• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.93-98
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• 2009

In this study, bovine Dnmt1 cDNA was sequenced and detected Dnmt1 mRNA level in bovine tissues by northern blot, methylation pattern of genome by southern blot, specific localization of Dnmt1 in mouse and bovine preimplantation embryos by immunocytostaining and Dnmt1 protein level in ovary and testis by western blot. Bovine Dnmt1 cDNA sequence showed more homology with that of human than mouse and rat. The RNA level of Dnmt1 was 10 times higher expression in placenta than other tissues. This indicates that placenta was hypermethylated compared to others organs. The genomic DNA could not be cut by a specific restriction enzyme (HpaII) in placenta, lung and liver of bovine. It suggests that Dnmt1 in some somatic cells was already methylated. Dnmt1, which has the antibody epitope 1316~1616, was distributed in nucleus and cytoplasm including the stage of pronuclear stage and maturation of oocyte and gradually weaken to blastocyst stage compare to negative. In addition, Dnmt1 was strongly expressed in tetraploid embryo and cloned 8-cell than IVF 8-cell. An aberrant pattern of DNA methylation in cloned embryo may be abnormal development of fetus, embryonic lethality and placenta dysfunction. The somatic specific band (190kDa) was appeared in ovary and testis, but oocyte specific band (175kDa) was not. Further investigations are necessary to understand the complex links between the methyltransferases and the transcriptional activity of genes in the cloned bovine tissues.

• Journal of Animal Science and Technology
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• v.57 no.4
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• pp.16.1-16.7
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• 2015

A well-characterized sperm specific protein of the Member of immunoglobulin superfamily, IZUMO1, has crucial role in fertilization by mediating sperm binding to the egg plasma membrane in the mouse. However little is known about IZUMO1 in bovine. Here, we describe the molecular cloning and expression analysis of bovine IZUMO1 (bIZUMO1). RT-PCR and Western blot analysis of the bovine tissues indicated that bIZUMO1 was specifically expressed in the testis and sperm, Furthermore, the result of our biotinylation assay from ejaculated bovine sperm strongly suggest the assumption that bIZUMO1 is localized on the cell surface. These data imply the potential role of bovine IZUMO1 in mammalian fertilization.

• Journal of Genetic Medicine
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• v.1 no.1
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• pp.51-56
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• 1997

Spermatogenesis is known to be regulated by a number of genes and several factors such as hormones, growth factors, cytokines and others. This study was done to evaluate the relationship between HSPs and DAZ genes in human spermatogenesis; we observed the expression pattern of HSP gene in azoospermia men with DAZ gene that regulated the gene expression related with human spermatogenesis. RT-PCR method was used to detect DAZ, HSP70A, and HSP70B transcripts in all RNA samples. Total RNA was extracted from 21 testis tissues using TRIZOL reagent. cDNAs were synthesized with reverse transcriptase, AMV. All PCR reaction were performed on a PCR themocycler with DAZ, HSP70A, and HSP70B-specific primers. Semen analysis, karyotyping and testis histology were performed. DAZ gene, known as a candidate gene of azoospermia factor(AZF), was deleted in 2 of 21 patients. To evaluate the only effects of HSPs in this patients, 2 DAZ deleted cases were removed. We observed the mRNA of HSP70B in 5 whereas none could be seen with regard to HSP70A. Furthermore, the sperm of these 5 men were discovered to be immature. In conclusion, HSP70B as well ad DAZ gene seem to be involved causing spermatogenic failure. We suggest that HSP70B plays an important role in spermatogenesis and it is one of factors induced sperm maturation in human.

• Development and Reproduction
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• v.26 no.2
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• pp.59-69
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• 2022

Many efforts have been made to study the expression of aquaporins (AQP) in the mammalian reproductive system, but there are not enough data available regarding their localized expression to fully understand their specific roles in male reproduction. The present study investigated the expression and localization patterns of different AQP subtypes in the adult mouse testes and testicular spermatozoa using an immunofluorescence assay. All the studied AQPs were expressed in the testes and revealed subtype-specific patterns in the intensity and localization depending on the cell types of the testes. AQP7 was the most abundant and intensive AQP subtype in the seminiferous tubules, expressing in Leydig cells and Sertoli cells as well as all stages of germ cells, especially the spermatids and testicular spermatozoa. The expression pattern of AQP3 was similar to that of AQP7, but with higher expression in the basal and lower adluminal compartments rather than the upper adluminalcompartment. AQP8 expression was limited to the spermatogonia and Leydig cells whereas AQP9 expression was exclusive to tails of the testicular spermatozoa and elongated spermatids. Taken together, the abundance and distribution of the AQPs across the different cell types in the testes indicating to their relavance in spermatogenesis, as well as in sperm maturation, transition, and function.

• Korean Journal of Poultry Science
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• v.46 no.4
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• pp.287-296
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• 2019

Avian sex determination system involves the male ZZ and female ZW chromosomes. However, very few studies are reported the expression, functional role and importance of genes on the W chromosome because of its small and highly heterochromatic genomic regions. Recent studies demonstrated that the W chromosome may have critical roles in physiology, sex determination and subsequent sexual differentiation in chickens. Therefore, gene annotation, including describing the expression and function of genes in the chicken W chromosome, is needed. In this study, we have searched the W chromosome of chickens and selected a total of 36 genes to evaluated their specific expression in the testis and ovary at various developmental stages such as embryonic day 6 (E6), hatch and adult. Interestingly, out of 36 genes in chicken W chromosome, we have found seven female-specific expression at E6.5 day, indicating that they are functionally related to female chicken gonadal differentiation. In addition, we have identified the stage specific gene expression from the sex specific genes. Furthermore, we analyzed the relative location of genes in the chicken W chromosome. Collectively, these results will contribute molecular insights into the sexual determination, differentiation and female development based on the W chromosome.

• Journal of Life Science
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• v.27 no.7
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• pp.739-745
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• 2017

In all mammalian species, progesterone is essential in the preparation for and maintenance of pregnancy. $20{\alpha}$-hydroxysteroid dehydrogenase ($20{\alpha}$-HSD) predominantly converts progesterone into its biologically inactive form $20{\alpha}$-hydroxyprogesterone ($20{\alpha}$-OHP), and plays a crucial role in the termination of pregnancy and initiation of parturition. In this study, we characterized the expression and localization of $20{\alpha}$-HSDinthe testis of MediKinetics $Micropigs^{(R)}$. The testes were collected at days 6, 9, 12, 18, and 21 after birth. The $20{\alpha}$-HSD mRNA was found to be expressed in the testis at day 6 after birth by RT-PCR. The highest level of mRNA expression in the testis was detected on day 21 after birth. However, the mRNA was not detected in the placenta after parturition. Western blot for $20{\alpha}$-HSD reveal that the specific 37-kDa band was detected in immature pig testis. However, this band was not detected in testis tissue at day 6 after birth. In the immunohistochemical analysis of the testis, $20{\alpha}$-HSD was detected in the Sertoli cells and Leydig cells. Taken together, our study shows for the first time that the $20{\alpha}$-HSD mRNA and protein are expressed in pig testis after birth. Further investigation is required to elucidate the functional mechanisms of $20{\alpha}$-HSD in pig testis after birth.

• Journal of Animal Science and Technology
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• v.46 no.3
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• pp.315-324
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• 2004

Cytochrome P450 aromatase is the enzyme responsible for biosynthesis of female sex hormone(estrogen) and 19-nortestosterone(nandrolone), a unique steroid hormone endogenously synthesized in the pig. By use of RT-PCR coupled with DHPLC technique (WAVE analysis), expression pattern of isoforms of porcine cytochrome P450 aromatase gene was investigated. Relatively higher expression of aromatase mRNA was observed in testis than in ovary and this result accounted for the previous findings of higher blood estrogen level in male compared with female in this species. The result from the DHPLC demonstrated that PCR amplified DNA fragments of ovary and testis tissues. using unique PCR primers for all three types of aromatase genes, were different from those of type II and ill genes. Further nucleotide sequence analyses of the plasmid clones containing the PCR products revealed that nucleotide sequences of all clones were identical to type I aromatase gene(ovary type). Thus, the result from the present study indicates that the ovary and testis express the same type of aromatase gene. Therefore, the efficacy of DHPLC techniques used for this study helped us to analyze tissue-specific expression of isoform of genes containing the nucleotide sequences with high homology.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.35.1-35.13
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• 2022

Background: The testis has been reported to be a naturally O2-deprived organ, dimethyloxaloylglycine (DMOG) can inhibit hypoxia inducible factor-1alpha (HIF-1α) subject to degradation under normal oxygen condition in cells. Objectives: The objective of this study is to detect the effects of DMOG on the proliferation and differentiation of spermatogonial stem cells (SSCs) in Bama minipigs. Methods: Gradient concentrations of DMOG were added into the culture medium, HIF-1α protein in SSCs was detected by western blot analysis, the relative transcription levels of the SSC-specific genes were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Six days post-induction, the genes related to spermatogenesis were detected by qRT-PCR, and the DNA content was determined by flow cytometry. Results: Results revealed that the levels of HIF-1α protein increased in SSCs with the DMOG treatment in a dose-dependent manner. The relative transcription levels of SSC-specific genes were significantly upregulated (p < 0.05) by activating HIF-1α expression. The induction results showed that DMOG significantly increased (p < 0.05) the spermatogenesis capability of SSCs, and the populations of haploid cells significantly increased (p < 0.05) in DMOG-treated SSCs when compared to those in DMOG-untreated SSCs. Conclusion: We demonstrate that DMOG can promote the spermatogenesis activity of SSCs.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5865-5869
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• 2012

RHOXF1 has been shown to be expressed in embryonic stem cells, adult germline stem cells and some cancer lines. It has been proposed as a candidate gene to encode transcription factors regulating downstream genes in the human testis with antiapoptotic effects. Its expression in cancer cell lines has implied a similar role in the process of tumorigenesis. The human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in DMEM medium and transfected with a pGFP-V-RS plasmid bearing an RHOXF1 specific shRNA. Quantitative real-time RT-PCR was performed for RHOXF1, CASP8, BCL2 and HPRT genes. Decreased RHOXF1 expression was confirmed in cells after transfection. shRNA knock down of RHOXF1 resulted in significantly decreased BCL2 expression in both cell lines but no change in CASP8 expression. shRNA targeting RHOXF1 was shown to specifically mediate RHOXF1 gene silencing, so RHOXF1 can mediate transcriptional activation of the BCL2 in cancers and may render tumor cells resistant to apoptotic cell death induced by anticancer therapy. shRNA mediated knock down of RHOXF1 can be effective in induction of apoptotic pathway in cancer cells via BCL2 downregulation, so it can have potential therapeutic utility for human breast cancer.

• Animal cells and systems
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• v.2 no.4
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• pp.495-500
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• 1998

D-type G1 cyclins are known to be crucial for the progression of mitotic cell cycle in mammals. Although many studies have been performed to elucidate the roles of D-type cyclins, it is largely unknown whether D-type cyclins are directly involved in the regulation of meiotic germ cell development. In the present study, we examined the expression patterns of D-type cyclins (cyclin D1 and D3) during male germ cell development by northern blot and in situ Hybridization analyses. In the adult testes, we detected a 4.2 kb cyclin D1 mRNA and two different sizes (2.3 kb and 1.8 kb) of cyclinD3 mRNAs. The short form of the cyclin D3 transcript was testis-specific. Along with the testicular development, expression of cyclin D3 mRNA was increased whereas cyclin D1 mRNA was gradually decreased. in situ hybridization study also revealed that the expression of cyclin D3 was restricted to the postmeiotic germ cells. Furthermore, the 2.3 kb transcript was highly expressed in the round spermatids and decreased in the elongated spermatids/residual bodies, while the 1.8 kb transcript was expressed in elongated spermatids/residual bodies more abundantly. Sucrose-gradient separation of polysomal RNA fractions demonstrated that some portions of the 2.3 kb transcript are translationally active, while the 1.8 kb transcript is likely to be inactive. Taken together, the present data suggest a functional importance of cyclin D3 expression in the differentiated postmeiotic male germ cells.