-line velocity, linearity of forward progression, maximum and mean lateral head amplitude, and beat cross frequency) were independent (p greater than 0.05) of temperature (24 vs. 37 degrees C). In washed sperm samples prepared by in vitro fertilization procedures, the percentage of HA cells was inversely related to measurement temperature and to albumin concentration in the culture medium. The effect of temperature on percentage of HA in washed sperm was reversible. HA analysis of washed sperm from 6 donors (3 ejaculates per donor) under defined DIA assay conditions showed no intra-donor variability (p greater than 0.05) but significant inter-donor variability (donor mean percentages ranging from a low of 3.6 to a high of 28.2). These results indicate that HA is influenced by measurement conditions, that HA is donor-dependent, and that each donor shows a characteristic level of HA.

Culture Media↗
PubMed1988

Quantitation of specific parameters of motility in large numbers of human sperm by digital image processing.

The CellSoft computer-assisted digital image analysis system was validated for quantitating specific motility parameters in large numbers of human sperm. Motility patterns ranging from linear head trajectories (Type 1) to nonlinear, asymmetric patterns with overlapping trajectory (Type 5) were subjectively identified in semen and washed samples prepared for in vitro fertilization. A representative of each type was used for optimizing the digital imaging set-up parameters, tracking rate, and frequency. Each cell type was also characterized according to the following motility parameters: curvilinear velocity (Vcl), straight line velocity (Vsl), linearity of forward progression (Lin), maximum and mean lateral head amplitude (maxLHA; mean LHA), and beat cross frequency (BCF). Comparison of all parameters that could be determined both digitally and manually (Vcl, Vsl, Lin, and BCF) indicated no differences (p greater than 0.05) in Vcl, Lin, or BCF and only slight differences (5-6%) in Vsl measurements. After validation of the digital imaging technique, populations of seminal and washed cells were studied. Replicate analysis of the same sample demonstrated no significant intraassay variability. A comparison of semen and washed cells from 10 different donors indicated that all of the motility parameters, with the exception of Lin, were significantly higher (p less than 0.05) in washed cells. It was concluded that the digital imaging system can adequately and rapidly quantitate a large number of cells with heterogeneous motility patterns. This technique may prove to be useful in defining motility characteristics associated with capacitation, the acrosome reaction, and fertility of human sperm.

Fertilization in Vitro↗