Jinping Luo | University of Utah (original) (raw)
Papers by Jinping Luo
Cell, 2015
Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutati... more Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.
Reproduction Fertility and Development, 2006
Molecular reproduction and development, 2006
Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, ... more Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P < 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attach...
Spermatogenesis is a fundamental biological process that ensures the transition of a gene from on... more Spermatogenesis is a fundamental biological process that ensures the transition of a gene from one ganeration to another via male gametes. This process relies on a rare population of testicular cells called spermatogonial stem cells (SSCs), which self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding study of SSCs, their biological properties remain largely unknown, which is partly due to the very limited availability of these cells. Here, we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs that can be successfully used to enrich them.
Reproduction, Fertility and Development, 2008
Biology of reproduction, 2012
FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function... more FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation wa...
The FASEB Journal, 2007
We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a... more We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild-type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV-transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV-mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research-Honaramooz, A., Megee, S., Zeng, W., Destrempes, M.M., I. Adeno-associated virus (AAV) -mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J.
Reproduction, Fertility and Development, 2010
Fyn kinase is highly expressed in oocytes, with inhibitor and dominant-negative studies suggestin... more Fyn kinase is highly expressed in oocytes, with inhibitor and dominant-negative studies suggesting a role in the signal transduction events during egg activation. The purpose of the present investigation was to test the hypothesis that Fyn is required for calcium signalling, meiosis resumption and pronuclear congression using the Fyn-knockout mouse as a model. Accelerated breeding studies revealed that Fyn-null females produced smaller litter sizes at longer intervals and exhibited a rapid decline in pup production with increasing age. Fyn-null females produced a similar number of oocytes, but the frequency of immature oocytes and mature oocytes with spindle chromosome abnormalities was significantly higher than in controls. Fertilised Fyn-null oocytes frequently (24%) failed to undergo pronuclear congression and remained at the one-cell stage. Stimulation with gonadotropins increased the number of oocytes ovulated, but did not overcome the above defects. Fyn-null oocytes overexpressed Yes kinase in an apparent effort to compensate for the loss of Fyn, yet still exhibited an altered pattern of protein tyrosine phosphorylation. In summary, Fyn-null female mice exhibit reduced fertility that appears to result from actin cytoskeletal defects rather than calcium signalling. These defects cause developmental arrest during oocyte maturation and pronuclear congression.
Reproduction, 2006
Fyn and other Src-family kinases play an essential role at several steps during egg activation fo... more Fyn and other Src-family kinases play an essential role at several steps during egg activation following fertilization of externally fertilizing species, such as marine invertebrates, fish, and frogs. Recent studies demonstrate that the requirement for Src-family kinases in activation of the mammalian egg is different from lower species, and the objective of this study was to test the role of the Fyn kinase in the mouse egg activated by intracytoplasmic sperm injection (ICSI). An Src homology 2 (SH2) domain containing fusion protein was used to suppress Fyn function in the mouse zygote following ICSI. Eggs injected with the Fyn SH2 domain at an intracellular concentration of 4-8 mM exhibited reduced developmental potential with 100% of the zygotes being arrested following the first or the second cleavage. At higher concentrations, the protein blocked pronuclear congression and the zygotes remained at the pronuclear stage. The SH2 domain had no effect on sperm-induced calcium oscillations in distinct contrast to its effect on the eggs of lower species. The results indicate that the SH2 domain of Fyn kinase plays an important role in pronuclear congression as well as early cleavage events and that this effect appears not to involve disruption of calcium oscillations. Reproduction (2006) 132 413-421 q 2006 Society for Reproduction and Fertility
Molecular Reproduction and Development, 2006
Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, ... more Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attached to the culture dish (fraction II) (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) after differential plating. Cells in fraction II appeared to be superior for future experiments due to higher viability (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;90%) than in fraction I ( approximately 50%). Velocity sedimentation plus differential plating achieved cell populations containing up to 70% spermatogonia with good viability (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;80%). Enriched spermatogonia from 2wo and 10wo testes could be maintained in a simple culture medium without additional growth factors for at least 2 weeks and continued to express PGP 9.5. These data provide the basis for future studies aimed at refining conditions of germ cell culture and manipulation prior to germ cell transplantation in pigs.
Molecular Reproduction and Development, 2009
Molecular Reproduction and Development, 2009
The objective of the present study was to determine whether Fyn kinase participated in signaling ... more The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm-egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI-606 (Bosutinib) and by analysis of Fyn-null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule-free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par-3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn-null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II. Pronuclei, however, eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally.
Journal of Cellular Physiology, 2009
Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, di... more Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is expressed in type A (A s , A pr , and A al ) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH-L1 was associated with self-renewal versus differentiation divisions of SSCs as defined by co-localization of UCH-L1 high and PLZF, a known determinant of undifferentiated SSCs, versus co-localization of UCH-L1 low/À with proteins expressed during SSC differentiation (DAZL, DDX4, c-KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH-L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH-L1 and PLZF in situ in seminiferous tubules. Expression level of UCH-L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH-L1-positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self-renewal and differentiation in mammalian spermatogenesis.
International Journal of Experimental Pathology, 2008
Cell Research, 2002
A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5&amp;amp;amp;am... more A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;t. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.
Biochemical and Biophysical Research Communications, 2014
Cloning, 2001
This study was designed to produce cloned goats from cumulus cells. Cloning donor nuclei were fro... more This study was designed to produce cloned goats from cumulus cells. Cloning donor nuclei were from cumulus cells either freshly isolated or cultured in vitro. Enucleated oocytes were either injected with cumulus cell nuclei without piezo-driven manipulator (injection method) or fused with cumulus cells (fusion method). The survival rate of cloned embryos, obtained by injection, was higher than that derived from fusion (62.7 and 45.9%, respectively). Two cloned goats were derived by fusion with in vitro cultured cumulus cells without starvation, but died shortly after natural birth, from respiratory difficulties. Their birth weights (2.23 kg and 2.03 kg) were within the normal range (2.0-2.7 kg) and postmortem analysis revealed no morphological abnormalities. The third cloned goat, derived by injection of nuclei from freshly isolated cumulus cells, weighed 3.3 kg at birth, and was 37% overweight compared with the average weight of the same species. This goat is healthy and well as this paper is being prepared. Nested PCR-RFLP analysis confirmed that all the cloned goats were derived from the donor cells.
Cell, 2015
Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutati... more Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.
Reproduction Fertility and Development, 2006
Molecular reproduction and development, 2006
Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, ... more Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P < 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attach...
Spermatogenesis is a fundamental biological process that ensures the transition of a gene from on... more Spermatogenesis is a fundamental biological process that ensures the transition of a gene from one ganeration to another via male gametes. This process relies on a rare population of testicular cells called spermatogonial stem cells (SSCs), which self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding study of SSCs, their biological properties remain largely unknown, which is partly due to the very limited availability of these cells. Here, we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs that can be successfully used to enrich them.
Reproduction, Fertility and Development, 2008
Biology of reproduction, 2012
FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function... more FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation wa...
The FASEB Journal, 2007
We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a... more We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild-type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV-transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV-mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research-Honaramooz, A., Megee, S., Zeng, W., Destrempes, M.M., I. Adeno-associated virus (AAV) -mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J.
Reproduction, Fertility and Development, 2010
Fyn kinase is highly expressed in oocytes, with inhibitor and dominant-negative studies suggestin... more Fyn kinase is highly expressed in oocytes, with inhibitor and dominant-negative studies suggesting a role in the signal transduction events during egg activation. The purpose of the present investigation was to test the hypothesis that Fyn is required for calcium signalling, meiosis resumption and pronuclear congression using the Fyn-knockout mouse as a model. Accelerated breeding studies revealed that Fyn-null females produced smaller litter sizes at longer intervals and exhibited a rapid decline in pup production with increasing age. Fyn-null females produced a similar number of oocytes, but the frequency of immature oocytes and mature oocytes with spindle chromosome abnormalities was significantly higher than in controls. Fertilised Fyn-null oocytes frequently (24%) failed to undergo pronuclear congression and remained at the one-cell stage. Stimulation with gonadotropins increased the number of oocytes ovulated, but did not overcome the above defects. Fyn-null oocytes overexpressed Yes kinase in an apparent effort to compensate for the loss of Fyn, yet still exhibited an altered pattern of protein tyrosine phosphorylation. In summary, Fyn-null female mice exhibit reduced fertility that appears to result from actin cytoskeletal defects rather than calcium signalling. These defects cause developmental arrest during oocyte maturation and pronuclear congression.
Reproduction, 2006
Fyn and other Src-family kinases play an essential role at several steps during egg activation fo... more Fyn and other Src-family kinases play an essential role at several steps during egg activation following fertilization of externally fertilizing species, such as marine invertebrates, fish, and frogs. Recent studies demonstrate that the requirement for Src-family kinases in activation of the mammalian egg is different from lower species, and the objective of this study was to test the role of the Fyn kinase in the mouse egg activated by intracytoplasmic sperm injection (ICSI). An Src homology 2 (SH2) domain containing fusion protein was used to suppress Fyn function in the mouse zygote following ICSI. Eggs injected with the Fyn SH2 domain at an intracellular concentration of 4-8 mM exhibited reduced developmental potential with 100% of the zygotes being arrested following the first or the second cleavage. At higher concentrations, the protein blocked pronuclear congression and the zygotes remained at the pronuclear stage. The SH2 domain had no effect on sperm-induced calcium oscillations in distinct contrast to its effect on the eggs of lower species. The results indicate that the SH2 domain of Fyn kinase plays an important role in pronuclear congression as well as early cleavage events and that this effect appears not to involve disruption of calcium oscillations. Reproduction (2006) 132 413-421 q 2006 Society for Reproduction and Fertility
Molecular Reproduction and Development, 2006
Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, ... more Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attached to the culture dish (fraction II) (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) after differential plating. Cells in fraction II appeared to be superior for future experiments due to higher viability (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;90%) than in fraction I ( approximately 50%). Velocity sedimentation plus differential plating achieved cell populations containing up to 70% spermatogonia with good viability (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;80%). Enriched spermatogonia from 2wo and 10wo testes could be maintained in a simple culture medium without additional growth factors for at least 2 weeks and continued to express PGP 9.5. These data provide the basis for future studies aimed at refining conditions of germ cell culture and manipulation prior to germ cell transplantation in pigs.
Molecular Reproduction and Development, 2009
Molecular Reproduction and Development, 2009
The objective of the present study was to determine whether Fyn kinase participated in signaling ... more The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm-egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI-606 (Bosutinib) and by analysis of Fyn-null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule-free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par-3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn-null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II. Pronuclei, however, eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally.
Journal of Cellular Physiology, 2009
Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, di... more Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is expressed in type A (A s , A pr , and A al ) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH-L1 was associated with self-renewal versus differentiation divisions of SSCs as defined by co-localization of UCH-L1 high and PLZF, a known determinant of undifferentiated SSCs, versus co-localization of UCH-L1 low/À with proteins expressed during SSC differentiation (DAZL, DDX4, c-KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH-L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH-L1 and PLZF in situ in seminiferous tubules. Expression level of UCH-L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH-L1-positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self-renewal and differentiation in mammalian spermatogenesis.
International Journal of Experimental Pathology, 2008
Cell Research, 2002
A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5&amp;amp;amp;am... more A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;t. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.
Biochemical and Biophysical Research Communications, 2014
Cloning, 2001
This study was designed to produce cloned goats from cumulus cells. Cloning donor nuclei were fro... more This study was designed to produce cloned goats from cumulus cells. Cloning donor nuclei were from cumulus cells either freshly isolated or cultured in vitro. Enucleated oocytes were either injected with cumulus cell nuclei without piezo-driven manipulator (injection method) or fused with cumulus cells (fusion method). The survival rate of cloned embryos, obtained by injection, was higher than that derived from fusion (62.7 and 45.9%, respectively). Two cloned goats were derived by fusion with in vitro cultured cumulus cells without starvation, but died shortly after natural birth, from respiratory difficulties. Their birth weights (2.23 kg and 2.03 kg) were within the normal range (2.0-2.7 kg) and postmortem analysis revealed no morphological abnormalities. The third cloned goat, derived by injection of nuclei from freshly isolated cumulus cells, weighed 3.3 kg at birth, and was 37% overweight compared with the average weight of the same species. This goat is healthy and well as this paper is being prepared. Nested PCR-RFLP analysis confirmed that all the cloned goats were derived from the donor cells.