72 PRESERVATION OF PORCINE GONOCYTES AT 4°C AND IN LIQUID NITROGEN - A PRESERVATION MODEL OF GENETIC RESOURCES IN DOMESTIC ANIMALS AND IN ENDANGERED SPECIES

2008 ◽  
Vol 20 (1) ◽  
pp. 117
Author(s):  
M. Fujihara ◽  
S. Goel ◽  
Y. Kimura ◽  
N. Minami ◽  
M. Yamada ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endangered Species ◽  
Cell Viability ◽  
Genetic Resources ◽  
Liquid Nitrogen ◽  
Germ Cells ◽  
Domestic Animals ◽  
Spermatogonial Stem Cells ◽  
Freezing And Thawing ◽  
Neonatal Testis

Gonocytes are primitive germ cells that reside in neonatal testis and are believed to be progenitor-type stem cells that differentiate into spermatogonial stem cells. Because of their self-renewal ability, gonocytes may be one of the targets for cryopreservation of genetic resources in domestic animals and in endangered species. However, there are only a few reports regarding the preservation of gonocytes and spermatogonial stem cells isolated from the testis. In this experiment, porcine gonocytes were used as a model for preservation of genetic resources. Porcine testes were collected at 2–6 days after birth. They were divided into the 5 experimental groups for storage: (1) DMEM/F12 medium, (2) DMEM/F12 + 15 mm HEPES, (3) PBS, (4) PBS + 15 mm HEPES, and (5) Liquid-Free, and stored at 4�C for 24 h. The testes were minced by scissors and digested with 2-step enzyme treatments. The gonocytes were isolated by Percoll density gradients and recovered from the fraction between 50 and 60%. The viability of cells was assessed using trypan blue dye exclusion. To determine optimum cryopreservation conditions for gonocytes, 10% DMSO, 10% glycerol, and 0.07 mm sucrose were used as cryoprotectants. The isolated gonocytes were suspended in DMEM/F12 + 10% FBS containing cryoprotectant at 4�C, kept at –80�C overnight, and finally immersed in liquid nitrogen. After freezing and thawing of gonocytes, cells were examined for viability and then cultured in DMEM/F12 + 10% FBS in 5% CO2, 95% air at 37�C in humidified atmosphere. Identification of gonocytes was performed using a specific marker of gonocytes, a lectin Dolichos biflorus agglutinin (DBA; Goel et al. 2007 Biol. Reprod. 77, 127–137). The gonocytes were recovered from testes at the purity level of around 70%. Cell viability in average after storage of testes at 4�C was significantly higher in DMEM/F12 + HEPES (95.3%) and PBS + HEPES (89.8%) than in DMEM/F12 (73.9%), PBS (79.7%), and Liquid-Free (72.2%) (P < 0.05; ANOVA). The addition of HEPES in storage medium seemed to be effective for improving cell viability. The use of 10% DMSO and 0.07 mm sucrose as cryoprotectants supported high cell viability (74.4%) of gonocytes after freezing and thawing. The addition of glycerol had an adverse effect on cell viability after freezing (18.3%). When cells were cultured, gonocytes started to form colonies after 3 days and continued to proliferate for at least 7 days in culture. These colonies showed DBA affinity and maintained their nature as gonocytes. The viability of gonocytes can be maintained in the testis at 4�C for at least 24 h and after freezing and thawing. The stored gonocytes successfully proliferated in culture for at least 7 days. In conclusion, these results may provide useful information for short-term storage of primitive germ cells and preservation of genetic resources in domestic animals and in endangered species. It may also have implications for assisted reproductive technology in humans.

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).

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.

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.

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.

scholarly journals In VitroCulture-Induced Pluripotency of Human Spermatogonial Stem Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Jung Jin Lim ◽  
Hyung Joon Kim ◽  
Kye-Seong Kim ◽  
Jae Yup Hong ◽  
Dong Ryul Lee
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Embryoid Bodies ◽  
Pluripotent Cells ◽  
Induced Pluripotency ◽  
Derivatives Of ◽  
Direct Induction

Unipotent spermatogonial stem cells (SSCs) can be transformed into ESC-like cells that exhibit pluripotencyin vitro. However, except for mouse models, their characterization and their origins have remained controversies in other models including humans. This controversy has arisen primarily from the lack of the direct induction of ESC-like cells from well-characterized SSCs. Thus, the aim of the present study was to find and characterize pluripotent human SSCs inin vitrocultures of characterized SSCs. Human testicular tissues were dissociated and plated onto gelatin/laminin-coated dishes to isolate SSCs. In the presence of growth factors SSCs formed multicellular clumps after 2–4 weeks of culture. At passages 1 and 5, the clumps were dissociated and were then analyzed using markers of pluripotent cells. The number of SSEA-4-positive cells was extremely low but increased gradually up to ~ 10% in the SSC clumps during culture. Most of the SSEA-4-negative cells expressed markers for SSCs, and some cells coexpressed markers of both pluripotent and germ cells. The pluripotent cells formed embryoid bodies and teratomas that contained derivatives of the three germ layers in SCID mice. These results suggest that the pluripotent cells present within the clumps were derived directly from SSCs duringin vitroculture.

scholarly journals Does co-transplantation of mesenchymal and spermatogonial stem cells improve reproductive efficiency and safety in mice?

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Prashant Kadam ◽  
Elissavet Ntemou ◽  
Jaime Onofre ◽  
Dorien Van Saen ◽  
Ellen Goossens
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Transforming Growth Factor Beta ◽  
Dna Methyltransferase ◽  
Transforming Growth Factor ◽  
Spermatogonial Stem Cells ◽  
Reproductive Efficiency ◽  
Control Group ◽  
Low Efficiency

Abstract Background Spermatogonial stem cell transplantation (SSCT) is a promising therapy in restoring the fertility of childhood cancer survivors. However, the low efficiency of SSCT is a significant concern. SSCT could be improved by co-transplanting transforming growth factor beta 1 (TGFβ1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGFβ1-induced MSCs. Methods A mouse model for long-term infertility was used to transplant SSCs (SSCT, n = 10) and a combination of SSCs and TGFβ1-treated MSCs (MSi-SSCT, n = 10). Both transplanted groups and a fertile control group (n = 7) were allowed to mate naturally to check the reproductive efficiency after transplantation. Furthermore, the testes from transplanted males and donor-derived male offspring were analyzed for the epigenetic markers DNA methyltransferase 3A (DNMT3A) and histone 4 lysine 5 acetylation (H4K5ac). Results The overall tubular fertility index (TFI) after SSCT (76 ± 12) was similar to that after MSi-SSCT (73 ± 14). However, the donor-derived TFI after MSi-SSCT (26 ± 14) was higher compared to the one after SSCT (9 ± 5; P = 0.002), even after injecting half of the number of SSCs in MSi-SSCT. The litter sizes after SSCT (3.7 ± 3.7) and MSi-SSCT (3.7 ± 3.6) were similar but differed significantly with the control group (7.6 ± 1.0; P < 0.001). The number of GFP+ offspring per litter obtained after SSCT (1.6 ± 0.5) and MSi-SSCT (2.0 ± 1.0) was also similar. The expression of DNMT3A and H4K5ac in germ cells of transplanted males was found to be significantly reduced compared to the control group. However, in donor-derived offspring, DNMT3A and H4K5ac followed the normal pattern. Conclusion Co-transplanting SSCs and TGFβ1-treated MSCs results in reproductive efficiency as good as SSCT, even after transplanting half the number of SSCs. Although transplanted males showed lower expression of DNMT3A and H4K5ac in donor-derived germ cells, the expression was restored to normal levels in germ cells of donor-derived offspring. This procedure could become an efficient method to restore fertility in a clinical setup, but more studies are needed to ensure safety in the long term.

scholarly journals Identification of Niche Conditions Supporting Short-term Culture of Spermatogonial Stem Cells Derived from Porcine Neonatal Testis

2014 ◽  
Vol 29 (3) ◽  
pp. 221-228
Author(s):  
Min Hee Park ◽  
Ji Eun Park ◽  
Min Seong Kim ◽  
Kwon Young Lee ◽  
Jung Im Yun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Spermatogonial Stem Cells ◽  
Short Term ◽  
Neonatal Testis ◽  
Short Term Culture

74 GERM CELLS AND TESTICULAR SOMATIC CELLS HAVE DIFFERENT SENSITIVITY TO CRYOPRESERVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 184
Author(s):  
H. Robbins ◽  
C. Dores ◽  
K. Coyle ◽  
I. Dobrinski
Keyword(s):  
Cell Viability ◽  
Germ Cell ◽  
Cell Population ◽  
Germ Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Somatic Cells ◽  
Freezing And Thawing ◽  
Fetal Bovine ◽  
Better Than

Spermatogonial stem cells (SSC) are the foundation of spermatogenesis. Undifferentiated spermatogonia, containing SSC, represent only 2 to 5% of cells recovered from immature mammalian testis. Cryopreservation in liquid nitrogen allows for long-term storage of cells. Preservation of germ cells can serve as a means of genetic preservation from immature males when sperm storage is not an option. Studies have investigated the effects of cryopreservation on the spermatogenic potential of SSC and the efficiency of various cryopreservation protocols. Preliminary observations indicated that germ cells may survive cryopreservation better than testicular somatic cells, resulting in a post-thaw cell population enriched in germ cells. However, this has not been critically evaluated. The objective of this study was to test the hypothesis that germ cells are less susceptible to cryo-damage than testicular somatic cells. Cells were harvested from the testes of 1-wk-old piglets by 2-step enzymatic digestion. The initial cell suspension was subjected to differential adhesion to enrich the cell population for germ cells. Cells were plated in DMEM + 5% fetal bovine serum and incubated at 37°C in 5% CO2 in air. After 18 h, cells in suspension and cells slightly attached were recovered by trypsinization (1 : 10 trypsin-ethylenediaminetetraacetic acid) for 30 s and replated. This was repeated 24 and 36 h after initial plating. The enriched population was placed into cryovials at a concentration of 30 × 106 cells in freezing media (70% DMEM + 20% fetal bovine serum + 10% dimethyl sulfoxide), kept for 24 h at –80°C in a cryogenic freezing container and transferred to liquid nitrogen for 1 week. Aliquots of cells before freezing and after thawing at 37°C followed by incubation at 37°C in 5% CO2 in air for 1 h were analyzed for viability by propidium iodide (PI) exclusion and immunofluorescence for the germ cell marker VASA to identify viable germ cells (VASA+/PI–), nonviable germ cells (VASA+/PI+), viable somatic cells (VASA–/PI–), and nonviable somatic cells (VASA–/PI+). The percentage of viable germ cells after freezing and thawing was compared to the percentage of viable somatic cells by ANOVA. After enrichment by differential plating, the cell population had 95.6 ± 0.9% viability and contained 27.1 ± 7.4% germ cells (n = 3 replicates). After cryopreservation, the overall cell viability was 77.5 ± 1.6%, and 25.8 ± 8.0% were germ cells. The overall viability after cryopreservation could potentially have benefited from the 1-h incubation prior to analysis. The viability of the germ cell population after freezing and thawing was higher (92.1 ± 3.1%) than somatic cell viability (72.3 ± 1.7%; P < 0.01). These results indicate that porcine germ cells survive cryopreservation better than do testicular somatic cells. Therefore, cryostorage of germ cells can be an efficient means for preservation of male genetic material. Supported by NIH ORIP/DCM RR17359.

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.

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