Age-related histopathological and biochemical testicular damages were ameliorated by vitamin C administration / Los daños testiculares histopatológicos y bioquímicos relacionados con la edad mejoraron con la administración de vitamina C

Introduction and objectives: Aging is an irreversible process associated with decreased biological functions that can lead to the reduction of reproductive organs capacities in males and females. Paternal age is a significant predictor of offspring health and development. So, the aim of this study was to evaluate the effects of vitamin C on histopathological and biochemical testicular changes following aging process with a focus on stereological methods. Material and methods: For this study, 48 adult male NMRI mice were divided into two control and experimental groups. Mice in experimental group were supplemented with vitamin C (150mg/kg) including 24-h interval by oral gavage for 33 weeks. Same regime was performed for animals in control group except that vitamin C was replaced by water. Then, right testes were extracted for stereological and left testes were used for molecular analyses on weeks 8, 12, and 33. (AU)

Introducción y objetivos: El envejecimiento es un proceso irreversible asociado a una disminución de las funciones biológicas que puede conducir a la reducción de la capacidad de los órganos reproductivos en hombres y mujeres. La edad paterna es un predictor significativo de la salud y el desarrollo de la descendencia. Por lo tanto, el objetivo de este estudio fue evaluar los efectos de la vitamina C sobre los cambios testiculares histopatológicos y bioquímicos posteriores al proceso de envejecimiento con un enfoque en los métodos estereológicos.Material y métodos: Para este estudio, 48 ratones NMRI machos adultos se dividieron en dos grupos de control y experimentales. Los ratones del grupo experimental se suplementaron con vitamina C (150mg/kg), incluido un intervalo de 24 horas mediante sonda oral durante 33 semanas. Se realizó el mismo régimen para los animales del grupo de control, excepto que se reemplazó la vitamina C por agua. Luego, se extrajeron los testículos derechos para estereología y los testículos izquierdos se utilizaron para análisis moleculares en las semanas 8, 12 y 33. (AU)

Inactivation of Cops5 in Smooth Muscle Cells Causes Abnormal Reproductive Hormone Homeostasis and Development in Mice.

COP9 constitutive photomorphogenic homolog subunit 5 (COPS5), also known as Jab1 or CSN5, has been implicated in a wide variety of cellular and developmental processes. By analyzing male germ cell-specific COPS5-deficient mice, we have demonstrated previously that COPS5 is essential to maintain male germ survival and acrosome biogenesis. To further determine the role of Cops5 in peritubular myoid cells, a smooth muscle lineage surrounding seminiferous tubules, we herein derived mice conditionally deficient for the Cops5 gene in smooth muscle cells using transgenic Myh11-Cre mice. Although these conditional Cops5-deficient mice were born at the expected Mendelian ratio and appeared to be normal within the first week after birth, the homozygous mice started to show growth retardation after 1 week. These mice also exhibited a variety of developmental and reproductive disorders, including failure of development of reproductive organs in both males and females, spermatogenesis defects, and impaired skeletal development and immune functions. Furthermore, conditional Cops5-deficient mice revealed dramatic impairment of the endocrine system associated with testicular functions, including a marked reduction in serum levels of gonadotropins (follicle-stimulating hormone, luteinizing hormone), testosterone, insulin-like growth factor 1, and glucose, but not vasopressin. All homozygous mice died before age 67 days in the study. Collectively, our results provide novel evidence that Cops5 in smooth muscle lineage plays an essential role in postnatal development and reproductive functions.

Morphology of the male rat gonad after in Utero exposure to immunosuppressants.

The protocol for immunosuppression of pregnant women is based on immunosuppressant panels. The aim of the study was to determine the influence of commonly applied combinations of immunosuppressants to pregnant rats on the morphology of the offspring' testes. Pregnant rats were treated with cyclosporin A (CsA), mycophenolate mofetil (MMF) and prednisone (Pred) (CMG); tacrolimus (Tc), MMF and Pred (TMG); CsA, everolimus (Ev) and Pred (CEG). Testes of mature offspring underwent morphological analysis. Mainly in the testes of CMG and TMG rats the morphological and functional changes were observed: immature germ cells (GCs) in the seminiferous tubule (ST) lumen, invaginations of the basement membrane, infolding to the seminiferous epithelium (SE), the ST wall thickening, increased acidophilia of Sertoli cells' (SCs) cytoplasm, large residual bodies near the lumen, dystrophic ST and tubules look like the Sertoli cell-only syndrome, Leydig cells with abnormal cell nucleus, hypertrophy of the interstitium, blurring of the boundary between ST wall and interstitium, a reduced number of GCs in the SE, vacuolation of the SE. In the CEG there were only a reduced number of GCs in some tubules and vacuolization of SCs. The safest combination of drugs was CEG, while the TMG and CMG were gonadotoxic.

Immunohistochemistry and immunocytochemistry analysis of PLZF and VASA in mice testis during spermatogenesis.

Spermatogonial stem cells (SSCs) are the basis of male spermatogenesis and fertility. SSCs are distinguished by their ability to self-renew and differentiate into spermatozoa throughout the male reproductive life and pass genetic information to the next generation. Immunohistochemistry (IHC), immunocytochemistry (ICC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze the expression of PLZF and VASA in mice testis tissue. In this experimental study, whereas undifferentiated spermatogonial cells sharply expressed PLZF, other types of germ cells located in the seminiferous tubule were negative for this marker. Conversely, the germ cells near the basal membrane of the seminiferous tubule showed VASA expression, whereas the undifferentiated germ cells located on the basal membrane were negative. The ICC analysis indicated higher expression of PLZF in the isolated undifferentiated cells compared with differentiated germ cells. Fluidigm real-time RT-PCR results demonstrated a significant expression (P < 0.05) of VASA in the SSCs compared with differentiated cells and also showed expression of PLZF in undifferentiated spermatogonia. These results clearly proved the role of PLZF as a specific marker for SSCs, and can be beneficial for advanced research on in vitro differentiation of SSCs to functional sperms.

STRUCTURAL PECULIARITIES OF RATS' TESTES DEVELOPMENT AFTER INTRODUCTION OF FEMALE HORMONES DURING PREGNANCY.

OBJECTIVE: The aim: To de!ne regularities of testicular construction of the rats' offspring at 1-90 days of postnatal life after the introduction of female sex hormones to pregnant rats during the second and third periods of pregnancy. PATIENTS AND METHODS: Materials and methods: The study was conducted on the testes of white laboratory rats' offspring during three months of life. Pregnant rats were exposed to intravaginal injection of Utrozhestan during the second and third periods of pregnancy. histological methods were used. Analysis of the obtained results was conducted by means of statistical methods with the use of computer license program «Statistica for Windows 13¼ (StatSoft Inc., # JPZ804I382130ARCN10-J). RESULTS: Results: Administration of female sex hormones to pregnant female rats leads to a reducing of the relative area, occupied by the convoluted seminiferous tubules with lumen, and increasing in relative area, occupied by extracellular matrix, starting from the 30th and up to the 90th observation day in the offsprings' testes. During the third month after birth, in experimental group a decreasing of the testicles' spermatids differentiation degree is determined. CONCLUSION: Conclusions: During the study, the following results and conclusions were obtained: decreasing of the relative area, occupied by convoluted seminiferous tubules, increasing in relative area, occupied by extracellular matrix, also decreasing Leydig cells relative amount and a delaying of spermatid differentiation process after exposing to female sex hormones during pregnancy, especially during third period, can lead to disruption of spermatogenesis and spermiogenesis in the future.

In vitro spermatogenesis in isolated seminiferous tubules of immature mice.

Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.

[Clinicopathological characteristics of Klinefelter syndrome: a testicular biopsy analysis of 87 cases].

Objective: To investigate the clinicopathological characteristics of testicular biopsies from Klinefelter syndrome (KS) patients. Methods: The testicular biopsy specimens of 87 patients with KS (a total of 107 biopsy specimens) were collected from the Department of Pathology, Peking University Third Hospital, Beijing, China from January 2017 to July 2022. All patients were diagnosed as KS by peripheral blood karyotyping analysis. The testicular histopathologic features, testicular volume and hormone levels were evaluated retrospectively. The histopathologic analysis was used to assess the quantity and morphology of Leydig cells, the spermatogenic state of seminiferous tubules, the thickening of the basement membrane of seminiferous tubules and the changes of stroma. Results: Leydig cell proliferative nodules were seen in 95.3% (102/107) of KS testicular biopsy tissues. The eosinophilic inclusion bodies and lipofuscin in Leydig cells were found in 52.3% (56/107) and 57.9% (62/107) of specimens, respectively. The Sertoli cell only seminiferous tubules and the hyalinized tubules were found in 66.4% (71/107) and 76.6% (82/107) of the examined tissues, respectively. The tubules with complete spermatogenic arrest were found in 15.9% (17/107) of specimens, and 5.6% (6/107) of the specimens showed low spermatogenesis or incomplete spermatogenic arrest. In 85.0% (91/107) of the specimens, increased thick-walled small vessels with hyaline degeneration were identified. Conclusions: The most common features of KS testicular specimens are Leydig cell proliferative nodules, hyaline degeneration of seminiferous tubules and proliferation of thick-walled blood vessels. Testicular biopsy specimens of KS are rare. The pathologists can make a tentative diagnosis of KS based on the histological findings, combined with the ultrasound and laboratory results, which is helpful for further diagnosis and treatment of KS.

[Short-term exposure to gossypol causes reversible reproductive toxicity and nephrotoxicity in mice].

OBJECTIVE: To study the toxic effects of short-term exposure to gossypol on the testis and kidney in mice and whether these effects are reversible. METHODS: Twenty 7 to 8-week-old male mice were randomized into blank control group, solvent control group, gossypol treatment group and drug withdrawal group. In the former 3 groups, the mice were subjected to daily intragastric administration of 0.3 mL of purified water, 1% sodium carboxymethylcellulose solution, and 30 mg/mL gossypol solution for 14 days, respectively; In the drug withdrawal group, the mice were treated with gossypol solution in the same manner for 14 days followed by treatment with purified water for another 14 days. After the last administration, the mice were euthanized and tissue samples were collected. The testicular tissue was weighed and observed microscopically with HE and PAS staining; the kidney tissue was stained with HE and examined for mitochondrial ATPase activity. RESULTS: Compared with those in the control group, the mice with gossypol exposure showed reduced testicular seminiferous epithelial cells with rounded seminiferous tubules, enlarged space between the seminiferous tubules, interstitium atrophy of the testis, and incomplete differentiation of the spermatogonia. The gossypol-treated mice also presented with complete, non-elongated spermatids, a large number of cells in the state of round spermatids, and negativity for acrosome PAS reaction; diffuse renal mesangial cell hyperplasia, increased mesangial matrix, and adhesion of the mesangium to the wall of the renal capsule were observed, with significantly shrinkage or even absence of the lumens of the renal capsules and reduced kidney mitochondrial ATPase activity. Compared with the gossypol-treated mice, the mice in the drug withdrawal group showed obvious recovery of morphologies of the testis and the kidney, acrosome PAS reaction and mitochondrial ATPase activity. CONCLUSIONS: Shortterm treatment with gossypol can cause reproductive toxicity and nephrotoxicity in mice, but these toxic effects can be reversed after drug withdrawal.

Sertoli cell-only syndrome: advances, challenges, and perspectives in genetics and mechanisms.

Male infertility can be caused by quantitative and/or qualitative abnormalities in spermatogenesis, which affects men's physical and mental health. Sertoli cell-only syndrome (SCOS) is the most severe histological phenotype of male infertility characterized by the depletion of germ cells with only Sertoli cells remaining in the seminiferous tubules. Most SCOS cases cannot be explained by the already known genetic causes including karyotype abnormalities and microdeletions of the Y chromosome. With the development of sequencing technology, studies on screening new genetic causes for SCOS are growing in recent years. Directly sequencing of target genes in sporadic cases and whole-exome sequencing applied in familial cases have identified several genes associated with SCOS. Analyses of the testicular transcriptome, proteome, and epigenetics in SCOS patients provide explanations regarding the molecular mechanisms of SCOS. In this review, we discuss the possible relationship between defective germline development and SCOS based on mouse models with SCO phenotype. We also summarize the advances and challenges in the exploration of genetic causes and mechanisms of SCOS. Knowing the genetic factors of SCOS offers a better understanding of SCO and human spermatogenesis, and it also has practical significance for improving diagnosis, making appropriate medical decisions, and genetic counseling. For therapeutic implications, SCOS research, along with the achievements in stem cell technologies and gene therapy, build the foundation to develop novel therapies for SCOS patients to produce functional spermatozoa, giving them hope to father children.

es-Arp3 and es-Eps8 regulate spermatogenesis via microfilaments in the seminiferous tubule of Eriocheir sinensis.

Spermatogenesis is a complicated process that includes spermatogonia differentiation, spermatocytes meiosis, spermatids spermiogenesis and final release of spermatozoa. Actin-related protein 3 (Arp3) and epidermal growth factor receptor pathway substrate 8 (Eps8) are two actin binding proteins that regulate cell adhesion in seminiferous tubules during mammalian spermatogenesis. However, the functions of these two proteins during spermatogenesis in nonmammalian species, especially Crustacea, are still unknown. Here, we cloned es-Arp3 and es-Eps8 from the testis of Chinese mitten crab Eriocheir sinensis. es-Arp3 and es-Eps8 were located in spermatocytes, spermatids and spermatozoa. Knockdown of es-Arp3 and es-Eps8 in vivo caused morphological changes to seminiferous tubules including delayed spermatozoa release, shedding of germ cells and vacuoles. Filamentous-actin (F-actin) filaments network was disorganized due to deficiency of es-Arp3 and es-Eps8. Accompanying this, four junctional proteins (α-catenin, ß-catenin, pinin and ZO1) displayed abnormal expression levels as well as penetrating biotin signals in seminiferous tubules. We also used the Arp2/3 complex inhibitor CK666 to block es-Arp3 activity and supported es-Arp3 knockdown results. In summary, our study demonstrated for the first time that es-Arp3 and es-Eps8 are important for spermatogenesis via regulating microfilament-mediated cell adhesion in Eriocheir sinensis.

ARHGEF15 is expressed in undifferentiated spermatogonia but is not required for spermatogenesis in mice.

Spermatogenesis is a continual process that relies on the activities of undifferentiated spermatogonia, which contain spermatogonial stem cells (SSCs) that serve as the basis of spermatogenesis. The gene expression pattern and molecular control of fate decisions of undifferentiated spermatogonia are not well understood. Rho guanine nucleotide exchange factor 15 (ARHGEF15, also known as EPHEXIN5) is a guanine nucleotide-exchange factor (GEF) that activates the Rho protein. Here, we reported that ARHGEF15 was expressed in undifferentiated spermatogonia and spermatocytes in mouse testes; however, its deletion did not affect spermatogenesis. Arhgef15-/- mice were fertile, and histological examination of the seminiferous tubules of Arhgef15-/- mice revealed complete spermatogenesis with the presence of all types of spermatogenic cells. Proliferation and differentiation of the undifferentiated spermatogonia were not impacted; however, further analysis showed that Arhgef15 deletion resulted in decreased expression of Nanos2, Lin28a and Ddx4. Together, these findings suggest that ARHGEF15 was specifically enriched in undifferentiated spermatogonia and regulated gene expression but dispensable for spermatogenesis in mice.

The Molecular Signature of Human Testicular Peritubular Cells Revealed by Single-Cell Analysis.

Peritubular cells of the human testis form a small compartment surrounding the seminiferous tubules. They are crucial for sperm transport, and they emerge as contributors to the spermatogonial stem cell niche. They are among the least known cell types of the human body. We employed single-cell RNA sequencing of cultured human testicular peritubular cells (HTPCs), which had been isolated from testicular samples of donors with normal spermatogenesis. The significant overlap between our results and recently published ex vivo data indicates that HTPCs are a highly adequate cellular model to define and study these cells. Thus, based on the expression of several markers, HTPCs can be classified as testicular smooth muscle cells. Small differences between the in vivo/in vitro expressed genes may be due to cellular plasticity. Plasticity was also shown upon addition of FCS to the culture medium. Based on transcriptome similarities, four cellular states were identified. Further analyses confirmed the presence of known stem cell niche-relevant factors (e.g., GDNF) and identified unknown functions, e.g., the ability to produce retinoic acid. Therefore, HTPCs allow us to define the signature(s) and delineate the functions of human testicular peritubular cells. The data may also serve as a resource for future studies to better understand male (in)fertility.

SATINN: an automated neural network-based classification of testicular sections allows for high-throughput histopathology of mouse mutants.

MOTIVATION: The mammalian testis is a complex organ with a cellular composition that changes smoothly and cyclically in normal adults. While testis histology is already an invaluable tool for identifying and describing developmental differences in evolution and disease, methods for standardized, digital image analysis of testis are needed to expand the utility of this approach. RESULTS: We developed SATINN (Software for Analysis of Testis Images with Neural Networks), a multi-level framework for automated analysis of multiplexed immunofluorescence images from mouse testis. This approach uses residual learning to train convolutional neural networks (CNNs) to classify nuclei from seminiferous tubules into seven distinct cell types with an accuracy of 81.7%. These cell classifications are then used in a second-level tubule CNN, which places seminiferous tubules into one of 12 distinct tubule stages with 57.3% direct accuracy and 94.9% within ±1 stage. We further describe numerous cell- and tubule-level statistics that can be derived from wild-type testis. Finally, we demonstrate how the classifiers and derived statistics can be used to rapidly and precisely describe pathology by applying our methods to image data from two mutant mouse lines. Our results demonstrate the feasibility and potential of using computer-assisted analysis for testis histology, an area poised to evolve rapidly on the back of emerging, spatially resolved genomic and proteomic technologies. AVAILABILITY AND IMPLEMENTATION: The source code to reproduce the results described here and a SATINN standalone application with graphic-user interface are available from http://github.com/conradlab/SATINN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Profound Effects of Dexamethasone on the Immunological State, Synthesis and Secretion Capacity of Human Testicular Peritubular Cells.

The functions of human testicular peritubular cells (HTPCs), forming a small compartment located between the seminiferous epithelium and the interstitial areas of the testis, are not fully known but go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR) indicates that they may be regulated by glucocorticoids (GCs). Herein, we studied the consequences of the GC dexamethasone (Dex) in cultured HTPCs, which serves as a unique window into the human testis. We examined changes in cytokines, mainly by qPCR and ELISA. A holistic mass-spectrometry-based proteome analysis of cellular and secreted proteins was also performed. Dex, used in a therapeutic concentration, decreased the transcript level of proinflammatory cytokines, e.g., IL6, IL8 and MCP1. An siRNA-mediated knockdown of GR reduced the actions on IL6. Changes in IL6 were confirmed by ELISA measurements. Of note, Dex also lowered GR levels. The proteomic results revealed strong responses after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h of Dex exposure (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were extracellular matrix (ECM) and basement membrane components (e.g., FBLN2, COL1A2 and COL3A1), as well as PTX3 and StAR. These results pinpoint novel, profound effects of Dex in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may occur in men upon treatment with Dex for medical reasons.

A novel pipeline for computerized mouse spermatogenesis staging.

MOTIVATION: Differentiating 12 stages of the mouse seminiferous epithelial cycle is vital towards understanding the dynamic spermatogenesis process. However, it is challenging since two adjacent spermatogenic stages are morphologically similar. Distinguishing Stages I-III from Stages IV-V is important for histologists to understand sperm development in wildtype mice and spermatogenic defects in infertile mice. To achieve this, we propose a novel pipeline for computerized spermatogenesis staging (CSS). RESULTS: The CSS pipeline comprises four parts: (i) A seminiferous tubule segmentation model is developed to extract every single tubule; (ii) A multi-scale learning (MSL) model is developed to integrate local and global information of a seminiferous tubule to distinguish Stages I-V from Stages VI-XII; (iii) a multi-task learning (MTL) model is developed to segment the multiple testicular cells for Stages I-V without an exhaustive requirement for manual annotation; (iv) A set of 204D image-derived features is developed to discriminate Stages I-III from Stages IV-V by capturing cell-level and image-level representation. Experimental results suggest that the proposed MSL and MTL models outperform classic single-scale and single-task models when manual annotation is limited. In addition, the proposed image-derived features are discriminative between Stages I-III and Stages IV-V. In conclusion, the CSS pipeline can not only provide histologists with a solution to facilitate quantitative analysis for spermatogenesis stage identification but also help them to uncover novel computerized image-derived biomarkers. AVAILABILITY AND IMPLEMENTATION: https://github.com/jydada/CSS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dose dependent intra-testicular accumulation of silver nanoparticles triggers morphometric changes in seminiferous tubules and Leydig cells and changes the structural integrity of spermatozoa chromatin.

Citrate-coated silver nanoparticles were synthesized in one step method using tri-sodium citrate and silver nitrate at pH 6.1. After synthesis, the resulting silver nano-suspension was characterized using UV-visible spectroscopy, dynamic light scattering, high resolution-scanning electron microscopy, energy dispersive x-ray spectroscopy and fourier transform infrared-spectroscopy. The particles were intraperitoneally injected into Swiss albino male mice for a period of one complete spermatogenic cycle. The LD50 was determined following the procedure of Dixon's Up-and-Down method. The intra-testicular level of silver was measured using the technique of inductively coupled plasma-mass spectrometry. The intra-testicular location of accumulated nanoparticles was observed using auto-metallography. The cytology and volume of Leydig cells were assessed and analysed. Following the exposure to silver nanoparticles, it was found that accumulation of nanoparticles inside the seminiferous tubules is a dose-dependent process. The deposition of silver agglomerate induced morphometric changes in the lumen of seminiferous tubules and Leydig cells. The exposure also caused significant changes at the level of structural integrity of sperm chromatin material and variable damages to sperm DNA.

Fractal methods applied to the seminiferous lumen images can quantify testicular changes induced by heat stress.

Male infertility affects many couples around the world and can be related to environmental factors such as exposure to high temperatures. Even so, automated methods evaluating the seminiferous tubules to detect testicular damage are still scarce. In search of new approaches to automation in the microscopic analysis of the testis; the present study used the fractal dimension, lacunarity, multifractality and quantitative morphometry to quantify changes in microphotographs of the seminiferous lumen in testicles reversibly damaged by heat stress (43 °C, 12 min). The parameters fractal dimension, lacunarity, multifractality (Dq and α), perimeter, feret and circularity were able to detect changes in the seminiferous lumen at 7, 15 and 30 days after the testicular damage. These methods also detected the recovery of spermatogenesis at 60 days after heat stress. Area, f(α), centroid X and Y, roundness, rectangle height and width were unable to detect changes caused by heat stress. In conclusion, computer assisted methods applied to the seminiferous lumen images can be a useful new viewpoint to analyze microscopic changes in the testicles, a fast low-cost tool to assist in the automated quantification of testicular damage.

Dhh signaling pathway regulates reconstruction of seminiferous tubule-like structure.

The reconstruction of a tubule-like structure in vitro has provided a promising system to analyze factors that drive morphogenesis and the underlying mechanisms. In this study, we took advantage of the inhibitor cyclopamine and a smoothened agonist to detect the role of the Dhh signaling pathway in the reconstructed tubule-like structure. Sertoli cells did not show polarity, rounded peritubular myoid cells and scattered Leydig cells were observed, combined with less laminin and lower proliferation of Leydig, peritubular myoid, germ, and Sertoli cells. However, in the presence of SAG, elongated peritubular myoid cells gathered at the bottom of polarized Sertoli cells, and most of the Leydig cells gathered at the outer part of the elongated peritubular myoid cells. Moreover, SAG promoted the secretion of laminin, assisting in the formation of the basal membrane and promoting the proliferation of Leydig, peritubular myoid, and germ cells. The level of Gli1 was significantly downregulated when treated with cyclopamine, whereas it was significantly upregulated when treated with SAG. These results indicate that the Dhh signaling pathway regulates the reconstruction of tubule-like structures by regulating the expression of Gli1.

Stage specific expression of angiotensin-converting enzyme and thickened lamina propria in relation to male fertility.

INTRODUCTION: The testis is an immune privileged organ that provides a specific environment for germ cell development. Various factors responsible for inflammatory changes can lead to deterioration of the immune tolerant model found in the testis. As a result, the thickness of the proper membrane of seminiferous tubules changes and the process of spermatogenesis is disturbed.

Connexin43 represents an important regulator for Sertoli cell morphology, Sertoli cell nuclear ultrastructure, and Sertoli cell maturation.

The Sertoli cell (SC)-specific knockout (KO) of connexin43 (Cx43) was shown to be an effector of multiple histological changes in tubular morphology, resulting in germ cell loss through to a Sertoli-cell-only (SCO) phenotype and vacuolated seminiferous tubules containing SC-clusters. Our present study focused on the effects of Cx43 loss on SC ultrastructure. Using serial block-face scanning electron microscopy (SBF-SEM), we could confirm previous results. Ultrastructural analysis of Sertoli cell nuclei (SCN) revealed that these appear in clusters with a phenotype resembling immature/proliferating SCs in KO mice. Surprisingly, SCs of fertile wild type (WT) mice contained SCN with a predominantly smooth surface instead of deep indentations of the nuclear envelope, suggesting that these indentations do not correlate with germ cell support or spermatogenesis. SBF-SEM facilitated the precise examination of clustered SCs. Even if the exact maturation state of mutant SCs remained unclear, our study could detect indications of cellular senescence as well as immaturity, emphasising that Cx43 affects SC maturation. Moreover, Sudan III staining and transmission electron microscopy (TEM) demonstrated an altered lipid metabolism in SCs of Cx43 deficient mice.