Effects of maternal consumption of morphine on rat skeletal system development

Background Opioid abuse is among the most ubiquitous issues world-wide, and when it happens in mothers, it puts them at risk of diseases that can be transferred to the next generation. Previous studies have indicated that morphine addiction during pregnancy could inhibit development in rat embryos and infants. The present study focused on the effects of maternal consumption of morphine on rat skeletal system development and also investigate the molecular pathway of chondrogenesis and osteogenesis of infants from control and addicted rat groups. Methods Thirty-two female rats were randomly assigned to four groups. The groups consisted of one- and seven-day-old female infants which were born of morphine-dependent mothers and a control group for each of them. Experimental groups received oral morphine at the final dose of 0.4 mg/ml/day. Withdrawal signs were confirmation of morphine dependency. Female rats were crossed with male rats and coupling time was recorded. Fixed bones of all groups were processed and then stained by hematoxyline-eosin method. Thickness and cell number of proximal and distal growth plate of bones were measured. The cartilage and bone cells were stained by alcian blue/alizarin red method. Additionally, the gene expression of alkaline phosphatase, osteocalcin, and COLL2 and SOX9 gene expression were studied immuno-histochemically. Results Unfavorable effects of morphine on histological measurements were observed in one-day and seven-day infants, with more effects on seven-day infants. The thickness and cell number of the proximal and distal growth plate of morphine-dependent rat offsprings were reduced significantly. Furthermore, morphine reduced growth of primary and secondary ossification centers, and thus, longitudinal bone growth was reduced. Moreover, a decrease in the alkaline phosphatase, osteocalcin, COLL2 and SOX9 gene expression, and the number of stained cells was observed. More adverse effects of morphine in seven-day infants compared to one-day infants which showed the time dependent of morphine to the time length of administration. Conclusion Histochemistry and immunohistochemistry findings on cartilage and bone matrix formation, as well as protein expression of chondrogenic and osteogenic markers suggest that morphine dependence in pregnant mothers may impair intra-cartilaginous osteogenesis in post-natal rats.

Up-regulation of sex-determining region Y-box 9 (SOX9) in growth hormone-secreting pituitary adenomas

Background Pituitary adenomas are benign brain tumors that cause considerable morbidity and neurological symptoms. SOX9 as a regulatory transcriptional mediator affects normal and tumor cell growth with an undefined role in pituitary adenomas pathogenesis. Thus, in the present study, the expression pattern of SOX9 in GH-secreting pituitary tumors and normal pituitary tissues is investigated. Methods The SOX9 gene expression level was evaluated in 60 pituitary tissues including different types of GH-secreting adenomas and normal pituitary tissues through Real-Time PCR. The protein level of SOX9 was assessed using immunohistochemistry. The correlations of SOX9 gene and protein expression level with the patient’s clinical and pathological features were considered. Results The SOX9 over-expression was detected in GH-secreting adenomas tumor tissues compared to normal pituitary tissues which were accompanied by overexpression of SOX9 protein in tumor tissues. The over-expression of SOX9 had a significant impact on GH-secreting adenomas tumor incidence with the odds ratio of 8.4 and the diagnostic value of SOX9 was considerable. The higher level of SOX9 expression was associated with invasive and macro tumors in GH-secreting pituitary adenoma patients. The positive correlation of SOX9 gene and protein level was observed and the tumor size and tumor invasive features were valuable in predicting SOX9 expression level in GH-producing pituitary tumors. Conclusion The study provided the first shreds of evidence regarding the expression pattern of SOX9 in the GH- secreting pituitary adenomas at both gene and protein levels which may emphasize the possible involvement of SOX9 as a mediator in pituitary adenoma tumor formation also open up new intrinsic molecular mechanism regarding pituitary adenoma pathogenesis.

Determination of SOX9 Gene Expression In Brain Tumours Among East Coast Malaysia Population

Introduction: SOX9, a members of SOX family, plays a significant roles in developmental processes during embryogenesis, including brain tissue. Few studies have shown that SOX9 has been involved in tumourigenesis of several types of cancer including brain tumour. However, such studies are still lacking in the Malaysian population. The aim of this study was to determine SOX9 expression level in several types of brain tumours in East Coast Malaysia. Materials and Methods: Five formalin-fixed pariffin-embedded brain tumour samples of Malay descendants were sectioned by using microtome. RNA extraction was performed with slight modification by adding Trizol during tissue lysis. The RNA was converted to cDNA using reverse transcription technique before SOX9 expression was detected using RT q-PCR assay in brain tumours normalized to non-neoplastic brain tissues. Results: Overall results displayed that SOX9 gene in all samples were up-regulated. SOX9 overexpression was found in both high and low grade glioma (anaplastic and pilocytic astrocytoma respectively). This is consistence with both low grade (benign) and atypical meningioma. Secondary brain tumour also showed up-regulation when compared to normal brain tissue. Conclusion: Up-regulation in SOX9 expression in selected brain tumours in Malay patients revealed its significant roles in brain tumourigenesis. Functional studies should be carried out to observe the SOX9 functions and mechanism whether they should reflect their diverse roles in Malaysia population.

Detection of a 4 bp Mutation in the 3′UTR Region of Goat Sox9 Gene and Its Effect on the Growth Traits

The SRY-type HMG box 9 (Sox9) gene plays an important role in chondrocyte development as well as changes in hypertrophic chondrocytes, indicating that Sox9 can regulate growth in animals. However, no studies to date have examined the correlation between variations in Sox9 and growth traits in goats. Here, we found a 4 bp indel in the 3′UTR of Sox9 and verified its association with growth traits in Shaanbei white cashmere goats (n = 1109). The frequencies of two genotypes (ID and II) were 0.397 and 0.603, respectively, and polymorphic information content (PIC) values showed that the indel had a medium PIC (PIC > 0.25). The 4 bp indel was significantly correlated with body length (p = 0.006), heart girth (p = 0.001), and hip width (p = 4.37 × 10 −4). Notably, individuals with the ID genotype had significantly superior phenotypic traits compared with individuals bearing the II genotype. Hence, we speculated that the 4 bp indel is an important mutation affecting growth traits in goat, and may serve as an effective DNA molecular marker for marker-assisted selection in goat breeding programs.

SUN-034 Combined CNV, Haplotyping and Whole Exome Sequencing Implicates Inherited Novel SRY Mutations Not Account for Familial 46,XY Sex Reversal

SRY is one of the important genes involved in the process of human sex determination. The disturbed sex determination caused by SRY mutation accounts for 10-15% cases with complete gonadal dysplasia (CGD), also known as 46, XY sex reversal. Recently, three distal enhancers are disclosed in the upstream of SOX9 gene. In an inherited 46, XY sex reversal pedigree with 5 patients, p.Arg76Leu mutation of SRY and p.G212S mutation of NR5A1were identified from the proband who present with primary amenorrhea and lack of puberty development. The missense mutation of NR5A1was found to be derived from the mother. Interestingly, the paternal inherited p.Arg76Leu mutation of SRY was revealed from other 2 CGD patients, as well as from apparent normal male family members with fertility. P.Arg76Leu variation was found have no effect to the transcriptional activity of target gene SOX9, neither alteration of the nuclear translocation of SRY. Whole exome sequencing also found SRY mutation, FGF10 mutation, GJB4 gene mutation, etc. with no segregation in the family, which suggested SNVs are not main cause of the disease in this pedigree. By copy number variation and SNP haplotype analysis, SOX9 gene far upstream deletion of 68kb was disclosed from the 3 patients in this family, containing one of the enhancers of SOX9. Real-time PCR confirmed that the heterozygous deletion of the region result in loss of SR-XY, but not eSR-B and eALDI. Therefore, single nucleotide variation (SNV) of SRY and NR5A1 are not main causes of severe phenotype of CGD, the enhancers of SOX9 should be investigated carefully in such patients.

Inherited Missense Mutation Occurring in Arginine76 of the SRY Gene Does Not Account for Familial 46, XY Sex Reversal

Background SRY (sex determining region of Y) is one of the important genes involved in the process of human sex determination. The disturbed sex determination caused by an SRY mutation accounts for 10% to 15% of cases with 46, XY sex reversal. Recently, 3 distal enhancers were identified upstream of the SOX9 gene. Objectives The purpose of this study was to investigate the molecular etiology of 46, XY sex reversal in 3 familial patients and a sporadic patient. Design Next-generation sequencing was used to reveal the genotype and inherited pattern. Copy number variations and single nucleotide polymorphism haplotyping were analyzed to observe the alteration of enhancers of SOX9. Transcriptional activity of SRY mutation were assessed by a dual luciferase reporting system, and nuclear translocation was observed by confocal microscopy. Results Two novel SRY gene mutations, p.Arg76Leu and p.Glu89flx15, were identified. In the pedigree with multiple patients, p.Arg76Leu mutation in SRY and p.Gly212Ser mutation in NR5A1 were identified in the proband. The heterozygous deletion far upstream of the SOX9 gene in chromosome 17 was identified in the 3 patients in this family, containing the distal enhancer eSR-A of SOX9 but not eSR-B and eALDI. The frameshift mutation p.Glu89flx15 was revealed to inhibit the transcriptional activity of the target gene, whereas the missense mutation p.Arg76Leu barely showed an effect. Conclusion In contrast to sporadic cases, inherited single nucleotide variations of SRY are not the main cause of the severe phenotype of 46, XY sex reversal, and the enhancers of SOX9 should be investigated carefully in such patients.