Basement Membrane Of Seminiferous Tubules Research Articles

HIV1-Nef perturbs the integrity of blood testis barrier in rat model

ABSTRACT The blood-testis barrier is a specialized feature within the mammalian testis, located in close proximity to the basement membrane of seminiferous tubules. This barrier serves to divide the seminiferous epithelium into distinct basal and adluminal (apical) compartments. The selectivity of the BTB to foreign particles makes it a safe haven for the virus, and the high affinity of HIV for testis might lead to the vertical transmission of the virus. In the present study, recombinant HIV1-Nef (rNef) protein was injected intravenously to examine the effect of rNef on BTB. SD male rats received 250 µg and 500 µg of rNef along with 2% Evans blue dye within 1 ml through the tail vein. After 1 hour of perfusion, the animals were sacrificed for analysis. The dye migration assay and ELISA confirmed a significant impairment in the blood-testis barrier (BTB) and the manifestation of rNef in testes tissues, respectively. Moreover, a decline in the expression of tight junction proteins, including ZO1 and Occludin, was observed during rNef-induced BTB disruption. Overall, our findings demonstrated that rNef induces BTB disruption through various signaling events. At the site of ectoplasmic specialization of the seminiferous epithelium, the localization of cadherins was found to be disrupted, making the testis a vulnerable site. In conclusion, rNef perturbs the integrity of the blood-testis barrier in rat models; hence, it can also serve as a suitable model for studying the dynamics of the blood-testis barrier.

Rosa26 Locus Is Inactive in Spermatogenesis of C57BL/6 Mice

ABSTRACT Background: The mouse Rosa26 locus demonstrates ubiquitous transcriptional activity, encoding long noncoding RNAs (LncRNAs) with no functional significance in various types of tissues and cells. It is widely employed in targeting transgene knock-in studies. However, the expression pattern of Rosa26 in the testes remains elusive. In this study, we conducted a detailed transcriptional analysis of the Rosa26 locus during mouse spermatogenesis. Methods: The transcriptional activity of the Rosa26 locus in the testes was assessed through immunohistochemistry or direct fluorescence observation in two Rosa26 locus-targeting knock-in mouse lines, Rosa26-Flag-Cas9 and Rosa26-mT/mG, both with a C57BL/6 background. The two mouse lines feature constitutively expressed Flag-tagged Cas9 and red fluorescent membrane-tethered tdTomato (mTomato) proteins. Results: In Rosa26-Flag-Cas9 mice, immunostaining of testis sections revealed robust expression of the Flag-Cas9 transgene in spermatogonia adjacent to the basement membrane of seminiferous tubules. However, its expression was absent in germ cells undergoing spermatogenesis in seminiferous tubules and in maturing spermatids in the epididymis. The transgene was also observed in intertubular Leydig cells and epididymal epithelia but was absent in Sertoli cells. Similarly, in Rosa26-mT/mG mice, fluorescence microscopy showed a complete absence of mTomato expression in the spermatogonial lineage and Sertoli cells. However, robust mTomato expression was observed in intertubular Leydig cells out of the seminiferous tubules and epididymal epithelia. Conclusion: Our findings suggest that the Rosa26 locus is inactive during spermatogenesis, indicating that functional gene studies by targeting knock-in transgenes at the Rosa26 locus in the spermatogonial lineage may not be applicable in C57BL/6 mice.

Androgen Receptor (AR) Depletion Underlies the Reproductive Dysfunctions in Male Rats Exposed to Alcohol and Combination Antiretroviral Therapy (cART)

The prevalence of alcohol abuse and HIV/AIDS is high in males of reproductive age; unfortunately, a high incidence of alcohol intake has been reported in HIV/AIDS patients including those on combination antiretroviral therapy (cART). Incidentally, alcohol and cART use have each been implicated in male reproductive dysfunction. Therefore, the interactive effects of alcohol and cART on reproductive hormone levels, testicular connective tissue, and androgen receptor and Ki-67 expression were evaluated in HIV naïve adult male Sprague Dawley rats. Adult male rats were divided into four groups: control, alcohol (A), cART, and A+cART. Animals were terminated after 90 days of treatment; then, the blood and testis were extracted for analysis. Special staining for testicular connective tissue, immunoassay for reproductive hormones, and immunohistochemistry for androgen receptor and Ki-67 were conducted. The study found significantly ( p < 0.05 ) increased thickness of seminiferous tubule basement membrane in the cART group and testicular capsule in the A+cART group. Collagen, reticulin, and elastin fibers decreased significantly in all the treated groups, except for reticulin in the testis of A group animals. With exception of luteinizing hormone in the cART group, all the treated groups had significantly elevated levels of luteinizing and follicle-stimulating hormones, but no significant ( p > 0.05 ) difference was found in their testosterone and inhibin B levels. The number of Sertoli and Leydig cells expressing androgen receptor reduced significantly in all the treated groups, except for A group’s number of Sertoli cells expressing androgen receptor. Further, in all the treated groups, Ki-67 expression significantly reduced relative to control. In this study, with exception of seminiferous tubule basement membrane, all study parameters were greatly altered in the A+cART group; we suggest that the exacerbated androgen receptor depletion recorded in the A+cART group might have led to the observed severe testicular structural alteration and spermatogenesis derangement.

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.

A Fundamental Research in In Vitro Spermatogonial Stem Cell Culturing: What Are Clump Cells?

Spermatogonial stem cells (SSCs) are a small group of testicular cells located in the basement membrane of seminiferous tubules and can balance self-renewal and differentiation during spermatogenesis. Our in vitro culture experiments of mouse SSCs indicated heterogeneity of cultured cells. Highly compact colonies were observed next to SSC colonies, which we call clump cells. We used immunocytochemical staining to identify SSCs and somatic cells with VASA and Vimentin antibodies. Subsequently, we compared mRNA expression levels of VASA, DAZL, PLZF, GFRA1, Lin28, Kit, Myc and Vimentin genes using Fluidigm real-time RT-polymerase chain reaction in clump cells, SSCs, and testicular stromal cells. To better understand the functions of selected genes, we created a protein-protein interaction network and performed an enrichment analysis using different databases. Based on the data collected, we state that clump cells do not express the molecular markers of SSCs, so we cannot consider them as SSCs; however, we claim that these cells are altered SSCs. The molecular mechanism of this conversion is still obscure. Therefore, this study can support the analysis of germ cell development both in vitro and in vivo. In addition, it can be effective in finding new and more efficient treatments for male infertility.

Macro and Micro Anatomical Observation on the Testes of Local Dog (Canis lupusfamiliaris) of Assam

Background: The study on testes of local dog of Assam is of great value in regard to germplasm conservation. The aim of the study was to evaluate the gross and histomorphological examination of testes of male reproductive system. Methods: The testes were collected at the time of castration from Department of Surgery and Radiology, College of Veterinary Science, Assam Agricultural University, Khanapra, Guwahati, Assam, India. The research was carried out for a period of one year in Department of Anatomy and Histology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam. Then gross anatomical studies were made on it and the tissue samples were fixed in 10% neutral buffered formalin solution and were processed as per the standard technique of procedure (Luna, 1968). The paraffin blocks were sectioned in Shandon Finesse microtome at 5 µm thickness and the sections were stained with Mayer’s Haematoxylin and Eosin staining technique for Cellular details as per the method of Luna (1968). Result: Grossly, the testes of local dog consisted of two surface viz., lateral and medial and two ends i.e. upper end and lower end. The upper end of the testes was occupied by the head of the epididymis and the lower end of the testes was occupied by the tail of the epididymis. Mediastinum testis was observed in the centre of testes of local dog. Histologically, the testes were covered by serous layer (Tunica vaginalis), connective tissue layer (Tunica albugenia) and vascular layer (Tunica vasculosa) from outside to inwards. Spermatogenic cells like spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa, and sertoli cells were observed in seminiferous tubules. The sertoli cells were attached to the basement membrane of seminiferous tubules. Cluster of Leyding cells were found between the semineniferous tubules and it contained large spherical nuclei. The epididymides were lined by pseudo stratified ciliated columnar epithelium.

Diabetes up-regulated collagen IV and laminin α5 genes in mRNA and protein levels in seminiferous tubules of C57BL/6 adult mice

Diabetes is a disease associated with impairment of the male reproductive system that causes complications such as decreased testosterone, the diameter of the seminiferous tubule, libido, and fertility. Extracellular matrix (ECM) molecules are involved in testicular development and spermatogenesis. Laminin and collagen are key proteins in seminiferous tubule basement membrane and play an important role in spermatogenesis. The present study was conducted to investigate the effect of diabetes on collagen IV and laminin α5 changes in mice testis. In this experimental study, 40 mice (C57BL/6) were divided randomly into 4 groups: 1) Control group: without intervention, 2) Diabetic group: treated mice with 50 mg/kg streptozotocin (STZ), 3) Diabetic + Insulin group: treated mice with STZ and insulin, and 4) Sham group: received citrate buffer. After 35 days, the left testes of all specimens were used for Real-Time PCR while their right testes were applied for immunohistochemical study and Periodic acid-Schiff (PAS) staining. This study showed that gene expression and immunoreactivity of laminin α5 and collagen IV were significantly increased in diabetic mice compared to other groups (P<0.05). Also, PAS staining showed the thickness of seminiferous tubule basement membrane in the Diabetic group compared to other group increased significantly (p<0.05). In Diabetic + Insulin compared to Diabetic group, gene expression, the intensity of immunoreactivity and thickness of seminiferous tubule basement membrane decreased significantly (P<0.05). Our findings indicated that diabetes causes up-regulation of collagen IV and laminin α5 in mRNA and protein levels in the seminiferous tubule basement membrane and may cause disorder in spermatogenesis in mice.

Histological, Micrometrical and Histochemical Studies on the Testes of Large White Yorkshire Pig (Sus scrofa domesticus)

The study has been conducted on the testes of 10 adult Large White Yorkshire pig (1.5-2) years of age. The testis was surrounded by a capsule made up of dense irregular connective tissue comprising three layers viz., outer visceral layer of tunica vaginalis, middle tunica albugenia and inner tunica vasculosa. The connective tissue trabeculae were extended from the capsule and divided the parenchyma of the testis into number of lobules and consisted of collagen, elastic and reticular fibers along with rete testis and blood vessels. The Periodic Acid–Schiff shows strong affinity in basement membrane of seminiferous tubules and tunica albuginea with a moderate activity in Leydig cells. The Alcian blue shows strong affinity in basement membrane of seminiferous tubule and weak activity was observed in tunica albuginea and interstitial tissue. The different histomorphometrical parameters with regard to thickness of testicular capsule, diameter and height of seminiferous tubules were found to be higher in the left testicle.

Three-dimensional co-culture of human spermatogonial stem cells with Sertoli cells in soft agar culture system supplemented by growth factors and Laminin

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.

Effects of Experimental Hyperthyroidism on Collagen IV and Laminin-α5 Gene Expression in Balb/C Mouse Seminiferous Tubules

Background: Hyperthyroidism is one of the disorders of the thyroid gland, an organ that controls the cellular and molecular behaviors of the seminiferous tubule basement membrane (BM), and ultimately, influences its evolutionary process. We aimed to investigate the effects of hyperthyroidism on immunohistochemical characteristics and gene expression levels of collagen IV and laminin-α5 in seminiferous tubules BM of Balb/C mice. Materials and Methods: Twenty male Balb/C mice were divided into experimental and control groups. The experimental group received 500 mg/l of levothyroxine (L-thyroxine) diluted in drinking water for two months to inducing hyperthyroidism, which was confirmed by radioimmunoassay. At the end of the study, the mice were sacrificed, and their testes were extracted for immunohistochemistry and real-time polymerase chain reaction assays. Results: Although a weak reaction was observed in the experimental specimens, no significant enhancement was noted in color intensity of type IV collagen in the seminiferous tubules BM of the experimental group as compared to the control group (P>0.05). Collagen IV gene expression results in the experimental group were not significantly different from the controls (P>0.05). Thus, there was a significant increase in laminin α5 gene expression compared to the control group (P=0.016). Conclusion: Considering the key role of collagen IV and laminin-α5 in the seminiferous tubule BM in the testes, the results of this study indicated that hyperthyroidism has important effects on both structures and functions of these two components. [GMJ.2019;8:e1369]

Effects of Experimental Hyperthyroidism on Collagen IV and Laminin-α5 Gene Expression in Balb/C Mouse Seminiferous Tubules.

Background: Hyperthyroidism is one of the disorders of the thyroid gland, an organ that controls the cellular and molecular behaviors of the seminiferous tubule basement membrane (BM), and ultimately, influences its evolutionary process. We aimed to investigate the effects of hyperthyroidism on immunohistochemical characteristics and gene expression levels of collagen IV and laminin-α5 in seminiferous tubules BM of Balb/C mice. Materials and Methods: Twenty male Balb/C mice were divided into experimental and control groups. The experimental group received 500 mg/l of levothyroxine (L-thyroxine) diluted in drinking water for two months to inducing hyperthyroidism, which was confirmed by radioimmunoassay. At the end of the study, the mice were sacrificed, and their testes were extracted for immunohistochemistry and real-time polymerase chain reaction assays. Results: Although a weak reaction was observed in the experimental specimens, no significant enhancement was noted in color intensity of type IV collagen in the seminiferous tubules BM of the experimental group as compared to the control group (P>0.05). Collagen IV gene expression results in the experimental group were not significantly different from the controls (P>0.05). Thus, there was a significant increase in laminin α5 gene expression compared to the control group (P=0.016). Conclusion: Considering the key role of collagen IV and laminin-α5 in the seminiferous tubule BM in the testes, the results of this study indicated that hyperthyroidism has important effects on both structures and functions of these two components.

Spermatogenesis Recovery Potentials after Transplantation of Adipose Tissue-Derived Mesenchymal Stem Cells Cultured with Growth Factors in Experimental Azoospermic Mouse Models.

Objective Approximately 1% of the male population suffers from obstructive or non-obstructive azoospermia. Previous in vitro studies have successfully differentiated mesenchymal stem cells (MSCs) into germ cells. Because of immune- modulating features, safety, and simple isolation, adipose tissue-derived MSCs (AT-MSCs) are good candidates for such studies. However, low availability is the main limitation in using these cells. Different growth factors have been investigated to overcome this issue. In the present study, we aimed to comparatively assess the performance of AT-MSCs cultured under the presence or absence of three different growth factors, epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF), following transplantation in testicular torsion-detorsion mice Materials and Methods This was an experimental study in which AT-MSCs were first isolated from male Naval Medical Research Institute (NMRI) mice. Then, the mice underwent testicular torsion-detorsion surgery and received bromodeoxyuridine (BrdU)-labeled AT-MSCs into the lumen of seminiferous tubules. The transplanted cells had been cultured in different conditioned media, containing the three growth factors and without them. The expression of germ cell-specific markers was evaluated with real-time polymerase chain reaction (PCR) and western-blot. Moreover, immunohistochemical staining was used to trace the labeled cells. Results The number of transplanted AT-MSCs resided in the basement membrane of seminiferous tubules significantly increased after 8 weeks. The expression levels of Gcnf and Mvh genes in the transplanted testicles by AT-MSCs cultured in the growth factors-supplemented medium was greater than those in the control group (P<0.001 and P<0.05, respectively). The expression levels of the c-Kit and Scp3 genes did not significantly differ from the control group. ConclusionOur findings showed that the use of EGF, LIF and GDNF to culture AT-MSCs can be very helpful in terms of MSC survival and localization.

Meiosis, spermatogenesis and ultrastructure of the basement membrane of seminiferous tubules in patients with azoospermia

The study objective is to determine the connection between meiosis abnormalities and changes in the basement membrane (BM) ultrastructure of the seminiferous tubules in patients with spermatogenesis disorders: azoospermia or severe oligospermia. Materials and methods. Electron microscopy of BMstructure of the seminiferous tubules of 31 patients with azoospermia or severe oligospermia and normal male karyotype (46, XY) without microdeletions of the Y chromosome or structural changes in chromosomes was performed. The group 1 included 18 patients with preserved spermatogenesis including germinal cell differentiation into mature spermatids; the group 2 consisted of13 patients with either Sertoli cells and spermatogonia or only Sertoli cells. Results. Six main types of ultrastructural changes in BM were observed. The most prominent were BM invaginations in the form of basket weaves that were observed in 8 of 13patients in the group 2. In the group 1, individual basket weaves were observed in 2 of 18patients. Im-munocytochemical examination of I order spermatocyte spread nuclei in 14 patients was performed. In patients of the group 2, spermatocytes weren’t discovered. In 10 patients of the group 1, meiosis arrest at the zygotene and pachytene stages was observed in 18—50 % of spermatocytes. In 6patients, 30 to 50 % of spermatocytes with impaired formation of the structure of the sex body were revealed. Conclusion . The morphological changes of the BM of the seminiferous tubules (“basket weaves” and duplications of the BM) are associated with marked disorders of meiosis and spermatogenesis. It is suggested about the possibility of mutations of the genes encoding the proteins of BM.

Effect of Lead Acetate on Basement Membrane of Seminiferous Tubules of Adult Rat Testis and Protective Effects of Ficus Carica: A Histological Study.

To determine the effects of lead acetate and Ficus carica on disruption of basement membrane in seminiferous tubules of adult rat testis. An experimental study. Department of Anatomy, Army Medical College, Rawalpindi in collaboration with National Institute of Health (NIH), Islamabad, from March to November 2017. Thirty male adult Sprague Dawley rats were selected and divided into three groups, each with ten animals. All treatments were given once daily for a period of eight weeks. Control was labelled as group A. Group B was administered lead acetate at a dose of 30 mg/kg body weight. Group C was treated with lead acetate at a dose of 30 mg/kg body weight and Ficus carica at a dose of 80 mg/kg body weight. Animals were dissected 24 hours after the last dose. Testis were treated, fixed and stained for histological study. Disruption of basement membrane in seminiferous tubules was scored morphometrically on a scale of 0 (normal) to 3 (>70% tubules showing disruption) and statistically analysed. Significant number of seminiferous tubules showed disruption of basement membrane in group B (20%) as compared to group A (0%). Less severe disruption of membrane was seen in group C as compared to group B, which was statistically not significant (p=0.082). Lead acetate causes significant disruption of basement membrane in seminiferous tubules of testis of adult rats but subsequent administration of Ficus carica reduces the effects on short term.

Successful Human Spermatogonial Stem Cells Homing in Recipient Mouse Testis after In Vitro Transplantation and Organ Culture.

Objective In vitro transplantation (IVT) of spermatogonial stem cells (SSCs) is one of the most recent methods in transplantation in recent decades. In this study, IVT and SSCs homing on seminiferous tubules of host testis in organ culture have been studied. Materials and Methods In this experimental study, human SSCs were isolated and their identities were confirmed by tracking their promyelocytic leukemia zinc finger (PLZF) protein. These cells were transplanted to adult azoospermia mouse testes using two methods, namely, IVT and in vivo transplantation as transplantation groups, and testes without transplantation of cells were assigned in the control group. Then histomorphometric, immunohistochemical and molecular studies were done after 2 weeks. Results After two weeks, histomorphometric studies revealed that the number of subsided spermatogonial cells (SCs) and the percentage of tubules with subsided SCs in IVT and in vivo groups were significantly more than those in the control group (P<0.05). Immunohistochemical studies in the transplantation groups confirmed that the PLZF protein was expressed in the cells subsided on the seminiferous tubule. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) demonstrated that the PLZF gene expression was only positive in the transplantation groups, but it was not significantly different between the IVT group and the in vivo group (P>0.05). ConclusionTesticular tissue culture conditions after SSC transplantation can help these cells subside on the seminiferous tubule basement membrane.

In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses

SummaryAdeno-associated virus (AAV) penetrates the blood-brain barrier, but it is unknown whether AAV penetrates other tight junctions. Genetic manipulation of testis has been hampered by the basement membrane of seminiferous tubules and the blood-testis barrier (BTB), which forms between Sertoli cells and divides the tubules into basal and adluminal compartments. Here, we demonstrate in vivo genetic manipulation of spermatogonial stem cells (SSCs) and their microenvironment via AAV1/9. AAV1/9 microinjected into the seminiferous tubules penetrated both the basement membrane and BTB, thereby transducing not only Sertoli cells and SSCs but also peritubular cells and Leydig cells. Moreover, when congenitally infertile KitlSl/KitlSl-d mouse testes with defective Sertoli cells received Kitl-expressing AAVs, spermatogenesis regenerated and offspring were produced. None of the offspring contained the AAV genome. Thus, AAV1/9 allows efficient germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for the development of gene therapies for reproductive defects.

Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment.

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research.

Three-dimensional analysis of seminiferous tubules and spermatogenic waves in mice.

The aim of the present study was to reconstruct seminiferous tubules and analyze spermatogenic waves in seminiferous epithelia in developing and adult mice using serial paraffin sections and high-performance three-dimensional (3D) reconstruction software. By labeling the basement membrane of seminiferous tubules with fluorescent immunohistochemistry or periodic acid-Schiff-hematoxylin staining, all seminiferous tubules were reconstructed in 9 testes from 9 different mice, 3 each at 0, 21 and 90 days (adult) postpartum. The 3D structure of seminiferous tubules, including the number and length of tubules as well as the number of connections with the rete testis, branching points and blind ends, was assessed accurately. Although tubules showed marked variations among individual mice, their overall structure was regular and retained from newborn to adult mice. Some seminiferous tubules contained inner portions running distant from the testis surface. In a representative testis at 21 days, the sites at which spermatids initially occurred were examined by labeling acrosomes and were found to be preferentially distributed in the upper and medial portions of the testis close to the rete testis. In a representative adult testis, 76 complete waves with an average length of 16.9 mm were found and their directions were analyzed. The methods used in the present study will be useful for investigating the structure and function of seminiferous tubules in mice and humans under normal and pathological conditions, such as infertility.

N-nitrosamines induced infertility and hepatotoxicity in male rabbits.

N-nitrosamines are widely spread environmental pollutants of well-known toxicity and carcinogenicity in various animal species. These compounds are metabolically activated by cytochrome P450 system predominantly in the liver and in other tissues into more active metabolites leading to generation of both alkylating agents that alkylate DNA and reactive oxygen species. In the current study, we investigated the influence of four types of N-nitrosamines that are commonly present in the environment [methyethylnitrosamine, (MEN), diethylnitrosamine (DEN), diphenylnitroasamine (DPN) and dimethylnitrosamine (DMN)] on both livers and testes of male rabbits through assessment of 17 β-hydroxysteroid dehydrogenase (17 β-HSD) activity. The protein expression of the three cytochrome P450s (CYP11A1, CYP19A1, and CYP21A2) is involved in the steroidogenesis. The levels of testosterone (T) and estradiol (E2) were also determined in the plasma of N-nitrosamines-treated rabbits after one, four-, eight- and twelve weeks of treatment of male New Zealand rabbits with an oral dose of 0.5 mg/kg B.W/day of each compound. In addition, activities of glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT) and levels of free radicals measured as thiobarbituric acid reactive substances (TBARS), and reduced glutathione (GSH) level were quantified in both livers and testes. The present study showed that levels of free radicals (TBARS) were markedly increased, whereas GSH levels were depleted in the tissues of both livers and testes after treatment of rabbits with any of N-nitrosamines. In addition, all tested N-nitrosamines inhibited the activities of antioxidant enzyme activities (GR, GST, SOD, and CAT) in hepatic and testicular tissues of rabbits after 12 weeks of treatment. Histopathological examination showed that N-nitrosamines caused lymphocytic infiltration with vascular degeneration and necrosis, congestion of central vein with RBCs hemolysis, dilated sinusoids, as well as fibrosis around portal areas were seen in hepatic tissues. In the testes, histopathological examination displayed disorganized seminiferous tubules with degeneration of germinal epithelium and Sertoli cells. Also, spermatogenic cells had pyknotic nuclei and others were detached from basement membranes of seminiferous tubules, edema was seen between seminiferous tubules. Moreover, the present data showed that MEN and DEN down-regulated the protein expression of both CYP19A1 and 21A2 in both livers and testes of male rabbits. In addition, both MEN and DEN decreased levels of testosterone and estradiol in plasma of treated rabbits. On the one hand, DMN and DPN markedly up-regulated the protein expression of CYP19A1 in both hepatic and testicular tissues of treated rabbits. These compounds potentially increased estradiol and decreased testosterone levels. On the other hand, no correlation was found between the expression of CYP11A1 and levels of both testosterone and estradiol. It is concluded that most of tested N-nitrosamines induce different changes, which could be a new mechanism of infertility due to exposure to N-nitrosamines from different environmental sources.

Transcriptional regulatory networks underlying the reprogramming of spermatogonial stem cells to multipotent stem cells

Spermatogonial stem cells (SSCs) are germline stem cells located along the basement membrane of seminiferous tubules in testes. Recently, SSCs were shown to be reprogrammed into multipotent SSCs (mSSCs). However, both the key factors and biological networks underlying this reprogramming remain elusive. Here, we present transcriptional regulatory networks (TRNs) that control cellular processes related to the SSC-to-mSSC reprogramming. Previously, we established intermediate SSCs (iSSCs) undergoing the transition to mSSCs and generated gene expression profiles of SSCs, iSSCs and mSSCs. By comparing these profiles, we identified 2643 genes that were up-regulated during the reprogramming process and 15 key transcription factors (TFs) that regulate these genes. Using the TF-target relationships, we developed TRNs describing how these TFs regulate three pluripotency-related processes (cell proliferation, stem cell maintenance and epigenetic regulation) during the reprogramming. The TRNs showed that 4 of the 15 TFs (Oct4/Pou5f1, Cux1, Zfp143 and E2f4) regulated cell proliferation during the early stages of reprogramming, whereas 11 TFs (Oct4/Pou5f1, Foxm1, Cux1, Zfp143, Trp53, E2f4, Esrrb, Nfyb, Nanog, Sox2 and Klf4) regulated the three pluripotency-related processes during the late stages of reprogramming. Our TRNs provide a model for the temporally coordinated transcriptional regulation of pluripotency-related processes during the SSC-to-mSSC reprogramming, which can be further tested in detailed functional studies.