Human Testicular Biopsies Research Articles

Evaluation of CHD5, H3K9me3, and H4K12ac in Human Testes with Spermatogenic Maturation Arrest: A Cross-Sectional Study.

Spermatogenic maturation arrest is thought to be caused by epigenetic defects, specifically in chromatin remodeling and histone modification. This study evaluated the status of chromatin remodeling chromodomain helicase DNA binding protein 5 (CHD5) and histone modifications histone 4 lys-12 acetylation (H4K12ac) and histone 3 lys-9 trimethylation (H3K9me3) in human testicular biopsies, based on maturation arrest type. The cross-sectional study utilized 18 Bouin-fixed paraffin-embedded (BFPE) specimens prepared from residual tissue from routine laboratory tests of infertile patients. The expression of CHD5, H4K12ac, and H3K9me3 was examined through immunohistochemistry (IHC). The intensity was measured using ImageJ with IHC Profiler and StarDist plugins. Statistical analysis was performed using Python with Scipy.Stats module. The data were tested with Shapiro- Wilk for normality and Levene test for homogeneity. The differences in the intensity of spermatogenic cells were assessed using Kruskal-Wallis and Mann-Whitney tests. A difference was considered statistically significant if P<0.05. We found three types of maturation arrest, including Sertoli cell only (n=5), spermatocyte arrest (n=4), and spermatid arrest (n=9). CHD5 was positive in spermatogonia and round spermatids but absent in spermatocytes. The mean grey value (MGV) of CHD5 in spermatogonia was generally weak in spermatocyte arrest (157.4 ± 16.6) and spermatid arrest (155.3 ± 16.8), and there was no significant difference between them [P=0.49, 95% confidence interval (CI): (-4.3, 6), effect size (r): 0.02]. Although there was a significant difference in the expression of H3K9me3 and H4K12ac (P<0.001), both histone modifications were found in all observed spermatogenic cells. The expressions of CHD5, H3K9me3, and H4K12ac in different spermatogenic cell types produce similar results, indicating that they cannot be used as markers to determine the type of spermatogenic maturation arrest in humans. The significant finding in this research is the expression of CHD5 in human spermatogonia cells, which requires further study for elaboration.

A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies

To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS). Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA-sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes. A laboratory study. Three men with a diagnosis of obstructive azoospermia (age range, 30-40 years). None. Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue. Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%-8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1-/doublesex and Mab-3 related transcription factor 1-/STRA8+ spermatogonia as well as SYCP3+/protamine 2- spermatocytes. This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and invitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies

BackgroundGerm cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression.MethodsFS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines’ representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling.ResultsCx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells.ConclusionThe junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression.

The murine male reproductive organ at a glance: Three-dimensional insights and virtual histology using label-free light sheet microcopy.

The unique anatomy of the male reproductive organ reflects its complex function from sperm maturation to their storage for months until emission. Since light microscopy in two dimensions (2d) cannot sufficiently demonstrate its complex morphology, a comprehensive visualization is required to identify pathologic alterations in its entire anatomical context. Aim of this study was to use three-dimensional (3d) light sheet fluorescence microscopy (LSFM) to visualize entire murine testes in 3d, label-free and at subcellular resolution, and to assign local autofluorescence to testicular and deferent structures. Murine testes were fixed with four different fixatives and subsequently cleared with benzoic acid/benzyl benzoate. Hereafter, complete murine testes were scanned with LSFM with different fluorescence filter sets and subsequently embedded in paraffin for further conventional planar histology. Autofluorescence signals of the murine reproductive organ allowed the unambiguous identification of the testicular anatomy from the seminiferous tubules to the vas deferens with their specific stratification independent of the used fixative. Blood vessels were visualized from the pampiniform plexus to the small capillaries of single tubules. Moreover, due to the specific intrinsic fluorescence properties of the efferent ducts and the epididymis, luminal caliber, the epithelial stratification and retronuclear cytoplasmic inclusions gave a unique insight into the interface of both morphological structures. Subsequent 2d histology confirmed the identified morphological structures. LSFM analysis of the murine reproductive organ allows due to its intrinsic fluorescence a simple, label-free 3d assessment of its entire duct morphology, the epithelial composition, and the associated blood supply in its anatomical relation. LSFM provides the technical basis for comprehensive analyses of pathologically altered murine testes in its entirety by depicting specific autofluorescence. Thereby it facilitates mouse studies of testicular disease or their drug-related alterations in more detail potentially for clinical translation assessing human testicular biopsies.

PD36-08 BLOOD TESTIS BARRIER-MIMETIC DROPLET INTERFACE BILAYERS (DIBS) FOR PASSIVE PERMEABILITY PREDICTION

You have accessJournal of UrologyCME1 May 2022PD36-08 BLOOD TESTIS BARRIER-MIMETIC DROPLET INTERFACE BILAYERS (DIBS) FOR PASSIVE PERMEABILITY PREDICTION Jaime Korner, Meghan Robinson, Katherine Elvira, Ryan Flannigan, and Stephanie Willerth Jaime KornerJaime Korner More articles by this author , Meghan RobinsonMeghan Robinson More articles by this author , Katherine ElviraKatherine Elvira More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Stephanie WillerthStephanie Willerth More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002594.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The blood testis barrier (BTB) serves as a “gatekeeper” for the apical compartment of the seminiferous epithelium where spermatogenesis takes place. Tight junctions between adjacent Sertoli cells create the BTB, regulate transport of molecules, and protect the luminal compartment from toxins and pathogens. Investigating the impact of toxins and drugs, and the regulation of human spermatogenesis requires realistic in vitro models. The purpose of this study was to develop an artificial cellular membrane to mimic the BTB using droplet interface bilayers (DIBs) derived from human Sertoli cell lipid extracts. METHODS: Human Sertoli cells were collected from a dissociated human testicular biopsy using magnetic activated sorting for Follicle-Stimulating Hormone Receptor (FSHR). Sertoli cells were expanded in vitro and lipids were extracted using a Lipid Extraction Kit (Cell Biolabs). Permeability experiments were conducted using a poly(dimethylsiloxane) (PDMS) microfluidic device which allows for the controlled formation of pairs of differing droplet compartments connected by a phospholipid bilayer. A solution of Sertoli lipids in squalene (10 mg/mL) was used as the outer phase. Donor droplets contained fluorophore (100 µM in buffer) and acceptor droplets contained only buffer (both pH 7.4). The DIB-connected droplet pairs were formed at 50 °C and the temperature was decreased to 37 °C for observation. RESULTS: These results represent the first DIBs to be made using human lipid extracts and the first use of DIBs to model permeability of the BTB. We found that the BTB-mimetic DIBs were impermeable to both fluorophores tested: fluorescein and calcein. Calcein is normally membrane-impermeable and serves as a control experiment to confirm the presence of an intact phospholipid bilayer. Fluorescein has been observed to permeate DIBs made using single, synthetic lipids and intestine-mimetic DIBs but not most blood-brain barrier-mimetic DIBs. CONCLUSIONS: DIBs can be made from human Sertoli cell lipid extracts and show great potential to more accurately model BTB permeability. Source of Funding: Collaborative Healthy Grant © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e635 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jaime Korner More articles by this author Meghan Robinson More articles by this author Katherine Elvira More articles by this author Ryan Flannigan More articles by this author Stephanie Willerth More articles by this author Expand All Advertisement PDF DownloadLoading ...

Using clinically derived human tissue to 3-dimensionally bioprint personalized testicular tubules for in vitro culturing: first report

To study the feasibility and spermatogenic potential of 3-dimensional (3D) bioprinting personalized human testicular cells derived from a patient with nonobstructive azoospermia (NOA). A human testicular biopsy from a single donor with NOA was dissociated into single cells, expanded invitro, and 3D bioprinted into tubular structures akin to the seminiferous tubule using AGC-10 bioink and an RX1 bioprinter with a CENTRA coaxial microfluidic printhead from Aspect Biosystems. Three-dimensional organoid cultures were used as a nonbioprinted invitro control. Academic medical center. A 31-year-old man with NOA with testis biopsy demonstrating Sertoli cell-only syndrome. Three-dimensional bioprinting and invitro culturing of patient-derived testis cells. Cellular viability after printing was determined, along with the expression of phenotypic and spermatogenic functional genetic markers after 12 days of invitro culture. Testicular cultures were expandable invitro and generated sufficiently large numbers for 3D bioprinting at 35 million cells per mL of bioink. Viability 24 hours after printing was determined to be 93.4% ± 2.4%. Immunofluorescence staining for the phenotype markers SRY-Box transcription factor 9, insulin-like 3, actin alpha 2 smooth muscle, and synaptonemal complex protein 3 after 12 days was positive, confirming the presence of Sertoli, Leydig, peritubular myoid, and meiotic germ cells. Reverse transcription qualitative polymerase chain reaction analysis showed that after 12 days in spermatogenic media, the bioprints substantially up-regulated spermatogenic gene expression on par with nonbioprinted controls and showed a particularly significant improvement in genes involved in spermatogonial stem cell maintenance: inhibitor of deoxyribonucleic acid binding 4 by 365-fold; fibroblast growth factor 3 by 94,152-fold; stem cell growth factor receptor KIT by twofold; stimulated by retinoic acid 8 by 125-fold; deleted in azoospermia-like by 114-fold; synaptonemal complex protein 3 by sevenfold; zona pellucida binding protein by twofold; transition protein 1 by 2,908-fold; and protamine 2 by 11-fold. This study demonstrates for the first time the feasibility of 3D bioprinting adult human testicular cells. We show that the bioprinting process is compatible with high testicular cell viability and without loss of the main somatic phenotypes within the testis tissue. We demonstrate an increase in germ cell markers in the 3D bioprinted tubules after 12 days of invitro culture. This platform may carry future potential for disease modeling and regenerative opportunities in a personalized medicine framework.

PD39-09 A NOVEL 3-DIMENSIONAL BIOPRINTED MODEL OF HUMAN SPERMATOGENESIS FOR HIGH THROUGHPUT APPLICATIONS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD39)1 Sep 2021PD39-09 A NOVEL 3-DIMENSIONAL BIOPRINTED MODEL OF HUMAN SPERMATOGENESIS FOR HIGH THROUGHPUT APPLICATIONS Meghan Robinson, Ryan Flannigan, and Luke Witherspoon Meghan RobinsonMeghan Robinson More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Luke WitherspoonLuke Witherspoon More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002049.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Male factor infertility affects 15% of couples, and yet the molecular basis of most cases remains unknown. To address this issue physiologic in vitro models must be developed to investigate the underlying mechanisms of male factor infertility. This study set out to generate a 3-D bioprinted model of spermatogenesis. METHODS: Human testicular biopsies were dissociated and sorted into germ and somatic fractions by differential plating, and expanded. Germ and somatic cells were combined 1:4 and bioprinted into hollow microfibers using an Aspect Biosystems RX1 bioprinter with a coaxial printhead. Bioprinted fibers were placed in media with Epidermal Growth Factor (EGF), Leukemia Inhibitory Factor (LIF), Bone Morphogenic Protein 4 (BMP4), Stem Cell Factor (SCF), Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), metribolone, and retinoic acid, and incubated at 5% CO2 and 34°C for 12 days. Live-dead staining was performed with SYBR14 and ethidium homodimer dyes. Gene expression was assessed by real time quantitative polymerase reaction. Organization was analyzed using hematoxylin and eosin staining. RESULTS: Human primary testicular cells proved to be expandable in culture, and highly viable after bioprinting (Figure 1B). Sertoli cell cords formed within the bioprinted fibers and the organoids, illustrating an intrinsic ability to self-organize (Figure 1D-E). Germ cells in the bioprinted fibers upregulated gene expression associated with spermatogonial stem cell quiescence, proliferation, meiosis, and spermiogenesis (Figure 1F-G). Overall, their ability to self-organize and support spermatogenic activity was on par with organoids. CONCLUSIONS: This study provides a proof-of-principle 3-D bioprinted models of spermatogenesis. We show that bioprinted testicular cells retain their phenotypes and functionality, opening the door to high throughput applications. Source of Funding: VCHRI, CUASF, CIHR, UBC. © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e671-e671 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Meghan Robinson More articles by this author Ryan Flannigan More articles by this author Luke Witherspoon More articles by this author Expand All Advertisement Loading ...

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

A fertility-oriented method for histological processing of testicular biopsies in men with azoospermia

ABSTRACT The present study was designed to evaluate whether tissue preparation by glutaraldehyde and glycol methacrylate (G/GMA) improves the diagnostic assessment of testicular biopsies from azoospermic men when compared to the standard tissue preparation using Bouin’s solution and paraffin. We prospectively included a total of 21 testicular biopsies of sexually mature men aged 29–50 years with infertility and azoospermia. One testicular biopsy fragment from each patient was processed by the G/GMA method, whereas another tissue fragment was contemporarily processed by the conventional Bouin/paraffin (B/P) method. The G/GMA method provided better resolution of cytological details of the seminiferous epithelium, changing the final diagnosis in four cases. The medians of Bergmann’s spermatogenesis scores were 0.25 (interquartile range 0.04–0.88) for B/P preparations and 0.79 (interquartile range 0.17–0.96) for G/GMA preparations. Both techniques allowed accurate prediction of sperm recovery from the biopsies (B/P, area under the receiver operating characteristics [ROC] curve 0.88, 95% confidence interval [CI] 0.75–1.00; G/GMA, area under the ROC curve 0.94, 95% CI 0.86–1.00). We conclude that human testicular biopsy preparation with G/GMA improved image resolution under light microscopy and produced more reliable results for the evaluation of spermatogenesis in comparison with B/P, allowing a more precise fertility-oriented diagnosis in azoospermic men. Abbreviations: B/P: Bouin/paraffin; GMA: glycol methacrylate; G/GMA: glutaraldehyde and glycol methacrylate; ICSI: intracytoplasmic sperm injection; OA: obstructive azoospermia; NOA: nonobstructive azoospermia; TESE: testicular sperm extraction.

A preliminary study of sperm identification in microdissection testicular sperm extraction samples with deep convolutional neural networks.

Sperm identification and selection is an essential task when processing human testicular samples for in vitro fertilization. Locating and identifying sperm cell(s) in human testicular biopsy samples is labor intensive and time consuming. We developed a new computer-aided sperm analysis (CASA) system, which utilizes deep learning for near human-level performance on testicular sperm extraction (TESE), trained on a custom dataset. The system automates the identification of sperm in testicular biopsy samples. A dataset of 702 de-identified images from testicular biopsy samples of 30 patients was collected. Each image was normalized and passed through glare filters and diffraction correction. The data were split 80%, 10%, and 10% into training, validation, and test sets, respectively. Then, a deep object detection network, composed of a feature extraction network and object detection network, was trained on this dataset. The model was benchmarked against embryologists' performance on the detection task. Our deep learning CASA system achieved a mean average precision (mAP) of 0.741, with an average recall (AR) of 0.376 on our dataset. Our proposed method can work in real time; its speed is effectively limited only by the imaging speed of the microscope. Our results indicate that deep learning-based technologies can improve the efficiency of finding sperm in testicular biopsy samples.

Detection of chlamydia infection within human testicular biopsies.

STUDY QUESTIONCan Chlamydia be found in the testes of infertile men?SUMMARY ANSWERChlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men.WHAT IS KNOWN ALREADYMale chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility.STUDY DESIGN, SIZE, DURATIONWe collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling.PARTICIPANTS/MATERIALS, SETTING, METHODSThe diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques.MAIN RESULTS AND THE ROLE OF CHANCEChlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia.LIMITATIONS, REASONS FOR CAUTIONNo reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples.WIDER IMPLICATIONS FOR THE FINDINGSApplication of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure.STUDY FUNDING/COMPETING INTEREST(S)Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest.TRIAL REGISTRATION NUMBERN/A

Assessment of Sertoli Cell Proliferation by 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide and Sulforhodamine B Assays

The correct functioning of Sertoli cells (SCs) is pivotal for successful spermatogenesis. They are major targets for hormones, endocrine disruptors, and other substances that men are subjected to every day. One of the main SC functions that quickly responds to a deleterious stimulus is proliferation. This is directly related to the in vivo capacity of these cells to sustain a good number of developing germ cells. The protocols in this article can be tested on SCs of different origin. For the case of human SCs from small human testicular biopsies, a short and simple protocol to isolate and culture these cells is provided. The other protocols discussed herein represent two different procedures, somewhat complementary, to assess SC proliferation. In brief, the sulforhodamine B assay allows the investigator to dye healthy fixed SCs maintained in culture. In the MTT assay, on the other hand, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced by live SCs. These methods are mostly used to evaluate how SC proliferative activity responds to exposure to compounds such as toxicants or hormones. © 2019 by John Wiley & Sons, Inc.

Investigation of activin A in inflammatory responses of the testis and its role in the development of testicular fibrosis.

Does activin A contribute to testicular fibrosis under inflammatory conditions? Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts. Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease. This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n= 6), 50 (n= 6) and 80 (n= 5) days after first immunization. Age-matched mice injected with adjuvant alone (n= 14) and untreated mice (n= 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n= 3) or vector with an empty cassette (empty vector controls; n= 2) 30days prior to the first immunization, as well as appropriate adjuvant (n= 2) and untreated (n = 2) controls, were also examined.Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n= 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n= 7).Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated. Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence. Production of activin A by SC was stimulated by 25 and 50ng/ml TNF (P< 0.01, P< 0.001, respectively) as compared to untreated cells. INHBA mRNA was increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis, compared with control biopsies (P< 0.05), accompanied by increased total collagen (P< 0.01) and fibronectin deposition. Total testicular collagen (P< 0.0001) and fibronectin protein expression (P< 0.05) were also increased in EAO, and fibronectin expression was correlated with the severity of the disease (r= 0.9028). In animals pre-treated with rAAV-FST315 prior to immunization with TH, protein expression of fibronectin was comparable to control. Stimulation of PTC and NIH 3T3 cells with activin A increased fibronectin mRNA (P< 0.05) and the production of collagen I (P< 0.001; P< 0.01) and fibronectin (P< 0.05). Moreover, activin A also increased collagen IV mRNA (P< 0.05) in PTC, while αSMA mRNA (P< 0.01) and protein (P< 0.0001) were significantly increased by activin A in NIH 3T3 cells. N/A. A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts. Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process. This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government's Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1-2) on `Molecular pathogenesis on male reproductive disorders' funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests.

Immunolocalization of DMRTB1 in human testis with normal and impaired spermatogenesis.

The transcription factor DMRTB1 plays a pivotal role in coordinating the transition between mitosis and meiosis in murine germ cells. No reliable data are available for human testis. The present study aims to examine the testicular expression pattern of DMRTB1 in men showing normal and impaired spermatogenesis. Immunohistochemistry was performed using 54 human testicular biopsy specimens and a commercial rabbit polyclonal anti-DMRTB1 primary antibody. RT-PCR complemented immunohistochemistry. To further characterize immunopositive cells and possible co-localization, the proliferation marker Ki-67, the tumor marker PLAP, and an anti-DMRT1 antibody were used. In men with normal spermatogenesis, a strong immunoreactivity was detectable in a subset of spermatogonia (38.34±2.14%). Some spermatocytes showed a weak immunostaining. Adjacent Sertoli cells were immunonegative. Compared with a hematoxylin and eosin overview staining, these immunopositive cells were almost exclusively identified as Apale and B spermatogonia and primary spermatocytes in (pre-)leptotene, zygotene, and pachytene stages. In patients with spermatogenic arrest at spermatogonial level, an altered staining pattern was found. No immunoreactivity was detected in Sertoli cells in Sertoli cell-only syndrome. In germ cell neoplasia insitu (GCNIS) tubules, except for a few (0.4±0.03%), pre-invasive tumor cells were immunonegative. Seminoma cells showed no immunostaining. According to previous findings in mice, it seems reasonable that DMRTB1 is expressed in these normal germ cell populations. Moreover, altered staining pattern in spermatogenic arrest at spermatogonial stage suggests a correlation with mitosis and transformation into B spermatogonia. The absence of DMRTB1 in GCNIS cells and tumor cells might be associated with uncontrolled neoplastic cell proliferation and progression into invasive germ cell tumors. Further research is required to elucidate, for example, the role of DMRTB1 in the malignant transformation of human germ cells. Our data indicate a relevant role for DMRTB1 regarding the entry of spermatogonia into meiosis in men.

Expression of ciliated bronchial epithelium 1 during human spermatogenesis

To define the precise cellular localization of ciliated bronchial epithelium 1 (CBE1) in the human testis and test its relationship to impaired spermatogenesis. Gene expression analysis, and histologic and immunohistochemical evaluation. University research laboratories and andrologic outpatient clinic. Forty-three human testicular biopsies: 12 biopsies showing normal spermatogenesis (NSP), 8 with maturation arrest at level of spermatocytes (STA), 8 with maturation arrest at level of spermatids (SDA), 4 with scattered elongating spermatids, and 12 with Sertoli cell-only syndrome, with an additional 5 semen samples from healthy donors. None. Evaluation of CBE1 expression in normal as well as impaired spermatogenesis on mRNA (quantitative reverse-transcription polymerase chain reaction and in situ hybridization) and protein level (immunohistochemistry, Western blot analysis). In normal spermatogenesis, CBE1 mRNA was expressed in late pachytene spermatocytes, and the protein was localized within the flagellum of elongating spermatids from stage V up to the spermiation in stage II. Immunoelectron microscopy showed CBE1 clearly associated with microtubules at the manchette, the head-tail coupling apparatus, and the flagellum, but the protein was absent in spermatozoa. Compared with normal spermatogenesis, CBE1 mRNA was statistically significantly reduced in samples with a maturation arrest at the level of round spermatids and primary spermatocytes, and was absent in samples showing Sertoli cell-only syndrome. CBE1 protein was completely missing in SDA samples showing few elongating spermatids. Our data strongly suggest an influence of CBE1 in ciliogenesis in spermatids due to the localization at the microtubules of the elongating spermatids, indicating a role in the intramanchette and/or intraflagellar transport mechanism. The absence of CBE1 in spermatozoa suggests that CBE1 is important for the spermatid development but not for the maintenance of mature spermatozoa as a component of the flagellum.

PD08-12 IN VITRO EXPANSION AND MANIPULATION OF HUMAN SPERMATOGONIAL STEM CELLS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology II1 Apr 2017PD08-12 IN VITRO EXPANSION AND MANIPULATION OF HUMAN SPERMATOGONIAL STEM CELLS Hye-Won Song, Tung-Chin Hsieh, and Miles Wilkinson Hye-Won SongHye-Won Song More articles by this author , Tung-Chin HsiehTung-Chin Hsieh More articles by this author , and Miles WilkinsonMiles Wilkinson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.549AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Spermatogonial stem cells (SSCs) are essential for the generation of sperm, an event that occurs throughout adult life. Moreover, SSCs have therapeutic potential. A prime application for SSC transplantation is cancer survivors who went through gonadotoxic therapy during their prepubertal period, and thus no mature sperm could be cryopreserved prior to treatment. Despite the importance of human SSCs, remarkably little is known about them, including the mechanisms driving their self-renewal and expansion. Here, we demonstrate effective ways to culture, expand, and manipulate gene expression in human SSCs. METHODS Human testicular biopsies were obtained from fertile donors and cultured either as slices (organ culture) or as dispersed cells. RHOXF2 was depleted using a RHOXF2 small hairpin (sh) RNA that we cloned into a lentiviral vector. Immunostaining, FACS, and quantitative (q) RT-PCR were used to assess gene expression. RESULTS Human testicular organ cultures exhibited a modest increase in SSC and spermatogonia markers and a dramatic decline in advanced germ cell markers (Fig. 1A). This suggests a proliferative expansion of spermatogonia, including SSCs, and loss of more differentiated germ cells. Cultured dissociated germ cells exhibited a similar pattern of marker expression except that advanced germ cell markers did not decline in level (Fig. 1B). Clusters of germ cells were observed in these cultures, possibly indicative of proliferative expansion (Fig. 1C). FACS analysis with the human SSC marker, SSEA4, revealed that both culture conditions increased the number of SSEA4+ cells, confirming expansion of SSCs/undifferentiated spermatogonia. Lentivirus infection with a RHOXF2 shRNA successfully depleted RHOXF2 expression (Fig. 1D), demonstrating the ability to manipulate gene expression in human spermatogonial cell cultures. CONCLUSIONS We show that human spermatogonia expressing SSC markers can be cultured and modestly expanded in vitro, either as tissue slices or dissociated cells. Our methods to culture and manipulate gene expression in human spermatogonia can be used to decipher mechanisms underlying human SSC self-renewal as a means to expand SSCs in vitro for clinical application. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e193 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Hye-Won Song More articles by this author Tung-Chin Hsieh More articles by this author Miles Wilkinson More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression of katanin p80 in human spermatogenesis.

To define the stage-by-stage expression of KATNB1 during human spermatogenesis. Gene expression analysis, histologic and immunohistochemical evaluation. University research laboratories and andrological clinic. Eighty human testicular biopsy samples: 43 showing normal spermatogenesis, 9 with maturation arrest at level of spermatocytes, 8 with maturation arrest at level of spermatogonia, and 20 with a Sertoli cell only syndrome. None. Evaluation of katanin p80 expression in normal as well as impaired spermatogenesis on mRNA (RT-PCR, RT-qPCR, and in situ hybridization) and protein level (immunohistochemistry/immunofluorescence). KATNB1 messenger RNA is exclusively expressed in germ cells, and quantitatively reduced in maturation arrests at the level of spermatogonia. The KATNB1 protein was detected in type B spermatogonia entering meiosis and in the Golgi complex of pachytene spermatocytes. Immediately before the first meiotic division, it is colocalized with the cleaving centriole. It was also detected in early round spermatids in the dictyosome. The expression and localization of KATNB1 support a role in spindle formation. The localization of KATNB1 in early round spermatids suggests an involvement in the formation of microtubule-based structures during spermiogenesis (manchette and flagellum). These data are consistent with the demonstrated role of KATNB1 in mouse meiosis, nuclear shaping, and flagellum formation of sperm and suggest the strong conservation of function even between distantly related species.

Identification and characterization of Xenopus tropicalis common progenitors of Sertoli and peritubular myoid cell lineages.

ABSTRACTThe origin of somatic cell lineages during testicular development is controversial in mammals. Employing basal amphibian tetrapod Xenopus tropicalis we established a cell culture derived from testes of juvenile male. Expression analysis showed transcription of some pluripotency genes and Sertoli cell, peritubular myoid cell and mesenchymal cell markers. Transcription of germline-specific genes was downregulated. Immunocytochemistry revealed that a majority of cells express vimentin and co-express Sox9 and smooth muscle α-actin (Sma), indicating the existence of a common progenitor of Sertoli and peritubular myoid cell lineages. Microinjection of transgenic, red fluorescent protein (RFP)-positive somatic testicular cells into the peritoneal cavity of X. tropicalis tadpoles resulted in cell deposits in heart, pronephros and intestine, and later in a strong proliferation and formation of cell-to-cell net growing through the tadpole body. Immunohistochemistry analysis of transplanted tadpoles showed a strong expression of vimentin in RFP-positive cells. No co-localization of Sox9 and Sma signals was observed during the first three weeks indicating their dedifferentiation to migratory-active mesenchymal cells recently described in human testicular biopsies.

MP70-03 RHOXF2 AS A GUARDIAN OF THE HUMAN MALE GERMLINE STEM CELLS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2016MP70-03 RHOXF2 AS A GUARDIAN OF THE HUMAN MALE GERMLINE STEM CELLS Hye-Won Song, David Skarbrevik, Eric Babajanian, Tung-Chin Hsieh, and Miles Wilkinson Hye-Won SongHye-Won Song More articles by this author , David SkarbrevikDavid Skarbrevik More articles by this author , Eric BabajanianEric Babajanian More articles by this author , Tung-Chin HsiehTung-Chin Hsieh More articles by this author , and Miles WilkinsonMiles Wilkinson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1428AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The human germline is essential for the maintenance of our species and thus must be protected from insults. A candidate to have a role in germline protection is the X-linked reproductive homeobox (RHOX) gene cluster, which encodes a large set of transcription factors expressed in the reproductive tract, including male and female germ cells. While the RHOX transcription factors have been well studied in rodents, little is known about their function in humans. Of particular interest is human RHOXF2, which we previously showed is expressed in human spermatogonia and regulates genes that have the potential to provide genome protective roles, including GDAP1 and UNC5C. Here we further test the hypothesis that RHOXF2 serves as a genome protector in male germ cells. We examine its effect on LINE1 elements, which are deleterious transposable elements constituting 17% of the human genome that must be suppressed to maintain genome integrity METHODS Human germ cells were obtained from testicular biopsies and cultured under different conditions. RHOXF2 expression was depleted using a RHOXF2 small hairpin (sh)RNA we cloned into the lentiviral vector. Immunostaining and quantitative (q)RT-PCR were used to test the expression of RHOXF2, as well as spermatogonial and proliferation markers. LINE1 transposition was assayed in vitro in HEK-293T cells using LINE1 luciferase reporters RESULTS Modest forced RHOXF2 expression (to a level similar to that in germ cells) repressed LINE1 transposition, as assayed using both mouse and human in vitro transposition assays, in HEK-293 cells. A mutant version of RHOXF2 present in human infertility patients failed to have this activity. Loss of the mouse RHOXF2 putative ortholog, Rhox10, led to increased expression of LINE1 and substantial reduction in the number of germline stem cells in vivo. To elucidate the underlying mechanism of RHOXF2 action, we optimized conditions for culturing spermatogonia from human testicular biopsies. We successfully obtained germ cells expressing spermatogonial markers that undergo proliferation in vitro. Using RNA interference (RNAi), we depleted RHOXF2 in these spermatogonia cultures CONCLUSIONS We provide evidence that human RHOXF2 and its putative mouse ortholog, Rhox10, serve to repress the transposition of deleterious transposable elements in male germ cells. Coupled with previous finding that RHOXF2 regulates genes critical for genome defense mechanisms, it suggests that RHOXF2 serves as a guardian of genome integrity in male germ cells © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e906-e907 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hye-Won Song More articles by this author David Skarbrevik More articles by this author Eric Babajanian More articles by this author Tung-Chin Hsieh More articles by this author Miles Wilkinson More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Targeting high mobility group box protein 1 ameliorates testicular inflammation in experimental autoimmune orchitis.

Does high mobility group box protein 1 (HMGB1) regulate inflammatory reactions in a rat model of experimental autoimmune orchitis (EAO)? HMGB1 appears to be involved in regulating inflammatory reactions in testes, as HMGB1 is translocated from testicular cells during the course of EAO and blocking its action by ethyl pyruvate (EP) reduces disease progression and spermatogenic damage. Despite its immune privileged status, the human testis is prone to inflammatory lesions associated with male factor infertility. Accumulating evidence shows that HMGB1 plays an important role in onset and progression of autoimmune diseases. This is a cross sectional and longitudinal study involving Wistar male rats immunized with testicular homogenates to induce EAO 50 (EAO50; n = 10) and 80 (EAO80; n = 10) days after first immunization. Control adjuvant animals received saline instead of testicular homogenate (n = 16). Untreated animals (n = 10) were also studied. An interventional study was performed to block the action of HMGB1 starting 20 days after first immunization in EAO animals and respective controls (n = 17). Rats were treated i.p. with EP and the effect of EP treatment on testicular pathogenesis was evaluated 30 days later. Moreover, human testicular biopsies from infertile men with focal lymphocytic infiltrates (n = 7) and sections with intact spermatogenesis (n = 6) were probed with antibodies against HMGB1. Testicular RNA and protein extracts from EAO animals, EAO animals treated with EP and relevant controls were used for analysis of cytokine expression by real-time RT-PCR and enzyme-linked immunosorbent assay. HMGB1 was co-localized on rat testicular cross sections with antibodies against testicular macrophages (TM), peritubular cells (PTC) and Sertoli cells (SC). Interaction of HMGB1 and its receptors (RAGE, TLR4) as well signaling pathways after HMGB1 stimulation were studied in isolated TM, PTC and SC by proximity ligation assay and western blot, respectively. Furthermore, HMGB1 immunofluorescence on human testicular biopsies was performed. HMGB1 was translocated from the nuclei in EAO testes and testes of infertile men with impaired spermatogenesis and lymphocytic infiltrates. Elevated HMGB1 levels were observed during late phase of EAO. In testicular somatic cells HMGB1 receptors Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) were differentially expressed: HMGB1-TLR4 binding was predominant in TM, while HMGB1-RAGE interaction was prevalent in SC and PTC. In support, HMGB1 triggered extracellular signal regulated kinase (ERK)1/2 and cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) activation in SC and PTC, while TM responded to HMGB1 stimulation with p38 mitogen-activated protein kinase (MAPK) and p65 nuclear factor Kappa B (NF-ĸB) phosphorylation followed by increased tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) mRNA levels. In vivo treatment of EAO animals with EP 20 days after induction of disease revealed beneficial effects, as documented by reduced disease progression and spermatogenic damage, lower macrophage numbers, as well as decreased concentrations of HMGB1 and IL-6 in the testis compared with EAO controls. The ability of HMGB1 to bind to a wide range of receptors makes it difficult to prevent its action by blockade of a specific receptor; therefore we applied EP, a drug preventing HMGB1 release from cells. Due to its mode of action EP decreases also the secretion of some other pro-inflammatory cytokines. Using isolated primary cells imposes limitations for cell transfection studies. As a compromise between purity and yield primary cells need to be isolated from animals of different age, which has to be considered when comparing their responses. HMGB1 could be a promising target in attenuating testicular damage caused by inflammatory reactions.