Evaluating the anti-fertility potential of α-chlorohydrin on testis and spermatozoa in the adult male wild Indian house rat (Rattus rattus)

To examine the effects of α-chlorohydrin on testis and cauda epididymis in the male house rat (Rattus rattus), 24 adult male rats were segregated into two groups. Group I rats were force-fed daily by intragastric intubation with α-chlorohydrin at a single dose of 1.0 mg/100 g body weight/d for 5, 15, and 45 days. Another group was fed with distilled water, which served as the control. The treated male rats were paired with 24 adult proestrus female rats for 5 days after the last oral treatment and fertility was tested. At the end of the experiments, all of the male rats were weighed and killed by cervical dislocation. The right testes were removed, weighed, and processed for ultrastructural changes of spermatozoa from the cauda epididymis and testis under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The seminiferous tubular area, nuclear diameter of the Sertoli and Leydig cells, percentage of spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, spermatozoa, and Sertoli cells in each group were compared morphometrically. Our results showed that the percentages of primary spermatocytes steadily increased from 5 to 15 days, but primary and secondary spermatocytes decreased significantly at 45 days. There was a steady decline in the percentages of spermatozoa and spermatids at all fixation intervals in the treated animals, but the percentages of spermatogonia and Sertoli cells increased significantly at 15 and 45 days. Seminiferous tubular areas, nuclear diameter of Leydig and Sertoli cells, and fertility rates were reduced after 45 days of treatment. SEM and TEM studies revealed severe morphological abnormalities in the spermatozoa, including deglutination of the acrosomal part, loss of head capsules, and fragmentation of tail fibrils. There was an enhanced anti-fertility effect and a lower number of implantation sites in the rats treated for 5 days. Our results validate α-chlorohydrin as a successful anti-fertility agent that prevents spermatogenesis.     

Immunohistochemical studies on the proliferative marker Ki-67 and estrogen receptor alpha (ERα) in the uterus of neonatal and immature pigs following exposure to flutamide

The development of uterine glands is characterized by the proliferation of epithelial cells and by estrogen receptor alpha (ERα) expression in the nascent glandular epithelium. It is known that androgen receptors are present in the porcine uterus during prenatal development and the neonatal window, when adenogenesis occurs. Therefore, the objective of the study was to determine whether the effects of maternal or neonatal administration of the anti-androgen, flutamide, could entail changes in the presence of ERα and proliferation of uterine cells in neonatal and three-month-old pigs. Following prenatal flutamide exposure, morphological differences and the acceleration of uterus differentiation marked by ERα expression in epithelial crypts were observed in the neonatal piglets. In the three-month-old pig uterus, the proliferation of stromal cells was observed only after prenatal exposure to flutamide, whereas ERα staining was weaker. The neonatal administration of flutamide caused a significant decrease in the proliferation of the surface epithelium and diminished intensity of ERα staining in the stromal cells of the uterus of three-month-old pigs, which paralleled decreased estrogen levels in these animals. Overall, prenatal flutamide exposure promoted growth and development of the neonatal porcine uterus. Moreover, in three-month-old pigs, flutamide application during the neonatal period decreased surface epithelium proliferation and stromal ERα expression, which confirmed the importance of epithelial-stromal interactions in the adenogenesis.     

Expression and ligand-binding function of the integrin α4β1 (VLA-4) on neural-crest-derived tumor cell lines

Human neural-crest-derived tumor cell lines, including three neuroblastomas, an astrocytoma, a glioblastoma, a rhabdomyosarcoma and a melanoma were screened for the expression of the integrin ₄₁ (VLA-4). The neuroblastomas IMR-32 and SK-N-SH, the astrocytoma 131-INI, the glioblastoma Fogerty and the rhabdomyosarcoma TE-671 expressed ₄₁ as determined by cytofluorometry and immunoprecipitation. Another neuroblastoma line, LA-N-1, did not express ₄₁. Analysis of immunoprecipitated ₄₁ showed that the ₄ subunit from the various cell types differed in relative molecular weight (M _r ). The variability in the observed M _r could be accounted for by differences in the levels of N-linked glycosylation. The observed variability in M _r did not appear to affect function since intact cells and solubilized ₄₁ bound to a synthetic peptide identical in sequence to the CS-1 region of the alternatively spliced IIICS domain of fibronectin, a known ₄₁, ligand.     

An iontophoretic study of the effects of α-amino-hydroxy-5-methyl-4-isoxazole propionic acid lesions of the nucleus basalis magnocellularis on cholinergic and GABAergic influences on frontal cortex neurones of rats

Unilateral lesions of the nucleus basalis magnocellularis (NBM) produced by -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid in rats caused, 8–10 weeks after the lesion, a 94% reduction in cortical acetylcholinesterase fibres and reduced activities of acetylcholinesterase and choline acetyltransferase by 70–80% in the frontal cortex ipsilateral to the lesion. In anaesthetized unlesioned control rats, iontophoretic administration of acetylcholine and carbachol produced atropine-sensitive inhibition and excitation of frontal cortical neurones, effects similar to those produced by electrically stimulating the NBM. The lesion reduced cortical neuronal firing rates but increased the percentage and sensitivity of neurones responding to acetylcholine, the predominant response changing from inhibition to excitation; response duration increased but latency was unaffected. The size of the response of individual neurones to carbachol, but not the percentage of sensitive neurones, was also increased in lesioned animals. The proportion of neurones responding to bicuculline and their individual sensitivities were increased by the lesion, suggesting that the lesion increased GABAergic tone; responses to glutamate were unchanged. The lesion did not affect the proportion of neurones in which acetylcholine modulated neuronal responses but reversed the nature of the modulation to predominantly excitatory; excitation was the predominant response to electrical forepaw stimulation in unlesioned control animals. This suggests a possible interaction between GABAergic and cholinergic mechanisms in selective attention and processing of cognitive information. Acute administration of di-isopropyl fluorophosphate to unlesioned animals significantly increased the number of frontal cortical neurones responding to acetylcholine, without affecting individual neuronal sensitivity or responses to carbachol and glutamate. The similarity of these effects to those of acetylcholine in lesioned animals suggests that the increased sensitivity to acetylcholine in the latter was due to loss of acetylcholinesterase, enabling diffusion of acetylcholine to more distant neurones. However, acetylcholinesterase does not hydrolyse carbachol and therefore it is necessary to postulate a different post-synaptic mechanism to explain the lesion-induced increases in the sensitivities of individual neurones to carbachol and to acetylcholine; interpretation of experimental findings should take these two mechanisms into account.     

Poly(ester amide)s based on (L)-lactic acid oligomers and α-amino acids: influence of the α-amino acid side chain in the poly(ester amide)s properties

Novel biodegradable and low cytotoxic poly(ester amide)s (PEAs) based on α-amino acids and (L)-lactic acid (L-LA) oligomers were successfully synthesized by interfacial polymerization. The chemical structure of the new polymers was confirmed by spectroscopic analyses. Further characterization suggests that the α-amino acid plays a critical role on the final properties of the PEA. L-phenylalanine provides PEAs with higher glass transition temperature, whereas glycine enhances the crystallinity. The hydrolytic degradation in PBS (pH?=?7.4) at 37?°C also depends on the α-amino acid, being faster for glycine-based PEAs. The cytotoxic profiles using fibroblast human cells indicate that the PEAs did not elicit an acute cytotoxic effect. The strategy presented in this work opens the possibility of synthesizing biodegradable PEAs with low citotoxicity by an easy and fast method. It is worth to mention also that the properties of these materials can be fine-tuned only by changing the α-amino acid.     

Neonatal dopamine lesion in the rat results in enhanced adenylate cyclase activity without altering dopamine receptor binding or dopamine- and adenosine 3′∶5′-monophosphate-regulated phosphoprotein (DARPP-32) immunoreactivity

Summary Newborn male Sprague-Dawley rats were treated neonatally with an intracisternal injection of 75 g 6-hydroxydopamine (6-OHDA) following desipramine pretreatment in order to induce a permanent selective dopamine (DA) lesion. At 60–70 days of age a massive loss of tyrosine hydroxylase (TH) immunoreactive (IR) cells was seen in substantia nigra. The TH-IR terminal density was reduced by 92% in striatum, 77% in nucleus accumbens and by 72% in tuberculum olfactorium. Quantitative autoradiography using ³H-SCH-23390 and ³H-spiperone did not reveal any alteration of DA D₁ and D₂ receptor binding in the denervated regions studied. Furthermore, no change in the B_max or K_d of ³H-SCH-23390 or ³H-spiperone in vitro binding was observed in membrane preparations of striatum following the neonatal DA lesion. Basal and DA-stimulated accumulation of cAMP was increased in striatal membrane preparations of the neonatally DA-lesioned rats. No alteration of the immunoreactivity of the D₁ receptor associated phosphoprotein dopamine- and adenosine 35-monophosphate-regulated phospho-protein (DARPP-32), was observed as visualized using quantitative immunohistochemistry. Thus, neonatal DA lesions seem to induce a selective functional supersensitivity reflected by an enhanced activity of D₁ receptor-coupled adenylate cyclase, without any alteration in the number or affinity of D1 and D₂ receptor sites. Further-more, the appearance of DARPP-32 seems to be independent of intact DA input during development.     

Resolving the Effects of Maternal and Offspring Genotype on Dyadic Outcomes in Genome Wide Complex Trait Analysis (“M-GCTA”)

Genome wide complex trait analysis (GCTA) is extended to include environmental effects of the maternal genotype on offspring phenotype (“maternal effects”, M-GCTA). The model includes parameters for the direct effects of the offspring genotype, maternal effects and the covariance between direct and maternal effects. Analysis of simulated data, conducted in OpenMx, confirmed that model parameters could be recovered by full information maximum likelihood (FIML) and evaluated the biases that arise in conventional GCTA when indirect genetic effects are ignored. Estimates derived from FIML in OpenMx showed very close agreement to those obtained by restricted maximum likelihood using the published algorithm for GCTA. The method was also applied to illustrative perinatal phenotypes from ~4,000 mother-offspring pairs from the Avon Longitudinal Study of Parents and Children. The relative merits of extended GCTA in contrast to quantitative genetic approaches based on analyzing the phenotypic covariance structure of kinships are considered.     

The effects of coadministration of ghrelin agonist (GHRP-2) and GH on TNF-α, IL-6, and iNOS genes expression induced by LPS in mouse liver

Growth hormone (GH)-releasing peptide-2 (GHRP-2), a ghrelin receptor agonist, has been reported to bear an anti-inflammatory effect. The aim of this study is to assess the impact of GHRP-2 and GH on the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) genes induced by lipopolysaccharide (LPS) in mouse liver tissues. Thirty-five male NMRI mice (25?±?5 g) were used. Mice were divided into five groups (n?=?7). GHRP-2 (100 μg/kg), GH (25 μg/kg), and (GHRP-2 + GH) were injected through the mice tail vein 30 min before the injection of LPS. Then, inflammation was induced by intraperitoneal injection of LPS (5 mg/kg) while the control animals received sterile saline. Changes in the levels of expression of TNF-α, IL-6, and iNOS genes in the mice liver induced by LPS injection for 2 h were studied by a semiquantitative RT-polymerase chain reaction method. Administration of LPS increased hepatic TNF-α, IL-6, and iNOS mRNAs. The results showed that intravenous administration of GHRP-2and GH significantly reduced the elevated TNF-α, IL-6, and iNOS mRNA levels 2 h after the injection. In contrast, injection of GHRP-2 prior to injection of LPS reduced IL-6. Injection of GH reduced the expression of iNOS in liver tissues. Coadministration of two drugs had a positive effect on the reduction of TNF-α, IL-6, and iNOS expression.     

Effects of different angiotensin Ⅱ type 1 receptor blockers on regulation of ACE-Ang Ⅱ-AT1 and ACE2-Ang( 1-7)-Mas axes in pressure overload-induced cardiac remodeling in male mice

AIM: To examine and compare the effects of several ARBs that are widely used in clinics,on the ACE-Ang II-AT1 receptor and the ACE2-Ang(1-7)-Mas axis during the development of cardiac remodeling after pressure overload. METHODS: All of the mice used in the study underwent transverse aortic constriction( TAC) or sham operation for 2 or 4 weeks. A solution of either ARBs or saline was administered through a stomach tube 3 days before the operation. Meanwhile,to eliminate the influence of Ang II,a recombinant adenovirus expressing small interfering RNAs targeting angiotensinogen( Ad-ATG si RNA) was injected via the tail vein. The surgery was then performed and the drug was administered as mentioned above. Cardiac function and remodeling were evaluated by echocardiography,hemodynamic measurements and cardiac histology. Western blotting was used to determine the protein expression levels.Meanwhile,we performed similar experiments using ARBs with or without ATG si RNA in cardiomyocytes induced by mechanical stretch. RESULTS: Although all of the six ARBs,none of which repressed the elevation of left ventricular pressure after TAC,attenuated the development of cardiac hypertrophy and heart failure in the wild-type mice,the degree of attenuation by Olmesartan,Candesartan and Losartan tended to be larger than that of the other three drugs tested. Additionally,the degree of downregulation of the ACEAng II-AT1 axis and upregulation of the ACE2-Ang(1-7)-Mas axis was higher in response to Olmesartan,Candesartan and Losartan administration in vivo and in vitro. Additionally,Olmesartan had a larger influence when administered long term. However,the expression of ACE was not influenced by the administration of ARBs in vivo and in vitro. Moreover,in angiotensinogen-knockdown mice,TAC-induced cardiac hypertrophy and heart failure were inhibited by Olmesartan,Candesartan and Losartan but not by Telmisartan,Valsartan and Irbesartan administration. Furthermore,only Olmesartan and Candesartan could downregulate the ACE-Ang II-AT1 axis and upregulate the ACE2-Ang(1-7)-Mas axis in vitro. CONCLUSION: Olmesartan,Candesartan and Losartan could effectively inhibit pressure overload-induced cardiac remodeling even when with knockdown of Ang II,possibly through upregulation of the expression of the ACE2-Ang(1-7)-Mas axis and downregulation of the expression of the ACE-Ang II-AT1 axis. In contrast,Telmisartan,Valsartan and Irbesartan only played a role in the presence of Ang II,and Losartan had no effect in the presence of Ang II in vitro.     

Adhesion of Human Myelomonocytic (HL-60) Cells Induced by 1, 25-dihydroxyvitamin D3 and Phorbol Myristate Acetate is Dependent on Osteopontin Synthesis and the αvβ3 Integrin

A key event in bone resorption is the attachment of osteoclasts on the bone surface. Accumulating data supports the notion that this interaction involves the matrix protein osteopontin on the bone surface interacting with a receptor of the integrin family (cx_vP₃) at the osteoclast clear zone. Based on the recent observation that osteopontin phosphorylation appears to be required for this interaction, it is of considerable interest to delineate the structural requirements for osteopontin-mediated cell attachment. Although binding of isolated osteoclasts to osteopontin-coated surfaces involves the Ovp₃ integrin, this system suffers from considerable disadvantages to allow detailed studies in this respect. We have therefore turned to another cell system, HL-60 promyelocyte leukemia cells, to address these questions. In the presence of the phorbol ester TPA (10 nM) and 1, 25-dihydroxyvitamin D₃ (100 nM), 90% of the HL-60 cells became adherent within 24 hours and exhibited a macrophage-like appearance. Under these conditions, the osteopontin mRNA levels was elevated around 60-fold compared to the control, non-adherent cells. The absolute requirement of de novo osteopontin synthesis for the TPA and 1, 25-vit 1), -induced HL-60 cell adhesion was demonstrated by neutralisation of adhesion using an anti-osteopontin polyclonal antibody as well as following transfection of an anti-sense osteopontin phosphorothioate-modified oligonucleotide. Finally, inhibition of induced HL-60 cell adhesion by an RGD-containing peptide or by an antibody to the α_vβ₃ integrin complex suggested that the cell-derived osteopontin interacts with this integrin. It is concluded that HL-60 cells induced to differentiate with the combination of TPA and 1, 25-vit D₃ can be utilised as a model system to delineate structural requirements involved in the interaction between osteopontin and the αvβ₃ integrin.     

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α confined to the surface of lipid droplets and adjacent narrow cytoplasm in progesterone-producing cells of in situ ovaries of adult mice

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) produced by phosphatidylinositol phosphate 5 kinase (PIP5K) plays not only as a precursor of second messengers in the phosphoinositide signal transduction, but also multiple roles influencing a variety of cellular activities. From this viewpoint, the present study attempted to localize PIP5Kα in the ovaries in situ of adult mice. PIP5Kα-immunoreactivity was confined to the surfaces of lipid droplets (LDs) and their adjacent cytoplasm in progesterone-producing cells of the interstitial glands, corpora lutea and theca interna. The LDs often contained membranous tubules/lamellae along their surfaces and within their interior whose membranes were continuous with those delineating LDs composed of a monolayer of phospholipids and were partially PIP5Kα-immunoreactive. Although granulosa cells of healthy-looking follicles were immunonegative, as the atresia progressed, PIP5Kα-immunoreactivity first appeared in sparsely dispersed dot forms in mural cells of the follicular epithelia, and then were dominant in almost all mural cells that remained after desquamation of the antral cells. The present study provides evidence suggesting that PI(4,5)P2 locally synthesized by PIP5K in LDs is involved in the lipid transfer between lipid droplets (LDs) and the endoplasmic reticulum, which eventually regulates ovarian progesterone production through control of multiple dynamic activities of LDs. It is also suggested that PIP5Kα and PI(4,5)P2 are implicated in the modulation of programmed cell death and/or acquiring the ability of progesterone production in some follicular cells surviving atresia.     

Generalized resistance to thyroid hormone: Identification of a novel c-erbAβ thyroid hormone receptor variant (leu450) in a Japanese family and analysis of its secondary structure by the Chou and Fasman method

Summary Generalized resistance to thyroid hormone (GRTH) is characterized by elevated circulating levels of thyroid hormone in the presence of a eumetabolic state and failure to respond to triiodothyronine. Various point mutations in the c-erbA thyroid hormone receptor gene are known to be responsible for different phenotypes of GRTH. We herein report a new c-erbA variant in a Japanese family. The variant consisting of a cytosine to adenine base substitution at nucleotide position 1650 altered phenylalanine to leucine in codon 450 in the T₃-binding domain of c-erbA. This base substitution was found in one allele of the 2 affected members of the family. Thein vitro translation products of this mutant c-erbA gene demonstrated a significantly reduced T₃-binding affinity. The secondary structure of this mutant thyroid hormone receptor predicted by the Chou and Fasman method included a new turn in the helix structure in the T₃-binding domain. We also discuss the secondary structures of the previously reported mutant receptors.This paper was previously presented in part at the 1st International Union of Biochemistry and Molecular Biology Conference, 1–6 June, 1992, in Nagoya, Japan.