Detection of Male Reproductive Abnormalities by Flow Cytometry Measurements of Testicular and Ejaculated Germ Cells

1984 ◽  
pp. 99-109 ◽  
Author(s):  
Donald P. Evenson ◽  
Paul J. Higgins ◽  
Myron R. Melamed
Keyword(s):  
Flow Cytometry ◽  
Germ Cells

Evaluation of 2‐methoxyacetic acid toxicity on mouse germ cells by flow cytometry

1991 ◽  
Vol 34 (1) ◽  
pp. 157-176 ◽  
Author(s):  
Marcello Spanò ◽  
Roberto Amendola ◽  
Cecilia Bartoleschi ◽  
Serena Emiliani ◽  
Eugenia Cordelli ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Methoxyacetic Acid ◽  
Acid Toxicity

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells

Zygote ◽  
2019 ◽  
Vol 27 (02) ◽  
pp. 82-88 ◽  
Author(s):  
Vivek Pandey ◽  
Anima Tripathi ◽  
Pawan K. Dubey
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Germ Cells ◽  
Expression Patterns ◽  
Intracellular Localization ◽  
Primordial Germ Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Single Type ◽  
Rt Pcr

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

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.

The Rapid and Sensitive Detection of Perturbations in Spermatogenesis: Assessment by Quantitative Dual Parameter (DNA/RNA) Flow Cytometry

1989 ◽  
Vol 8 (3) ◽  
pp. 507-523 ◽  
Author(s):  
Jules R. Selden ◽  
Richard T. Robertson ◽  
Judith E. Miller ◽  
Chris Vetter ◽  
David H. Minsker ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Biomedical Research ◽  
State Of The Art ◽  
The State ◽  
Sensitive Detection ◽  
Sprague Dawley ◽  
Adult Rats ◽  
Single Bolus ◽  
Preliminary Study

This presentation will concentrate on the emerging field of flow cytometry. The first portion will be a review of the state-of-the-art applications of flow cytometry in the field of biomedical research; the second portion will describe the results of a preliminary study using a published technique that is useful in detecting cellular perturbations in germ cells. The model used in this study was the testis from the Sprague-Dawley rat. Adult rats received a single bolus of busulfan, and their testes were examined up to 56 days postadministration.

scholarly journals Efficient GFP-labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry

2018 ◽  
Author(s):  
Leah L. Zagore ◽  
Cydni C. Akesson ◽  
Donny D. Licatalosi
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Molecular Mechanisms ◽  
Control Cell ◽  
Cell Types ◽  
Cell Development ◽  
Specific Gene ◽  
Haploid Male ◽  
Human Reproductive ◽  
Development And Differentiation

AbstractSpermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue-specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre-lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.

323. MEASURING CHANGES IN TESTICULAR CELL POPULATIONS USING FLOW CYTOMETRY

2010 ◽  
Vol 22 (9) ◽  
pp. 123
Author(s):  
G. Morin ◽  
K. Loveland
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Cell Biology ◽  
Leydig Cells ◽  
Cell Populations ◽  
Published Data ◽  
Wild Type ◽  
Proteomics Analysis ◽  
Kit Receptor ◽  
Different Levels

Spermatogenesis is first established during the first two weeks postpartum by the transition of undifferentiated (Kit–) into differentiated spermatogonia (Kit+). We recently showed that changes in the level of the growth factor activin alters the proportion of spermatogonial subtypes (1). However, detection of this transition by histology is unreliable. This project objective is to develop methods to efficiently measure changes in somatic and germ cell populations at the onset of spermatogenesis. Using surface (Kit receptor) and internal (mouse vasa homologue {MVH}) markers, we evaluated the proportion of differentiating germ cells in wild type Swiss mice by flow cytometry. Whole testes of mice at 7, 10, 14 days postpartum (dpp) were enzymatically dissociated and single cell suspensions were labelled with anti-Kit receptor antibody to detect Leydig cells and differentiating spermatogonia. These suspensions were then fixed and permeabilized in order to detect MVH, allowing spermatogonia to be distinguished from Leydig cells. Our present results show that combined Kit and MVH labelling is effective for evaluating the proportion of undifferentiating and differentiating germ cells. Our preliminary observations identified an elevation in the proportion of Kit+MVH+ cells between 7 and 10 days from 0.37 to 18%, indicating that spermatogonial differentiation advances dramatically between these ages. At day 14, the proportion of Kit+MVH+ cells decreased to 11%, as the emerging spermatocytes dilute spermatogonial numbers. These findings agree with published data (2). We have also used surface markers to discriminate between spermatogonia and Leydig cells without fixation or permeabilization, allowing us to isolate these cells for molecular and proteomics analysis. This will facilitate comparative profiling of germ cells with different levels of Kit, including those in mice with altered levels of growth factors (2) and hormones that govern the progression of testis development. (1) Mithraprabhu, 2010 Biology of Reproduction.(2) Bellve, 1977 Journal of Cell Biology.

Male germ cells of the Pacific oysterCrassostrea gigas: flow cytometry analysis, cell sorting and molecular expression

2011 ◽  
Vol 24 (3) ◽  
pp. 237-245 ◽  
Author(s):  
Alban Franco ◽  
Kristell Kellner ◽  
Michel Mathieu ◽  
Christophe Lelong ◽  
Didier Goux ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Cell Sorting ◽  
Flow Cytometry Analysis ◽  
Male Germ Cells ◽  
The Pacific ◽  
Molecular Expression

Mouse primordial germ cells lacking beta1 integrins enter the germline but fail to migrate normally to the gonads

Development ◽  
1999 ◽  
Vol 126 (8) ◽  
pp. 1655-1664 ◽  
Author(s):  
R. Anderson ◽  
R. Fassler ◽  
E. Georges-Labouesse ◽  
R.O. Hynes ◽  
B.L. Bader ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Migration ◽  
Germ Cell ◽  
Germ Cells ◽  
Fluorescent Protein ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Integrin Subunit ◽  
Integrin Beta1 ◽  
Germ Cell Migration

Primordial germ cells are the founder cells of the gametes. They are set aside at the initial stages of gastrulation in mammals, become embedded in the hind-gut endoderm, then actively migrate to the sites of gonad formation. The molecular basis of this migration is poorly understood. Here we sought to determine if members of the integrin family of cell surface receptors are required for primordial germ cell migration, as integrins have been implicated in the migration of several other motile cell types. We have established a line of mice which express green fluorescent protein in germline cells that has enabled us to efficiently purify primordial germ cells at different stages by flow cytometry. We have catalogued the spectrum of integrin subunit expression by primordial germ cells during and after migration, using flow cytometry, immunocytochemistry and RT-PCR. Through analysis of integrin beta1(−/−)-->wild-type chimeras, we show that embryonic cells lacking beta1 integrins can enter the germline. However, integrin beta1(−/−) primordial germ cells do not colonize the gonad efficiently. Embryos with targeted deletion of integrin subunit alpha3, alpha6, or alphaV show no major defects in primordial germ cell migration. These results demonstrate a role for beta1-containing integrins in the development of the germline, although an equivalent role for * integrin subunit(s) has yet to be established.

Establishment of novel monoclonal antibodies for identification of type A spermatogonia in teleosts†

2019 ◽  
Vol 101 (2) ◽  
pp. 478-491 ◽  
Author(s):  
Makoto Hayashi ◽  
Kensuke Ichida ◽  
Sakiko Sadaie ◽  
Misako Miwa ◽  
Ryo Fujihara ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Rainbow Trout ◽  
Cell Surface ◽  
Germ Cell ◽  
Cell Transplantation ◽  
Germ Cells ◽  
Type A ◽  
Germ Cell Transplantation ◽  
Type A Spermatogonia

AbstractWe recently established a germ cell transplantation system in salmonids. Donor germ cells transplanted into the body cavity of recipient embryos migrate toward and are incorporated into the recipient gonad, where they undergo gametogenesis. Among the various types of testicular germ cells, only type A spermatogonia (A-SG) can be incorporated into the recipient gonads. Enriching for A-SG is therefore important for improving the efficiency of germ cell transplantation. To enrich for A-SG, an antibody against a cell surface marker is a convenient and powerful approach used in mammals; however, little is known about cell surface markers for A-SG in fish. To that end, we have produced novel monoclonal antibodies (mAbs) against cell-surface molecules of rainbow trout (Oncorhynchus mykiss) A-SG. We inoculated mice with living A-SG isolated from pvasa-GFP transgenic rainbow trout using GFP-dependent flow cytometry. By fusing lymph node cells of the inoculated mice with myeloma cells, we generated 576 hybridomas. To identify hybridomas that produce mAbs capable of labeling A-SG preferentially and effectively, we screened them using cell ELISA, fluorescence microscopy, and flow cytometry. We thereby identified two mAbs that can label A-SG. By using flow cytometry with these two antibodies, we could enrich for A-SG with transplantability to recipient gonads from amongst total testicular cells. Furthermore, one of these mAbs could also label zebrafish (Danio rerio) spermatogonia. Thus, we expect these monoclonal antibodies to be powerful tools for germ cell biology and biotechnology.

scholarly journals Flow cytometric analysis of apoptosis in cryoconserved chicken primordial germ cells

2015 ◽  
Vol 20 (1) ◽  
Author(s):  
Dorota Sawicka ◽  
Luiza Chojnacka-Puchta ◽  
Marcin Zielinski ◽  
Grazyna Plucienniczak ◽  
Andrzej Plucienniczak ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Marker Gene ◽  
Flow Cytometric Analysis ◽  
Slow Freezing ◽  
Ex Situ ◽  
Blue Staining

AbstractOur research aimed to compare the effects of four cryoprotectants and four slow freezing programs on the viability and apoptosis of primordial germ cells (PGCs) in vitro. PGCs were collected from chicken embryonic blood at Hamburger and Hamilton (HH) stages 14-16 and purified by Percoll density gradient centrifugation and then subjected to cryopreservation. We applied microscopy to determine the survival of PGCs after trypan blue staining and flow cytometry to examine apoptosis and viability after annexin V kit staining. We also examined the functionality of cryopreserved PGCs in vivo. Significant differences in viability of PGCs determined via microscopy and flow cytometry were observed. The most unfavorable combination for slow freezing PGCs was program 3 and MIX H (10% DMSO and 5% glycerol in Hank’s solution supplemented with 10% FBS) as the cryoprotectant (48.43 and 15.37% live and early apoptotic PGCs, respectively). The highest average percentage of live PGCs (93.1%) and the lowest percentage of early apoptotic PGCs (6.5%) were achieved by slow freezing PGCs in the presence of DMSO F (10% DMSO in FBS) via program 1. Therefore, this method was chosen for the in vivo test. Cryopreserved (group 1) and freshly isolated (group 2) PGCs were transfectedwith a pEGFP-N1 plasmid, cultured under antibiotic selection, and then injected into 3-day-old embryos. After 5 days of incubation, we identified the EGFP marker gene in the gonads of 40 and 45% of recipients in groups 1 and 2, respectively. This is the first study to apply flow cytometry to examine the apoptosis and viability of cryopreserved PGCs. The in vitro and in vivo findings showed that the developed PGC cryoconservation method, depending on slow freezing at the rate of 2°C/min (program 1) in the presence of 10% DMSO F, is an improvement over previous cryoconservation methods and may be a useful tool for the ex situ strategy of poultry biodiversity preservation.

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