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Ralph L. Brinster - One of the best experts on this subject based on the ideXlab platform.

  • Isolation of the Spermatogonial Stem Cell-Containing Fraction from Testes.
    Cold Spring Harbor protocols, 2017
    Co-Authors: Shaun M. Goodyear, Ralph L. Brinster
    Abstract:

    Abstract A Testis Cell fraction enriched for spermatogonial stem Cells (SSCs) compared with the unfractionated total Testis Cell population can be isolated from testes after birth and adult stages. This protocol involves dissociating the seminiferous tubules into a single-Cell suspension, using enzymes and mechanical disruption. This is followed by the selection of the Thy1(+) Cell fraction that contains nearly the entire SSC population and is enriched for these Cells compared with the total Testis Cell population. This isolated fraction can be used for molecular studies, transfer into culture to establish primary cultures, or transplantation into the testes of recipients for spermatogenesis. Typically, if the SSCs are destined for transplantation, a marker is included to visualize the clones of spermatogenesis (e.g., lacZ or green fluorescent protein [GFP] transgenes). It is important to note that the isolated Thy1(+) Cell fraction does not consist of pure SSCs and that most germ Cells are non-stem-Cell progenitors, and a smaller portion of the population is somatic Cells.

  • colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem Cell self renewal
    Development, 2009
    Co-Authors: Jon M. Oatley, Mary R. Avarbock, Melissa J Oatley, John W Tobias, Ralph L. Brinster
    Abstract:

    Self-renewal and differentiation of spermatogonial stem Cells (SSCs) provide the foundation for Testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expressions are augmented in the SSC-enriched Thy1+ germ Cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1+ germ Cell fraction with the Thy1-depleted Testis Cell population identified 202 genes that are expressed 10-fold or higher in Thy1+ Cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions. These analyses revealed that colony stimulating factor 1 receptor (Csf1r) gene expression is enriched in Thy1+ germ Cells. Addition of recombinant colony stimulating factor 1 (Csf1), the specific ligand for Csf1r, to culture media significantly enhanced the self-renewal of SSCs in heterogeneous Thy1+ spermatogonial cultures over a 63-day period without affecting total germ Cell expansion. In vivo, expression of Csf1 in both pre-pubertal and adult testes was localized to clusters of Leydig Cells and select peritubular myoid Cells. Collectively, these results identify Csf1 as an extrinsic stimulator of SSC self-renewal and implicate Leydig and myoid Cells as contributors of the testicular stem Cell niche in mammals.

  • Male germ-line stem Cell potential is predicted by morphology of Cells in neonatal rat testes.
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Kyle E. Orwig, Buom-yong Ryu, Mary R. Avarbock, Ralph L. Brinster
    Abstract:

    Gonocytes are a transient population of male germ-line stem Cells that are derived from primordial germ Cells in the embryo and give rise to spermatogonial stem Cells, which establish and maintain spermatogenesis in the postnatal Testis. In contrast to spermatogonial stem Cells, gonocytes can be identified easily in neonatal rat Testis Cell suspensions based on their large size and distinct morphology. Furthermore, histological analysis of testes from neonatal transgenic rats demonstrated that gonocytes are the only Cells that express a lacZ reporter transgene. Two gonocyte subpopulations, designated pseudopod and round, were identified and isolated from neonatal (0–4 days postpartum) rat Testis Cell suspensions. Male germ-line stem Cells, identified by their ability to produce and maintain colonies of spermatogenesis upon transplantation into infertile recipient testes, were present almost exclusively in the pseudopod gonocyte subpopulation. In contrast, annexin V staining indicated that the majority of round gonocytes undergo apoptosis. These results indicate that a nearly pure population of male germ-line stem Cells can be prospectively identified in neonatal rat Testis Cell suspensions by morphological criteria. Together, the pseudopod and round gonocyte populations will provide powerful tools for the study of Cellular mechanisms that control Cell fates and the establishment of spermatogenesis in the postnatal Testis.

  • primate spermatogonial stem Cells colonize mouse testes
    Biology of Reproduction, 2001
    Co-Authors: Makoto Nagano, John R Mccarrey, Ralph L. Brinster
    Abstract:

    In mice, transplantation of spermatogonial stem Cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human Testis Cells to mice have led to conflicting claims that no donor germ Cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which Testis Cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem Cells readily establish germ Cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ Cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem Cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these Cells and the Testis environment have been preserved for 100 million years of evolutionary separation. Because germ Cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.

Kate L Loveland - One of the best experts on this subject based on the ideXlab platform.

  • Activin A Balances Sertoli and Germ Cell Proliferation in the Fetal Mouse Testis
    Biology of Reproduction, 2010
    Co-Authors: Sirisha Mendis, Mai A Sarraj, Sarah J Meachem, Kate L Loveland
    Abstract:

    Activin affects many aspects of Cellular development, including those essential for reproductive fitness. This study examined the contribution of activin A to murine fetal testicular development, revealing contrasting outcomes of activin actions on Sertoli Cells and gonocytes. Shortly after sex determination, from Embryonic Day 12.5 (E12.5) through to birth (0 dpp), the activin A subunit transcript (Inhba) level rises in Testis but not ovary, followed closely by the Inha transcript (encoding the inhibitory inhibin alpha subunit). Activin receptor transcript levels also change, with Acvr1 (encoding ALK2) and Acvr2b (ActRIIB) significantly higher and lower, respectively, at 0 dpp compared with E13.5 and E15.5. Transcripts encoding the signaling mediators Smad1, Smad3, and Smad4 were higher at 0 dpp compared with E13.5 and E15.5, whereas Smad2, Smad5, and Smad7 were lower. Detection of phosphorylated (P-)SMAD2/3 in nearly all Testis Cell nuclei indicated widespread transforming growth factor beta (TGFB) and/...

  • fetal Testis dysgenesis and compromised leydig Cell function in tgfbr3 betaglycan knockout mice
    Biology of Reproduction, 2010
    Co-Authors: Mai A Sarraj, Kate L Loveland, Ruth M Escalona, Alexandra Umbers, Hui Kheng Chua, Chris Small, Michael D Griswold, Jock K Findlay, Kaye L Stenvers
    Abstract:

    Abstract Betaglycan (Tgfbr3) is a coreceptor for transforming growth factor-beta (TGFB) superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5–13.5 days postcoitum (dpc) betaglycan null murine Testis. Immunohistochemistry with antibodies against Cell-specific markers revealed defects in somatic Cell populations. To confirm these data, quantitative real-time PCR was performed to determine changes in the expression levels of genes involved in fetal Testis Cell differentiation and function. The expression levels of the Leydig Cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout Testis compared to wild-type Testis, beginning at 12.5 dpc. Whole mount in situ hybridization confirmed that Cyp11a1 expression was reduced in the null Testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of betaglycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed...

  • fetal Testis dysgenesis and compromised leydig Cell function in tgfbr3 betaglycan knockout mice short title phenotype of betaglycan null Testis summary sentence the absence of murine tgfbr3 betaglycan causes a disruption to fetal testicular cord stru
    2009
    Co-Authors: Mai A Sarraj, Kate L Loveland, Ruth M Escalona, Alexandra Umbers, Hui Kheng Chua, Chris Small, Jock K Findlay, Kaye L Stenvers
    Abstract:

    Betaglycan (Tgfbr3) is a coreceptor for TGFB superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5-13.5 dpc betaglycan null murine Testis. Immunohistochemistry with antibodies against Cell-specific markers revealed defects in somatic Cell populations. To confirm these data, quantitative real time PCR was performed to determine changes in the expression levels of genes involved in fetal Testis Cell differentiation and function. The expression levels of the Leydig Cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout Testis compared to wild-type Testis, beginning at 12.5 dpc. Wholemount in situ hybridization confirmed that Cyp11a1 expression was reduced in null Testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of betaglycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed no changes in Leydig Cell counts between the wild-type and the knockout Testis at 14.5 dpc, indicating that fetal Leydig function, rather than number, was affected by the loss of betaglycan. The expression levels of Sertoli Cell markers Dhh, Sox9, and Amh were also reduced in the knockout Testis at 14.5 dpc. However, the expression of fetal germ Cell markers Pou5f1 and DDX4 were not changed across the genotypes at any age examined. Our data show that the presence of betaglycan is required for normal cord formation, normal fetal Leydig Cell development and the establishment of fetal Testis endocrine function, thus implicating TGFB superfamily members as regulators of early fetal Testis structure and function.

  • Gene expression study in the juvenile mouse Testis: identification of stage-specific molecular pathways during spermatogenesis
    Mammalian Genome, 2006
    Co-Authors: Emily J. Clemente, Kate L Loveland, Robert A. Furlong, Nabeel A. Affara
    Abstract:

    A gene expression time course in the juvenile mouse Testis was established using cDNA microarrays derived from a variety of isolated Testis Cell types. In conjunction with the use of four germ Cell-deficient mouse models, a stage and Cell-type classification over nine time points has been obtained and analyzed for differential expression of genes. The expression profiles have been clustered into nine groups and subjected to detailed analysis of associated gene ontology. This has allowed the correlation of particular Cellular processes and functions with different expression clusters. Focused analysis of transcripts involved in Cell number regulation (apoptosis and proliferation) and their spatiotemporal expression patterns are presented. The findings indicate that for genes involved in both apoptosis and proliferation, several distinct pathways regulating these processes are active in somatic and germ Cell lineages.

Mai A Sarraj - One of the best experts on this subject based on the ideXlab platform.

  • Activin A Balances Sertoli and Germ Cell Proliferation in the Fetal Mouse Testis
    Biology of Reproduction, 2010
    Co-Authors: Sirisha Mendis, Mai A Sarraj, Sarah J Meachem, Kate L Loveland
    Abstract:

    Activin affects many aspects of Cellular development, including those essential for reproductive fitness. This study examined the contribution of activin A to murine fetal testicular development, revealing contrasting outcomes of activin actions on Sertoli Cells and gonocytes. Shortly after sex determination, from Embryonic Day 12.5 (E12.5) through to birth (0 dpp), the activin A subunit transcript (Inhba) level rises in Testis but not ovary, followed closely by the Inha transcript (encoding the inhibitory inhibin alpha subunit). Activin receptor transcript levels also change, with Acvr1 (encoding ALK2) and Acvr2b (ActRIIB) significantly higher and lower, respectively, at 0 dpp compared with E13.5 and E15.5. Transcripts encoding the signaling mediators Smad1, Smad3, and Smad4 were higher at 0 dpp compared with E13.5 and E15.5, whereas Smad2, Smad5, and Smad7 were lower. Detection of phosphorylated (P-)SMAD2/3 in nearly all Testis Cell nuclei indicated widespread transforming growth factor beta (TGFB) and/...

  • fetal Testis dysgenesis and compromised leydig Cell function in tgfbr3 betaglycan knockout mice
    Biology of Reproduction, 2010
    Co-Authors: Mai A Sarraj, Kate L Loveland, Ruth M Escalona, Alexandra Umbers, Hui Kheng Chua, Chris Small, Michael D Griswold, Jock K Findlay, Kaye L Stenvers
    Abstract:

    Abstract Betaglycan (Tgfbr3) is a coreceptor for transforming growth factor-beta (TGFB) superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5–13.5 days postcoitum (dpc) betaglycan null murine Testis. Immunohistochemistry with antibodies against Cell-specific markers revealed defects in somatic Cell populations. To confirm these data, quantitative real-time PCR was performed to determine changes in the expression levels of genes involved in fetal Testis Cell differentiation and function. The expression levels of the Leydig Cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout Testis compared to wild-type Testis, beginning at 12.5 dpc. Whole mount in situ hybridization confirmed that Cyp11a1 expression was reduced in the null Testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of betaglycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed...

  • fetal Testis dysgenesis and compromised leydig Cell function in tgfbr3 betaglycan knockout mice short title phenotype of betaglycan null Testis summary sentence the absence of murine tgfbr3 betaglycan causes a disruption to fetal testicular cord stru
    2009
    Co-Authors: Mai A Sarraj, Kate L Loveland, Ruth M Escalona, Alexandra Umbers, Hui Kheng Chua, Chris Small, Jock K Findlay, Kaye L Stenvers
    Abstract:

    Betaglycan (Tgfbr3) is a coreceptor for TGFB superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5-13.5 dpc betaglycan null murine Testis. Immunohistochemistry with antibodies against Cell-specific markers revealed defects in somatic Cell populations. To confirm these data, quantitative real time PCR was performed to determine changes in the expression levels of genes involved in fetal Testis Cell differentiation and function. The expression levels of the Leydig Cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout Testis compared to wild-type Testis, beginning at 12.5 dpc. Wholemount in situ hybridization confirmed that Cyp11a1 expression was reduced in null Testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of betaglycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed no changes in Leydig Cell counts between the wild-type and the knockout Testis at 14.5 dpc, indicating that fetal Leydig function, rather than number, was affected by the loss of betaglycan. The expression levels of Sertoli Cell markers Dhh, Sox9, and Amh were also reduced in the knockout Testis at 14.5 dpc. However, the expression of fetal germ Cell markers Pou5f1 and DDX4 were not changed across the genotypes at any age examined. Our data show that the presence of betaglycan is required for normal cord formation, normal fetal Leydig Cell development and the establishment of fetal Testis endocrine function, thus implicating TGFB superfamily members as regulators of early fetal Testis structure and function.

Kaye L Stenvers - One of the best experts on this subject based on the ideXlab platform.

  • fetal Testis dysgenesis and compromised leydig Cell function in tgfbr3 betaglycan knockout mice
    Biology of Reproduction, 2010
    Co-Authors: Mai A Sarraj, Kate L Loveland, Ruth M Escalona, Alexandra Umbers, Hui Kheng Chua, Chris Small, Michael D Griswold, Jock K Findlay, Kaye L Stenvers
    Abstract:

    Abstract Betaglycan (Tgfbr3) is a coreceptor for transforming growth factor-beta (TGFB) superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5–13.5 days postcoitum (dpc) betaglycan null murine Testis. Immunohistochemistry with antibodies against Cell-specific markers revealed defects in somatic Cell populations. To confirm these data, quantitative real-time PCR was performed to determine changes in the expression levels of genes involved in fetal Testis Cell differentiation and function. The expression levels of the Leydig Cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout Testis compared to wild-type Testis, beginning at 12.5 dpc. Whole mount in situ hybridization confirmed that Cyp11a1 expression was reduced in the null Testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of betaglycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed...

  • fetal Testis dysgenesis and compromised leydig Cell function in tgfbr3 betaglycan knockout mice short title phenotype of betaglycan null Testis summary sentence the absence of murine tgfbr3 betaglycan causes a disruption to fetal testicular cord stru
    2009
    Co-Authors: Mai A Sarraj, Kate L Loveland, Ruth M Escalona, Alexandra Umbers, Hui Kheng Chua, Chris Small, Jock K Findlay, Kaye L Stenvers
    Abstract:

    Betaglycan (Tgfbr3) is a coreceptor for TGFB superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5-13.5 dpc betaglycan null murine Testis. Immunohistochemistry with antibodies against Cell-specific markers revealed defects in somatic Cell populations. To confirm these data, quantitative real time PCR was performed to determine changes in the expression levels of genes involved in fetal Testis Cell differentiation and function. The expression levels of the Leydig Cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout Testis compared to wild-type Testis, beginning at 12.5 dpc. Wholemount in situ hybridization confirmed that Cyp11a1 expression was reduced in null Testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of betaglycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed no changes in Leydig Cell counts between the wild-type and the knockout Testis at 14.5 dpc, indicating that fetal Leydig function, rather than number, was affected by the loss of betaglycan. The expression levels of Sertoli Cell markers Dhh, Sox9, and Amh were also reduced in the knockout Testis at 14.5 dpc. However, the expression of fetal germ Cell markers Pou5f1 and DDX4 were not changed across the genotypes at any age examined. Our data show that the presence of betaglycan is required for normal cord formation, normal fetal Leydig Cell development and the establishment of fetal Testis endocrine function, thus implicating TGFB superfamily members as regulators of early fetal Testis structure and function.

Gwonhwa Song - One of the best experts on this subject based on the ideXlab platform.

  • butylated hydroxyanisole induces testicular dysfunction in mouse Testis Cells by dysregulating calcium homeostasis and stimulating endoplasmic reticulum stress
    Science of The Total Environment, 2020
    Co-Authors: Gwonhwa Song
    Abstract:

    Abstract Butylated hydroxyanisole (BHA), a synthetic phenolic antioxidant (SPA), has been used as a food additive. However, BHA acts as an environmental hormone, i.e., endocrine disruptor. Here, we investigated BHA-induced male reproductive dysfunction in mouse Leydig and Sertoli Cells. We found that BHA suppressed proliferation and induced Cell cycle arrest in TM3 and TM4 Cells. Furthermore, we investigated mitochondrial permeabilization, expression profiles of pro-apoptotic and anti-apoptotic proteins, calcium influx, and endoplasmic reticulum (ER) stress in testicular Cells after BHA treatment. The results indicated that BHA-mediated calcium dysregulation and ER stress downregulated steroidogenesis- and spermatogenesis-related genes in mouse Testis Cell lines. Additionally, proliferation of both TM3 and TM4 Cells in response to BHA treatment was regulated via the Mapk and Akt signaling pathways. Therefore, constant BHA exposure may lead to testicular toxicity via mitochondrial dysfunction, ER stress, and abnormal calcium levels in the Testis.

  • alpha solanine inhibits Cell proliferation via mitochondrial dysfunction and inhibin synthesis in mouse Testis in vitro and in vivo
    Chemosphere, 2019
    Co-Authors: Sunwoo Park, Min Young Park, Gwonhwa Song
    Abstract:

    Abstract Sertoli and Leydig Cells provide key supporting roles in spermatogenesis. Various toxins have been studied in the TM3 and TM4 mouse Testis Cell lines to identify their regulatory effects. Alpha-solanine (α-solanine), a toxic compound found in the potato, has cytotoxic effects on various Cells, including cancer Cells. However, the effect of α-solanine on Testis function has not been identified. In this study, we verified for the first time the anti-proliferative effect of α-solanine in mouse testes. α-Solanine reduced Cell viability in TM3 and TM4 Cells and reduced the expression of the Cell cycle checkpoint genes Ccnd1 and Ccne1. We also detected changes in the mitochondrial membrane potential (MMP) and in the cytosolic calcium and intraCellular signal pathways in both Cell lines. α-Solanine induced AKT, P70S6K, S6, ERK1/2, and JNK activation in mouse Testis Cells. In addition, the inhibition of AKT with a pharmacological inhibitor (LY294002) demonstrated more synergic anti-proliferative effects than in the TM3 and TM4 Cell lines treated only with α-solanine. Inha and Inhba mRNA expression also decreased in both Cell lines and α-solanine i.p. injected mouse testes. Collectively, the results from this study verify the toxic effects of α-solanine on testes and male reproductive function.

  • Gossypol Induces Disruption of Spermatogenesis and Steroidogenesis in Male Mice.
    Journal of Agricultural and Food Chemistry, 2019
    Co-Authors: Sunwoo Park, Gwonhwa Song
    Abstract:

    Gossypol, commonly found in cotton seeds, is hazardous to male reproductive physiology. Although several studies have indicated the toxicity of gossypol in human and animal reproduction, the mechanism of gossypol action in testes has not yet been elucidated. In the present study, we investigated the effects of gossypol in normal mouse Testis Cells, TM3 and TM4 Cells, and in gossypol-treated C57BL/6 mice. We confirmed the antiproliferative effects of gossypol using Cell viability assays, with PCNA as a proliferation marker, and Cell cycle analysis. We also verified mitochondrial dysfunction and Ca2+ dysregulation in the cytosol of TM3 and TM4 Cells, using JC-1 and Fluo-4 dyes. To confirm the Cellular signaling mechanisms in Testis Cell lines, we performed Western blot analysis to assess the changes in MAPK and PI3K/Akt signal transduction, using their pharmacological inhibitors. Moreover, we screened the mRNA expression of genes involved in spermatogenesis and steroidogenesis in TM3 and TM4 Cells. We also ...

  • Gossypol Induces Disruption of Spermatogenesis and Steroidogenesis in Male Mice
    2019
    Co-Authors: Whasun Lim, Sunwoo Park, Jiyeon Ham, Hyocheol Bae, Seungkwon You, Gwonhwa Song
    Abstract:

    Gossypol, commonly found in cotton seeds, is hazardous to male reproductive physiology. Although several studies have indicated the toxicity of gossypol in human and animal reproduction, the mechanism of gossypol action in testes has not yet been elucidated. In the present study, we investigated the effects of gossypol in normal mouse Testis Cells, TM3 and TM4 Cells, and in gossypol-treated C57BL/6 mice. We confirmed the antiproliferative effects of gossypol using Cell viability assays, with PCNA as a proliferation marker, and Cell cycle analysis. We also verified mitochondrial dysfunction and Ca2+ dysregulation in the cytosol of TM3 and TM4 Cells, using JC-1 and Fluo-4 dyes. To confirm the Cellular signaling mechanisms in Testis Cell lines, we performed Western blot analysis to assess the changes in MAPK and PI3K/Akt signal transduction, using their pharmacological inhibitors. Moreover, we screened the mRNA expression of genes involved in spermatogenesis and steroidogenesis in TM3 and TM4 Cells. We also confirmed the mRNA expression and localization of genes regulating Testis function in gossypol-treated and untreated mice testes. Collectively, we suggest that gossypol induces negative effects on Testis function by reducing Cell viability, mitochondrial membrane potential, and Testis development-related genes in vitro and in vivo as well as by modulating the MAPK and PI3K signaling pathways