Beta-cell mass and proliferation following late fetal and early postnatal malnutrition in the rat

We have recently shown that maternal food restriction during late pregnancy in rats decreased beta-cell mass in the offspring at birth, without altering beta-cell proliferation. The aim of the present work was to determine: 1) whether sustained maternal undernutrition until weaning (R group) more dramatically alters beta-cell mass in the offspring and if normal food supply from weaning until adulthood could reverse the deleterious effects and; 2) if altered beta-cell proliferation was responsible for the decreased beta-cell mass. Beta-cell fraction and proliferative capacity were determined during the suckling period and at adult age after ad libitum feeding from weaning in the R animals and in age-matched controls (C group). At day 21, the offspring born and nursed by food-restricted mothers (R animals) showed a 66% reduction in beta-cell mass and number, which did not increase from birth to weaning, although beta-cell proliferation remained normal. At 3 months of age, R animals had 35% decreased beta-cell fraction, with a 50% decrease in the head of the pancreas. In that area, beta-cell proliferation was similar to that of the controls. In the tail of the pancreas, beta-cell fraction was only slightly impaired but beta-cell proliferation was increased by 37%, as compared with the controls. This increase was associated with a shift in islet size distribution towards medium and large islets compared with the head of pancreas from these R animals. No regional variations of beta-cell fraction, proliferation or islet size distribution were observed in adult control animals. In conclusion, prolonged malnutrition until weaning impairs beta-cell development but not beta-cell proliferation. Subsequent re-nutrition is followed by increased beta-cell proliferation but this is insufficient to fully restore beta-cell mass.     

Identification of type 1 5alpha-reductase in myelin membranes of male and female rat brain

Following its release from cells during infection and inflammation, calreticulin (CRT) can act as an autoantigen in diseases such as SLE. Why CRT is a target of protective immunity and whether it may interfere with innate immunity once released from cells during inflammation is unclear. In the present study, we found that CRT was detected more frequently in SLE sera and in higher amounts than found in control sera. Approximately 40% of SLE sera tested contained autoantibodies against CRT as detected by ELISA and immunoblotting. CRT was found to be predominantly in the sera of SLE patients associated with immune complexes and C1q, and only bound to the surfaces of neutrophils in the presence of low levels of calcium and magnesium. In order to further investigate the C1q-CRT interaction, recombinant CRT and its discrete domains (N-, P-, and C-domains) were produced in Escherichia coli. CRT binds to globular head region of C1q primarily via its N- and P-domains. The N-domain was shown to be the most autoantigenic region of CRT, as the anti-CRT autoantibodies from most patients reacted against this region. CRT also altered C1q-mediated immune functions. The P-domain of CRT bound to C1q and reduced the binding of immune complexes in SLE sera to immobilized C1q. Full length CRT and its N- and P-domains were able to reduce the C1q-dependent binding of immune complexes to neutrophils and solid-phase bound C1q. We conclude that CRT, once released from leucocytes during inflammation, may not only induce an antigenic reaction, but also interfere with C1q-mediated inflammatory processes.     

Cell-type-specific expression of rat steroid 5 alpha-reductase isozymes

The enzyme steroid 5 alpha-reductase (EC 1.3.99.5) is a component of an intercellular signaling pathway that determines cell fate in the primordium of the mammalian reproductive tract. During male phenotypic sexual differentiation, the dihydrotestosterone product of this enzyme binds to the androgen receptor and initiates development of the external genitalia and prostate. Genes encoding two isozymes of steroid 5 alpha-reductase with different biochemical properties and tissue distributions have recently been isolated. In the current study, we utilize in situ hybridization analysis to determine cell-type-specific expression patterns of the 5 alpha-reductase isozyme mRNAs in two androgen target tissues (regenerating ventral prostate and epididymis) and a peripheral tissue (liver). In regenerating ventral prostate, the type 1 mRNA is expressed in basal epithelial cells whereas expression of the type 2 mRNA is largely confined to stromal cells. These results were confirmed by immunohistochemical analysis and are consistent with distinct roles played by the isozymes in the prostate. In the epididymis, both 5 alpha-reductase isozyme mRNAs are expressed in epithelial cells. Only the type 1 mRNA is present in the liver. This mRNA is distributed in a striking spatial gradient extending from hepatocytes surrounding the portal triad (high expression) to those surrounding the central vein (low to absent expression). These findings demonstrate cell-type-specific expression of the steroid 5 alpha-reductase isozymes and underscore their distinct and overlapping functions in androgen physiology.     

Expression and regulation of steroid 5 alpha-reductase in the genital tubercle of the fetal rat

Nociceptin/orphanin FQ is a heptadecapeptide which was recently isolated from brains. It induces hyperalgesia, in contrast to the analgesic effects of opioid ligands, although it and its receptor structurally resemble opioid peptides and opioid receptors, respectively. To investigate the molecular mechanism underlying nociceptin/orphanin FQ actions, we performed Xenopus oocyte expression assays, in situ hybridization histochemistry and electrophysiological analyses of neurons. We found that the nociceptin/orphanin FQ receptor is functionally coupled with the G-protein-activated K+ (GIRK) channel in Xenopus oocytes, and that the receptor mRNA and GIRK1 mRNA co-exist in various neurons, including hippocampal pyramidal cells. Furthermore, we found that nociceptin/orphanin FQ induces hyperpolarizing currents via inward-rectifier K+ channels in hippocampal pyramidal cells, suggesting that the nociceptin/orphanin FQ receptor couples with the GIRK channel in this region. We conclude that the nociceptin/orphanin FQ receptor couples with the GIRK channel in various neurons, including hippocampal pyramidal cells, thereby modulating neuronal excitability.     

Localization by in situ hybridization of steroid 5alpha-reductase isozyme gene expression in the human prostate and preputial skin

PURPOSE: The synthesis of dihydrotestosterone is catalyzed by steroid 5alpha-reductase isozymes, designated as types 1 and 2. Controversial results have been reported on the identification of the cell types expressing each isozyme in the human prostate and genital skin. The objective of the present study was to clearly identify at the cellular level the cells containing each type of 5alpha-reductase isozyme. MATERIALS AND METHODS: We used for the first time an in situ hybridization technique involving use of [35S]-labeled oligonucleotide probes to determine the cell type expression patterns of the two 5alpha-reductase isozymes in human prostate and preputial skin. RESULTS: In the prostate, hybridization with types 1 and 2 5alpha-reductase antisense probes led to a positive reaction in both epithelial and stromal cells, while the sense probes did not generate any signal. The silver grain counts for both isozymes revealed a higher labeling in the epithelial cells. In fact, the ratio epithelial cells/stroma was around 2 for both 5alpha-reductase types 1 and 2. In the preputial skin, 5alpha-reductase type 1 was found to be highly expressed in all the layers of the epidermis with the exception of the stratum corneum while a lower labeling was observed in some fibroblasts as well as in the secretory cells of sebaceous glands and excretory duct cells of sweat glands. Type 2 5alpha-reductase showed a very similar pattern of distribution. CONCLUSION: It appears that, in two human tissues, the two 5alpha-reductase isozymes mRNAs are expressed by the same cell types. This new observation should help clarify the physiological role of each type of 5alpha-reductase isozyme.     

Nonisotopic single strand conformation analysis of the 5 alpha-reductase type 2 gene for the diagnosis of 5 alpha-reductase deficiency

5 alpha-Reductase deficiency is a rare autosomal recessive disorder of defective virilization in karyotypic males due to reduced conversion of testosterone to dihydrotestosterone. The gene encoding the affected 5 alpha-reductase type 2 enzyme has recently been cloned, and mutations within the coding region have been discovered as the cause of this disease. We address the possibility of a rapid nonradioactive molecular genetic screening technique for initial diagnosis and report different point mutations in this gene in eight unrelated patients with clinical features of 5 alpha-reductase deficiency. For molecular genetic analysis, DNA from peripheral blood leukocytes was studied. The coding region of the 5 alpha-reductase type 2 gene was characterized by exon-specific PCR amplification, nonradioactive single strand conformation analysis, and direct sequencing. In seven patients, homozygous point mutations were identified (Leu55-Gln, delta Met157, Gly196-Ser, Arg227-Gln, Ala228-Thr, and His231-Arg). One individual was a compound heterozygote carrier of two mutations (Ile112-Asn and Gln126-Arg). We conclude that molecular genetic characterization of point mutations in the 5 alpha-reductase type 2 gene may be used as an additional valuable procedure for the diagnosis of this disorder.     

Steroid 5alpha-reductase type 1 immunolocalized in the rat peripheral nervous system and paraganglia

Steroid 5alpha-reductase is an enzyme that converts a number of steroids with a C-4, 5 double bond and C-3 ketone to 5alpha-reduced metabolites. This enzyme has been suggested to play a role in brain development and myelination in the rat nervous system. In the present study, we examined the cellular and subcellular localization of the enzyme immunocytochemically in the rat peripheral nervous system and paraganglia using a polyclonal antibody against rat 5alpha-reductase type 1. Light and electron microscopical studies localized 5alpha-reductase in the Schwann cells of myelinated and unmyelinated nerve fibres, the satellite cells of the ganglia, the enteric glial cells and the supporting/sustentacular cells of the paraganglia. In the myelinated nerve fibres, immunoreactivity was observed in the outer loops, the nodes of Ranvier and the Schmidt-Lanterman incisures. Subcellularly, the immunoreactivity was localized in the cytoplasm of various glial cells. No immunoreactivity was observed in the myelin membrane, the axon or the neuronal perikaryon. These findings suggest that 5alpha-reductase is widely distributed in glial cells, and that, in addition to myelination, 5alpha-reduced steroids play a role in some glial functions in the peripheral nervous system.     

Regional distribution of 5alpha-reductase type 1 and type 2 mRNA along the human epididymis

BACKGROUND AND PURPOSE: In women, symptoms of coronary artery disease are delayed by 10 to 15 years in comparison with men, most likely because of the protective effect of ovarian hormones. This report compares the prevalence and degree of carotid atherosclerosis between 292 premenopausal women and 294 women at 5 to 8 years after menopause. METHODS: Scans were performed in the same laboratory over the same time period for both groups. Intima-media thickness (IMT) was averaged across the common, bulb, and internal carotids. The plaque index summarized degree of focal plaque based on the size and number of plaques throughout both carotid systems. RESULTS: Mean IMT was 0.69 mm for premenopausal women and 0.77 mm for postmenopausal women (P < 0.001). Prevalence of plaque was 25% among premenopausal women and 54% among postmenopausal women (P < 0.001). In both premenopausal and postmenopausal women, risk factors measured before menopause were associated with carotid atherosclerosis. Premenopausal risk factors independently associated with IMT were higher pulse pressure (P < 0.001), triglycerides (P = 0.002), body mass index (P < 0.001), and study group (a surrogate for both age and menopausal status; P < 0.001). Premenopausal risk factors independently associated with focal plaque were ever smoking (P = 0.002), higher pulse pressure (P = 0.028), higher LDL (P = 0.003), age at baseline (P = 0.050), and study group (P < 0.001). CONCLUSIONS: Subclinical carotid atherosclerosis can be observed in middle-aged women. Risk factors measured before menopause are clearly associated with subclinical disease measured both concurrently and at 5 to 8 years after menopause.     

Acinar cell responsiveness to urecholine in the rat pancreas during fetal and early postnatal growth

These studies were performed to determine when, during fetal or postnatal life, rat pancreatic acinar cells acquire their capacity to respond to cholinergic drugs. The release of amylase, lipase, and chymotrypsin from rat pancreas in response to urecholine was measured in vitro. Fetal and newborn pancreas did not respond to 10(-5) M urecholine; 3-day- to 15-day-old pancreas demonstrated an increasing response to this dose. Peak sensitivity when compared to adult pancreas was present between days 15 and 21. Our results indicate that the exocrine pancreas acquired a responsiveness to urecholine on the 3rd day of postnatal life and becomes more sensitive to stimulation from the 15th day.     

Expression of type 2 11beta-hydroxysteroid dehydrogenase and corticosteroid hormone receptors in early human fetal life

In adult life, the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11betaHSD2) protects the mineralocorticoid receptor (MR) from glucocorticoid by inactivating cortisol to cortisone. 11betaHSD2 activity has been reported in human fetal tissues, where glucocorticoids may impair fetal growth yet are also required for normal fetal development. Using digoxigenin-labeled complementary ribonucleic acid (RNA) probes and an in-house 11betaHSD2 antiserum, we have analyzed the expression of 11betaHSD2, MR, and glucocorticoid receptor (GR) in human fetal tissues of gestational age 6-17 weeks (n=15). 11BetaHSD2 expression was absent at gestational age 6+ weeks, but was expressed in abundance in many fetal tissues between 8-12 weeks. At this time, 11betaHSD2 colocalized with GR messenger RNA (mRNA) expression in metanephros, gut, muscle, spinal cord and dorsal root ganglia, periderm, sex chords of testis, and adrenal. In particular within fetal kidney, intense expression of 11betaHSD2 and GR mRNA was observed over Bowman's capsule and the vascular tufts of developing glomeruli as they migrated from the surface of the kidney to the inner cortex. Only lung and adrenal medullary rests demonstrated high levels of GR mRNA but low levels of 11betaHSD2. 11BetaHSD2 mRNA and immunoreactivity staining patterns were similar, with the exception of the fetal adrenal, where mRNA was localized to the outer definitive zone but immunoreactivity was localized to the inner fetal zone. Colocalization of 11betaHSD2 (and GR mRNA) with MR mRNA was observed principally within epithelial cells of collecting ducts, particularly after 16 weeks gestation when the pattern of distribution of 11betaHSD2 became more adult in nature. High levels of MR mRNA were observed within developing bone. The data indicate that 11betaHSD2 in fetal life principally modulates ligand access to the GR in most fetal tissues, notably glomeruli and tubules in the developing kidney, testis, and periderm, and this may be have ramifications for fetal sodium homeostasis and differentiation. The development of tissues previously shown to have a critical requirement for glucocorticoids, such as lung and adrenal medulla, is facilitated by the expression of GR mRNA, but not 11betaHSD2. The expression of MR mRNA in high abundance in bone suggests a role for corticosteroids in human bone development, and the low/absent expression of 11betaHSD2 at this site suggests that it is functionally acting as a GR.     

Molecular characterization of 5 alpha-reductase type 2 deficiency and fertility in a Swedish family

The molecular background of 5 alpha-reductase type 2 deficiency was investigated in a Swedish family with no known consanguinity and in which the affected males were fertile. The three male siblings were born with ambiguous external genitalia, and the diagnosis of 5 alpha-reductase deficiency was established at the ages of 16, 14, and 10 yr, respectively. All three siblings underwent surgery for hypospadias repair. At least two of the brothers are demonstrably fertile. Molecular analysis showed the three brothers to be compound heterozygotes, carrying two different mutations in exon 4 of the 5 alpha-reductase type 2 gene. The two mutations (G196S and H231R) have been described previously and reported to give rise to partially functioning enzymes, which may explain the milder phenotype and perhaps the fertility in the preset three patients.     

Systemic interleukin-1 induces early and late patterns of c-fos mRNA expression in brain

This study was designed to examine the mechanisms by which systemic interleukin-1 affects neuroendocrine systems in the brain. Intraperitoneal injections of interleukin-1 beta (1.25 micrograms/rat) were administered to rats. One or three hours after injection, the expression levels of the immediate-early gene c-fos and of genes for several neuropeptides, receptors, and enzymes were examined by in situ hybridization histochemistry. In the brainstem at 1 hr, c-fos mRNA was elevated in the area postrema and nucleus of the solitary tract, but not in the locus coeruleus. At 3 hr, the c-fos mRNA levels had increased further in the nucleus of the solitary tract. Rostrally, elevations in c-fos mRNA levels were found in the hypothalamic and thalamic paraventricular nuclei, central nucleus of amygdala, bed nucleus of the stria terminalis, and medial preoptic area, peaking at 1 hr and diminishing at 3 hr. In addition, at 3 hr a new pattern of c-fos activity emerged--the arcuate nucleus and cells at the external margins throughout the brain now expressed c-fos mRNA. Corticotropin-releasing hormone mRNA levels were doubled in the paraventricular nucleus at 1 and 3 hr, concomitant with elevations in plasma adrenocorticotrophic hormone (ACTH) and corticosterone. Tyrosine hydroxylase mRNA levels in the brainstem did not change. The c-fos mRNA induction patterns reveal a temporally dynamic response to interleukin-1 administration. We propose that the early set of structures responding to interleukin-1 initiates the neuroendocrine response to cytokines. Coactivation of the area postrema and nucleus of the solitary tract may reflect entry into the brain and neural transduction of the peripheral signal. The late set--including the nucleus of the solitary tract, arcuate nucleus, and the brain's edge--may reflect cellular activation along the diffusion routes traveled by interleukin-1 or a bioactive transduction product, because the pattern of edge labeling is similar to the autoradiographic pattern of flow lf radiolabeled tracer substances in the cerebrospinal fluid. The late c-fos mRNA response to interleukin-1, therefore, may represent a demonstration of information transfer in the parasynaptic mode, also known as volume transmission.     

Indole and benzimidazole derivatives as steroid 5alpha-reductase inhibitors in the rat prostate

A novel series of indole and benzimidazole derivatives were synthesized and evaluated for their inhibitory activity of rat prostatic 5alpha-reductase. Among these compounds, 4-?2-[1-(4,4'-dipropylbenzhydryl)indole-5-carboxamido]phenoxy?buty ric acid (15) and its benzimidazole analogue 25 showed potent inhibitory activities for rat prostatic 5alpha-reductase (IC50 values of 9.6+/-1.0 and 13+/-1.5 nM, respectively), with the potency very close to that of finasteride. Compound 30, in which the moiety between the benzene ring and amide bond was replaced by quinolin-4-one ring, showed almost equipotent activity (IC50= 19+/-6.2nM) with the correspondent amide derivative 13. This result was consistent with the previous observation that the coplanarity of this moiety might contribute to the potent inhibitory activity.     

The 5alpha-reductase in the central nervous system: expression and modes of control

The present paper will summarize two important aspects of the interactions between steroids and the brain, which have recently been studied in the authors' laboratory. In particular the paper will consider data on: (1) the significance of the two isoforms of the 5alpha-R during brain ontogenesis and development, and (2) the cross-talk between glial and neuronal elements, particularly in relation to the metabolism of sex hormones. (1) The data obtained have shown that the 5alpha-R type 1 enzyme is constitutively expressed in the rat CNS at all stages of brain development. Moreover, the expression of the 5alpha-R type 1 is similar in males and in females, and does not appear to be controlled by androgens. The gene expression of the 5alpha-R type 2 is totally different. This isoform appears to be expressed in the rat brain almost exclusively in the late fetal/early post-natal life and is controlled by testosterone. (2) The present data show that two cell lines derived respectively from a rat glioma (C6 cell line) and from a human astrocytoma (1321N1 cell line) are able to convert testosterone and progesterone into their corresponding 5alpha-reduced metabolites dihydrotestosterone and dihydroprogesterone. The possibility that secretory products of normal and tumoral brain cells might be able to influence steroid metabolism occurring in the two glial cell lines previously mentioned has been considered.     

Vitamin and mineral transfer during fetal development and the early postnatal period in pigs

There are periods during pregnancy when sows may have a temporally high requirement for certain vitamins and minerals. Proteins transferring retinol and Fe to the developing pig fetus have been discovered, whereas transport mechanisms for other vitamins and minerals are probably present but have not yet been identified. Sow body tissues can serve as a reservoir for many micronutrients, but it is not known whether these reserves can supply an adequate quantity during critical fetal developmental periods. There is a low placental transfer of vitamin E to the fetus even if the dietary concentration fed to a gestating animal is high, but colostrum and milk concentrations can be increased when the nutrient is fed to sows. If the dam's diet contains inadequate Ca or P, the concentration of these elements in the developing fetus and milk will not be affected. Consequently, sow bone demineralization will occur under conditions of dietary inadequacy of Ca and P. Other nutrients can be depleted from sow tissue reservoirs over several parities (e.g., Se), resulting in low quantities being provided in the milk for nursing pigs. Scientific information involving adequate vitamin and mineral nutrition for female pigs to improve conception rate and embryonal survival that will result in optimum fetal and postnatal pig development can be considered to be in its infancy.     

Ontogenic expression of natriuretic peptide mRNAs in postnatal rat brain: implications for development?

The central natriuretic peptide system is composed of at least three structurally homologous and uniquely distributed peptides and receptors which are thought to be involved in the central regulation of cardiovascular and autonomic function and more recently been shown to affect cellular growth and proliferation, processes pertinent to mammalian development. As such, following our initial mapping of preproatrial natriuretic peptide (ppANP) mRNA in adult brain [M.C. Ryan, A.L. Gundlach, Anatomical localization of preproatrial natriuretic peptide mRNA in the rat brain by in situ hybridization histochemistry: in olfactory regions, J. Comp. Neurol., 356 (1995) 168-182], it was of interest to determine the ontogenic expression of natriuretic peptide mRNAs in the developing rat brain. Using in situ hybridization histochemistry of specific [35S]- or [33P]-labeled oligonucleotides, ppANP and preproC-type natriuretic peptide (ppCNP) mRNAs were detected in the developing rat brain from postnatal day 4 to day 60 (adult). PpANP mRNA was observed in many hindbrain, but only some forebrain, regions at postnatal day 4. Regional differences in the temporal expression of ppANP mRNA were apparent with ppANP mRNA detected in the medial preoptic area, mammillary nuclei and medial habenular nucleus at postnatal day 4 and in other areas including the arcuate and dorsomedial hypothalamic nuclei and in olfactory and limbic regions at postnatal day 10. A number of regions also exhibited transient expression of ppANP mRNA such as the bed nucleus of the stria terminalis and the medial cerebellar nucleus. In contrast, ppCNP mRNA was detected at relatively high levels in several regions on postnatal day 4 including olfactory nuclei, the hippocampus and particularly the pontine nucleus. The level of expression appeared to increase markedly in most regions including forebrain olfactory and hippocampal areas and in brainstem regions including the pontine nucleus, the parvocellular and lateral reticular and spinal trigeminal nuclei by postnatal days 10 and 13, but decreased from this peak to equivalent to adult levels by postnatal day 28. The differential and transient expression of the natriuretic peptides during postnatal development, together with previous reports of the ontogenic regulation of natriuretic peptide receptor expression and binding patterns, further suggests their involvement in developmental processes in the rat CNS and provides information relevant to the likely functional development of natriuretic peptide-utilizing pathways.     

Infraorbital nerve transection alters serotonin transporter expression in sensory pathways in early postnatal rat development

OBJECTIVE: To determine the relationship between sperm motility and sperm morphology parameters and IVF and pregnancy rates. DESIGN: Pre- and postpreparation analysis of semen samples from infertile couples undergoing IVF-ET. SETTING: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland. PATIENT(S): One hundred fifty couples undergoing IVF-ET treatment at the Regional Fertility Centre. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The ability of human sperm to achieve IVF and pregnancy was investigated in relation to motility parameters (assessed with computer-aided sperm analysis [Integrated Visual Optical System] and percent normal morphology (determined with the strict criteria). RESULT(S): Significant differences were observed in motility parameters and percent normal morphology in samples that achieved > or =50% fertilization compared with < or =50% fertilization and between samples that achieved a pregnancy compared with those that did not. Significant positive correlations were observed between percent progressive motility, the velocity of sperm movement, and morphology parameters and both IVF and pregnancy. CONCLUSION(S): Both sperm motility parameters and percent normal morphology are significant factors in predicting fertilization and pregnancy rates in IVF.