• BMB Reports
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• 제40권5호
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• pp.749-756
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• 2007

Phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization. In the present work, we reported the isolation and characterization of mouse testis-specific serine/threonine kinase 5 (Tssk5), which contains four alternatively spliced variants including, Tssk5$\alpha$, Tssk5$\beta$, Tssk5$\gamma$ and Tssk5$\delta$. Moreover, the locus of Tssk5 is on chromosome 14qC3 and the four variants had a similar high expression in the testis and the heart; however, had a low expression in other tissues, except for Tssk5$\alpha$ which also had comparably high expression in the spleen. Each variant of Tssk5 expression began in the testis 16 days after birth. Aside from TSSK5$\alpha$, the other isoforms have an insertion of ten amino acid residues (RLTPSLSAAG) in region VIb (HRD domain) (His-Arg-Asp). Moreover, only TSSK5$\alpha$ exhibited kinase activity and consistently, a further Luciferase Reporter Assay demonstrated that TSSK5$\beta$, TSSK5$\gamma$ and TSSK5$\delta$ cannot be stimulated at the CREB/CRE responsive pathway in comparison to TSSK5$\alpha$. These findings suggest that TSSK5$\beta$, TSSK5$\gamma$, TSSK5$\delta$ may be pseudokinases due to the insertion, which may damage the structure responsible for active kinase activity. Pull-down assay experiments indicated that TSSK5$\beta$, TSSK5 $\gamma$ and TSSK5$\delta$ can directly interact with TSSK5$\alpha$. In summary, these four isoforms with similar expression patterns may be involved in spermatogenesis through a coordinative way in testis.

• Journal of Genetic Medicine
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• 제2권1호
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• pp.31-34
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• 1998

The N-methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-methylpurine and other damaged purines induced in DNA. Tissue-specific mRNA levels of the N-methylpurine-DNA glycosylase (MPG) were investigated in Balb/c mice of four different growing stages; newborn, 1, 4 and 8-weeks postpartum. MPG expressions in the newborn and the 8-week-old mice were the highest in thymus and testis, respectively. The tested tissues of the newborn mice had consistently higher MPG mRNA level than 8-week-old adults except in testis and thymus. The MPG mRNA level in testis was the lowest in the newborn mice, but it attained the highest in the 8-week-old mice. The levels of MPG mRNA among the different tissues in the newborn and the 8-week-old mice were more than 9.0 and 19.0-fold respectively. These results suggest that the of MPG expression was dependent on the growing stage and had tissue-specificity.

• BMB Reports
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• 제47권2호
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• pp.86-91
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• 2014

Tissue-specific gene expression is regulated by epigenetic modification involving trans-acting factors. Here, we identified that the human MAGEB16 gene and its mouse homolog, Mageb16, are only expressed in the testis. To investigate the mechanism governing their expression, the promoter methylation status of these genes was examined in different samples. Two CpG islands (CGIs) in the 5' upstream region of MAGEB16 were highly demethylated in human testes, whereas they were methylated in cells without MAGEB16 expression. Similarly, the CGI in Mageb16 was hypomethylated in mouse testes but hypermethylated in other tissues and cells without Mageb16 expression. Additionally, the expression of these genes could be activated by treatment with the demethylation agent 5'-aza-2'-deoxycytidine (5'-aza-CdR). Luciferase assays revealed that both gene promoter activities were inhibited by methylation of the CGI regions. Therefore, we propose that the testis-specific expression of MAGEB16 and Mageb16 is regulated by the methylation status of their promoter regions.

• Molecules and Cells
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• 제25권2호
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• pp.317-321
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• 2008

Members of the large family of Asb proteins are ubiquitously expressed in mammalian tissues; however, the roles of individual Asb and their function in the developmental testes have not been reported. In this report, we isolated a murine Asb4 from mouse testis. Northern blot analysis revealed that mAsb-4 was expressed only in testes and produced in a stage-specific manner during spermatogenesis. It was expressed in murine testes beginning in the fourth week after birth and extending into adulthood. Pachytene spermatocytes had the highest level of expression. Interestingly, the human homologue of mAsb-4, ASB-4 (hASB-4) was also expressed in human testis. These results suggest that ASB-4 plays pivotal roles in mammalian testis development and spermatogenesis.

• 한국발생생물학회지:발생과생식
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• 제21권3호
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• pp.327-333
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• 2017

Gonadotropins are heterodimers consisting an alpha chain ($Cg{\alpha}$) and a beta chain. Interestingly, presence of complicated $LH-{\beta}$ transcripts in rat testis was accidently found; testicular $LH-{\beta}$ transcripts were confined in seminiferous tubules to spermatids, and the translated products were localized in the elongated spermatids. We hypothesized that mouse testis has potential to produce the tissue specific $LH-{\beta}$ with similar structure to the rat testicular forms. To verify our hypothesis, we examined the adult mouse (ICR) testis using RT-PCR and immunohistochemistry. The PCR revealed the presence of the identical products in the reactions for three LH subunit types. The expected product sizes for mouse $Cg{\alpha}$ and $LH-{\beta}$ known as pituitary type were 224 bp and 503 bp, respectively. The testicular type $LH-{\beta}$ products were produced by a primer set based on the rat sequences, with unexpected size of 800 bp. Sequencing revealed that the proximal and distal parts (2-82 and 661- 773 bp, respectively) were homologous to rat testicular $LH-{\beta}$ cDNA, and middle part (83-660 bp) was a unique mouse-specific region. Both $Cg{\alpha}$ and $LH-{\beta}$ positive signals were in the round and elongated spermatids and mature sperms, and the $LH-{\beta}$ signals were more intense. In conclusion, our study demonstrated that the presence and localization of the LH subunits in mouse testis. Further studies will be needed to understand the precise structure and function of mouse testicular LH.

• Journal of Animal Science and Technology
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• 제54권3호
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• pp.157-163
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• 2012

The male reproduction is precisely controlled by a number of intrinsic and extrinsic factors. These factors usually involve in expressional regulation of various molecules influencing on sperm production in the testis. A number of ways are employed to control the transcription of specific genes, including epigenetic modifications of DNA and histone molecules. DNA methylation of CpG dinucleotides is a commonly used regulatory mechanism for testicular genes associated with the fertility. Previous studies have demonstrated the infertility induced by improper DNA methylation of these genes. In the present research, we attempted to determine transcriptional expression of some of these genes in the rat testis at different postnatal ages using real-time PCR analysis. These genes include neurotrophin 3 (Ntf3), insulin-like growth factor II (Igf2), JmjC-domain-containing histone demethylase 2A 1 (Jhm2da), paired box 8 transcription factor (Pax8), small nuclear ribonucleoprotein polypeptide N (Snrpn), and 5,10-methylenetetrahydrofolate reductase (Mthfr). The expression levels of Ntf3, Igf2, and Snrpn genes were the highest at the neonatal age, followed by transient decreases at the prepubertal age. Expression of Jhm2da and Mthfr genes were continuously increased from the neonate to 1 year of age. The levels of Pax8 mRNA at the early ages were higher than those at the later ages of postnatal development. These findings suggest that expression of some fertility-associated testicular genes in the rat during postnatal period could be differentially regulated by the control of the degree of DNA methylation.

• Molecules and Cells
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• 제28권1호
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• pp.31-36
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• 2009

Centrobin/Nip2 was initially identified as a centrosome protein that is critical for centrosome duplication and spindle assembly. In the present study, we determined the expression and subcellular localization of centrobin in selected mouse tissues. Immunoblot analysis revealed that the centrobin-specific band of 100 kDa was detected in all tissues tested but most abundantly in the thymus, spleen and testis. In the testis, centrobin was localized at the centrosomes of spermatocytes and early round spermatids, but no specific signal was detected in late round spermatids and elongated spermatids. Our results also revealed that the centrosome duplication occurs at interphase of the second meiotic division of the mouse male germ cells. The centrobin protein was more abundant in the mitotically active ovarian follicular cells and thymic cortex cells than in non-proliferating corpus luteal cells and thymic medullary cells. The expression pattern of centrobin suggests that the biological functions of centrobin are related to cell proliferation. Consistent with the proposal, we observed reduction of the centrobin levels when NIH3T3 became quiescent in the serum-starved culture conditions. However, a residual amount of centrobin was also detected at the centrosomes of the resting cells, suggesting its role for maintaining integrity of the centrosome, especially of the daughter centriole in the cells.

• Clinical and Experimental Reproductive Medicine
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• 제35권3호
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• pp.181-191
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• 2008

목 적: 본 연구에서는 에스트로겐 수용체의 agonist로 작용할 수 있는 내분비계 교란물질인 bisphenol A (BPA)가 정소 내 세포들의 유전자 발현 및 세포자멸사에 미치는 영향을 관찰하였다. 연구방법: 그룹 I의 7주령 수컷 생쥐들에는 sesame oil에 녹인 BPA를 1 mg/kg, 10 mg/kg, 100 mg/kg를 1회 복강주사하여 급성 영향을 조사하였고, 그룹 II의 생쥐들에는 BPA 10 ${\mu}g/kg$, 1 mg/kg, 100 mg/kg를 하루에 1회씩 14일간 피하주사하여 장기적 효과를 보았다. 생쥐의 정소를 채취하여 정소의 다양한 표지유전자들의 발현을 RT-PCR로 조사하였고, 조직학적 관찰, 형광면역염색법과 TUNEL 염색을 수행하였다. 결 과: RT-PCR 결과, 정소의 표지유전자 중 특이적으로 Leydig cell의 표지유전자인 LHR가 고농도의 BPA 처리군에서 감소되어 있었다. 정원세포 표지유전자인 ERM은 장기간 BPA를 주사 받은 생쥐들의 정소에서 감소되었다. 1회의 BPA를 주사 받은 그룹에서는 조직학적 영향은 없는 것으로 보였다. 또한 세포자멸사의 표지인 Bax가 Leydig cell이 존재하는 정소의 interstitial area에서 BPA에 의해 증가된 것을 관찰하였으며 이는 TUNEL 염색이 positive하게 나타난 부분과 일치하였다. 결 론: 본 연구는 BPA가 정소에서 주로 Leydig cell에 영향을 주며 이는 Leydig cell이 에스트로겐 수용체와 에스트로겐 합성효소를 발현하는 내분비 세포라는 사실에 부합되는 것으로, 차후 그 분자적 기작을 밝히는 연구로 이어져야 할 것이다.

• 대한의생명과학회지
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• 제10권4호
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• pp.385-389
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• 2004

A novel gene Jpk, originally isolated as a trans-acting factor associating with the position-specific regulatory element of murine Hox gene has been reported to be expressed differentially in the liver of diabetic animals. Therefore, in an attempt to develop a possible diagnostic marker and/or new therapeutic agent for the Diabetes Mellitus, we analysed the expression pattern of Jpk among organs of normal and diabetic Sprague-Dawley (SD) rats. Total RNAs were isolated from each organs (brain, lung, heart, liver, spleen, kidney, muscle, blood, and testis) of diabetic and normal rats in both normal feeding and after fasting condition. And then RT (reverse transcription) PCR has been performed using Jpk­specific primers. The Jpk gene turned out to be expressed in all organs tested, with some different expression profiles among normal and diabetes, though. Upon fasting, Jpk expressions were reduced in all organs tested except kidney, muscle and brain of normal rat. Whereas in diabetes, Jpk expressions were increased in all organs except heart, muscle and testis when fasted. Compared to the normal rat, the Jpk expression level in blood was remarkably upregulated (about 15-30times) in diabetic rat whether in normal feeding or fasting conditon, suggesting that the Jpk could be a candidate gene for the possible blood diagnostic marker for the Diabetes Mellitus.

• Clinical and Experimental Reproductive Medicine
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• 제23권3호
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• pp.269-275
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• 1996

Biosynthesis and secretion of anterior pituitary hormones are under the control of specific hypothalamic stimulatory and inhibitory factors. Among them, Growth Hormone Releasing Hormone (GHRH) is the major stimulator of pituitary somatotrophs activating GH gene expression and secretion. Human GHRH is a polypeptide of 44 amino acids initially isolated from pancreatic tumors, and the gene for the hypothalamic form of GHRH is organized into 5 exons spanning over 10 kilobases (kb) on genomic DNA and encodes a messenger RNA of 700-750 nucleotides. Several neuropeptides classically associated with the hypothalamus have been found in the extrahypothalamic regions, suggesting the existence of novel sources, targets and functions. GHRH-like immunoreactivity has been found in several peripheral sites, including placenta, testis, and ovary, indicating that GHRH may also have regulatory roles in peripheral reproductive organs. Furthermore, higher molecular weight forms of the GHRH transcripts were identified from these organs (1.75 kb in testis; 1.75 and >3 kb in ovary). These tissue-specific expression of GHRH gene suggest the existence of unique regulatory mechanism of GHRH expression and function in these organs. In fact, placenta-specific and testis-specific promoters for GHRH transcripts which are located in about 10 kb upstream region of hypothalamic promoter were reported. The use of unique promoters in extrahypothalamic sites could be refered in a different control of GHRH gene and different functions of the translated products in these tissues. Somatotrophs and lactotrophs have been thought to be derived from a common bipotential progenitor, the somatolactotrophs, which give origins to either phenotypes. Although the precise mechanism responsible for the lactotroph differentiation in the anterior pituitary gland has not been yet clalified, there are several candidators for the generation of lactotrophs. In human, the presence of GHRH peptides with different size from authentic hypothalamic form in the normal anterior pituitary and several types of adenoma were demonstrated. Recently our group found the existence of immunoreactive GHRH and its transcript from the normal rat anterior pituitary (gonadotroph> somatotroph> lactotroph), and the GHRH treatment evoked the increased proliferation rate of anterior pituitary cells in vitro. The transgenic mouse models clearly shown that GHRH or NGF overexpression by anterior pituitary cells induced development of pituitary hyperplasia and adenomas particularly GH-oma and prolactinoma. Taken together, we hypothesize that the pituitary GHRH could serve not only as a modulator of hormone secretion but as a paracrine or autocrine regulator of anterior pituitary cell proliferation and differentiation. Interestingly enough, the expression of Pit-1 homeobox gene (the POU class transcription factor) was confined to somatotrophs, lactotrophs and somatolactotrophs in which GHRH receptors are expressed commonly. Concerning the mechanism of somatolactotroph and lactotroph differentiation in the anterior pituitary, we have focused following two possibilities; (1) changes in the relative levels or interactions of both hypothalamic and intrapituitary factors such as dopamine, VIP, somatostatin, NGF and GHRH; (2) alterations of GHRH-GHRH receptor signaling and Pit-1 activity may be the cause of lactotroph differentiation or pituitary hyperplasia and adenoma formation. Extensive further studies will be necessary to solve these complicated questions.