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.

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 The role of the NMD factor UPF3B in olfactory sensory neurons

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kun Tan ◽  
Samantha H Jones ◽  
Blue B Lake ◽  
Jennifer N Dumdie ◽  
Eleen Y Shum ◽  
...  
Keyword(s):  
Human Cognition ◽  
Cell Populations ◽  
Rna Seq ◽  
Wild Type ◽  
Single Cell Rna Sequencing ◽  
Nonsense Mediated Rna Decay ◽  
Different Levels ◽  
Selection Of

The UPF3B-dependent branch of the nonsense-mediated RNA decay (NMD) pathway is critical for human cognition. Here, we examined the role of UPF3B in the olfactory system. Single-cell RNA-sequencing (scRNA-seq) analysis demonstrated considerable heterogeneity of olfactory sensory neuron (OSN) cell populations in wild-type (WT) mice, and revealed that UPF3B loss influences specific subsets of these cell populations. UPF3B also regulates the expression of a large cadre of antimicrobial genes in OSNs, and promotes the selection of specific olfactory receptor (Olfr) genes for expression in mature OSNs (mOSNs). RNA-seq and Ribotag analyses identified classes of mRNAs expressed and translated at different levels in WT and Upf3b-null mOSNs. Integrating multiple computational approaches, UPF3B-dependent NMD target transcripts that are candidates to mediate the functions of NMD in mOSNs were identified in vivo. Together, our data provides a valuable resource for the olfactory field and insights into the roles of NMD in vivo.

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 Functions of TAM RTKs in regulating spermatogenesis and male fertility in mice

Reproduction ◽  
2009 ◽  
Vol 138 (4) ◽  
pp. 655-666 ◽  
Author(s):  
Yongmei Chen ◽  
Huizhen Wang ◽  
Nan Qi ◽  
Hui Wu ◽  
Weipeng Xiong ◽  
...  
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Tyrosine Kinases ◽  
Male Fertility ◽  
Leydig Cells ◽  
Seminiferous Tubules ◽  
Wild Type ◽  
Male Sterile ◽  
Triple Mutant ◽  
Insight Into

Mice lacking TYRO3, AXL and MER (TAM) receptor tyrosine kinases (RTKs) are male sterile. The mechanism of TAM RTKs in regulating male fertility remains unknown. In this study, we analyzed in more detail the testicular phenotype of TAM triple mutant (TAM−/−) mice with an effort to understand the mechanism. We demonstrate that the three TAM RTKs cooperatively regulate male fertility, and MER appears to be more important than AXL and TYRO3. TAM−/− testes showed a progressive loss of germ cells from elongated spermatids to spermatogonia. Young adult TAM−/− mice exhibited oligo-astheno-teratozoospermia and various morphological malformations of sperm cells. As the mice aged, the germ cells were eventually depleted from the seminiferous tubules. Furthermore, we found that TAM−/− Sertoli cells have an impaired phagocytic activity and a large number of differentially expressed genes compared to wild-type controls. By contrast, the function of Leydig cells was not apparently affected by the mutation of TAM RTKs. Therefore, we conclude that the suboptimal function of Sertoli cells leads to the impaired spermatogenesis in TAM−/− mice. The results provide novel insight into the mechanism of TAM RTKs in regulating male fertility.

scholarly journals Fertility and spermatogenesis are altered in α1b-adrenergic receptor knockout male mice

2007 ◽  
Vol 195 (2) ◽  
pp. 281-292 ◽  
Author(s):  
Sakina Mhaouty-Kodja ◽  
Anne Lozach ◽  
René Habert ◽  
Magali Tanneux ◽  
Céline Guigon ◽  
...  
Keyword(s):  
Germ Cells ◽  
Adrenergic Receptor ◽  
Male Fertility ◽  
Leydig Cells ◽  
Critical Role ◽  
Wild Type ◽  
Cell Death Pathway ◽  
Testicular Weight ◽  
Circulating Levels

To examine whether norepinephrine, through activation of α1b-adrenergic receptor, regulates male fertility and testicular functions, we used α1b-adrenergic receptor knockout (α1b-AR-KO) mice. In the adult stage (3–8 months), 73% of the homozygous males were hypofertile with relatively preserved spermatogenesis. Of the remaining males, 27% exhibited a complete infertility with a drastic reduction in testicular weight and spermatogenesis defect with germ cells entering a cell death pathway at meiotic stage. In both phenotypes, circulating levels of testosterone were highly reduced (−55 and −81% in hypofertile and infertile males respectively versus wild-type males). Consequently, circulating levels of LH were significantly elevated in α1b-AR-KO infertile mice. When incubated in vitro, the whole testes from infertile KO mice released significantly lower levels of testosterone (−40%). This, together with the fact that the mean absolute volume of Leydig cells per testis was not changed, suggests a compromised steroidogenic capacity of Leydig cells in infertile animals. In addition, RNA in situ hybridization study indicated an apparent higher expression of inhibin α- and βB-subunits in Sertoli cells of infertile α1b-AR-KO mice. This was correlated with a higher intra-testicular content of inhibin B (+220% above wild-type mice). Using specific primers, mRNA encoding α1b-AR was localized in early spermatocytes of wild-type testes. Our results indicate, for the first time, that α 1b-AR signaling plays a critical role in the control of male fertility, spermatogenesis, and steroidogenic capacityof Leydig cells. It is thus hypothesized that the absence of α1b-AR alters either directly germ cells or indirectly Sertoli cell/Leydig cell communications in infertile α1b-AR-KO mice.

scholarly journals Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice

2006 ◽  
Vol 27 (3) ◽  
pp. 1146-1157 ◽  
Author(s):  
Chantal Kress ◽  
Carole Gautier-Courteille ◽  
H. Beverley Osborne ◽  
Charles Babinet ◽  
Luc Paillard
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Leydig Cells ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Cell Number ◽  
Pcr Analysis ◽  
Wild Type ◽  
Mammalian Development ◽  
Males And Females

ABSTRACT CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1 − / − mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1 − / − males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1 − / − males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.

249. Regulated expression of KIT protein in juvenile and adult germ cells of the rodent testis

2008 ◽  
Vol 20 (9) ◽  
pp. 49
Author(s):  
S. Mithraprabhu ◽  
C. W. Brown ◽  
M. M. Matzuk ◽  
K. L. Loveland
Keyword(s):  
Protein Expression ◽  
Germ Cells ◽  
Wild Type Mouse ◽  
Erythroid Differentiation ◽  
Activin A ◽  
Mrna Levels ◽  
Wild Type ◽  
Kit Receptor ◽  
Murine Erythroleukemia

KIT receptor is an established marker of differentiating spermatogonia and its activation is required to trigger spermatogonial maturation. KIT mRNA, however, can be detected in undifferentiated spermatogonia in the absence of protein expression, as previously established by us in the irradiated adult rat testes [1]. This differential regulation of mRNA and protein is presumably modulated by either local hormone action or by cues from the adult testicular microenvironment. Endogenous regulatory factors known to stimulate KIT synthesis in juvenile male germ cells in vitro are bone morphogenetic protein 4 (BMP4) and retinoic acid (RA), while factors known to suppress KIT at the onset of spermatogenesis have not yet been identified. Activin A, implicated in KIT downregulation in a murine erythroleukemia cell line [2], is produced within the juvenile mammalian testis and influences activities of spermatogonia and Sertoli cells. We hypothesised that activin acts to repress KIT expression in spermatogonia and therefore modulate spermatogonial behaviour. Evidence for this was first derived from Sertoli and germ cell co-cultures of day 8 wild type mouse testes in which exogenous activin addition caused a dose-dependent reduction of KIT mRNA. Whole testes mRNA analyses of two activin transgenic mouse models, the newborn Inhba−/− (lacking activin A) and postnatal InhbaBK/BK (decreased bioactive activin), revealed a significant elevation in KIT expression relative to wild type littermates. In the postnatal day 7 InhbaBK/BK testes, an elevated proportion of differentiated spermatogonia, increased cell surface KIT protein levels, enhanced mRNA levels of a known downstream target of KIT signalling pathway, cyclind3 and a meiotic marker, Sycp3, were observed. These data provide the first comprehensive evidence for activin modulation of KIT expression at spermatogenesis onset, in germ cells of the juvenile testis. This finding is of fundamental importance to other KIT-dependent processes. (1) Prabhu, S.M., et al. Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis. Reproduction, 2006. 131(3): p. 489–99. (2) Hino, M., et al. Down-modulation of c-kit mRNA and protein expression by erythroid differentiation factor/activin A. FEBS Lett, 1995. 374(1): p. 69–71.

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.

Evaluation of flow-cytometry for the monitoring of infectious human rotavirus in water

1997 ◽  
Vol 35 (11-12) ◽  
pp. 451-453
Author(s):  
F. X. Abad ◽  
A. Bosch ◽  
J. Comas ◽  
D. Villalba ◽  
R. M. Pintó
Keyword(s):  
Flow Cytometry ◽  
Water Samples ◽  
Wild Type ◽  
Cell Monolayers ◽  
Naturally Occurring ◽  
Human Rotavirus ◽  
Faecal Samples

A method has been developed for the detection of infectious human rotavirus (HRV), based on infection of MA104 and CaCo-2 cell monolayers and ulterior flow cytometry. The sensitivity of the flow cytometry procedure for the cell-adapted HRV enabled the detection of 200 and 2 MPNCU in MA104 and CaCo-2 cells, respectively. Flow cytometry performed five days after infection of CaCo-2 enabled the detection of naturally occurring wild-type HRV in faecal samples and concentrated water samples.

Aromatase and 11β-hydroxysteroid dehydrogenase 2 localisation in the testes of pigs from birth to puberty linked to changes of hormone pattern and testicular morphology

2008 ◽  
Vol 20 (4) ◽  
pp. 505 ◽  
Author(s):  
A. Wagner ◽  
R. Claus
Keyword(s):  
Blood Plasma ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Blood Collection ◽  
Testicular Development ◽  
Post Mortem ◽  
Cell Numbers ◽  
Testicular Morphology

Oestrogens and glucocorticoids are important for spermatogenesis and are regulated via aromatase for oestradiol synthesis and 11β-hydroxysteroid dehydrogenase 2 (11β-HSD 2) as an inactivator of cortisol. In the present study postnatal changes of these two enzymes were monitored together with testicular development and hormone concentrations. Pigs were assigned to three periods: Weeks 0–5, Weeks 5–11 or Weeks 11–17. In Period 1, groups of four piglets were killed after each week. Blood plasma and testes were sampled immediately post mortem. For Periods 2 and 3, groups of six pigs were fitted with vein catheters for daily blood collection. Testes from all pigs were obtained after killing. Levels of testosterone, oestradiol, LH, FSH and cortisol were determined radioimmunologically. The 11β-HSD 2- and aromatase-expressing cells were stained immunocytochemically. All hormones were maximal 2 weeks after birth. A rise of LH, testosterone and oestradiol occurred again at Week 17. FSH and cortisol remained basal. Parallel to the first postnatal rise, the presence of aromatase and 11β-HSD 2 in Leydig cells increased, together with germ and Sertoli cell numbers. Expression was low from 3 to 5 weeks, was resumed after Week 5 and was maximal at Week 17. The amount of 11β-HSD 2 in germ cells was greatest at birth, decreased thereafter and was absent after Week 3.

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