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The early detection of tumors improves chances of decreased morbidity and prolonged survival. Serum biomarkers are convenient to use and have several advantages over other approaches, such as accuracy and straightforward protocols. Reliable biomarkers from easily accessible sources are warranted for the development of cost-effective assays for routine screening, particularly in veterinary medicine. Extracellular c-AMP-dependent protein kinase A (ECPKA) is a cytosolic leakage enzyme. The diagnostic accuracy of detecting autoantibodies against ECPKA was found to be higher than that of ECPKA activity from enzymatic assays, which use a complicated method. Here, we investigated the diagnostic significance of measuring serum ECPKA autoantibody levels using an in-house kit (AniScan cancer detection kit; Biattic, Anyang, Korea). We used sera from 550 dogs, including healthy dogs and those with malignant and benign tumors. Serum ECPKA and immunoglobulin G were determined using the AniScan cancer detection kit. ECPKA autoantibody levels were significantly higher (p < 0.01) in malignant tumors than in benign tumors, non-tumor diseases, and healthy controls. On the basis of sensitivity and specificity values, AniScan ECPKA is a rapid and easy-to-use assay that can be applied to screen malignant tumors from benign tumors or other diseases in dogs.

Maintenance of adult tissues depends on stem cell self-renewal in local niches. Spermatogonial stem cells (SSC) are germline adult stem cells necessary for spermatogenesis and fertility. We show that testicular endothelial cells (TECs) are part of the SSC niche producing glial cell line-derived neurotrophic factor (GDNF) and other factors to support human and mouse SSCs in long-term culture. We demonstrate that FGF-2 binding to FGFR1 on TECs activates the calcineurin pathway to produce GDNF. Comparison of the TEC secretome to lung and liver endothelial cells identified 5 factors sufficient for long-term maintenance of human and mouse SSC colonies in feeder-free cultures. Male cancer survivors after chemotherapy are often infertile since SSCs are highly susceptible to cytotoxic injury. Transplantation of TECs alone restores spermatogenesis in mice after chemotherapy-induced depletion of SSCs. Identifying TECs as a niche population necessary for SSC self-renewal may facilitate fertility preservation for prepubertal boys diagnosed with cancer., Self-renewal of spermatogonial stem cells (SSC) is necessary for spermatogenesis and male fertility. Here the authors identify testicular endothelial cells (TECs) as a source of 5 key growth factors for self-renewal and expansion of human and mouse SSCs.

Protein kinase A, a cyclic adenosine monophosphate (AMP)-dependent enzyme, normally exists within mammalian cells; however, in cancer cells, it can leak out and be found in the serum. Extracellular cyclic AMP-dependent protein kinase A (ECPKA) has been determined to increase in the serum of cancer-bearing dogs. However, there have been no reports in the veterinary literature on serum ECPKA autoantibody (ECPKA-Ab) expression in dogs with cancer. The aim of this study was to evaluate ECPKA-Ab and C-reactive protein (CRP) as serum biomarkers for cancer in dogs. ECPKA-Ab and CRP levels were detected by an enzyme-linked immunosorbent assay in serum samples from dogs with malignant tumours (n = 167), benign tumours (n = 42), or non-tumour disease (n = 155) and from healthy control dogs (n = 123). ECPKA-Ab and CRP levels were significantly higher in the dogs with malignant tumours than in those with benign tumours or non-tumour diseases, as well as in the healthy controls (P < 0.001, Kruskal-Wallis test). There was a significant positive correlation between the neoplastic index, which was developed using ECPKA-Ab and CRP levels, and the presence of cancer in dogs (P < 0.001); the area under the receiver-operating characteristic curve was estimated to be >0.85 (P < 0.001). In conclusion, ECPKA-Ab is a potential serum biomarker for a broad spectrum of cancers. Combined measurement of CRP and ECPKA-Ab levels in serum improves the sensitivity and accuracy of a diagnosis of cancer in dogs.

Spermatogonial stem cells (SSCs) are adult male germ cells that develop after birth. Throughout the lifetime of an organism, SSCs sustain spermatogenesis through self-renewal and produce daughter cells that differentiate into spermatozoa. Several studies have demonstrated that SSCs can acquire pluripotency under appropriate culture conditions, thus becoming multipotent germline stem cells (mGSCs) that express markers of pluripotency in culture and form teratomas following transplantation into immunodeficient mice. In the present study, we generated neural precursor cells expressing CD24, a neural precursor marker, from pluripotent stem cell lines and demonstrated that these cells effectively differentiated along a neural lineage in vitro. In addition, we found that paracrine factors promoted CD24 expression during the neural differentiation of mGSCs. Our results indicated that the expression of CD24, enhanced by a combination of retinoic acid (RA), noggin and fibroblast growth factor 8 (FGF8) under serum-free conditions promoted neural precursor differentiation. Using a simple cell sorting method, we were able to collect neural precursor cells with the potential to differentiate from mGSCs into mature neurons and astrocytes in vitro.

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 200 mM 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 200 mM 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 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout postnatal life in male and have the ability to transmit genetic information to the subsequent generation. In this study, we have optimized the transduction efficiency of SSCs using a lentiviral vector by considering different multiplicity of infection (MOI), duration of infection, presence or absence of feeder layer and polycationic agents. We tested MOI of 5, 10 or 20 and infection duration of 6, 9 or 12 h respectively. After infection, cells were cultured for 1 week and as a result, the number of transduced SSCs increased significantly for MOI of 5 and 10 with 6 h of infection. When the same condition (MOI of 5 with 6 hours) was applied in presence or absence of STO feeder layer and infected SSCs were cultured for 3 weeks on the STO feeder layer, a significant increase in the number of transduced cells was observed for without the feeder layer during infection. We subsequently studied the effects of polycationic agents, polybrene and dioctadecylamidoglycyl spermine (DOGS), on the transduction efficiency. Compared with the polybrene treatment, the recovery rate of the transduced SSCs was significantly higher for the DOGS treatment. Therefore, our optimization study could contribute to the enhancement of germ-line modification of SSCs using lentiviral vectors and in generation of transgenic animals.

Spermatogonial stem cells (SSCs) are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male's lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM) resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media.

Oriental natural plants have been used as medical herbs for the treatment of various diseases for over 2,000 years. In this study, we evaluated the effect of several natural plants on the preservation of male fertility by assessing the ability of plant extracts to stimulate spermatogonial stem cell (SSC) proliferation by using a serum-free culture method. In vitro assays showed that Petasites japonicus extracts, especially the butanol fraction, have a significant effect on germ cells proliferation including SSCs. The activity of SSCs cultured in the presence of the Petasites japonicus butanol fraction was confirmed by normal colony formation and spermatogenesis following germ cell transplantation of the treated SSCs. Our findings could lead to the discovery of novel factors that activate SSCs and could be useful for the development of technologies for the prevention of male infertility.

Gonocytes are long-lived primary germ cells that reside in the center of seminiferous cords until differentiation into spermatogonia that drive spermatogenesis. In pigs, gonocytes have research value in the production of transgenic offspring through germline modification and transplantation. However, the rarity of pig gonocytes has raised the need for an efficient isolation method. Therefore, in this study we use components of extracellular matrix, laminin, fibronectin, and collagen type IV and their derivative, gelatin, to establish a negative selection system for functionally viable gonocytes in neonatal pig. We then demonstrate functional analysis with genetic modification using lentiviral transduction and successfully transplant the donor gonocytes, which colonized the seminiferous tubules of the recipient mouse. The most effective selection method was established by sequential use of laminin and gelatin, in which the purity of gonocytes was 80% and the recovery rate of gonocytes was 78%. The selected gonocytes were labeled with fluorescent dye PKH26 and transplanted into busulfan-treated immunodeficient mouse testes. The fluorescent gonocytes colonized the recipient testes, and the resultant germ cell colonies were visible up to 4 mo after transplantation. When gonocytes were transplanted after transduction with an enhanced green fluorescent protein marker gene using lentiviral vectors, the transduced germ cell colonies were visible up to 6 mo and displayed an estimated transduction efficiency of 11.1%. These results can be applied and extended to isolate and enrich gonocytes of other species for in vitro and in vivo studies and to assist in genetic modification of male germline stem cells of livestock species.

Objective To study the influence of sugars and establish a serum-free freezing method for the cryopreservation of spermatogonial stem cells (SSCs). Design Animal study. Setting University laboratory. Animal(s) C57BL/6-TgEGFP, C57BL/6 mice. Intervention(s) Germ cells enriched from testis cells were frozen using standard freezing medium containing sugars, including monosaccharides, disaccharides, and trisaccharides at 50, 100, and 200 mM, respectively. To study the feasibility of establishing a serum-free freezing method, fetal bovine serum was substituted with knockout serum replacement. Main Outcome Measure(s) Freeze-thawed germ cells were evaluated for recovery rate, proliferation capacity, and stem cell activity after transplantation to recipient testes. Result(s) Supplementation of freezing medium with 200 mM disaccharide is an effective method for cryopreservation of SSCs. Trehalose is the most effective cryoprotectant among all the sugars tested and only lactose was comparable to trehalose. Our proliferation and transplantation data show that serum-free freezing can be achieved in freezing medium supplemented with 200 mM trehalose, 10% knockout serum replacement, and 10% dimethyl sulfoxide (DMSO) for cryopreservation of SSCs. Conclusion(s) These findings raise the possibility of effectively banking frozen SSCs from various species, including humans, in a traditional serum-free medium for germ cell research and male infertility treatments.

Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.