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.

288 THE EXPRESSION PATTERN OF ACETYLATED ALPHA-TUBULIN IS CONSERVED IN PORCINE AND MURINE SPERMATOGONIAL STEM CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 223
Author(s):  
J. Luo ◽  
S. Megee ◽  
I. Dobrinski
Keyword(s):  
Stem Cells ◽  
Basement Membrane ◽  
Expression Pattern ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Histone Deacetylase 6 ◽  
Pgp 9.5

During mammalian spermatogenesis, spermatogonial stem cells (SSCs) reside in the stem cell niche on the basement membrane where they undergo self-renewing divisions. Differentiating daughter cells are located progressively more toward the tubular lumen where they ultimately form spermatozoa. The mechanisms responsible for maintenance of SSCs at the basement membrane are unclear. Microtubules consisting of α/β-tubulin heterodimers are associated with many cellular functions. Reversible acetylation of α-tubulin at Lys40 has been implicated in regulating microtubule stability and function. Acetylation of α-tubulin is abundant in stable microtubules but absent from dynamic cellular structures. Deacetylation of α-tubulin is controlled by histone deacetylase 6 which is predominantly expressed in mouse testis. Here, we tested the hypothesis that differential acetylation of α-tubulin might be involved in maintenance of SSCs. Immunohistochemistry for acetylated α-tubulin (Ac-α-Tu) and the spermatogonia specific proteins PGP 9.5, DAZL, and PLZF were used to characterize the expression pattern of Ac-α-Tu in porcine and murine germ cells at different stages of testis development. In immature boar testes, Ac-α-Tu was present exclusively in gonocytes but not in other testicular cells at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating to the basement membrane of the seminiferous tubules, and Ac-α-Tu appeared to be polarized toward the basement membrane. In immature mouse testes, Ac-α-Tu was present in germ cells and Sertoli cells at 6 days of age, whereas at 2 weeks of age, Ac-α-Tu expression was stronger in spermatogonia co-expressing PGP 9.5 and in spermatocytes than in Sertoli cells or PGP 9.5-negative spermatogonia. In adult boar and mouse testes, Ac-α-Tu was detected in a few single or paired spermatogonia expressing PGP 9.5 localized on the basement membrane as well as in spermatocytes, spermatids, and spermatozoa. Spermatogonia with high levels of Ac-α-Tu expressed PLZF but did not express DAZL, suggesting that only undifferentiated spermatogonia maintain a high level of Ac-α-Tu. When seminiferous tubules from 1-week-old and adult boar testes were maintained in vitro for 1–2 days, high levels of Ac-α-Tu were detected in single or paired round spermatogonia with a large nucleus, compared to low levels in elongated paired and aligned spermatogonia. The unique expression pattern of Ac-α-Tu in undifferentiated germ cells during postnatal development appears to be conserved in mammalian testes. Since Ac-α-Tu is a component of long-lived stable microtubules and reducing acetylation of α-tubulin enhances cell motility, these results suggest that stabilization of microtubules might contribute to the maintenance of spermatogonial stem cells. This work was supported by 1R01 RR 17359-05.

scholarly journals Preservation of fertility in nature and ART

Reproduction ◽  
2002 ◽  
pp. 3-11 ◽  
Author(s):  
R Gosden ◽  
M Nagano
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Reproductive Technologies ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Natural Fertility ◽  
Fertilization In Vitro ◽  
Gonadal Failure

Individuals may regard reproduction as optional but sufficient number of them must be productive to perpetuate the species. The reproductive system is surprisingly vulnerable and depends, among other things, on a limited endowment of oocytes, controlled proliferation of spermatogonial stem cells and the genetic integrity of both. The developmental competence of oocytes and spermatogonial stem cells is maintained by evolved mechanisms for cellular detoxification and genomic stability, and excess or damaged cells are eliminated by apoptosis. Gonadal failure as a result of germ cell depletion can occur at any age, and from the effects of chemical cytotoxicity, disease and infection as well as genetic predisposition. Among extrinsic factors, alkylating agents and ionizing radiation are important causes of iatrogenic gonadal failure in young women and men. In animal models, there is evidence that hormonal manipulation, deletion of genes involved in apoptotic pathways and dietary manipulation can protect against natural and induced germ cell loss, but evidence in humans is absent or unclear. Assisted reproductive technologies (ARTs) provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Semen cryopreservation was the first technology for preserving male fertility, but this cannot serve prepubertal boys, for whom banking of testicular biopsies may provide a future option. In sterilized rodents, cryopreserved spermatogonial stem cells can recolonize seminiferous tubules and reinitiate spermatogenesis, and subcutaneous implantation of intact tubules can generate spermatozoa for fertilization in vitro by intracytoplasmic sperm injection. Transplantation of frozen-banked ovarian tissue is well-established for restoring cyclicity and fertility and is currently undergoing clinical evaluation for cancer patients. When restoration of natural fertility is unnecessary or reimplantation is unsafe, it is desirable to culture the germ cells from thawed tissue in vitro until they reach the stage at which they can be fertilized. Low temperature banking of immature germ cells is potentially very versatile, but storage of embryos and, to a lesser extent, mature oocytes is already practised in a number of species, including humans, and is likely to remain a mainstay for fertility preservation.

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.

Differentiation of Human Male Germ Cells From Human Umbilical Cord-Derived Mesenchymal Stem Cells in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4581-4581
Author(s):  
Lian Ma ◽  
Xiaoying Wu ◽  
Limin Lin ◽  
Guangyu Lin ◽  
Qiuling Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Umbilical Cord ◽  
Germ Cells ◽  
Seminiferous Tubules ◽  
Human Umbilical Cord ◽  
Cell Markers ◽  
Human Male

Abstract Abstract 4581 Introduction Infertility affects 15% of couples, about 50% infertility caused by male and growing evidence suggested an increasing problems in male reproductive. Recent studies have demostrated that adult stem cells have more flexible potentials than expected, and possessed the plasticity and capacity to transdifferentiate into mutilineage cells, including germ cells. Human umbilical cord-derived mesenchymal stem cells (HUCMSCs) possess stem cell properties. In this study, we cultured HUCMSCs, and assessed the possibility of HUCMSCs differentiated into human male germ cells in vivo and in vitro, and find a new source of cells for the transplantation to the male infertility. Methods The ethics committee of our institution approved this study. HUCMSCs were isolated from the Wharton's jelly of the umbilical cord, clonally expanded. To investigate the capacity of differentiation in vitro, HUCMSCs were treated with human menopausal gonadotropinn (HMG) and retinoid acid (RA) in vitro. While investigate the capacity of differentiation in vivo, HUCMSCs were transplanted into the seminiferous tubules of busulfan-treated mice testes after labeled with pIRES2-EGFP or bromodeoxyuridine (BrdU). After induction in vitro, the morphologic changes of the differentiated cells were detected by phase contrast microscopy?Aelectron microscopy and transmission electron microscope?Gthe male germ cell markers were detected by immunohistochemistry, Western-blot and PT-PCR. HUCMSCs were also transplanted into the seminiferous tubules of the busulfan-treated mice by microinjection. To assess the fate of HUCMSCs in the testis, the survival?Amigration and germ cell markers of the HUCMSCs in the infertility mice testis were detected by immunohistochemistry?A immunofluorescence. Results HUCMSCs can express some some molecular markers of germ cells after induction. Immunohistochemistry revealed that HUCMSCs can survive in the mice testis at least 120 days, and they can migrate from the lumens to the basement membrane then to lumens again. Immunofluorescence showed that HUCMSCs can go further differentiation in the mice favorable testicular environment, and express the germ cell markers. Conclusions These suggested that HUCMSCs may differentiate into male germ cell-like cells after induced by HMG and RA in vitro; and it could survive also in a favorable testicular environment, may differentiate into germ cell lineages. This finding may provide a new strategy for the treatment of male infertility. Disclosures: No relevant conflicts of interest to declare.

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.

428 PRODUCTION OF GERM-LINEAGE CELL AND FUNCTIONAL GAMETES FROM MULTI-POTENT SPERMATOGONIAL STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 371
Author(s):  
J. E. Lim ◽  
J. H. Eum ◽  
H. J. Kim ◽  
H. S. Lee ◽  
J. H. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Culture Medium ◽  
Genetically Modified ◽  
Spermatogonial Stem Cells ◽  
Specific Gene ◽  
Specific Marker ◽  
Male Gametes ◽  
Genetically Modified Mice

Multi-potent spermatogonial stem cells (mSSC), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESC). Similar to ESC, mSSC are capable of differentiating into 3-germ layers in vitro and teratoma formation in vivo. Additionally, mSSC proliferate rapidly and can be transfected more easily than SSC. In contrast to previous reports, we have found that mSSC also have germ-cell-specific micro (mi)RNA and gene expression profiles. Therefore, the aims of this study were to compare the efficiency of mSSC v. ESC to differentiate into germ lineage and produce male gametes, as well as to develop a novel system for the production of genetically modified mice. Mouse mSSC were transfected with a lentiviral vector expressing green fluorescent protein (GFP) and testis-specific gene and maintained in the ESC-culture medium containing leukemia inhibitory factor (LIF). Embryonic bodies (EB) were formed after the cells were detached from the feeder cells. Bone morphogenetic protein (BMP)-4 (10 ng mL˜1) and retinoic acid (RA, 0.1 μM) were added to the ESC-culture medium for 3 days in order to induce differentiation into germ lineage cells. Then, these cells were changed to germ cell-culture medium (Stem-Pro™ containing GDNF; Invitrogen, Carlsbad, CA, USA) and cultured for 3 days. After 6 days, cultured cells were sorted by magnetic activating cell sorting system using specific marker for germ cells, CD-9. Isolated germ lineage cells were transplanted into a busulfan-treated mouse testis for the production of male germ cells. Three to 6 weeks later, the testis and epididymis were collected, and half of the sample was used to perform histological analysis and the other half for the production of intracytoplasmic sperm injection (ICSI)-derived embryos. The statistical significance of differences between the 2 groups was evaluated by Student’s t-test Immunocytochemical and flow cytometrical analysis performed 6 days after differentiation showed that the ratio of germ cell-specific markers in EB derived from mSSC was higher than those from ESC. Moreover, after 3 to 6 weeks of transplantation the testis produced sperms and germ cells expressing GFP. We have successfully produced embryos by ICSI and offspring by embryo transfer into uteri of poster mothers. These results demonstrate that mSSC can be easily differentiated into germ lineage cells compared with ESC and have the potential to generate functional gametes. Therefore, the differentiation and transgenesis of mSSC may be a useful model for production of genetically modified mice. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084923).

Xenogeneic and endogenous spermatogenesis following transplantation of rat germ cells into testes of immunocompetent mice

2012 ◽  
Vol 24 (2) ◽  
pp. 337 ◽  
Author(s):  
Ning Qu ◽  
Munekazu Naito ◽  
Jun Li ◽  
Hayato Terayama ◽  
Shuichi Hirai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Response ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Recipient Mouse ◽  
Immunocompetent Mice ◽  
Rat Spermatogenesis ◽  
Privileged Site

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and are characterised by their ability to self-renew and to produce differentiated progeny that form spermatozoa. It has been demonstrated that rat spermatogenesis can occur in the seminiferous tubules of congenitally immunodeficient recipient mice after transplantation of rat SSCs. However, the testis is often viewed as an immune-privileged site in that autoimmunogenic antigens on germ cells do not normally elicit an immune response in situ. In the present study, we tried to transplant rat SSCs into immunocompetent mice after depletion of their own germ cells by means of busulfan. The results showed that some transplanted SSCs could undergo complete spermatogenesis in recipient mouse testes, the rat spermatozoa being detected in 7 of 28 recipient epididymides. A significant increase in mouse spermatozoa was also noted in all 28 epididymides of recipient mice regardless of whether rat spermatozoa were concurrently present or not. These results suggest that transplanted rat SSCs can be tolerated in the testes of immunocompetent mice and that the transplantation of rat SSCs stimulates endogenous spermatogenesis in the recipient mice.

The development of the testis of the Merino ram, with special reference to the orgin of the adult stem cells

1962 ◽  
Vol 13 (3) ◽  
pp. 487 ◽  
Author(s):  
CS Sapsford
Keyword(s):  
Stem Cells ◽  
Basement Membrane ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Adult Stem Cells ◽  
Germ Line ◽  
Primordial Germ Cells ◽  
Seminiferous Tubules ◽  
Stages Of Development ◽  
Striking Change

In the ram, as in other mammals, the sex cords are made up of two types of cell: indifferent cells (derivatives of the coelomic epithelium) and primordial germ cells. In the cords, each type pursues a separate and independent line of development to become respectively the Sertoli cells and the stem cells (type A spermatogonia) of the adult testis. The principal changes taking place in the primordial germ cells (gonocytes) are a reduction in the size and number of the Feulgen-positive particles in the nuclei, the appearance and subsequent fusion of the nucleoli, and, finally, an increase in the size of the nuclei. While these changes are taking place, the cytoplasm increases in volume and inclusions become more numerous. Cells which have undergone all these transformations have been called prospermatogonia. The cells of the germ line are at first more centrally placed in the sex cords than the indifferent cells. Just before spermatogenesis begins, they migrate to the basement membrane of the seminiferous tubules. All germ cells in tubules in which spermatogenesis has been initiated are seen as prospermatogonia. These cells become flattened against the basement membrane, and their nuclei become more oval in shape. They thus become identical with the stem cells of the adult. Little change is evident in the nuclei of the indifferent cells until puberty. Feulgen-positive material is found in the form of coarse granules at earlier stages of development. At puberty, these granules become dispersed to give a much more homogeneous nucleus. Concurrently, nuclei increase in size, and single or double true nucleoli can be identified. During development, increases in cytoplasmic volume take place. Although cell boundaries between indifferent cells cannot be seen in fixed material, phase contrast observations of fresh material have demonstrated that some forms exist as mononucleate units. It could not be determined whether the same was true in the case of Sertoli cells. No striking change in the relative numbers of glandular interstitial cells could be observed at different stages of development.

229 LONG-TERM PROPAGATION AND CRYOPRESERVATION OF CAT SPERMATOGONIAL STEM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 246
Author(s):  
L. M. Vansandt ◽  
M. Dickson ◽  
R. Zhou ◽  
L. Li ◽  
B. S. Pukazhenthi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Domestic Cat ◽  
Feeder Cell ◽  
Cell Clusters ◽  
Fetal Fibroblasts

Spermatogonial stem cells (SSC) are unique adult stem cells that reside within the seminiferous tubules of the testis. As stem cells, SSC maintain the ability to self-replicate, providing a potentially unlimited supply of cells and an alternate source for preservation of the male genome. While self-renewing, long-term SSC culture has been achieved in mice, there is virtually no information regarding culture requirements of felid SSC. Therefore, the objectives of this study were to (1) evaluate the ability of 3 feeder cell lines to support germ cell colony establishment in domestic cats (Felis catus), and (2) assess long-term culture using the best feeder(s). Cells isolated enzymatically from peripubertal cat testes (n = 4) and enriched by differential plating were cultured on mouse embryonic fibroblasts (STO line), mouse-derived C166 endothelial cells, and primary cat fetal fibroblasts (cFF). Colony morphology was assessed every other day and immunocytochemistry (ICC) was performed to investigate expression of SSC markers. At 5 days in vitro (DIV), a cluster forming activity assay was used to estimate the number of SSC supported by each feeder cell line. Differences among treatments were compared using Tukey-Kramer adjustment for pair-wise mean comparisons. Data were expressed as mean cluster number ± SE per 105 cells input. When cultured on STO feeders, cat germ cells were distributed as individual cells. On both C166 cells and cFF feeders, germ cell clumps (morphologically consistent with SSC colonies in other species) were observed. Immunocytochemistry revealed that the single germ cells present on STO feeders were positive for UCHL1 and weakly expressed PLZF and OCT4. Cells within the germ cell clumps on C166 cells and cFF co-expressed all 3 SSC markers. The C166 cells supported a higher number of germ cell clusters (77.4 ± 13.8) compared with STO (3.5 ± 1.1, P = 0.0003) or cFF (22.7 ± 1.0, P = 0.0024). Therefore, subsequent subculture experiments were performed exclusively with C166 feeder layers. Cultures from 2 donors were passaged at 12 DIV and periodically as needed thereafter. Germ cell clumps consistently reestablished following each subculture and immunocytochemistry analysis confirmed maintenance of all 3 SSC markers. Cells were also positive for alkaline phosphatase activity. Cells that had been cryopreserved in culture medium with 5% (vol/vol) dimethyl sulphoxide after144 DIV (7 passages) were thawed and cultured for an additional 18 days. These cells continued to express SSC markers and form germ cell clusters. Taken together, these data demonstrate that C166 feeder cells can facilitate colony establishment and in vitro propagation of germ cell clumps in the domestic cat. This represents an important first step towards attainment and optimization of a long-term SSC culture system in the cat. This system would provide a mechanism to explore regulation of spermatogenesis, test species-specific drugs, and produce transgenic biomedical models.

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