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ABSTRACT: ADAM2, a member of the 'a disintegrin and metalloprotease' (ADAM) family, is a key protein in mammalian fertilization that is specifically expressed in testicular germ cells. Here, we investigated the transcriptional regulation of the mouse Adam2 gene. An in silico analysis identified two conserved non-coding sequences located upstream of the mouse and human ADAM2 genes. The upstream region of the mouse Adam2 gene was found to lack typical TATA and CAAT boxes, and to have a high GC content. Our in vitro transient transfection-reporter analysis identified a promoter in this region of the mouse Adam2 gene, along with regulatory regions that inhibit the activity of this promoter in somatic cells. Site-directed mutagenesis revealed that the caudal-type homeobox 1 and CCTC-binding factor motifs are responsible for the inhibitory activities of the repressor regions. Finally, electrophoretic mobility shift assays showed putative transcription factor-promoter DNA complexes, and DNA-affinity chromatography and proteomic analyses identified myelin gene regulatory factor as a binding partner of the Adam2 promoter. This provides the first identification and characterization of promoter and repressor regions that regulate the transcription of the mouse Adam2 gene, and offers insights into the regulation of this germ-cell-specific gene.
Molecular Biology Reports 10/2012; · 2.93 Impact Factor
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ABSTRACT: Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.
Journal of Biological Chemistry 06/2011; 286(33):29108-17. · 4.77 Impact Factor
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ABSTRACT: Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant
genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the
SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin
inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed
that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues.
Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot
analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular
spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated
mutant mice with diminished levels of SPINK2 using a gene-trap mutagenesis approach. Mutant male mice exhibit significantly
impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction
in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ-cell apoptosis
accompanied with elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required
for maintaining normal spermatogenesis, and potentially regulates serine protease-mediated apoptosis in male germ cells.
Journal of Biological Chemistry 06/2011; · 4.77 Impact Factor
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ABSTRACT: Demand for plasmid DNA of high purity and safety has increased with rapid advances in gene therapy and DNA vaccines in addition to basic DNA study. Using activated charcoal (AC), we have developed protocols for pure plasmid DNA. Plasmid DNA extracted by the alkaline lysis method was inevitably contaminated with nucleotide fragments. Treatment with AC during purification instead of RNase completely removed nucleotide fragments in the final plasmid DNA and the removing capability of AC was dose dependent on AC quantity. Of note is that nucleotide fragments less than 0.4 kbp were effectively removed by AC and purification up to 500 ml was easily achieved. Taken together, inexpensive AC effectively removed the troublesome nucleotide fragments and practically substituted for expensive RNase. The resultant plasmid DNA has enough quality needed for basic DNA study and application.
Journal of Bioscience and Bioengineering 11/2010; 110(5):608-13. · 1.79 Impact Factor
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ABSTRACT: The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model.
To this end, an expression plasmid, pCMV-rAct, with the activin betaA cDNA fused to the cytomegalovirus promoter, was introduced into muscle of the female adult mice by direct injection.
Activin betaA mRNA was detected in the muscle by RT-PCR and subsequent Southern blot analysis. Activin betaA was also detected, and western blot analysis revealed a relatively high level of serum activin with correspondingly increased FSH. In the pCMV-rAct-injected female mice, estrus stage within the estrous cycle was extended. Moreover, increased numbers of corpora lutea and a thickened granulosa cell layer with a small antrum in tertiary follicles within the ovary were observed. When injected female mice were mated with males of proven fertility, a subset of embryos died in utero, and most of those that survived exhibited increased body weight.
Taken together, our data reveal that activin betaA can directly influence the estrous cycle, an integral part of the reproduction in female mice and activin betaA can also influence the embryo development as an endocrine fashion.
Reproductive Biology and Endocrinology 01/2009; 6:63. · 2.05 Impact Factor
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ABSTRACT: Abstract
Background
The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model.
Methods
To this end, an expression plasmid, pCMV-rAct, with the activin betaA cDNA fused to the cytomegalovirus promoter, was introduced into muscle of the female adult mice by direct injection.
Results
Activin betaA mRNA was detected in the muscle by RT-PCR and subsequent Southern blot analysis. Activin betaA was also detected, and western blot analysis revealed a relatively high level of serum activin with correspondingly increased FSH. In the pCMV-rAct-injected female mice, estrus stage within the estrous cycle was extended. Moreover, increased numbers of corpora lutea and a thickened granulosa cell layer with a small antrum in tertiary follicles within the ovary were observed. When injected female mice were mated with males of proven fertility, a subset of embryos died in utero, and most of those that survived exhibited increased body weight.
Conclusion
Taken together, our data reveal that activin betaA can directly influence the estrous cycle, an integral part of the reproduction in female mice and activin betaA can also influence the embryo development as an endocrine fashion.
Reproductive Biology and Endocrinology. 01/2008;
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ABSTRACT: The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes.
We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein.
We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.
BMC Genomics 02/2007; 8:256. · 4.07 Impact Factor
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ABSTRACT: Prolactin (PRL) is a pituitary hormone involved in various physiological processes, including lactation, mammary development, and immune function. To further investigate the in vivo and comparative endocrine roles of PRL, mouse PRL cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Previously we studied the function of rat PRL using the same protocol. PRL mRNA was detected in the muscle following injection by RT-PCR and subsequent Southern blot analysis. PRL was also detected and Western blot analysis revealed a relatively high level of serum PRL. In the pCMV-mPRL-injected female mice, the estrous cycle was extended, especially in diestrus stage and the uterus thickening that was shown in normal estrous stage was not observed. In the pCMV-mPRL-injected male mice, new blood vessels were first found at 5 weeks of age and fully developed blood vessels were found after 8 weeks in the testis. The number of Leydig cells increased within the testis and the testosterone level in serum was observed high. Finally, the number of white blood cells (WBCs) increased in the pCMV-mPRL-injected mice. The augmentation of WBCs persisted for at least 20 days after injection. When injection was combined with adrenalectomy, there was an even greater increase in number of WBCs, especially lymphocytes. This increase was returned normal by treatment with dexamethansone. Taken together, our data reveal that intramuscularly expressed mouse PRL influences reproductive functions in female, induces formation of new blood vessels in the testis, and augments WBC numbers. Of notice is that the Leydig cell proliferation with increased testosterone was conspicuously observed in the pCMV-mPRL-injected mice. These results also suggest subtle difference in function of PRL between mouse and rat species.
Molecules and Cells 11/2006; 22(2):189-97. · 2.18 Impact Factor
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ABSTRACT: Inhibin is a gonadal hormone composed of an a-subunit and one of two beta-subunits (betaA, betaB), and its primary role is to inhibit FSH secretion by the pituitary. To investigate the roles of inhibin alpha in the reproductive system, an expression plasmid, pCMV-rINA, with the rat inhibin alpha cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Inhibin alpha mRNA was detected in the muscle by RT-PCR and Southern blot analysis. Inhibin protein was also detected, and Western blot analysis revealed a relatively high level of serum inhibin, but not of activin betaA. The estrous cycle of the pCMV-rINA-injected mice was extended, but there was no change in levels of pituitary FSH mRNA or serum FSH and no ovarian cysts were observed. When injected female mice were mated with males of proven fertility, litter size increased. Surprisingly, the embryos of pregnant females injected with pCMV-rINA, were retarded in growth and had defects in internal organs. When male mice were injected, testicle weight increased slightly without any noticeable change in the histology of the seminiferous tubules. Taken together, our data indicate that the inhibin alpha subunit influences a number of the reproductive functions of female mice.
Molecules and Cells 09/2004; 18(1):79-86. · 2.18 Impact Factor
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ABSTRACT: ADAM (a disintegrin and metalloprotease) family members with testis-specific or -predominant gene expression are divided phylogenically into two groups: ADAMs 2, 3, 5, 27, and 32 (the first group) and ADAMs 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the second group). We cloned and sequenced cDNAs for previously unidentified mouse Adams that belong to the second group. We found that all the Adam genes in the second phylogenic group are transcribed by both somatic and germ cells in mouse testis, representing a unique expression pattern different from that of the first-group Adams. Genomic analyses revealed that all the second-group Adam genes lack introns interrupting protein-coding sequences and many of them are present as multicopy genes, resulting in total of 14 functional mouse genes in this phylogenic group. Comparing the mouse and human ADAM genes, we found that a number of these mouse Adam genes do not have human orthologues and, even if they exist, some orthologues are pseudogenes in human. These results suggest the differential expansion of the second-group Adam genes in the mouse genome during evolution and a relationship between these Adams and male reproduction unique to mouse.
Genomics 05/2004; 83(4):636-46. · 3.02 Impact Factor
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ABSTRACT: Inhibin is a gonadal hormone which is composed of an alpha-subunit and one of two related beta-subunits (betaA, betaB). Inhibin is important for pituitary FSH regulation, normal follicle development and maintenance of the estrous cycle in the female, whereas the role of inhibin in the male is less clear. Thus, we examined the expression of the inhibin-alpha gene in testis during sexual maturation in male mice, to try to gain insight into its functions in the male. Male mice of the ICR strain attained fertility at 6 weeks of age, and histological analysis revealed that a functional testis was formed, with seminiferous tubules which contain mature sperm and with an abundant population of Leydig cells. Parallel with this sexual maturation, inhibin-alpha subunit protein synthesis increased, whereas synthesis of the activin betaA and activin betaB followed with a delayed time course. Inhibin-alpha mRNA also increased during this critical period, and this corresponded to a change in the methylation status of the inhibin-alpha gene. Taken together, our data reveal that activation of inhibin-alpha gene during testis development correlated with the histological maturation of the testis and the acquisition of fertility in male mice.
Molecules and Cells 03/2004; 17(1):67-72. · 2.18 Impact Factor
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ABSTRACT: Prolactin (PRL) is a pituitary hormone involved in a broad spectrum of physiological processes, including lactation, development, and immune function. To further investigate the in vivo roles of PRL, rat PRL cDNA, fused to the cytomegalovirus promoter, was introduced into mouse muscle by direct injection. Prolactin mRNA and protein were detected in the muscle following injection. As a result the number of white blood cells (WBC) increased. When injection was combined with adrenalectomy there was an even greater increase. The augmentation of WBCs persisted for at least 20 days after injection of the rPRL plasmid either on its own and after injection combined with adrenalectomy. The increase in WBCs was accompanied in both cases by an increase in blood cell DNA content. We also observed an increase in heart volume, particularly of the left ventricle. Evidence of marked angiogenesis was found in the testis of rPRL- injected mice. New blood vessels were first found at 8 weeks of age and fully developed blood vessels with complex branching patterns were found after 11 weeks. When PRL fused with EGFP was introduced into mice by intramuscular injection, the EGFP localized to areas of the testis that corresponded to the sites of new blood vessel formation. PRL inhibited this binding. Taken together, our data reveal that intramuscularly expressed PRL augments WBC numbers and induces formation of new blood vessels in the testis, suggesting important roles for PRL in hematopoiesis and angiogenesis. They also indicate that direct intramuscular injection of naked DNA can be used effectively to study the function of secreted proteins, including endocrine signaling molecules.
Molecules and Cells 05/2003; 15(2):262-70. · 2.18 Impact Factor
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ABSTRACT: The aim of this study was to investigate expression patterns of the prolactin (PRL) and c‐fos genes by 17β‐estradiol (17β‐E) and stress in the mouse pituitary. In the pituitary, the levels of PRL mRNA were found high with some fluctuation at 30, 60, and 90 min whereas the levels of PRL mRNA were low at 120 min when ovariectomized female mice were injected with 17β‐E or vehicle. PRL mRNA levels began to increase again at 4 h and remained high up to 24 h only in the 17β‐E‐treated mice. The overall changes in c‐fos mRNA by 17β‐E were very similar to those in PRL mRNA in the pituitary. Subsequent study revealed that these high initial levels of PRL and c‐fos mRNAs were caused by stress during injection, not by 17β‐E, since vehicle injection alone into the ovariectomized mice could increase the levels of PRL and c‐fos mRNAs. The stress‐induced elevations of PRL and c‐fos mRNAs were inhibited by bromocriptin, a dopamine agonist, suggesting that the dopaminergic system is involved in the action route of injection stress. In addition, the induced levels of c‐fos mRNA by 17β‐E and stress in the pituitary were very low compared with those in the uterus. The time course changes in c‐fos mRNA level were different between the pituitary and uterus. Taken together, these data indicate that PRL and c‐fos gene expression in the pituitary are regulated by 17β‐E and stress in a parallel manner, supporting the notion that c‐Fos plays a role in regulation of PRL gene expression.
Korean journal of biological sciences 01/2000; 4(1):71-76.
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Chongil Yi,
Jong-Min Woo,
Cecil Han,
Jeong Su Oh,
Inju Park,
Boyeon Lee,
Sora Jin,
Heejin Choi,
Jun Tae Kwon, Byung-Nam Cho,
Do Han Kim,
Chunghee Cho
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ABSTRACT: A number of members belonging to a disintegrin and metalloprotease (ADAM) family of cell surface proteins, including ADAM21, are expressed specifically or predominantly in the mammalian testis. Here, we investigated the transcriptional characteristics of the Adam21 gene. We found that Adam21 produces two types of transcripts with different developmental stages and cellular localizations. One type comprises germ cell-specific transcripts with both exons 1 and 2, while the other type corresponds to exon 2 and is expressed in testicular somatic cells. Further, regulatory and promoter regions responsible for the expression of Adam21 in testicular somatic cells were investigated using an in silico sequence analysis and an in vitro transient transfection assay. We identified an essential promoter and mapped regulatory regions that repress the transcription of Adam21. Finally, we confirmed the expression of Adam21 at the protein level in testicular somatic cells in which the promoter of the gene was found to be active. This is the first study to provide information regarding transcriptional regulation of a testicular ADAM family member, which will aid in elucidation of the transcriptional mechanisms of other testicular Adam genes.
Gene Expression Patterns 10(2-3):152-8. · 2.02 Impact Factor
