Normal Testis Research Articles

Distribution of extracellular matrix related proteins in normal and cryptorchid ziwuling black goat testes.

The Ziwuling black goat is an indigenously in China, their offspring are frequently affected by congenital cryptorchidism. The extracellular matrix (ECM) contains cytokines and growth factors that regulate the development of the testis, and component changes often result in pathological changes. Cryptorchidism is closely related to structural changes in ECM. In this study, the histochemical staining, immunohistochemical, immunofluorescence and Western blot combined with semi-quantitative analysis was used to describe the distribution of the important ECM components Collagen type IV (Col IV), laminin (LN)and heparan sulfate proteoglycans (HSPG) in the normal and cryptorchid testes of Ziwuling black goats. Results showed that: The histochemical staining showed that the dysplasia of seminiferous tubules and decreased number of Sertoli cells in cryptorchidism, as well as sparse collagen fiber. Meanwhile, the distribution of reticular fibers is relatively rich. Furthermore, the PAS and AB staining in the interstitial vessels and lamina propria of seminiferous tubules is weak. The immunohistochemical and immunofluorescence revealed that Col IV, LN was strongly expressed in Leydig, Sertoli cells of normal testes and moderately positive in the spermatogonia and spermatids, but HSPG was not expressed in the spermatogonia. However, cryptorchidism, the expression of Col IV, LN and HPSG in Leydig, Sertoli cells significantly decreased, as well as the expression of Col IV and LN in capillary endothelial cells, but HSPG was moderately expressed in spermatogonia. Based on these data, the underdevelopment of spermatogenic epithelium, decreased synthesis function of collagen fibers and Leydig cells develop usually in the cryptorchidism were shown to be closely related to the abnormal metabolism of Col IV and LN. The positive expressed of HSPG in the spermatogonia of cryptorchid testes is related to the compensatory development of spermatogonia.

Shear Wave Elastography for Assessment of Testicular Stiffness in Patients with Varicocele: A Prospective Comparative Study.

The purpose of this study was to use ultrasonography and shear wave elastography (SWE) to assess stiffness and volume of testes in adult patients with varicocele, to compare the results with those of normal contralateral testes of the same patients and healthy testes of control subjects. In this IRB-approved prospective comparative study, 58 patients with varicocele (116 testes) and 58 control patients (116 testes) were enrolled. A total of 66 testes with varicocele were added to group A, their 50 healthful contralateral testes were added to Group B, and 116 healthy control testes were added to Group C. Analysis of variance (one way ANOVA) test was used to compare groups and Student's t-test was used for their binary comparisons. The correlation between the testicular stiffness and volume was studied using Pearson's correlation test. There was not a significant difference in the mean SWE values between three groups and also, between two-group comparisons (P < 0.05). In the mean testicular volumes, a significant difference was observed between Groups A and C (P = 0.028). Conversely, there was not a significant difference between Groups A and B (P = 0.907) or Groups B and C (P = 0.094). A meaningful correlation between testicular stiffness and volume was not detected for each group. A significant correlation was not determined between SWE values and varicocele, and also between SWE values and testicular volume. We advise that more studies with larger populations of patients are required to verify the effectiveness of SWE in predicting testicular parenchymal damage.

Successful Production of an All-Female Common Carp (Cyprinus carpio L.) Population Using cyp17a1-Deficient Neomale Carp

Due to sexual dimorphism in the growth of certain cultured fish species, the production of monosex fishes is desirable for the aquaculture industry. Nowadays, the most widely practiced technique available for the mass production of monosex fish populations is sex steroid-induced sex reversal. Here, a novel strategy for the successful production of all-female (AF) common carp (Cyprinus carpio L.), to take advantage of the sexual dimorphism in growth documented in this species, has been developed using genetic engineering via single gene-targeting manipulation without any exogenous hormone treatments. Male and female heterozygous cyp17a1-deficient common carp were first obtained using the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) technique. An all-male phenotype for homozygous cyp17a1-deficient carp, regardless of the individuals’ sex-determination genotypes (XY or XX), has been observed. A male-specific DNA marker newly identified in our laboratory was used to screen the neomale carp population with the XX genotype from the cyp17a1-deficient carp. These neomale carp develop a normal testis structure with normal spermatogenesis and sperm capacity. The neomale common carp were then mated with wild-type (WT) females (cyp17a1+/+ XX genotype) using artificial fertilization. All the AF offspring sample fish from the neomale-WT female mating were confirmed as having the cyp17a1+/− XX genotype, and normal development of gonads to ovaries was observed in 100.00% of this group at eight months post-fertilization (mpf). A total of 1000 carp fingerlings, 500 from the WT male and female and 500 from the neomale and WT female mating, were mixed and reared in the same pond. The average body weight of cyp17a1+/− XX females was higher by 6.60% (8 mpf) and 32.66% (12 mpf) than that of the control common carp. Our study demonstrates the first successful production of a monosex teleost population with the advantages of sexual dimorphism in growth using genetic manipulation targeting a single locus.

Cancer testis antigen PASD1 expression and immunogenicity in human colorectal cancer and polyps.

Colorectal cancer (CRC) is a malignant tumor arising from a human inner colon lining that may spread to other organs such as the liver and lungs. Per ARNT Sim domain containing 1 (PASD1) is a cancer-testis antigen expressed in cancers including CRC but not in normal tissues except for normal testes. This study aims to study PASD1 protein as a potential target for CRC immunotherapy. A total of 90 CRC and polyps tissue samples were investigated for PASD1 RNA and protein expression using a real-time polymerase chain reaction and immunohistochemical staining, respectively. Matched patients' peripheral blood mononuclear cells were pulsed with PASD1 peptides and measured for immunogenicity, cell cytotoxicity, and cytokine assays. The clinical data were collected and analyzed accordingly. Our results show that PASD1_v2 mRNA expression was highly expressed in CRC (46.0%) and polyps samples (33.3%). Both PASD1-1 and PASD1-2 proteins were expressed in 31.7% of CRC and 29.4% of polyps samples. Protein expression was weak to moderate positive in the cytoplasm and/or nucleus of the tissues. Immune responses towards CD4-specific PASD1 peptides were detected in 21.7% of CRC and 23.5% of polyps patients. The most immunogenic peptide was PASD1 (1) in CRC while PASD1 (3) in polyps. Cytotoxicity effects were detected up to 57.20% observed in CRC samples while IL-17A and IL-6 cytokines were highly expressed. The demographic data suggest that Chinese female patients more than 60 years old, diagnosed with late-stage rectosigmoid tumors may benefit from the PASD1 peptide immunotherapy approach. This is the first report describing CD4-positive T-helper response to the PASD1 positive CRC patients and its cytotoxicity.

VARIED PRESENTATIONS OF SCROTAL PATHOLOGIES ON HIGH-FREQUENCY ULTRASOUND AND COLOUR DOPPLER WITH REVIEW OF CURRENT KNOWLEDGE.

Introduction: Ultrasonography gives anatomical details of the testicle and surroundings. It can characterize both intratesticular and extra testicular masses and other abnormalities. Vascular perfusion can be easily assessed using colour and spectral Doppler analysis. Purpose: To classify (etiologically) and evaluate various scrotal pathologies using ultrasonography and describe the role of High-resolution ultrasound and colour Doppler in their diagnosis and differentiation. Materials and Methods: The study was done on 75 patients who were referred to the Radiology department by the General Surgery and Urology departments for scrotal ultrasound and Doppler study. This study was conducted over a period of 10 months. Results: Of the 75 patients studied, the incidence of various pathologies was as follows -inflammatory pathology in 26%, congenital in 12%, scrotal hernia in 16%, infertility in 8%, torsion in 7%, tumours in 3.3%, trauma in 3%, and others in 26.7%. The imaging features of these conditions with differentiating hallmarks are described. Conclusion: ultrasonography enables clear demonstration of the normal testes and epididymis morphology, and any morphological alterations associated with acute or chronic scrotal inflammatory diseases. Colour Doppler is highly sensitive in diagnosing acute scrotal pathology and accurately differentiates testicular ischemia /torsion from acute inflammation.

A review of scrotal temperature regulation and its importance for male fertility

Male fertility is a very important but often neglected area in medical practice. Compared to what is available on female subfertility, there is a dearth in research on male fertility. Fertility in male depends on the normally functioning hypothalamopituitary gonadal axis, normal testis and the environment necessary for viable, motile sperm production. The production of healthy germ cells require a temperature which is slightly below the core body temperature and there are several temperature regulatory mechanisms in humans that ensure the maintenance of the scrotal temperature several degrees lower. The aim of the present review is to synthesise and summarise the literature available on scrotal temperature regulation in the healthy male and to recapitulate the disease conditions, environmental, occupational and lifestyle factors that affects scrotal temperature and thus male fertility.

Investigation of pathology associated with Chlamydia pecorum infection in the male reproductive tract, and the effect on spermatogenesis and semen quality in the koala (Phascolarctos cinereus)

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.

Laparoscopic –Assisted Orchidopaxy in Non-Palpable Testes in Adult Population

ABSTRACT:&#x0D; &#x0D; Objectives: To present our experience of managing impalpable undescended testes in post-pubertal patients with intention of testicular preservation using laparoscopic assisted orchidopaxy. &#x0D; &#x0D; Study Design: Descriptive Case series.&#x0D; &#x0D; Place &amp;&#x0D; Duration of&#x0D; Study: Department of Urology, Jinnah Postgraduate Medical Center (JPMC) Karachi, between Jan 2015 to Dec 2020 with minimal 6 months follow-up.&#x0D; &#x0D; Methodology: We included all patients with clinically impalpable testes with aged 12 and above. All patients had ultrasound abdomen performed as initial screening test followed by laparoscopic assessment and definite procedure. Patients with normal or hypo-plastic testes were underwent single staged orchidopaxy, while patients with testicular atrophy were planned for orchiectomy. Intraoperative testicular biopsy and Semen analysis were not routinely performed in our study.&#x0D; &#x0D; Results: Total 36 patients with 41 impalpable testes underwent laparoscopic localization and management. Mean age of patients was 20.75+ 5.98 years. 50% of testes were on the right side, 36.1% on left sided while 3.88% were bilateral. 5 (12.1%) patients had clinical inguinal hernia.. Diagnostic yield of ultrasound was (75.6%) and 100 % in laparoscopy. 33 testis were true intra-abdominal while 4 were inguinal and 4 were vanishing testes Normal looking testes were observed in 24 testes (64.86%) followed by hypoplastic in 11(29.72%) while 2 testes (5.40%) were atrophic. Two intra-operative complications included urinary bladder injury and vas deferens injury in one patient each. Postoperatively testicular ischemic necrosis in one (2.4%) and wound infection in 2 (4.8%) patients were noted. Late follow up revealed one port site hernia (2.4%) and one testicular atrophy (2.4%). Success rate of orchidopaxy in our study was 94.6%.&#x0D; &#x0D; Conclusions: Laparoscopic has been proved as excellent diagnostic and therapeutic tool for the management of intra- abdominal undescended testes in adolescent and post-pubertal patients.&#x0D; &#x0D; &#x0D; Key words: Impalpable testes, Laparoscopic-assisted orchidopaxy, Adolescent, Adult

Dihydrotestosterone through blockade of TGF-β/Smad signaling mediates the anti-fibrosis effect under hypoxia in canine Sertoli cells

The hypoxic microenvironment of cryptorchidism is an important factor to induce the impairment of the structure and function of Sertoli cells and thus lead to spermatogenesis loss or tumorigenesis. Dihydrotestosterone (DHT), as a potent nonaromatizable 5α-reduced androgen, has both positive and negative effect on pathological fibrosis process. However, it is still unknown whether DHT can regulate hypoxia-induced fibrosis of Sertoli cells. Herein, in this study, we evaluate the DHT level, two 5α-reductase isoforms, 5α-red1 and 5α-red2, as well as HIF-1α expression pattern in canine cryptorchidism and contralateral normal testis. Results showed that the abdominal testes presented low DHT levels and 5α-red1 and 5α-red2 expression, while significantly higher HIF-1α expression and ECM production compared with the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl2), and found that DHT inhibited the fibrosis of Sertoli cells in a dose-dependent manner. Meanwhile, DHT interfered with the TGF-β signaling by reducing the expression of TGF-βRI and TGF-βRII and inhibiting the expression and phosphorylation of Smad2 and Smad3, while flutamide (androgen receptor inhibitor) inhibited these effects of DHT. Furthermore, use of LY2109761 (TGF-β receptor type I/II inhibitor) to interfere with the TGF-β/Smad pathway showed a similar effect with DHT suppression of the fibrosis in Sertoli cells. Our research data demonstrated that cryptorchidism is located in a hypoxic and DHT deficiency microenvironment. Moreover, supplementing DHT can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with AR regulating the inhibition of TGF-β/Smad signaling.

Immunohistochemical Expression of Preferentially Expressed Antigen in Melanoma (PRAME) in the Uninvolved Background Testis, Germ Cell Neoplasia In Situ, and Germ Cell Tumors of the Testis.

Preferentially expressed antigen in melanoma (PRAME) has a key role in regulating pluripotency of primordial germ cells and in the development of germ cell tumors of the testis (GCTT). However, its immunohistochemical expression in normal testes and its neoplastic counterpart remain largely unknown. We retrospectively investigated the expression of PRAME in 26 cases of GCTT, 21 cases of germ cell neoplasia in situ (GCNIS), and 17 cases of uninvolved background testes. We found that PRAME was expressed more strongly by seminomatous rather than nonseminomatous GCTT (P = .000) and by pure seminoma rather than the seminoma component of seminomatous/nonseminomatous GCTT (P = .025). In addition, GCNIS and uninvolved background testes displayed high levels of PRAME expression. PRAME is an additional marker for the differential diagnosis of GCTT and could play a key role in the transition from seminomatous to nonseminomatous GCTT.

Different fixatives influence morphology, antigen preservation, and TUNEL staining in chicken (Gallus gallus) testis

The optimized fixative for testis is still controversial. This study investigated the effects of Modified Davidson’s Fluid (mDF), 4% Paraformaldehyde (4% PFA), and Bouin’s Fluid (BF) fixatives on chicken testes in normal/cadmium (Cd) feeding groups using hematoxylin and eosin (HE), immunohistochemistry (IHC), and Terminal Transferase dUTP Nick End Labeling (TUNEL) staining. Compared to the mDF, we established that the testes fixed with 4% PFA and BF in the normal group had severe shrinkage in tubular and interstitial compartments. Moreover, compared with 4% PFA, the number of GATA4-positive Sertoli cells/mm2 reduced by 67.61% in mDF and 80.57% in BF for one seminiferous tubule. The TUNEL assay illustrated that more positive cells/mm2 in mDF group (28.47 ± 11.38) than in 4% PFA (10.49 ± 7.89). In Cd-treated testes, mDF showed more morphological details than 4% PFA and BF. In contrast, the number of GATA4-positive Sertoli cells/mm2 of 4% PFA was higher than that of mDF by 65.78% and BF by 64.80% in a seminiferous tubule. The number of TUNEL positive cells/mm2 in mDF (272.60 ± 34.41) were higher than in 4% PFA (175.91 ± 19.87). These results suggest that mDF fixative is suitable for normal and Cd-treated testis fixation for HE and TUNEL staining in chicken, whereas 4% PFA fixative is better for IHC examination.

Serial Inhibin B Measurements in Boys with Congenital Monorchism Indicate Compensatory Testicular Hypertrophy in Early Infancy.

Congenital monorchism is considered a condition in which an initially normal testis has existed but subsequently atrophied and disappeared due to a third trimester catastrophe (presumably torsion). Since inhibin B concentrations appear related to Sertoli and germ cells number, we evaluated pre- and postoperative inhibin B of boys with congenital monorchism to determine whether the well-known hypertrophy of the contralateral testis was reflected in inhibin B concentrations. Twenty-seven boys consecutively diagnosed with congenital monorchism (median age 12 months) underwent follow-up with reproductive hormones 1 year postoperatively (median age 25 months). The results were compared with inhibin B of 225 boys with congenital nonsyndromic unilateral cryptorchidism, by converting values to multiple of the median (MoM) for age in normal boys. Ten boys (37%) had blind-ending vessels and ductus deferens (vanished testis) and the remaining (63%) had testicular remnants. At the time of diagnostic procedure, monorchid boys did not have significantly lower inhibin B (median 114, range 20-208) than unilateral cryptorchid boys (136, 47-393) (p = 0.27). During follow-up, MoM values of inhibin B increased in monorchid boys (median 0.59 to 0.98) and in unilateral cryptorchid boys (0.69 to 0.89) (both p < 0.0001). Compared with the concentration at surgery, an additional 44% monorchid boys had inhibin B MoM values higher than 1.0, whereas only additional 23% of unilateral cryptorchid boys exhibited such values (p = 0.04). Generally, inhibin B MoM values were normalized during follow-up in boys with congenital monorchism, reflecting compensatory hypertrophy within the first 2.5 years of life. The compensatory capacity to increase was better in monorchism than in unilateral cryptorchidism.

Comprehensive analysis of differences of N6-methyladenosine of lncRNAs between atrazine-induced and normal Xenopus laevis testis

BackgroundIncreasing evidence suggested N6-methyladenosine (m6A) modification is crucial for male germline development. However, m6A modification of lncRNAs gains a little attention in amphibians in recent years. Xenopus laevis (X. laevis) was chosen to be an ideal model organism for testing environmental endocrine disrupting chemicals (EDCs) exposure and resultant effects. Atrazine (AZ) as an endocrine disrupt can effect development of testis in amphibians. Our previous study revealed that m6A is a highly conserved modification across the species.ResultsThe results of m6A sequences showed that m6A-methylated lncRNAs enriched in intergenic region in testes of X. laevis. We further examined the differential expression of lncRNAs m6A sites in testes of AZ-exposed and compared with that in animals from control group. The results indicated that up to 198 differentially methylated m6A sites were detected within 188 lncRNAs, in which 89 significantly up-methylated sites and 109 significantly down-methylated sites. Data from KEGG pathway analysis indicated that AZ-affected lncRNAs m6A sites were mainly involved in 10 pathways in which 3 mutual pathways were found in the result of differentially m6A-methylated mRNAs.ConclusionsThese findings suggested that differentially m6A-methylated lncRNAs and these 3 pathways may act on regulatory roles in abnormal testis development of AZ-exposed X. laevis. This study for the first time provides insights into the profile of lncRNAs m6A modifications in amphibian species.

Novel expression of zona pellucida 3 protein in normal testis; potential functional implications

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

TFDP3 as E2F Unique Partner, Has Crucial Roles in Cancer Cells and Testis.

Transcription factor DP family member 3 (TFDP3) is a cancer-testis antigen, mainly expressed in normal testis and multiple cancers. TFDP3 gene (Gene ID: 51270) is located on the chromosome X and shares a high degree of sequence homology with TFDP1 and TFDP2, which can form heterodimers with E2F family members and enhance DNA-binding activity of E2Fs. In contrast to TFDP1 and TFDP2, TFDP3 downregulates E2F-mediated transcriptional activation. During DNA damage response in cancer cells, TFDP3 is induced and can inhibit E2F1-mediated apoptosis. Moreover, TFDP3 is involved in cell autophagy and epithelial-mesenchymal transition. Regarding cancer therapy opportunity, the transduction of dendritic cells with recombinant adenovirus-encoding TFDP3 can activate autologous cytotoxic T lymphocytes to target hepatoma cells. Here, we review the characterization of TFDP3, with an emphasis on the biological function and molecular mechanism. A better understanding of TFDP3 will provide new insights into the pathological mechanisms and therapeutic strategies for cancers.

Shear-wave elastography for the assessment of testicular involvement of hematologic malignancies in children and young adults: a feasibility study.

PurposeThis study aimed to assess the feasibility of shear-wave elastography (SWE) for assessing the testicular involvement of hematologic malignancies in children and young adults.MethodsEight patients (mean age, 11.0 years; range, 0.8 to 20 years) with biopsy-confirmed testicular involvement of hematologic malignancy between January 2018 and December 2020 were retrospectively evaluated. Multiparametric ultrasound examinations, including grayscale, color Doppler ultrasonography (CDUS), and SWE, were performed. Stiffness was measured in the involved testicular area and contralateral normal parenchyma. If there was bilateral testicular involvement, the stiffness of the involved area and the adjacent normal echoic parenchyma was measured on one testis. The Mann-Whitney U test was used to compare stiffness values.ResultsOn grayscale, the testicular lesions were noted as a solitary mass in one patient, multiple lesions in four patients, and diffuse involvement in three patients. On CDUS and SWE, all patients demonstrated increased vascularity, and the stiffness of the involved area was higher than the values of normal parenchyma (the involved area vs. normal parenchyma, 11.6 kPa [3.9-20.2 kPa] vs. 2.9 kPa [1.1-3.7 kPa], P=0.003). The ratio of stiffness between the involved area and normal parenchyma was 3.4, ranging from 1.9 to 5.1. One patient showed decreased stiffness on follow-up SWE after treatment (affected testis vs. normal testis: initial, 13.8 vs. 3.2 kPa; 1 year later, 2.2 vs. 2.4 kPa).ConclusionIncreased testicular stiffness on SWE in children and young adults with hematologic malignancies suggests the possibility of testicular involvement.

A magnetic resonance imaging study in etiology of nonobstructive azoospermia.

Testicular magnetic resonance imaging parameters, including apparent diffusion coefficient, fractional anisotropy, magnetization transfer ratio, and normalized metabolite concentrations represent useful noninvasive fingerprints of nonobstructive azoospermia. Nonobstructive azoospermia etiology might correlate with the spermatogenesis status. To assess the possible association between apparent diffusion coefficient, fractional anisotropy, magnetization transfer ratio, and normalised metabolite concentrations with nonobstructive azoospermia etiology. This retrospective study included 48 consecutive men with nonobstructive azoospermia and 18 age-matched controls. All participants underwent scrotal magnetic resonance imaging. The testicular apparent diffusion coefficient, fractional anisotropy, magnetization transfer ratio, and normalized metabolite concentrations were calculated. nonobstructive azoospermia men were classified into three groups, based on etiology: group 1, idiopathic; group 2, genetic causes; and group 3, non-genetic causes. Parametric and nonparametric statistical tests were used to evaluate differences in magnetic resonance imaging parameters between nonobstructive azoospermia groups and normal testes (group 4). Regression analysis was performed to assess the most predictive magnetic resonance imaging factor of nonobstructive azoospermia etiology. Differences in mean apparent diffusion coefficient (p<.001), fractional anisotropy (p<.001), magnetization transfer ratio (p<.001), and normalized concentrations of total choline (p=.005), glucose (p=.012), myo-inositol (p=.024), and lipids (p=.010) were observed among groups. Regression analysis failed to identify the most discriminating magnetic resonance imaging feature for nonobstructive azoospermia etiology. Apparent diffusion coefficient, fractional anisotropy, magnetization transfer ratio, and normalized concentrations of total choline, glucose, myo-inositol, and lipids are helpful in discriminating nonobstructive azoospermia etiology. Magnetic resonance imaging may provide useful, noninvasive information on the microstructural and biochemical milieu of nonobstructive azoospermia testes.

Evaluation of immune status in testis and macrophage polarization associated with testicular damage in patients with nonobstructive azoospermia.

Immune cells residing in the testicular interstitial space form the immunological microenvironment of the testis. They are assumed to play a role in maintaining testicular homeostasis and immune privilege. However, the immune status and related cell polarization in patients with nonobstructive azoospermia (NOA) remains poorly characterized. System evaluation of the testis immunological microenvironment in NOA patients may help to reveal the mechanisms of idiopathic azoospermia. The gene expression patterns of immune cells in normal human testes were systematically analyzed by single-cell RNA sequencing (scRNA-seq) and preliminarily verification by the human protein atlas (HPA) online database. The immune cell infiltration profiles and immune status of patients with NOA was analyzed by single-sample gene set enrichment analysis (ssGSEA) and gene set variation analysis (GSVA) based on four independent public microarray datasets (GSE45885, GSE45887, GSE9210, and GSE145467), obtained from Gene Expression Omnibus (GEO) online database. The relationship between immune cells and spermatogenesis score was further analyzed by Spearman correlation analysis. Finally, immunohistochemistry (IHC) staining was performed to identify the main immune cell types and their polarization status in patients with NOA. Both scRNA-seq and HPA analysis showed that testicular macrophages represent the largest pool of immune cells in the normal testis, and also exhibit an attenuated inflammatory response by expressing high levels of tolerance proteins (CD163, IL-10, TGF-β, and VEGF) and reduced expression of TLR signaling pathway-related genes. Correlation analysis revealed that the testicular immune score and macrophages including M1 and M2macrophages were significantly negatively correlated with spermatogenesis score in patients with NOA (GSE45885 and GSE45887). In addition, the number of M1 and M2macrophages was significantly higher in patients with NOA (GSE9210 and GSE145467) than in normal testis. GSVA analysis indicated that the immunological microenvironment in NOA tissues was manifested by activated immune system and pro-inflammatory status. IHC staining results showed that the number of M1 and M2macrophages was significantly higher in NOA tissues than in normal testis and negatively correlated with the Johnson score. Testicular macrophage polarization may play a vital role in NOA development and is a promising potential therapeutic target.

Generation of a Highly Biomimetic Organoid, Including Vasculature, Resembling the Native Immature Testis Tissue.

The creation of a testis organoid (artificial testis tissue) with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air–liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications.

Inactivation of testis-specific gene C4orf46 is dispensable for spermatogenesis and fertility in mouse.

Several genes have been reported to be involved in spermatogenesis but their functional importance in male fertility is yet needed to be elucidated. Therefore, in current research, we focused to explore the in vivo role of evolutionary conserved and testis-specifically expressed, C4orf46, gene in male mouse fertility and spermatogenesis. The expression profile of C4orf46 is specific to testes and expressed in testes from 7 days of postpartum to onward.Thus, we generated the C4orf46 knockout mice by utilizing CRISPR/Cas9 genome editing technology and examined gene function in spermatogenesis and fertility. Surprisingly, C4orf46 knockout mice were completely fertile, displayed normal testes morphology, however, higher sperm contents were observed in knockout mice compared to wild type (WT) littermates. Subsequently, intact testis histology and architecture of seminiferous tubules were observed in C4orf46 knockout and WT mice. Similarly, sperm morphology and swimming velocity of C4orf46 knockout mice were comparable with the WT littermates. Furthermore, all type of germ cells ranging from spermatogonia to mature spermatozoa were observed in the testes and epididymis sections of C4orf46 knockout mice suggesting that disruption of C4orf46 did not impact spermatogenesis. Moreover, meiotic prophase I progression was normal, and each type of cell population was comparable between knockout and WT mice. Overall, finding from this research indicates that C4orf46 is not an essential gene for fertility in mice. This study will help researchers to avoid the repetition and duplication of efforts, and to explore the genes that are indispensable for spermatogenesis and male fertility.