Post-Cryo Survival of Rat Testicular Interstitial Cells in Hydroxyethyl Starch-Based Media

The development of cryoprotective serum-free or xeno-free media is required for safe use of cryopreserved testicular material for transplantation. In this study, the solutions containing 10% fetal bovine serum (FBS) or 5 mg/ml bovine serum albumin (BSA) did not significantly enhance the general survival of interstitial cells (ICs) after cryopreservation but increase their metabolic activity and steroid producing cell (HSD+-cells) survival. The use of 50 and 100 mg/ml hydroxyethyl starch (HES) in DMSO-based cryoprotective solutions instead of BSA or FBS enabled the improvement of the IC general survival and the survival of HSD+-cells. The use of HES supplemented media allowed to decrease the dimethyl sulfoxide (DMSO) concentration from 1.4 to 0.7 M and to preserve the amount and metabolic activity of ICs. Thus, designing cryoprotective media containing DMSO and HES can facilitate the formulation of serum-free solutions for cryopreservation that in turn paves a way for implementation of the use of cryopreserved material for practical medicine.

Relevance of angiogenesis in autoimmune testis inflammation

Experimental autoimmune orchitis (EAO) is a useful model to study organ-specific autoimmunity and chronic testicular inflammation. This model reflects testicular pathological changes reported in immunological infertility in men. Progression of EAO in rodents is associated with a significantly increased percentage of testicular endothelial cells and interstitial testicular blood vessels, indicating an ongoing angiogenic process. Vascular endothelial growth factor A (VEGFA), the main regulator of physiological and pathological angiogenesis, can stimulate endothelial cell proliferation, chemotaxis and vascular permeability. The aim of this study was to explore the role of VEGFA in the pathogenesis of testicular inflammation. Our results found VEGFA expression in Leydig cells, endothelial cells and macrophages in testis of rats with autoimmune orchitis. VEGFA level was significantly higher in testicular fluid and serum of rats at the end of the immunization period, preceding testicular damage. VEGF receptor (VEGFR) 1 is expressed mainly in testicular endothelial cells, whereas VEGFR2 was detected in germ cells and vascular smooth muscle cells. Both receptors were expressed in testicular interstitial cells. VEGFR2 increased after the immunization period in the testicular interstitium and VEGFR1 was downregulated in EAO testis. In-vivo-specific VEGFA inhibition by Bevacizumab prevented the increase in blood vessel number and reduced EAO incidence and severity. Our results unveil relevance of VEGFA-VEGFR axis during orchitis development, suggesting that VEGFA might be an early marker of testicular inflammation and Bevacizumab a therapeutic tool for treatment of testicular inflammation associated with subfertility and infertility.

Primary human testicular PDGFRα+ cells are multipotent and can be differentiated into cells with Leydig cell characteristics in vitro

STUDY QUESTION Is it possible to differentiate primary human testicular platelet-derived growth factor receptor alpha positive (PDGFRα+) cells into functional Leydig cells? SUMMARY ANSWER Although human testicular PDGFRα+ cells are multipotent and are capable of differentiating into steroidogenic cells with Leydig cell characteristics, they are not able to produce testosterone after differentiation. WHAT IS KNOWN ALREADY In rodents, stem Leydig cells (SLCs) that have been identified and isolated using the marker PDGFRα can give rise to adult testosterone-producing Leydig cells after appropriate differentiation in vitro. Although PDGFRα+ cells have also been identified in human testicular tissue, so far there is no evidence that these cells are true human SLCs that can differentiate into functional Leydig cells in vitro or in vivo. STUDY DESIGN, SIZE, DURATION We isolated testicular cells enriched for interstitial cells from frozen–thawed fragments of testicular tissue from four human donors. Depending on the obtained cell number, PDGFRα+-sorted cells of three to four donors were exposed to differentiation conditions in vitro to stimulate development into adipocytes, osteocytes, chondrocytes or into Leydig cells. We compared their cell characteristics with cells directly after sorting and cells in propagation conditions. To investigate their differentiation potential in vivo, PDGFRα+-sorted cells were transplanted in the testis of 12 luteinizing hormone receptor-knockout (LuRKO) mice of which 6 mice received immunosuppression treatment. An additional six mice did not receive cell transplantation and were used as a control. PARTICIPANTS/MATERIALS, SETTING, METHODS Human testicular interstitial cells were cultured to Passage 3 and FACS sorted for HLA-A,B,C+/CD34−/PDGFRα+. We examined their mesenchymal stromal cell (MSC) membrane protein expression by FACS analyses. Furthermore, we investigated lineage-specific staining and gene expression after MSC trilineage differentiation. For the differentiation into Leydig cells, PDGFRα+-sorted cells were cultured in either proliferation or differentiation medium for 28 days, after which they were stimulated either with or without hCG, forskolin or dbcAMP for 24 h to examine the increase in gene expression of steroidogenic enzymes using qPCR. In addition, testosterone, androstenedione and progesterone levels were measured in the culture medium. We also transplanted human PDGFRα+-sorted testicular interstitial cells into the testis of LuRKO mice. Serum was collected at several time points after transplantation, and testosterone was measured. Twenty weeks after transplantation testes were collected for histological examination. MAIN RESULTS AND THE ROLE OF CHANCE From primary cultured human testicular interstitial cells at Passage 3, we could obtain a population of HLA-A,B,C+/CD34−/PDGFRα+ cells by FACS. The sorted cells showed characteristics of MSC and were able to differentiate into adipocytes, chondrocytes and osteocytes. Upon directed differentiation into Leydig cells in vitro, we observed a significant increase in the expression of HSD3B2 and INSL3. After 24 h stimulation with forskolin or dbcAMP, a significantly increased expression of STAR and CYP11A1 was observed. The cells already expressed HSD17B3 and CYP17A1 before differentiation but the expression of these genes were not significantly increased after differentiation and stimulation. Testosterone levels could not be detected in the medium in any of the stimulation conditions, but after stimulation with forskolin or dbcAMP, androstenedione and progesterone were detected in culture medium. After transplantation of the human cells into the testes of LuRKO mice, no significant increase in serum testosterone levels was found compared to the controls. Also, no human cells were identified in the interstitium of mice testes 20 weeks after transplantation. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was performed using tissue from only four donors because of limitations in donor material. Because of the need of sufficient cell numbers, we first propagated cells to passage 3 before FACS of the desired cell population was performed. We cannot rule out this propagation of the cells resulted in loss of stem cell properties. WIDER IMPLICATIONS OF THE FINDINGS A lot of information on Leydig cell development is obtained from rodent studies, while the knowledge on human Leydig cell development is very limited. Our study shows that human testicular interstitial PDGFRα+ cells have different characteristics compared to rodent testicular PDGFRα+ cells in gene expression levels of steroidogenic enzymes and potential to differentiate in adult Leydig cells under comparable culture conditions. This emphasizes the need for confirming results from rodent studies in the human situation to be able to translate this knowledge to the human conditions, to eventually contribute to improvements of testosterone replacement therapies or establishing alternative cell therapies in the future, potentially based on SLCs. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Amsterdam UMC, location AMC, Amsterdam, the Netherlands. All authors declare no competing interests.

Sonoelastography in differentiation of benign and malignant testicular lesion in dogs

Present study aimed to evaluae the elasticity of tumorous testicular lesions and usefulness of the elastographic examination for diagnosing lesions in the testes. The study was carried out on nine dogs in which tumorous testicular lesions had been found in the ultrasonographic examination. In all the animals examined, the elastographic examination of the lesions was performed and then castration and the histopathological examination of specimens in order to determine the type of the changes. On the basis of the results of the histopathological examination the dogs were divided into two groups: group I - consisted of three dogs in which nonneoplastic testicular lesions were found and group II comprised six dogs in which neoplasic lesions that began in testicular interstitial cells (Leydigoma) were detected. The lesions observed in dogs of group I showed low stiffness (average 11.25 kPa, range 6.1 to 16.4 kPa), whereas the lesions found in dogs of group II were characterized by high stiffness (average 91.85 kPa, range 52.3 to 131.4 kPa). On the basis of a scale proposed by Goodie et al. (2012), the lesions in group I were in the range of SC1,and in turn, the lesions in group II were in the range of SC 3 inverted. Based on the results obtained, it can be stated that the sonoelastographic examination is useful method for the screening diagnostics of testicular lesions.

Blocking Both E-Selectin and P-Selectin Inhibits Neutrophil Recruitment into the Murine Testis after Ischemia-Reperfusion-Induced Injury

Ischemia-reperfusion (IR) injury of the testis results in germ cell specific apoptosis, a process in which neutrophil recruitment to the testes plays a critical role. Adhesion molecules, in particular E- and P-selectins, play a critical role in this recruitment. The present study sought to characterize the inhibitory effect of function-blocking monoclonal anti-mouse E- and P-selectin antibodies on the migration of neutrophils into the IR-induced testis of the mouse. Mice were subjected to a 2 hr period of testicular torsion (ischemia) followed by detorsion (reperfusion). Ten minutes after the onset of reperfusion mice received either a mixture of 200 μg function-blocking monoclonal E-selectin and P-selectin antibody (FBMAb group; 100 μg; each) intravenously or 200 μg of isotype-matched control-antibody (IMCAb group). Separate groups of mice underwent shamoperation (SO group) or received 500 ng of TNFα (IF group) to induce inflammation. Mice were sacrificed 24 h after reperfusion and testicular interstitial cells were isolated and analyzed for the presence of neutrophils by means of flow cytometry. The mixture of function-blocking monoclonal E- and P-selectin antibody (FBMAb) decreased neutrophil recruitment to the IR-induced testis significantly (FBMAb group as compared to the IMCAb group 20.2 ± 2.8 vs. 51.9 ± 4.0 % Gr-1+CD11b+ of total leukocytes; p = 0.0002). Therefore, blocking both E- and P-selectin may be therapeutically beneficial to protect postischemic testis.