Experimental induction of testicular teratomas in dissociated—reaggregated chimaeric gonads

Development ◽  
1982 ◽  
Vol 72 (1) ◽  
pp. 153-167
Author(s):  
Urs Regenass ◽  
Thomas D. Friedrich ◽  
Leroy C. Stevens
Keyword(s):  
Germ Cells ◽  
Resistant Strain ◽  
Somatic Cells ◽  
Susceptible Strain ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Experimental Induction ◽  
High Incidence ◽  
Low Incidence ◽  
Male Genital

Testicular teratomas can be experimentally induced in some strains of mice by grafting12·5-day male genital ridges to the testes of adults. The grafts develop into testes and most of them have teratomas. The cells of 12·5-day foetal gonads were dissociated and the germ cells and somatic cells were separated. When germ cells were reaggregated with somatic cells and implanted in adult testes, they formed seminiferous tubules with teratomas. The somatic cell populations were contaminated with about 1 % germ cells, and when they were implanted in adult testes, they formed testes with a comparatively low incidence of teratomas. When germ cells of a highly susceptible strain were combined with somatic cells from a resistant strain, they formed chimaeric testes with a high incidence of teratomas. When germ cells from a resistant strain were combined with somatic cells from a susceptible strain they formed chimaeric gonads and the incidence of teratomas was low. This indicates that at 12·5 days the genotype of the germ cells is responsible for susceptibility. When germ cells from older foetal gonads were combined with somatic cells of 12·5-day gonads, the incidence of teratomas was low. This showed that 12·5-day somatic cells cannot ‘rejuvenate’ older germ cells in a way to regain their susceptibility. When 12·5-day germ cells of highly susceptible strains were combined with older somatic cells the incidence of tumours was low indicating that the age of the somatic cells influences susceptibility to teratocarcinogenesis.

286 EXPRESSION OF PLURIPOTENT STEM CELL MARKERS IN DOMESTIC CAT SPERMATOGONIAL CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 290 ◽  
Author(s):  
R. H. Powell ◽  
M. N. Biancardi ◽  
J. Galiguis ◽  
Q. Qin ◽  
C. E. Pope ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Basement Membrane ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Surface Markers ◽  
Cell Markers

Spermatogonial stem cells (SSC), progenitor cells capable of both self-renewal and producing daughter cells that will differentiate into sperm, can be manipulated for transplantation to propagate genetically important males. This application was demonstrated in felids by the successful xeno-transplantation of ocelot mixed germ cells into the testes of domestic cats, which resulted in the production of ocelot sperm (Silva et al. 2012 J. Androl. 33, 264–276). Spermatogonial stem cells are in low numbers in the testis, but have been identified and isolated in different mammalian species using SSC surface markers; however, their expression varies among species. Until recently, little was known about the expression of SSC surface markers in feline species. We previously demonstrated that many mixed germ cells collected from adult cat testes express the germ cell markers GFRα1, GPR125, and C-Kit, and a smaller population of cells expresses the pluripotent SSC-specific markers SSEA-1 and SSEA-4 (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222). In the present study, our goal was to identify germ cell and SSC-specific markers in SSC from cat testes. Immunohistochemical (IHC) localization of germ cell markers GFRα1, GPR125, and C-Kit and pluripotent SSC-specific markers SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 was detected in testis tissue from both sexually mature and prepubertal males. Testes were fixed with modified Davidson’s fixative for 24 h before processing, embedding, and sectioning. The EXPOSE Mouse and Rabbit Specific HRP/DAB detection IHC kit (Abcam®, Cambridge, MA, USA) was used for antibody detection. Staining for SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 markers was expressed specifically at the basement membrane of the seminiferous tubules in both adult and prepubertal testes. The GFRα1 and GPR125 markers were detected at the basement membrane of the seminiferous tubules and across the seminiferous tubule section. However, C-Kit was not detected in any cell. Using flow cytometry from a pool of cells from seven adult testes, we detected 45% GFRα1, 50% GPR125, 59% C-Kit, 18% TRA-1-60, 16% TRA-1-81 positive cells, and a very small portion of SSEA-1 (7%) and SSEA-4 (3%) positive cells. Dual staining of germ cells pooled from 3 testes revealed 3 distinct cell populations that were positive for GFRα1 only (23%), positive for both GFRα1 and SSEA-4 (6%), and positive for SSEA-4 only (1%). Our IHC staining of cat testes indicated that cells along the basement membrane of seminiferous tubules were positive for SSC-specific markers, and flow cytometry analysis revealed that there were different cell populations expressing both germ cell and SSC-specific markers. Flow cytometry results show overlapping germ cell populations expressing SSEA-4 and GFRα1, and IHC results reveal that SSEA-4 positive cells are spermatogonia, whereas GFRα1 positive cells include other stages of germ cells, indicating that the small population of cells positive only for SSEA-4 is undifferentiated cat SSC.

scholarly journals The single-cell epigenetic regulatory landscape in mammalian perinatal testis development

2021 ◽  
Author(s):  
Jinyue Liao ◽  
Hoi Ching Suen ◽  
Shitao Rao ◽  
Alfred Chun Shui Luk ◽  
Ruoyu Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Somatic Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cellular Heterogeneity ◽  
Cell Populations ◽  
Cell Type

AbstractSpermatogenesis depends on an orchestrated series of developing events in germ cells and full maturation of the somatic microenvironment. To date, the majority of efforts to study cellular heterogeneity in testis has been focused on single-cell gene expression rather than the chromatin landscape shaping gene expression. To advance our understanding of the regulatory programs underlying testicular cell types, we analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis. We showed that scATAC-Seq allowed us to deconvolve distinct cell populations and identify cis-regulatory elements (CREs) underlying cell type specification. We identified sets of transcription factors associated with cell type-specific accessibility, revealing novel regulators of cell fate specification and maintenance. Pseudotime reconstruction revealed detailed regulatory dynamics coordinating the sequential developmental progressions of germ cells and somatic cells. This high-resolution data also revealed putative stem cells within the Sertoli and Leydig cell populations. Further, we defined candidate target cell types and genes of several GWAS signals, including those associated with testosterone levels and coronary artery disease. Collectively, our data provide a blueprint of the ‘regulon’ of the mouse male germline and supporting somatic cells.

371 EVALUATION OF ENRICHMENT STRATEGIES FOR PORCINE SPERMATOGONIA BY EXPRESSION OF PROTEIN GENE PRODUCT 9.5, A SPERMATOGONIA-SPECIFIC MARKER IN THE PIG TESTIS

2006 ◽  
Vol 18 (2) ◽  
pp. 293
Author(s):  
J. Luo ◽  
S. Megee ◽  
R. Rathi ◽  
I. Dobrinski
Keyword(s):  
Gene Product ◽  
Germ Cells ◽  
Protein Gene ◽  
Velocity Sedimentation ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Specific Marker ◽  
Pgp 9.5 ◽  
Protein Gene Product ◽  
Fraction I

Transplantation of genetically altered male germ cells is under investigation as a novel route to generate transgenic animal models. Identification and isolation of spermatogonial stem cells are a prerequisite for this strategy. The objectives of this study were to validate a marker for identification of undifferentiated porcine spermatogonia, and to use this marker to develop a practical enrichment strategy for spermatogonia from pig testis. We established that expression of protein gene product (PGP) 9.5 is a spermatogonia-specific marker in porcine testis through analysis of its expression pattern in testis cells, by comparison with the expression of the cell-type specific proteins GATA-4 (expressed in Sertoli cells) or PLZF (expressed in undifferentiated mouse spermatogonia) in seminiferous tubules at different ages, and by comparison of expression levels of PGP 9.5 and the germ cell-specific protein VASA in different cell fractions after differential plating. Using expression of PGP 9.5 as a marker, we characterized enrichment of porcine spermatogonia from two-week-old (2wo) and 10-week-old (10wo) pigs by immunofluorescence either after differential plating only or after velocity sedimentation at unit gravity followed by differential plating. After differential plating with overnight culture to deplete testicular somatic cells that firmly attach to culture dishes, spermatogonia (mean � SEM per 1000 cells) were 5-fold enriched (P < 0.05) in cells remaining in suspension (fraction I) (2wo: 54.0 � 9.1; 10wo: 162.7 � 30.5) and in populations slightly attached to the culture plate (fraction II) (2wo: 92.7 � 8; 10wo: 159.5 � 22.5) compared to the initial samples (2wo: 12.3 � 2.7; 10wo: 27.2 � 2.9). Slightly attached spermatogonia appear to be superior for future experiments due to higher viability (>90%) than spermatogonia remaining in suspension (<50%). Cell populations containing up to 70% spermatogonia with good viability (>80%) were achieved by velocity sedimentation isolation followed by differential plating. These results indicate that expression of PGP 9.5 is a useful marker for identification of undifferentiated porcine germ cells. Simple differential adhesion culture of testis cells harvested from pre-pubertal boars can supply cell populations enriched in spermatogonia for subsequent genetic manipulation and transplantation. This work was supported by 1 R01 RR17359-01.

201 TRANSPLANTATION OF SSEA-1+ AND SSEA-4+ SPERMATOGONIAL CELL SUBPOPULATIONS IN UNTREATED SEXUALLY IMMATURE DOMESTIC CATS

2014 ◽  
Vol 26 (1) ◽  
pp. 215
Author(s):  
R. H. Powell ◽  
J. L. Galiguis ◽  
Q. Qin ◽  
M. N. Biancardi ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Cell Biology ◽  
Captive Breeding ◽  
Rete Testis ◽  
Domestic Cat ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Specific Cell ◽  
Surface Markers

Captive breeding efforts in felids, including assisted reproduction techniques, have had varied success depending on species. Spermatogonial stem cells (SSC), comprising a small percentage of germ cells in the testis, are progenitor cells with the ability to both self-renew and differentiate into spermatozoa throughout the life of the male. Manipulation of SSC for transplantation (SSCT) may allow the propagation of genetically important males, as demonstrated by the production of ocelot sperm following transplantation of ocelot mixed germ cells to domestic cat testes (Silva et al. 2012 J. Androl. 33, 264–276). Using specific cell surface markers, SSC have been isolated from mixed germ cells in several other species for SSCT, culture, and studying germ cell biology; however, expression may differ with species. Using the domestic cat as a model for exotic felids, we recently began evaluating the expression of surface markers in feline SSC. Previously, we determined that pluripotent markers SSEA-1, SSEA-4, TRA-1–60, and TRA-1–81 were more specific to cat spermatogonia than SSC surface markers GFRα1 and GPR125 used in other species, with SSEA-1 and SSEA-4 expressed in the fewest cells (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222; Powell et al. 2012 Reprod. Fertil. Dev. 25, 290–291). Our current goal was to 1) confirm the presence of SSC within SSEA-1+ and SSEA-4+ cell populations by the ability to colonize following SSCT; 2) compare the effectiveness of transplanting SSC purified by flow cytometry versus mixed germ cells; and 3) show that depletion of endogenous germ cells before SSCT, usually performed by irradiation or chemotherapy in other studies, is not necessary when using sexually immature recipients. Mixed germ cells from 8 to 12 adult testes were pooled, stained for SSEA-1 or SSEA-4, and sorted by flow cytometry. SSEA-1+, SSEA-4+, or mixed germ cells were then labelled with the membrane dye PKH26 (Sigma MINI26) and injected into the testes of six 5-month-old and six 6-month-old cats at the site of the external rete testis after carefully microdissecting the head of the epididymis away from the testis. Injections contained an average of 230 000 sorted or 10 × 106 mixed germ cells suspended in 80 μL of DMEM/F12 + 3 μL of Trypan Blue (T8154, Sigma, St. Louis, MO, USA). Testes were harvested 10 to 12 weeks post-SSCT and bisected, half snap-frozen for later cryosectioning and the other half enzymatically digested to loosen seminiferous tubules for immediate evaluation. Fluorescence was detected in the testes of both 6-month-old males that received injections of mixed germ cells, one 6-month-old male injected with SSEA-4+ cells, and two 5-month-old males, one injected with SSEA-4+ cells and one with SSEA-1+ cells. Results indicate that SSC are found in both SSEA-1+ and SSEA-4+ cell populations, but that purification of SSC is not necessary for successful SSCT. Additionally, SSC colonization in cats is possible without depletion of endogenous cells in sexually immature recipients.

scholarly journals Hypobaric hypoxia causes deleterious effects on spermatogenesis in rats

Reproduction ◽  
2010 ◽  
Vol 139 (6) ◽  
pp. 1031-1038 ◽  
Author(s):  
Weigong Liao ◽  
Mingchun Cai ◽  
Jian Chen ◽  
Jian Huang ◽  
Fuyu Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Hypobaric Hypoxia ◽  
Diploid Cell ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Control Group ◽  
Spermatogenic Cells ◽  
Tetraploid Cell ◽  
Normoxic Control

The study was conducted to explore the effects of hypobaric hypoxia on spermatogenesis in rats. Adult male Wistar rats were randomly divided into four groups: three hypoxia-exposed groups and one normoxic control group. Rats in the normoxic control group were raised at an altitude of 300 m, while rats in the 5-, 15-, and 30-day hypoxic groups were raised in a hypobaric chamber simulating a high altitude of 5000 m for 5, 15, and 30 days respectively. Flow cytometry was used to detect the DNA content of testicular spermatogenic cells in rats. The apoptosis of germ cells in testis was analyzed by using TUNEL assay. Spermatogenesis was also evaluated by morphology. Flow cytometry analysis revealed that 5–30 days of hypobaric hypoxia exposure significantly reduced the percentage of tetraploid cell population in rat testis. After rats were exposed to hypobaric hypoxia for 30 days, the ratio of haploid and diploid cell populations in testis reduced significantly. Seminiferous tubules with apoptotic germ cell increased after exposure to hypoxia. Most apoptotic germ cells were spermatogonia and spermatocytes. Hypoxia also caused decrease of cellularity of seminiferous epithelium, degeneration and sloughing of seminiferous epithelial cells occasionally. The data suggest that hypobaric hypoxia inhibits the spermatogenesis in rats. Decrease of tetraploid spermatogenic cells (primary spermatocytes) induced by hypoxia is an important approach to suppress spermatogenesis. The apoptosis of primary spermatocytes and spermatogonia may contribute to the loss of tetraploid cell populations.

scholarly journals Spatial distribution and risk assessment of toxic metals in agricultural soils from endemic nasopharyngeal carcinoma region in South China

2020 ◽  
Vol 12 (1) ◽  
pp. 568-579
Author(s):  
Liping Mo ◽  
Yongzhang Zhou ◽  
Gnanachandrasamy Gopalakrishnana ◽  
Xingyuan Li
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
South China ◽  
Toxic Metals ◽  
Agricultural Soils ◽  
Hazard Index ◽  
Toxic Metal ◽  
Carcinogenic Risk ◽  
Pollution Indices ◽  
High Incidence ◽  
Low Incidence

AbstractSihui city (South China) is much affected by nasopharyngeal carcinoma (NPC). To investigate the relationships between the toxic metals in soil and NPC incidence in Sihui, 119 surface soil samples were collected from agricultural fields and analyzed. The soil As–Cr contents in Longjiang (high-incidence area) are significantly lower than those in Weizheng and Jianglin (low-incidence areas), whereas the soil Pb content in Longjiang is significantly higher than that in Weizheng. The Nemerow pollution indices (PIN) of soils decrease in the order of Jianglin > Weizheng > Longjiang. The enrichment factor (EF) of Cd indicates that the Cd enrichment is contributed by human activities. Potential toxic metal-related ecological risk values decrease in the order of Jianglin > Weizheng > Longjiang. The mean hazard index (HI) value of Longjiang was lower than those of Weizheng and Jianglin. There are no adverse noncarcinogenic health effects of soil toxic metals to adults in the study areas. Carcinogenic risks of As and Cr via ingestion and dermal contact and total carcinogenic risk are within the warning range, from 10−6 to 10−4. Hence, we suggest that toxic metals in the soil may not be major geochemical carcinogenic factors of high NPC incidence in Sihui.

scholarly journals Polygenic and single gene responses to selection for resistance to diazinon in Lucilia cuprina.

Genetics ◽  
1992 ◽  
Vol 130 (3) ◽  
pp. 613-620 ◽  
Author(s):  
J A McKenzie ◽  
A G Parker ◽  
J L Yen
Keyword(s):  
Resistant Strain ◽  
Single Gene ◽  
Natural Populations ◽  
Susceptible Strain ◽  
Selection Line ◽  
Base Population ◽  
Gene Response ◽  
Biochemical Profiles ◽  
Resistant Strains ◽  
Selection For

Abstract Following mutagenesis with ethyl methanesulfonate, selection in a susceptible strain with a concentration of the insecticide diazinon (0.0004%, w/v) above that required to kill 100% of the susceptible strain, the LC100 of that strain, resulted in a single gene response. The resultant four mutant resistant strains have equivalent physiological, genetical and biochemical profiles to a diazinon-resistant strain derived from a natural population and homozygous for the Rop-1 allele. Modification of the microsomal esterase E3 is responsible for resistance in each case. The Rop-1 locus maps approximately 4.4 map units proximal to bu on chromosome IV. Selection within the susceptible distribution, at a concentration of diazinon [0.0001% (w/v)] less than the LC100, resulted in a similar phenotypic response irrespective of whether the base population had been mutagenized. The responses were polygenically based, unique to each selection line and independent of Rop-1. The relevance of the results to selection for insecticide resistance in laboratory and natural populations is discussed.

scholarly journals Could MicroRNAs Be Useful Tools to Improve the Diagnosis and Treatment of Rare Gynecological Cancers? A Brief Overview

2021 ◽  
Vol 22 (8) ◽  
pp. 3822
Author(s):  
Riccardo Di Fiore ◽  
Sherif Suleiman ◽  
Francesca Pentimalli ◽  
Sharon A. O’Toole ◽  
John J. O’Leary ◽  
...  
Keyword(s):  
Female Genital Tract ◽  
Germ Cell Tumors ◽  
Delayed Diagnosis ◽  
Public Health Issue ◽  
Gene Promoters ◽  
Gynecological Cancers ◽  
High Incidence ◽  
Low Incidence ◽  
Diagnosis Therapy ◽  
Malignant Germ Cell Tumors

Gynecological cancers pose an important public health issue, with a high incidence among women of all ages. Gynecological cancers such as malignant germ-cell tumors, sex-cord-stromal tumors, uterine sarcomas and carcinosarcomas, gestational trophoblastic neoplasia, vulvar carcinoma and melanoma of the female genital tract, are defined as rare with an annual incidence of <6 per 100,000 women. Rare gynecological cancers (RGCs) are associated with poor prognosis, and given the low incidence of each entity, there is the risk of delayed diagnosis due to clinical inexperience and limited therapeutic options. There has been a growing interest in the field of microRNAs (miRNAs), a class of small non-coding RNAs of ∼22 nucleotides in length, because of their potential to regulate diverse biological processes. miRNAs usually induce mRNA degradation and translational repression by interacting with the 3′ untranslated region (3′-UTR) of target mRNAs, as well as other regions and gene promoters, as well as activating translation or regulating transcription under certain conditions. Recent research has revealed the enormous promise of miRNAs for improving the diagnosis, therapy and prognosis of all major gynecological cancers. However, to date, only a few studies have been performed on RGCs. In this review, we summarize the data currently available regarding RGCs.

THE DIFFERENTIAL RESPONSE OF STRAINS OF WILD CARROT TO 2,4-D AND RELATED HERBICIDES

1963 ◽  
Vol 43 (3) ◽  
pp. 255-262 ◽  
Author(s):  
C. W. Whitehead ◽  
C. M. Switzer
Keyword(s):  
Resistant Strain ◽  
Differential Response ◽  
Susceptible Strain ◽  
Severe Injury ◽  
Radicle Growth ◽  
Cotyledon Stage ◽  
Wild Carrot

Studies were conducted on the effect of 2,4-D and some related herbicides on a susceptible and a resistant strain of wild carrot (Daucus corota L.). When treated with 2,4-D, 4-(2,4-DB) or 4-(MCPB), plants of the susceptible strain died within a few weeks while those of die resistant strain recovered after showing severe injury symptoms. Treatment with 2,4,5-T or silvex killed both the susceptible and resistant strains.Seeds of the two strains, placed in various concentrations of 2,4-D, showed no differences in germination or radicle growth after 7 days. However, sprays of 2,4-D on seedlings just after emergence produced the differential response. Resistance appeared to develop between germination and the cotyledon stage of growth.There were no differences in the effect of 2,4-D on the respiration of whole tissue of the susceptible and resistant wild carrots.

scholarly journals Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1813-1819 ◽  
Author(s):  
Eri Shiraishi ◽  
Norifumi Yoshinaga ◽  
Takeshi Miura ◽  
Hayato Yokoi ◽  
Yuko Wakamatsu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sex Differentiation ◽  
Somatic Cells ◽  
Oryzias Latipes ◽  
Cell Number ◽  
Gonadal Differentiation ◽  
Loss Of Function ◽  
Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

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