Bovine Spermatogonial Stem Cells Research Articles

Spermatogonial stem cell biology in the bull: development of isolation, culture, and transplantation methodologies and their potential impacts on cattle production

Widespread adoption of artificial insemination as a breeding practice has allowed for expanded use of desirable genetics from specific sires and greatly influenced production traits in dairy cattle populations worldwide. In fact, the average dairy cow in the US in 2009 produced 4.5 times more milk than in 1940 when commercialization of artificial insemination began. While many factors have contributed to this rapid increase in levels of milk production, genetic gain through expanded utilization of germlines from specific sires has been a major contribution. In comparison, use of artificial insemination in beef cattle populations has been limited due to challenges with implementing intensive management strategies required for success. Thus, there is need for alternative reproductive tools to expand use of desirable male genetics in the beef cattle industry. The process of sperm production, termed spermatogenesis, is supported by a tissue- specific stem cell population referred to as spermatogonial stem cells (SSCs). These unique cells have the capacity for infinite self-renewal and long-term regeneration of spermatogenesis following transplantation. In rodents, methods for isolating, culturing, and transplanting SSCs have been devised. For beef cattle, transplanting SSCs isolated from a donor male into the testes of recipient males in which donor-derived spermatogenesis occurs and offspring with donor genetics are produced from natural breeding has great potential as an alternative to artificial insemination. This potential reproductive strategy would allow for expansive use of genetics from desirable sires that overcomes the logistical challenges of artificial insemination. Translation of the methods devised for rodents to cattle is at the forefront of development. Devising means for isolating an SSC-enriched cell fraction from donor testes and identifying conditions that support long-term maintenance and proliferation of bovine SSCs in vitro are two tools that would greatly accelerate the pace at which transplantation will become a commercially viable option for cattle industries. Recent studies showed that expression of THY1 by SSCs is a conserved phenotype between rodents and cattle, and selection of the THY1+ fraction from donor testes can be used for isolating an SSC-enriched germ cell population. In addition, the conditions devised tor expanding the number of rodent SSCs in vitro continues to serve as the basis for developing conditions that support bovine SSCs. With these tools in hand major advances in developing implementable reproductive tools with SSCs for commercial cattle production will be made in the coming decade.

Optimization of <i>in vitro</i> culture and transfection condition of bovine primary spermatogonial stem cells

The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase (AP) staining and expression of vasa and thy1 genes as specific bovine SSC markers. To evaluate the effect of antioxidants on vitality, colony formation, and the expression of pro- and anti-apoptotic genes of bovine SSCs, various concentrations of vitamin C (5, 10, 25 and 50 μg/mL) and Trolox (a water soluble α-tocopherol analogue) (12.5, 25, 50 and 100 μg/mL) were added to the SSC culture medium. The results showed that SSCs treated with 50 μg/mL of vitamin C or 25 μg/mL of Trolox individually could increase cell viability and colony formation significantly in comparison with other concentrations and the control group. Additionally, the expressions of bax (as a pro-apoptotic gene) and bcl2 (as an anti-apoptotic gene) were significantly lower and higher than the control group, respectively. To optimize the transfection condition, the effective dosages of vitamin C or Trolox, with various concentrations of two transfection reagents (X-tremeGENE HP and Turbofect) and DNA, at day 8 of culture, were studied. Results showed that 1 μl X-tremeGENE HP or 0.5 μl Turbofect and 2 μg of DNA are the best concentrations for transfecting SSCs. However, X-tremeGENE HP expressed more potential for transfecting SSCs in comparison with Turbofect. Besides, no difference was observed between the use of defined doses of vitamin C or Trolox.Keywords: Apoptosis, gene transfer, primary cells, viability, vitamin C, Trolox

Evaluating the behavior of cultured sertoli cells in the presence and absence of spermatogonial stem cell.

The complex process of spermatogenesis is regulated by various factors. Several studies have been conducted to proliferate cells involved in the spermatogenesis process, in culture by used growth factors, different hormones and feeder cells. This study was conducted to evaluate the role of Sertoli cells on gene expression of fibroblast growth factor (FGF2) and glial cell derived neurotrophic factor (GDNF) after removal of spermatogonial stem cells (SSCs) from the culture medium. Following isolation, bovine SSCs were co-cultured with Sertoli cells and follicular stimulating hormone (FSH) for 12 days. In the treatment group, SSCs were removed from the culture medium; in the control group no intervention was done in the culture. Colony formation of SSCs was evaluated by using an inverted microscope. Then, the expression of factors genes were assessed by quantitative RT-PCR. Data was analyzed by using paired-samples t-test. The results showed that removal of SSCs led to the increase in expression of GDNF and FGF2. These findings suggest that loss of SSCs population or decline in its population leads to changing in behavior of somatic cells which forming niche and consequently stimulates self-renewal and inhibits differentiation of SSCs. The present study showed that removal of SSCs from the culture medium could be a model for damage to SSCs; the results revealed that niche cells respond to SSCs removal by upregulation of FGF2 and GDNF to stimulate self-renewal of SSCs and abrogation of differentiation.

192 Culture Conditions Supporting Long-Term Expansion of Bovine Spermatogonial Stem Cells Isolated from Adult and Immature Testes

Spermatogonial stem cells (SSC) self-renew and differentiate into spermatocytes to produce haploid sperm. Because SSC are a small population of adult stem cells in the testis, numerous studies have been reported to derive cell lines from cultured SSC. It has been reported that neonatal and adult mouse SSC can be cultured in vitro over the long term. Male germline stem (GS) cells, embryonic stem (ES)-like cells, and multipotent male germline stem (MGS) cells were derivated from mouse SSC. However, in domestic species including cattle, information about in vitro culture of SSC is mainly available in the neonatal and immature animal. To our knowledge, there are no reports about long-term culture of SSC isolated from adult bovine testis. In this report, we established culture conditions to maintain SSC isolated from adult and immature testes. The SSC were isolated by 3-step enzymatic digestion and enriched by Percoll gradient centrifugation. For adult testicular cell suspensions, SSC were further enriched by differential plating on precoated gelatin dish. After Percoll gradient centrifugation, we found differential expression of SSC markers (GFRα-1 and UCHL-1) in the isolated cells from immature and adult testis. The RT-PCR results also confirmed the expression of differentiated spermatogonia markers (SYCP3 and STRA-8) in adult testicular cell suspensions. It suggests that isolated testicular germ cell population from adult testis are more heterogeneous than those of immature testis. The SSC isolated from adult testes were cultured in low-serum media containing 6-bromoindirubin-3′-oxime (BIO), an inhibitor of glycogen synthase kinase-3α (GSK3), and subsequently the cultures were maintained in the medium containing glial cell line-derived neurotropic factor (GDNF). The cell lines have characteristics resembling mouse GS cell lines as confirmed by their grape-like shape morphology, the expression of SSC markers (UCHL-1, DBA, and GFRa-1), and pluripotent stem cell markers (POU5F1, SOX2, KLF4). The SSC from immature testes were proliferated for more than 3 months in serum-free culture conditions in the presence of GDNF and bovine leukemia inhibitory factor (LIF). The cell lines had ES-like cell morphology, expressed pluripotent stem cell markers and SSC-specific markers. They differentiated in vitro into 3 germ layers confirmed by the expression of ectoderm (NESTIN), mesoderm (BMP4), and endoderm (GATA-6) markers by RT-PCR and neuron like-cells confirmed by the expression of glial fibrillary acidic protein (GFAP) by immunofluorescence analysis. In conclusion, these findings indicate an efficient method to enrich SSC without cell sorting method and different long-term culture systems subsequently established to maintain SSC from adult and immature testes. Furthermore, our data would be useful for further studies that aim to preserve endangered species and improve livestock production through genome editing technology.

Evaluation of the effect of follicular stimulating hormone on the in vitro bovine spermatogonial stem cells self-renewal: An experimental study

Spermatogonial stem cells (SSCs) are undifferentiated cells which are highly reproducible and expandable. Several studies have been conducted to reproduce these cells in culture. They used growth factors, hormones and different feeder cells to improve survival and proliferation of SSCs. This study was conducted to evaluate the effects of follicular stimulating hormone (FSH) on gene expression of fibroblast growth factor (FGF2) and glial cell-derived neurotrophic factor (GDNF) in Sertoli cells. Sertoli cells and SSCs were isolated from 3-5 month-old calves. Bovine testicular cells were cultured for 15 days with or without FSH. Identification of these cells was confirmed by immunocytochemistry analysis. Colony formation of SSCs was evaluated using an inverted microscope. The gene expression of FGF2 and GDNF and the gene markers bcl6b, thy-1, and C-kit were evaluated using the quantitative RT-PCR technique. The results indicated that FSH increased colonization of SSCs. the expression of GDNF, FGF2, and markers of undifferentiated spermatogonia was increased following culture in control and FSH groups (p<0.05), this increase was more in FSH group. Conversely, the expression of C-kit was decreased in both groups (p<0.05). The results showed that FSH can increase the self-renewal of SSCs in vitro via upregulation of GDNF and FGF2 expression in Sertoli cells.

Transfection of bovine spermatogonial stem cells in vitro.

Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic information to the future generations. Considering the fact that a single SSC gives rise to a vast number of spermatozoa, genetic manipulation of these cells is a potential novel technology with feasible application to various animal species. The aim of this study was to evaluate enhanced green fluorescent protein (EGFP) gene transfection into bovine SSCs via liposome carrier and assess the best incubation day in uptake exogenous gene by SSCs. Transfection efficiency of EGFP gene with lipofectamine 2000 was determined in days following each three day of transfection (day 4, 6 and 8 of the culture) by fluorescent microscope. Results showed that the transfected cells through lipofection increased significantly (P<0.05) in each three days of transfection in comparison with those of the control groups. The transfected SSCs were higher in comparison with those of the free exogenous gene carrier groups (P<0.05). In comparison with these three days, the rate of infected cells was higher when transfection proceeds at day four. It was concluded that lipofectamine can be used safely for direct loading exogenous DNA to SSCs particularly during the fourth day of culture.

Conditions for Long-Term Culture of Cattle Undifferentiated Spermatogonia.

Continual and robust spermatogenesis relies on the actions of an undifferentiated spermatogonial population that contains stem cells. A remarkable feature of spermatogonial stem cells (SSCs) is the capacity to regenerate spermatogenesis following isolation from a donor testis and transplantation into a permissive recipient testis. This capacity has enormous potential as a tool for enhancing the reproductive capacity of livestock, which can improve production efficiency. Because SSCs are a rare subset of the undifferentiated spermatogonial population, a period of in vitro amplification in number following isolation from donor testicular tissue is essential. Here, we describe methodology for isolation of a cell fraction from prepubertal bull testes that is enriched for undifferentiated spermatogonia and long-term maintenance of the cells in both the feeder cell coculture and the feeder-free format. To achieve this method, we derived bovine fetal fibroblasts (BFF) to serve as feeders for optimizing medium conditions that promote maintenance of bovine undifferentiated spermatogonia for at least 2 mo. In addition, we devised a feeder-free system with BFF-conditioned medium that sustained bovine undifferentiated spermatogonia for at least 1 mo in vitro. The methodologies described could be optimized to provide platforms for exponential expansion of bovine SSCs that will provide the numbers needed for transplantation into recipient testes.

Enrichment and culture of spermatogonia from cryopreserved adult bovine testis tissue

Propagation of bovine spermatogonial stem cells (SSCs) from the cryopreserved testicular tissue is essential for the application of SSCs-related techniques. To explore the appropriate conditions for in vitro culture of bovine spermatogonia (containing putative SSCs), Sertoli cell monolayer and serum concentration were set as two main control factors. Morphological examination showed that the intactness and structure of adult bovine testicular tissue were well maintained after cryopreservation. The enriched bovine spermatogonia were large round CD9 and promyelocytic leukemia zinc finger protein (PLZF) positive cells, with high nucleocytoplasmic ratios and multiple types including single, paired-, aligned-cells or grape cluster-like colonies in vitro. In Sertoli cell co-culture system, bovine spermatogonia attached quickly and proliferated obviously faster than those in the system without Sertoli cells. Serum-free media was no good for the attachment and proliferation of bovine spermatogonia. When 2.5%, 5% and 10% fetal bovine serum (FBS) was employed in the media, spermatogonia attached easily and divided quickly to form paired-, chained-cells or grape cluster-like colonies with comparable percentages in all groups. However, the contaminated somatic cells proliferated robustly in groups containing 5% and 10% FBS. Together, bovine spermatognia isolated from cryopreserved adult testis tissue express CD9 and PLZF, can survive and proliferate conspicuously in Sertoli cell co-culture system, and low serum provides an optimal condition for the survival and proliferation of bovine spermatogonia because of avoiding the rapid growth of testis somatic cells.

In vitro spermatogenesis using bovine testis tissue culture techniques

Spermatogenesis is a complex process initiated by spermatogonial stem cells (SSCs) that have the ability to differentiate into mature spermatozoa or to self-renew to maintain the SSC population and long-term fertility. However, a technique for complete spermatogenesis in vitro using cell culture has not yet been developed. In the present study, we developed in vitro spermatogenesis techniques using bovine testis tissue culture. The effects of specific temperatures and different media on maintaining tubule and germ cell competency were investigated. We found that the optimal temperature and media were 37°C and mouse serum-free medium (mSFM), respectively. In addition, the efficacy of various hormones and growth factors on spermatogenesis in bovine testis tissues maintained in vitro was evaluated. We found that the addition of triiodothyronine (T3) and stem cell factor (SCF) induced spermatogenesis of bovine SSCs in vitro. Therefore, tissue fragments were cultured in the presence of T3 and SCF for three months to induce spermatogenesis in vitro. Overall, in vitro spermatogenesis was enhanced 2.4- to 2.7-fold. Our tissue culture technique may serve as a model system that leads to a more comprehensive understanding of the biology of SSCs as well as the factors that regulate male fertility. Furthermore, the results of this study will be integral for the continued refinement of techniques to manipulate bovine SSCs.

Effect of extracellular matrix on bovine spermatogonial stem cells and gene expression of niche factors regulating their development in vitro

Extracellular matrix (ECM) could influence cells function through providing structural and functional networks facilitating the cellular interactions. The present study was conducted to evaluate the effect of culture on ECM versus plastic on bovine spermatogonial stem cells (SSCs) and growth factors regulating their development. Following isolation, bovine testicular cells were cultured on ECM-coated or uncoated (control) plates for 12 days. The colonization of SSCs was assessed by inverted microscope and the gene expression was evaluated using quantitative real-time PCR. The colonization rate was greater in ECM than the control group (P<0.05). The expression of markers of undifferentiated spermatogonia increased in response to conventional culture (P<0.05). Conversely, the expression of ckit as a marker for differentiated spermatogonia was reduced following culture in the control and ECM groups (P<0.05), but this decrease was less in ECM group (P<0.05). Accordingly, while cells cultured on uncoated plates had greater expression of markers of undifferentiated spermatogonia (P<0.05), cells cultured on ECM-coated plates showed higher expression of ckit (P<0.05). Moreover, culture on ECM resulted in higher expression of kit ligand (Kitlg; P<0.05), whereas culture on plastic led to greater expression of glial cell line-derived neurotrophic factor (Gdnf; P<0.05). In conclusion, the present study revealed that the permissive effect of ECM on bovine SSCs differentiation in vitro, which was probably mediated through upregulation of KITLG expression. Moreover, the results imply that GDNF might contribute to germ cells self-renewal during conventional culture.

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200mM trehalose is an efficient cryopreservation protocol for bovine SSCs.

Corrigendum to: 198 AGE EFFECT ON RELATIVE GENE EXPRESSION OF BOVINE SPERMATOGONIA

Spermatogonial stem cells (SSCs) have important applications in treatments for infertility and animal transgenesis. However, the isolation of bovine SSCs is less efficient than for other species and it is lower for adult bulls. The goal of this study was to verify whether the relative gene expression of spermatogonia is affected by age after the laminin differential plating. Ten grams of parenchyma of testicles from pre-pubertal animals (5 months of age, n = 5) and bulls (3–4 years of age, n = 5) were minced and digested: colagenase (1 mg mL–1) for 30 min at 37°C and trypsin (2.5 mg mL–1) for 5 min at 37°C. Cells were plated (3 × 106 viable cells) in 60-mm culture dish covered with laminin (20 ng mL–1) and they were cultured for 15 min in high-humidity atmosphere with 5% of CO2 at 37°C. The adherent cells were recovered by enzymatic digestion with 0.1% trypsin for 1 min at 37°C. Viable cells were selected by the Trypan exclusion method, RNA was extracted from ~200.000 viable cells (Ilustra RNAspin Mini RNA, GE Healthcare, Waukesha, WI, USA) and cDNA synthesis was performed (SuperScript® VILO™, Invitrogen, Carlsbad, CA, USA). ITGA6 (integrin, α 6), SELP (Selectin P) and ICAM (Intercellular Adhesion Molecule 1) relative gene expression were determined by real-time RT-PCR (Mastercycler ep Realplex, Eppendorf International, Hamburg, Germany); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and ACTB (actin β) were used as housekeeping genes. The statistical analysis was performed by QPCR_MIXED (SAS®, SAS Institute Inc., Cary, NC, USA). An α level at 0.05 was always adopted. ITGA6 and ICAM1 relative expression weren't effected by age; respectively, P = 0.2367 and P = 0.3583. However, SELP was highly expressed in adult bulls (P = 0.0022). Previously, ICAM1 and SELP have also been shown to mark aging haematopoietic stem cells and were more highly expressed in spermatogonia from adult mice than younger. However, SELP is expressed in more differentiated spermatogenic cells such as human sperm. The expression levels of ITGA6 did not seem to be significantly altered by age, indicating that age affects only certain SSC properties. Financial support was provided by FAPESP (CEUA/FMVZ/USP 2509/2011).

Corrigendum to: 198 AGE EFFECT ON RELATIVE GENE EXPRESSION OF BOVINE SPERMATOGONIA

Spermatogonial stem cells (SSCs) have important applications in treatments for infertility and animal transgenesis. However, the isolation of bovine SSCs is less efficient than for other species and it is lower for adult bulls. The goal of this study was to verify whether the relative gene expression of spermatogonia is affected by age after the laminin differential plating. Ten grams of parenchyma of testicles from pre-pubertal animals (5 months of age, n = 5) and bulls (3–4 years of age, n = 5) were minced and digested: colagenase (1 mg mL–1) for 30 min at 37°C and trypsin (2.5 mg mL–1) for 5 min at 37°C. Cells were plated (3 × 106 viable cells) in 60-mm culture dish covered with laminin (20 ng mL–1) and they were cultured for 15 min in high-humidity atmosphere with 5% of CO2 at 37°C. The adherent cells were recovered by enzymatic digestion with 0.1% trypsin for 1 min at 37°C. Viable cells were selected by the Trypan exclusion method, RNA was extracted from ~200.000 viable cells (Ilustra RNAspin Mini RNA, GE Healthcare, Waukesha, WI, USA) and cDNA synthesis was performed (SuperScript® VILO™, Invitrogen, Carlsbad, CA, USA). ITGA6 (integrin, α 6), SELP (Selectin P) and ICAM (Intercellular Adhesion Molecule 1) relative gene expression were determined by real-time RT-PCR (Mastercycler ep Realplex, Eppendorf International, Hamburg, Germany); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and ACTB (actin β) were used as housekeeping genes. The statistical analysis was performed by QPCR_MIXED (SAS®, SAS Institute Inc., Cary, NC, USA). An α level at 0.05 was always adopted. ITGA6 and ICAM1 relative expression weren't effected by age; respectively, P = 0.2367 and P = 0.3583. However, SELP was highly expressed in adult bulls (P = 0.0022). Previously, ICAM1 and SELP have also been shown to mark aging haematopoietic stem cells and were more highly expressed in spermatogonia from adult mice than younger. However, SELP is expressed in more differentiated spermatogenic cells such as human sperm. The expression levels of ITGA6 did not seem to be significantly altered by age, indicating that age affects only certain SSC properties. Financial support was provided by FAPESP (CEUA/FMVZ/USP 2509/2011).

Study of insulin-like growth factor 1 effects on bovine type A spermatogonia proliferation and viability

Spermatogonial stem cells (SSCs) are the best candidates for exploitation in genetic improvement of cattle herds. In fact, SSCs transmit genetic information to future generations. In vitro improvement in proliferation and frozen-thawed SSCs numbers is therefore needed. In the present study, bovine SSCs were cocultured with Sertoli cells, with or without insulin-like growth factor 1 (IGF-1). During 16 days of incubation, 100 ng/mL IGF-1 showed higher efficacy in increasing the numbers and diameters of SSCs colonies than 40 and 150 ng/mL concentrations. Furthermore, 100 ng/mL IGF-1 increased the frozen/thawed cells’ viability rate. Overall, these data demonstrate that usage of 100 ng/mL IGF-1 in vitro improves proliferation and postfreezing viability rate of SSCs.

292 COMPARISON OF DIFFERENTIAL PLATING METHODS TO OBTAIN ENRICHED POPULATIONS OF BOVINE SPERMATOGONIAL STEM CELLS

Spermatogonial stem cells (SSC) represent only 0.1% of all germ cells found in adult animal testicles. The main goal of differential plating treatments is to isolate SSC and enrich the SSC population in a cell subculture. The aim of this study was to determine which differential plating treatment – laminin, lectin from Datura stramonium (DSA), Matrigel (BD Biosciences, San Jose, CA, USA), bovine serum albumin (BSA), or PBS/control – was the most efficient for isolating bovine SSC. Thus, 3 g of testicular parenchyma of adult Nelore bulls was minced and consecutively digested in two steps: collagenase (1 mg mL–1) and trypsin (2.5 mg mL–1). Cells from each testicle were plated (3 × 106 viable cells) in a culture dish coated with 1 mL of each of the following plating treatments: laminin (20 µg mL–1), DSA (20 µg mL–1), Matrigel (5%), BSA (0.5 mg mL–1), or PBS. Cells were cultured for 18 h in a high-humidity atmosphere containing 5% CO2 at 37°C. Supernatants were centrifuged and cell concentration and viability were assessed by Trypan Blue exclusion method. Then, levels of thiobarbituric acid reactive substances (TBARS) were evaluated in a 20 mM ascorbic acid solution and in 25µL of 4 mM ferrous sulfate solution immediately after incubation with 2 × 106 viable cells (n = 30) from each testicle (n = 6) for all treatments. The supernatant was collected and the same volume of 0.1% thiobarbituric acid was added. The TBARS level was quantified by spectrophotometry (532 nm). Approximately 1 × 106 viable cells (n = 55) from each testis (n = 11) for all treatments were fixed with 70% ethanol and incubated with an anti-α six integrin (A6Int) antibody, followed by Alexa Fluor 488 (BioLegends®, San Diego, CA, USA) and propidium iodide labelling. The percentage of marked cells with the anti-A6Int was determined by flow cytometry (Guava Technologies, Hayward, CA, USA). Kruskal-Wallis ANOVA and post hoc Bonferroni tests were performed; P &lt; 0.05 was considered significantly different (Stata version 12.0, Stata Corp., College Station, TX, USA). No treatment side effects were observed on percentage of A6Int-positive cells recovered, TBARS level, or rate of viable cells after and before differential plating. A difference in cell viability was observed between the Matrigel and laminin treatments (P = 0.04). In conclusion, our results showed no quantitative difference between enriched SSC populations purified by differential plating treatments, as was expected. Thus, this experiment needs to be complemented with better characterisation of purified bovine SSC for their molecular markers expression using real time RT-PCR and flow cytometry. Only then will we be able to reach a conclusion on the effectiveness of these treatments. Financial support: FAPESP (2011/20115-1).

α‐6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 × 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α-6 integrin expression. Flow cytometry analysis demonstrated no differences in the α-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to α-6 integrin, the expression of GFRa-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α-6 integrin expression.

Spermatogonial stem cell biology in the bull: development of isolation, culture, and transplantation methodologies and their potential impacts on cattle production

Widespread adoption of artificial insemination as a breeding practice has allowed for expanded use of desirable genetics from specific sires and greatly influenced production traits in dairy cattle populations worldwide. In fact, the average dairy cow in the US in 2009 produced 4.5 times more milk than in 1940 when commercialization of artificial insemination began. While many factors have contributed to this rapid increase in levels of milk production, genetic gain through expanded utilization of germlines from specific sires has been a major contribution. In comparison, use of artificial insemination in beef cattle populations has been limited due to challenges with implementing intensive management strategies required for success. Thus, there is need for alternative reproductive tools to expand use of desirable male genetics in the beef cattle industry. The process of sperm production, termed spermatogenesis, is supported by a tissue-specific stem cell population referred to as spermatogonial stem cells (SSCs). These unique cells have the capacity for infinite self-renewal and long-term regeneration of spermatogenesis following transplantation. In rodents, methods for isolating, culturing, and transplanting SSCs have been devised. For beef cattle, transplanting SSCs isolated from a donor male into the testes of recipient males in which donor-derived spermatogenesis occurs and offspring with donor genetics are produced from natural breeding has great potential as an alternative to artificial insemination. This potential reproductive strategy would allow for expansive use of genetics from desirable sires that overcomes the logistical challenges of artificial insemination. Translation of the methods devised for rodents to cattle is at the forefront of development. Devising means for isolating an SSC-enriched cell fraction from donor testes and identifying conditions that support long-term maintenance and proliferation of bovine SSCs in vitro are two tools that would greatly accelerate the pace at which transplantation will become a commercially viable option for cattle industries. Recent studies showed that expression of THY1 by SSCs is a conserved phenotype between rodents and cattle, and selection of the THY1 + fraction from donor testes can be used for isolating an SSC-enriched germ cell population. In addition, the conditions devised for expanding the number of rodent SSCs in vitro continues to serve as the basis for developing conditions that support bovine SSCs. With these tools in hand major advances in developing implementable reproductive tools with SSCs for commercial cattle production will be made in the coming decade.

Propagation of bovine spermatogonial stem cells in vitro

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

Immortalization of bovine germ line stem cells by c-myc and hTERT

The limited life span of bovine germ line stem cells in vitro is one of the obstacles to spermatogenesis analysis, genetic manipulation and generating transgenic animal. The aim of this study is to establish immortalized bovine germ line stem cells by c-myc or hTERT. We constructed pEMY and pETE expression vectors and transfected germ line cells from 5-month-old bovine. After G418 screening, four types of positive clones were obtained. The results showed that they expressed exogenous genes c-myc or hTERT at mRNA and protein level by RT-PCR and Western blotting detection. Presumable cell lines GM7, GT3, GMT5 all expressed germ-line-stem-cell-specific makers by immunocytochemical analysis, such as c-kit, Oct-4 and GFRα-1. The putative cell lines also had higher capacity of proliferation than freshly isolated bovine spermatogonial stem cells. So we can conclude that exogenous genes c-myc or hTERT have integrated into the genome of bovine germ cells and upregulated the expression of telomerase.

Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture.

Functional roles of spermatogonial stem cells in spermatogenesis are self-renewing proliferation and production of differentiated daughter progeny. The ability to recapitulate these actions in vitro is important for investigating their biology and inducing genetic modification that could potentially lead to an alternative means of generating transgenic animals. The objective of this study was to evaluate the survival and proliferation of frozen-thawed bovine spermatogonial stem cells in vitro and investigate the effects of exogenous glial cell line-derived neurotrophic factor (GDNF). In order to accomplish this objective we developed a bovine embryonic fibroblast feeder cell line, termed BEF, to serve as feeder cells in a coculture system with bovine germ cells. Bovine spermatogonial stem cell survival and proliferation in vitro were evaluated by xenogeneic transplantation into the seminiferous tubules of immunodeficient mice. Bovine germ cells cocultured for 1 wk resulted in significantly more round cell donor colonies in recipient mouse testes compared to donor cells transplanted just after thawing. Bovine germ cells cocultured for 2 wk had fewer colony-forming cells than the freshly thawed cell suspensions or cells cultured for 1 wk. Characterization of the feeder cell line revealed endogenous expression of Gdnf mRNA and protein. Addition of exogenous GDNF to the culture medium decreased the number of stem cells present at 1 wk of coculture, but enhanced stem cell maintenance at 2 wk compared to cultures without added GDNF. These data indicate that frozen-thawed bovine spermatogonial stem cells survive cryopreservation and can be maintained during coculture with a feeder cell line in which the maintenance is influenced by GDNF.