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.

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.

219 ISOLATION AND CHARACTERIZATION OF DOMESTIC CAT SPERMATOGONIAL CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 221 ◽  
Author(s):  
R. H. Powell ◽  
M. N. Biancardi ◽  
C. E. Pope ◽  
S. P. Leibo ◽  
G. Wang ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Population ◽  
Mammalian Species ◽  
Domestic Cat ◽  
Seminiferous Tubules ◽  
Specific Cell ◽  
Surface Markers ◽  
Isolation And Characterization ◽  
Positive Expression ◽  
Number Of Cells

Spermatogonial stem cells (SSC) have the capacity for self-renewal and the potential of producing progenitor spermatogonia that will differentiate into spermatozoa. SSC transplantation may be a valuable alternative for the propagation of genetically important males and preservation of endangered wild felids, as recently demonstrated by the production of ocelot spermatozoa after the xeno-transplantation of a mixed germ cell population into the testis of a domestic cat (Silva et al. 2011 J. Androl.). SSC are in low numbers in the testis and have been isolated in different mammalian species by using specific cell surface markers; however, the expression of SSC-surface markers in feline species has not been characterised. In the present study, testes of domestic cats were obtained from veterinary clinics. The selected testes (n = 4) ranged in size from 1.3 to 2.0 cm in length. To obtain a suspension of a mixed population of spermatogonial cells, seminiferous tubules were enzymatically dissociated using two digestion steps followed by dual filtration through 100-μm and 40-μm nylon mesh filters and a final separation over a 5-layer Percoll™ density gradient (35, 30, 27.5, 25 and 20%). Spermatogonial cells were morphologically identified by their characteristic large round nucleus with a homogenous appearance in the bands formed at the 30%, 27.5% and 25% layers. The mean number of cells/testis collected was ∼13 × 106 ± 12.2, with an overall percentage of live cells of 97%. After isolation, cells were fixed with 4% PFA, blocked overnight and stained with primary antibodies specific for SSC-surface markers (CD49f, CD9, C-Kit, GFRα1, GPR125 and Thy-1) and pluripotent stage-specific embryonic antigen markers (SSEA-1 and SSEA-4). Fluorescence microscopy showed a positive expression of GFRα1, GPR125 and C-Kit, but not for CD49f, CD9, or Thy-1. It also revealed that a low number of cells were positive for SSEA-1 and SSEA-4. For further characterisation, molecular detection of the pluripotent gene Oct-4 and the germ cell-specific genes BOLL, DAZL and VASA was performed in germ cells isolated from one testis of four individuals. For RT-qPCR, the Cells-to-cDNA™ II kit (Ambion) was used to produce cDNA from an aliquot of ∼30 000 cells directly after isolation. RT-qPCR showed that none had detectable levels of Oct-4 within the range of the standard. Three of the four testes expressed all three germ cell-specific genes, BOLL, DAZL and VASA, while only VASA was detected in the remaining testis. These results suggest that cat SSCs and spermatogonial cells express some of the SSC markers tested. However, the positive expression of SSEA-1 and SSEA-4 in a low number of cells further supports the stem cell-like state of cat SSCs and that these markers can be used in dual staining for purifying cat SSCs from a mixed germ cell population by fluorescence-activated cell sorting.

Male germ cell transplantation: promise and problems

2001 ◽  
Vol 13 (8) ◽  
pp. 609 ◽  
Author(s):  
Fang-Xu Jiang
Keyword(s):  
Germ Cell ◽  
Cell Transplantation ◽  
Germ Cells ◽  
Cell Biology ◽  
Direct Injection ◽  
Primordial Germ Cells ◽  
Rete Testis ◽  
Male Germ Cell ◽  
Seminiferous Tubules ◽  
Germ Cell Transplantation

Male germ cell transplantation is a novel technique in which donor male stem germ cells are surgically transferred to the seminiferous tubules of a recipient testis by direct injection or via the rete testis or efferent duct. All germ cells that are destined to become stem spermatogonia are defined as male stem germ cells, including primordial germ cells from the gonadal ridges, and gonocytes and stem spermatogonia from the testis, all of which are transplantable and capable of undergoing normal spermatogenesis. Xenotransplantation of male germ cells from one species into the testis of another species, including human testicular cells in the mouse, has so far proved to be unsuccessful. However, the immunodeficient mouse testis can support rat spermatogenesis and produce apparently normal rat spermatozoa. The underlying mechanisms remain elusive. The present mini-review will focus on the importance of stem spermatogonial transplantation for testicular stem cell biology and discuss the likelihood of immune rejection after transplantation, which may limit the success of all male germ cell transplantation.

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.

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 A novel Amh-Treck transgenic mouse line allows toxin-dependent loss of supporting cells in gonads

Reproduction ◽  
2014 ◽  
Vol 148 (6) ◽  
pp. H1-H9 ◽  
Author(s):  
Mai Shinomura ◽  
Kasane Kishi ◽  
Ayako Tomita ◽  
Miyuri Kawasumi ◽  
Hiromi Kanezashi ◽  
...  
Keyword(s):  
Germ Cells ◽  
Granulosa Cells ◽  
Sertoli Cells ◽  
Seminiferous Tubules ◽  
Specific Cell ◽  
Partial Recovery ◽  
Dependent Manner ◽  
Mouse Line ◽  
Cell Degeneration

Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti-Müllerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DT injection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads.

scholarly journals The rete testis harbors Sertoli-like cells capable of expressing DMRT1

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. 399-413 ◽  
Author(s):  
Ekaterina A Malolina ◽  
Andrey Yu Kulibin
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Adult Mouse ◽  
Rete Testis ◽  
Seminiferous Tubules ◽  
Rna Seq ◽  
Supporting Cells ◽  
Testicular Cells ◽  
Qpcr Analysis ◽  
Different Levels

Sertoli cells (SCs) are supporting cells in the mammalian testis that proliferate throughout fetal and postnatal development but exit the cell cycle and differentiate at puberty. In our previous study, we isolated a population of highly proliferative Sertoli-like cells (SLCs) from the region of the adult mouse testis containing the rete testis and adjacent seminiferous tubules. Here RNA-seq of the adult SLC culture as well as qPCR analysis and immunofluorescence of the adult and immature (6 dpp) SLC cultures were performed that allowed us to identify SLC-specific genes, including Pax8, Cdh1, and Krt8. Using these, we found that SLCs are mostly localized in the rete testis epithelium; however, some contribution of transitional zones of seminiferous tubules could not be excluded. The main feature of SLCs indicating their relationship to SCs is DMRT1 expression. More than 40% of both adult and immature SLCs expressed DMRT1 at different levels in culture. Only rare DMRT1+ cells were detected in the adult rete testis, whereas more than 40% of cells were positively stained for DMRT1 in the immature rete testis. One more SC protein, AMH, was found in some rete cells of the immature testis. It was also demonstrated that SLCs expressed such SC genes as Nr5a1, Dhh, Gdnf, and Kitl and interacted with germ cells in 3D co-culture with immature testicular cells. All these similarities between SLCs and rete cells on one the hand and SCs on the other, suggest that rete cells could share a common origin with SCs.

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.

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.

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