Assessing reproductive toxicity and antioxidant enzymes on beta asarone induced male Wistar albino rats: In vivo and computational analysis.

AIM: Beta asarone is the major constituent of oil obtained from Acorus calamus, the Indian traditional medicine plant. Several studies have shown that beta asarone causes liver and cardiac damages but the reproductive toxicity is not well understood. The present study was initiated to investigate whether beta asarone has the potential to cause reproductive toxicity by inducing oxidative stress in the testis of male Wistar albino rats. MATERIALS AND METHODS: For this study, the animals were divided into six groups: Group I was treated with saline (normal saline), Group II with DMSO (vehicle control) and Group III with cisplatin (10mg/kgb.wt.). Group IV, V and VI animals were administrated at three dose levels of beta asarone 12.5, 25 and 50mg/kgb.wt. The treatment was carried out for 14days and animals were sacrificed on 29th day and processed for sperm analysis, hormone assay, histopathological, and antioxidant enzymatic assays. We also used molecular docking studies to predict the binding nature of beta asarone with luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). KEY FINDINGS: Beta asarone administered at a dose of 50mg/kgb.wt. was responsible for inducing certain noticeable degenerative changes in histopathological analysis of the tissue. This was supported by altered sperm morphology and hormonal variations when compared to the control groups. Antioxidant enzyme levels were also found to be decreased. This was further validated by molecular docking studies. SIGNIFICANCE: The present study provides evidence that beta asarone administered at a dose of 50mg/kg b.wt. is capable enough in bringing about moderate amount of degenerative changes in rat testis and altered antioxidant status. Therefore provides a suitable evidence to prove that beta asarone causes reproductive toxicity.

Implications for molecular mechanisms of glycoprotein hormone receptors using a new sequence-structure-function analysis resource.

Comparison between wild-type and mutated glycoprotein hormone receptors (GPHRs), TSH receptor, FSH receptor, and LH-chorionic gonadotropin receptor is established to identify determinants involved in molecular activation mechanism. The basic aims of the current work are 1) the discrimination of receptor phenotypes according to the differences between activity states they represent, 2) the assignment of classified phenotypes to three-dimensional structural positions to reveal 3) functional-structural hot spots and 4) interrelations between determinants that are responsible for corresponding activity states. Because it is hard to survey the vast amount of pathogenic and site-directed mutations at GPHRs and to improve an almost isolated consideration of individual point mutations, we present a system for systematic and diversified sequence-structure-function analysis (http://www.fmp-berlin.de/ssfa). To combine all mutagenesis data into one set, we converted the functional data into unified scaled values. This at least enables their comparison in a rough classification manner. In this study we describe the compiled data set and a wide spectrum of functions for user-driven searches and classification of receptor functionalities such as cell surface expression, maximum of hormone binding capability, and basal as well as hormone-induced Galphas/Galphaq mediated cAMP/inositol phosphate accumulation. Complementary to known databases, our data set and bioinformatics tools allow functional and biochemical specificities to be linked with spatial features to reveal concealed structure-function relationships by a semiquantitative analysis. A comprehensive discrimination of specificities of pathogenic mutations and in vitro mutant phenotypes and their relation to signaling mechanisms of GPHRs demonstrates the utility of sequence-structure-function analysis. Moreover, new interrelations of determinants important for selective G protein-mediated activation of GPHRs are resumed.

The formation of a salt bridge between helices 3 and 6 is responsible for the constitutive activity and lack of hormone responsiveness of the naturally occurring L457R mutation of the human lutropin receptor.

The human lutropin receptor (hLHR) plays a pivotal role in reproductive endocrinology. A number of naturally occurring mutations of the hLHR have been identified that cause the receptor to become constitutively active. To gain further insights into the structural basis for the activation of the hLHR by activating mutations, we chose to examine a particularly strong constitutively activating mutation of this receptor, L457R, in which a leucine that is highly conserved among rhodopsin-like G protein-coupled receptors in helix 3 has been substituted with arginine. Using both disruptive as well as reciprocal mutagenesis strategies, our studies demonstrate that the ability of L457R to stabilize an active form of the hLHR is because of the formation of a salt bridge between the replacing amino acid and Asp-578 in helix 6. Such a lock between the transmembrane portions of helices 3 and 6 is concurrent with weakening the connections between the cytosolic ends of the same helices, including the interaction found in the wild-type receptor between Arg-464, of the (E/D)R(Y/W) motif, and Asp-564. This structural effect is properly marked by the increase in the solvent accessibility of selected amino acids at the cytosolic interfaces between helices 3 and 6. The integrity of the conserved amino acids Asn-615 and Asn-619 in helix 7 is required for the transfer of the structural change from the activating mutation site to the cytosolic interface between helices 3 and 6. The results of in vitro and computational experiments further suggest that the structural trigger of the constitutive activity of the L457R mutant may also be responsible for its lack of hormone responsiveness.

Three different turkey luteinizing hormone receptor (tLH-R) isoforms I: characterization of alternatively spliced tLH-R isoforms and their regulated expression in diverse tissues.

Using combinations of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends, three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms were characterized from ovarian mRNA. The first cDNA (tLH-R(intact)) showed 98% and 72-75% similarity with chicken and mammalian LH-R sequences, respectively. The second cloned cDNA isoform (tLH-R(insert)) contained an in-frame TGA stop codon within an 86-base pair insertion that was located in the extracellular domain of the seven-transmembrane region. The tLH-R(insert) isoform could encode a truncated soluble protein isoform that lacked the transmembrane region. The third cDNA isoform truncated the transmembrane region (tLH-R(trunc)) and was derived by the deletion of the last exon by incomplete splicing. Generation of multiple transcripts by alternative splicing was elucidated by partial characterization of tLH-R genomic sequences. The differentially regulated expression of the tLH-R mRNA isoforms in nongonadal tissues and ovarian stromal tissues during various reproductive stages was quantified and analyzed by Northern blot and/or RT-PCR. Alternatively spliced tLH-R isoforms were differentially expressed in a tissue-specific manner in most of the tissues examined. The steady-state levels of tLH-R mRNA isoforms were relatively high in the hypothalamus and optic nerve and relatively low in the cortex, pituitary, and cerebellum when compared to levels in ovarian follicles. In nongonadal reproductive tissues, the steady-state levels of tLH-R mRNA isoforms were relatively high in the uterus and infundibulum and relatively low in the isthmus, oviduct, and magnum. In addition, in the nongonadal peripheral tissues, the steady-state levels of tLH-R isoforms were relatively high in the thyroid gland and relatively low in the spleen, adrenal gland, kidney, skin, bursa, and muscle. The present study suggests that the alternative splicing of LH-R transcripts occurs in a tissue-specific manner and has been evolutionarily conserved (similar results were obtained in chicken and swine). These results raise fundamental questions as to the function of LH-R isoforms in nongonadal tissues.

Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions.

BACKGROUND: Glycoprotein hormones influence the development and function of the ovary, testis and thyroid by binding to specific high-affinity receptors. The extracellular domains of these receptors are members of the leucine-rich repeat (LRR) protein superfamily and are responsible for the high-affinity binding. The crystal structure of a glycoprotein hormone, namely human choriogonadotropin (hCG), is known, but neither the receptor structure, mode of hormone binding, nor mechanism for activation, have been established. RESULTS: Despite very low sequence similarity between exon-demarcated LRRs in the receptors and the LRRs of porcine ribonuclease inhibitor (RI), the secondary structures for the two repeat sets are found to be alike Constraints on curvature and beta-barrel geometry from the sequence pattern for repeated beta alpha units suggest that the receptors contain three-dimensional structures similar to that of RI. With the RI crystal structure as a template, models were constructed for exons 2-8 of the receptors. The model for this portion of the choriogonadotropin receptor is complementary in shape and electrostatic characteristics to the surface of hCG at an identified focus of hormone-receptor interaction. CONCLUSIONS: The predicted models for the structures and mode of hormone binding of the glycoprotein hormone receptors are to a large extent consistent with currently available biochemical and mutational data. Repeated sequences in beta-barrel proteins are shown to have general implications for constraints on structure. Averaging techniques used here to recognize the structural motif in these receptors should also apply to other proteins with repeated sequences.

Identification of two amino acid residues on the extracellular domain of the lutropin/choriogonadotropin receptor important in signaling.

The lutropin/choriogonadotropin receptor (LH/CG-R) is a G protein-coupled receptor with a relatively large extracellular domain. The cDNAs of LH/CG-R wild type and 15 point and double mutants, which encoded residues of opposite charge to that of wild type, were transiently transfected into COS-7 cells. Human choriogonadotropin (hCG) binding was determined, as was hCG-mediated cAMP production. Most of the replacements resulted in no substantive effect on the binding affinity of hCG to LH/CG-R or on hCG-stimulated cAMP production, although the mutants expressed at a lower level than LH/CG-R wild type. The most interesting observation was noted with two point mutants of LH/CG-R, Glu332-->Lys and Asp333-->Lys, which bound hCG but failed to give increased cAMP production. Several of the mutant forms of LH/CG-R that expressed at low levels were further analyzed by soluble binding assays and Western blots. There was no evidence of any significant degree of intracellular trapping of hCG-binding mutant receptors. The expected major (93 kDa) and minor (78 kDa) forms were found for LH/CG-R wild type and several of the mutants. The Lys235-->Asp and Asp333-->Lys mutants exhibited primarily the lower M(r) form, indicating that receptor processing was impaired or that the mutant higher M(r) form was more rapidly degraded than LH/CG-R wild type. These results demonstrate that Glu332 and Asp333, which are located near the first transmembrane helix, are important in receptor activation, while other conserved ionizable residues of LH/CG-R appear important in cell surface expression or stability but not in binding or signaling.

Genetic heterogeneity of constitutively activating mutations of the human luteinizing hormone receptor in familial male-limited precocious puberty.

Genomic DNA from 32 unrelated families with male-limited precocious puberty was examined for the previously described Asp-578-->Gly, Met-571-->Ile, and Thr-577-->Ile mutations in transmembrane helix 6 of the human luteinizing hormone receptor (hLHR). Twenty-eight families had the inherited form of the disorder, and of these, 24 were found to have the Asp-578-->Gly mutation. Four additional mutations were found among the remaining four families with the inherited form and in four sporadic cases of the disorder: an A-->C transversion resulting in substitution of leucine for Ile-542 in the fifth transmembrane helix, an A-->G transition resulting in substitution of glycine for Asp-564 in the third cytoplasmic loop, a G-->T transversion resulting in substitution of tyrosine for Asp-578 in the sixth transmembrane helix, and a T-->C transition resulting in substitution of arginine for Cys-581 in the sixth transmembrane helix. Human embryonic kidney cells transfected with cDNAs for each of the mutant hLHRs, created by PCR-based mutagenesis of the wild-type hLHR cDNA, exhibited increased levels of basal cAMP production in the absence of agonist, indicating constitutive activation of the mutation hLHRs. Three of the additional mutations had specific features: Ile-542-->Leu and Cys-581-->Arg appeared ligand-unresponsive, whereas Asp-578-->Tyr appeared to correlate genotype with phenotype. We conclude that the region spanning nt 1624-1741 of exon 11 is a hotspot for heterogeneous point mutations that constitutively activate the hLHR and cause male-limited precocious puberty.

A sporadic case of male-limited precocious puberty has the same constitutively activating point mutation in luteinizing hormone/choriogonadotropin receptor gene as familial cases.

Familial male-limited precocious puberty (FMPP) is an autosomal dominant disorder characterized by marked elevation of serum testosterone despite low levels of gonadotropin. Recently, a single point mutation in the LH/hCG receptor (LH/CGR) gene was found in FMPP families that constitutively activates the LH/CGR, causing Leydig cell activation and precocious puberty. Among the Japanese population, only four sporadic cases of male-limited precocious puberty have been reported. In the current study, we examined one of the four reported Japanese patients with sporadic male-limited precocious puberty and found the same mutation as that in the FMPP families. Genomic DNA was isolated, and the polymerase chain reaction (PCR) was performed to amplify a fragment of LH/CGR DNA encoding amino acid residues that include transmembrane helixes 5 and 6. Sequencing of the PCR products revealed a heterozygous adenosine-guanine transition at nucleotide 1733 in codon 578. The mutation encodes an aspartic acid578-glycine substitution in transmembrane helix 6. The mutant LH/CGR, created by site-directed mutagenesis in vitro, exhibited constitutively higher cAMP levels in transfected COS-7 cells than the wild-type LH/CGR, as described previously; however, basal inositol phosphate levels were not increased by transfection with complementary DNA for the mutant receptor. The concentration and affinity of [125I]hCG-binding sites were similar in cells transfected with the mutant and wild-type LH/CGR complementary DNAs, indicating that the mutant did not alter the production of receptor or its ability to bind human LH/CG. The sporadic occurrence of this case was confirmed by further studies. The mutation creates a recognition site for the restriction endonuclease MspI. Restriction digestion was positive for the mutant not digested by MspI, indicating that the patient's mutant allele was not inherited from his parents. DNA analysis of the patient and the parents, using microsatellite repeat markers, was compatible with biological paternity and maternity. We conclude that the aspartic acid578-->glycine mutation in the LH/CGR has arisen in the Japanese population and is the cause of a sporadic case of male-limited precocious puberty.