Background: Testis-specific protein on Y chromosome (TSPY) is the output of a tandem gene cluster. TSPY expression has been observed in gonadoblastoma and numerous distinct kinds of germ cell tumors, such as carcinoma in situ/intratubular germ cell neoplasia, seminoma, and extragonadal intracranial germ cell tumors (GCT). Myrtus communis extract rich in-pinene showed high antioxidant and anticancer activity against a TSPY. Methods: The molecular weight and theoretical isoelectric of the TSPY proteins were calculated, using the ExPASSY ProtParam tools. Some software like mega 6, BioEdit, NEB cutter (New England Biolabs), and CAP3 were used to analyze clustering and find restriction enzymes on the TSPY sequence. To evaluate the nucleotide diversity of all sequences, the number of diverse situations and Tajima’ s andWatterson’ s estimators of theta were assessed. Nucleotide polymorphism can be measured by several parameters, such as haplotypes diversity, nucleotide diversity, Theta using Dnasp software. To find interaction networks of protein-protein search tool for the retrieval of interacting genes/proteins (STRING) tools and to predict 3D structure, SWISS-MODEL was used; however, for docking protein-peptide based on interaction, Swiss Dock, Galaxy web, and CABS-dock software were employed. Results: We report a high (0. 91) dN/dS index, positive Tajima’ s D, Fu, and Li’ s tests, and a non-significant D test suggesting the occurrence of old modifications or a decrease of newborn mutations in the TSPY gene family. Interestingly, several hub proteins produced a strong chain or an operative module within their protein groups, such as nucleosome assembly protein (1NAP1L), RBMXL2, TBL1Y, and AMELY, which are all associated with the same cellular appliance elements and/or genetic uses. The docking of the TSPY target with -pinene using docking revealed that the computationally-prognosticated lowest energy networks of TSPY are established by intermolecular hydrogen bonds and stacking interactions. Conclusions: The results of this study demonstrated that -pinene interacts with the TSPY protein target and could be developed as a promising candidate for the new anticancer agent.

Background: Fetal DNA in maternal plasma and serum has been shown to be a useful material for prenatal fetal sex determination during early gestational ages. Non-invasive prenatal diagnosis is now possible at 8th week of pregnancy, by maternal blood sample testing.Objective: The purpose of this study was to evaluate two DNA extraction methods from mother plasma and its routine clinical application in bovine fetus gender determination with non-invasive method.Materials and Methods: Maternal blood samples were taken from 40 pregnant cows during the 8th-38th weeks of gestation. DNA was extracted from 350 ml of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A260 and purity (A260/A280) of extracted DNA were detected by ultraviolet spectrophotometer. Three ml of the extracted DNA with phenol-chloroform method was used as a template. The PCR reaction was carried out to amplify the fragments of X and Y chromosomes of amelogenin, TSPY and BC1.2 genes.Results: The difference between the mean absorption of DNA extracted by phenol-chloroform method and salting-out method was not significant in A260 (p>0.05, p=0.3549), but the difference between mean purity (A260/A280) of DNA extracted by phenol-chloroform method and salting-out method was significant (p<0.001). X chromosome fragment was detected in all 40 samples and Y chromosome fragments were detected in 25 plasma samples which were delivered a male calf. The sensitivity and specificity of test was 100% with no false negative and false positive results.Conclusion: The results showed that phenol-chloroform method is a simple and sensitive method for isolation of fetal DNA in maternal plasma.

Objective: Sexual dimorphism in mammals can be described as subsequent transcriptional differences from their distinct sex chromosome complements. Following X inactivation in females, the Y chromosome is the major genetic difference between sexes. In this study, we used a male embryonic stem cell line (Royan H6) to identify the potential role of the male-specific region of the Y chromosome (MSY) during spontaneous differentiation into embryoid bodies (EBs) as a model of early embryonic development. Materials and Methods: In this experimental study, RH6 cells were cultured on inactivated feeder layers and Matrigel. In a dynamic suspension system, aggregates were generated in the same size and were spontaneously differentiated into EBs. During differentiation, expression patterns of specific markers for three germ layers were compared with MSY genes. Results: Spontaneous differentiation was determined by downregulation of pluripotent markers and upregulation of fourteen differentiation markers. Upregulation of the ectoderm markers was observed on days 4 and 16, whereas mesoderm markers were upregulated on the 8th day and endodermic markers on days 12-16. Mesoderm markers correlated with 8 MSY genes namely DDX3Y, RPS4Y1, KDM5D, TBL1Y, BCORP1, PRY, DAZ, and AMELY, which were classified as a mesoderm cluster. Endoderm markers were co-expressed with 7 MSY genes, i. e. ZFY, TSPY, PRORY, VCY, EIF1AY, USP9Y, and RPKY, which were grouped as an endoderm cluster. Finally, the ectoderm markers correlated with TXLNGY, NLGN4Y, PCDH11Y, TMSB4Y, UTY, RBMY1, and HSFY genes of the MSY, which were categorized as an ectoderm cluster. In contrast, 2 MSY genes, SRY and TGIF2LY, were more highly expressed in RH6 cells compared to EBs. Conclusion: We found a significant correlation between spontaneous differentiation and upregulation of specific MSY genes. The expression alterations of MSY genes implied the potential responsibility of their gene co-expression clusters for EB differentiation. We suggest that these genes may play important roles in early embryonic development.