Basement Membrane Of Tubules Research Articles

Vitrified canine testicular cells allow the formation of spermatogonial stem cells and seminiferous tubules following their xenotransplantation into nude mice

Belgian Malinois (BM), one of the excellent military dog breeds in South Korea, is usually castrated before sexual maturation. Therefore, the transfer of their genetic features to the next generation is difficult. To overcome this, testicular cells from 4-month-old BMs were frozen. Testicular cells were thawed after 3 months and cultured in StemPro-34 medium. Spermatogonial stem cell (SSC) characteristics were determined by the transplantation of the cultured germ cell-derived colonies (GDCs) into empty testes, containing only several endogenous SSCs and Sertoli cells, of immunodeficient mice, 4 weeks after busulfan treatment. Following the implantation, the transplanted cells localized in the basement membrane of the seminiferous tubules, and ultimately colonized the recipient testes. Xenotransplantation of GDCs together with testicular somatic cells conjugated with extracellular matrix (ECM), led to the formation of de novo seminiferous tubules. These seminiferous tubules were mostly composed of Sertoli cells. Some germ cells were localized in the basement membrane of seminiferous tubules. This study revealed that BM-derived SSCs, obtained from the castrated testes, might be a valuable tool for the transfer of BM genetic features to the next generation.

Effects of Extracellular Matrix Protein-derived Signaling on the Maintenance of the Undifferentiated State of Spermatogonial Stem Cells from Porcine Neonatal Testis.

In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.

Derivation of male germ cells from ram bone marrow mesenchymal stem cells by three different methods and evaluation of their fate after transplantation into the testis.

Mesenchymal stem cells (MSCs) have the capacity to differentiate into germ cells (GCs). This research, for the first time, has evaluated the fate of in vitro MSC-derived GCs generated by three different induction methods and compared them after transplantation into the testes of rams. Passage-3 ram bone marrow (BM)-MSCs were divided into three treatment groups: (1) 14-d treatment with 10 μM retinoic acid (RA; RA14), (2) 21-d treatment with 10 μM RA (RA21), and (3) 21-d treatment with 10 ng/ml transforming growth factor beta-1 (TGFb1). After confirmation of the existence of germ-like cells in the culture, the treated cells were labeled and transplanted into the testes of ram lambs. After 2 mo, we conducted histological evaluations of the rams' testes. Results showed that in vitro-derived GCs from all treatment groups survived in the testes. Some of these GCs homed at the basement membrane of seminiferous tubules and formed colonies. The homed cells and cell colonies were similar to testicular native spermatogonia and expressed PGP9.5. TGFb1 exhibited the highest efficiency for in vitro production of GCs as well as the highest capability for homing and colony formation in the testes. RA21 was less efficient than TGFb1, particularly in colony formation. RA14 was the weakest group. No further differentiation of the transplanted GCs was observed. From our results, it could be concluded that a 21-d treatment period of BM-MSCs with TGFb1 is the most efficient method for in vitro generation of spermatogonia-like cells that survive, home, and form colonies in the testes.

Renal toxicity of slimming herbs (Aristolochia fangchi) containing aristolochic acid in rats

Introduction Aristolochic acid (AA) is a potent nephrotoxic and carcinogenic in both animals and humans. The present study supported the evidence that AA is nephrotoxic and recovery can be achieved after stopping the intake of slimming pills containing this chemical. Aim The present study aimed to show that natural products are not without risk and prompts nephrologists to investigate this possibility when faced with renal disease of unknown origin. Materials and methods Forty adult female albino rats were divided into four groups: the control group and three experimental groups. The control group received normal saline intraperitoneally, and the three experimental groups (1AA+1AAR, 2AA+2AAR, and 3AA+3AAR (Aristolochic Acid Recovery); each comprising 10 rats) received AA intraperitoneally (0.1 mg/kg) for 7, 14, and 35 days, respectively. At the end of each treatment period, five rats were sacrificed and five other rats represented the recovery groups. At the time of sacrifice the rats were anesthetized, and blood samples were taken for measuring kidney functions. Specimens from the right kidney of each rat were taken for light and electron microscopic examination. Results Light microscopic changes were congestion of glomerular capillaries, degeneration of proximal tubules, and increased deposition of collagen fibers around Bowman’s basement membrane and the basement membranes of proximal and distal tubules. The medulla showed hyalinization of collecting tubules, congestion of vasa recta, and inflammatory cells infiltration. Ultrastructural observations were fusion of podocytes, damaged mitochondria of proximal tubules, and destruction of its basolateral infoldings. Conclusion Our results strongly demonstrate that pure AA, which has been identified in slimming Chinese herb mixtures used in weight-reducing pills, is nephrotoxic.

Rituximab for the Treatment of IgG4-Related Tubulointerstitial Nephritis: Case Report and Review of the Literature.

Immunoglobulin type gamma 4 (IgG4)-related disease is a relatively newly described clinical entity characterized by a distinctive histopathological appearance, increased numbers of IgG4 positive plasma cells and often, but not always, elevated serum IgG4 concentrations. The most common renal manifestation of IgG4-related disease is tubulointerstitial nephritis marked with proteinuria, hematuria, decreased kidney function, hypocomplementemia, and radiologic abnormalities. Renal biopsy characteristics include dense lymphoplasmacytic tubulointerstitial nephritis that stains for IgG4, storiform fibrosis, and immune complex deposition in the interstitium and along tubule basement membranes. Treatment traditionally consists of prolonged glucocorticoids but cases refractory to glucocorticoids have been reported.We report a case of a 58-year-old Caucasian man who presented with fatigue, 50 pound weight loss, dyspnea, lymphadenopathy, and nephromegaly. The patient was first misdiagnosed as chronic interstitial nephritis secondary to renal sarcoid and was treated with repeated doses of prednisone. On his third relapse, he underwent a repeat renal biopsy and a diagnosis of IgG4-tubulointerstitial nephritis was confirmed. He was refractory to treatment with prednisone. The patient received Rituximab and had prompt sustained improvement in renal function. At 1 year post Rituximab treatment, his serum creatinine remains at baseline and imaging study revealed reduction in his kidney size.This is the first case report using Rituximab as a steroid sparing option for refractory IgG4-tubulointerstitial nephritis. More information is needed on the long-term effects of using of B-cell depleting agents for glucocorticoid resistant IgG4-tubulointerstitial nephritis.

The role of interstitial changes in the progression of chronic kidney disease.

Interstitium - the renal tubulointerstitial compartment - is located between the renal tubule basement membrane and microcirculation vessels. Interstitial fibroblasts produce the extracellular matrix and constitute the structure's cellular skeleton, regulating spatial relationships between its components (microenvironment). The tubular epithelium and endothelium cooperate within an integrated microenvironment. Structural or functional impairment of the extracellular matrix, microcirculation vessels or tubular epithelium results in disturbances of tubulointerstitial compartment components. In the course of glomerular kidney diseases, the intrarenal RAA system becomes activated and inflammatory mediators are released. Interstitial inflammation and microcirculatory disorders develop, inducing adverse consequences, manifested mainly through the process of hypoxia and inflammation. Inflammation-induced increase in interleukin-1 (TNF-α) expression leads to increased concentrations of VEGF, ICAM-1, angiotensin II, IL-6 and IL-8. Cytokines activate fibroblasts, myofibroblasts and endothelial cells. Fibrosis is also triggered by HIF-1alpha pathway activation, resulting in vascular growth and fibroblast proliferation. This reaction likewise occurs through activation of NF-ĸβ, EPO, GLUT-1, IGF-1 and INOS. Interstitial fibrosis is one of the factors determining the clinical course of kidney diseases. Apart from inducing fibrosis, microcirculatory disorders lead to the progression of hypoxia. Angiogenesis is a part of the repair process accompanying fibrosis. Its determinant is the normal function and structure of endothelial cells manifested by their ability to migrate and proliferate in response to, inter alia, angiopoietins, VEGF and nitric oxide synthase. Administering a three-drug RAAS-inhibiting therapy to patients with chronic glomerulopathies improves tubular function, measured by the decrease in excretion of NAG and propeptide of type III procollagen fibres, and contributes to the improvement in microcirculation functioning.

Effect of crude polyphenol extracted from black olive fruit (Olea europae) on male reproductive system of rats

The current study was conducted to evaluate the effect of olive polyphenols (200 mg/kg) on mature reproductive performance of male rats subjected to oxidative stress by 1%hydrogen peroxide. Twenty rats were divided randomly into four equal groups and treated for 30 days to control group C which were orally adminstrated ordinary tap water, while the first group (G1) was given 1% H2O2 in drinking water, the second group (G2) was treated orally alcoholic extract of polyphenol in olive (200mg/kg) by oral intubation. The third treated group (G3) was treated orally H2O2 and black olive fruit- polyphenol. Blood samples were collected after 30 days for measuring, the serum concentration of testosterone, follicular stimulating hormone (FSH) and leuitinizing hormone (LH). At the end of experiment, the animals were scarified and testis were removed, weighed as well as section from them were taken for histopathological study and the epididymis were taken for counting dead and live sperms. The results showed a significant increase in the weight of the testis in G2 group as compared to the control and significant decrease in the percentage of live sperms in (G1) group and significant increase in the percentage of dead sperms in H2O2 treated G1 group and control group. The results show significant differences in testosterone hormone concentration between treated groups and control group while there was known significant increased in concentration of luteinizing hormone between treated groups and control group. There was a significant decrease in FSH in treated (G1) group comparing with control group. The histological study showed edema and thickness of basement membrane of seminiferous tubules with incomplete spermatogenesis process in treated (G1) group, there were no lesions observed in other treated groups. Finally, the outcomes of of this study documented the advantageous effect of crude polyphenolic compounds of olive against the harmful effect of H2O2 on reproductive system of adult males rats.

Mammalian gonocyte and spermatogonia differentiation: recent advances and remaining challenges.

The production of spermatozoa relies on a pool of spermatogonial stem cells (SSCs), formed in infancy from the differentiation of their precursor cells, the gonocytes. Throughout adult life, SSCs will either self-renew or differentiate, in order to maintain a stem cell reserve while providing cells to the spermatogenic cycle. By contrast, gonocytes represent a transient and finite phase of development leading to the formation of SSCs or spermatogonia of the first spermatogenic wave. Gonocyte development involves phases of quiescence, cell proliferation, migration, and differentiation. Spermatogonia, on the other hand, remain located at the basement membrane of the seminiferous tubules throughout their successive phases of proliferation and differentiation. Apoptosis is an integral part of both developmental phases, allowing for the removal of defective cells and the maintenance of proper germ-Sertoli cell ratios. While gonocytes and spermatogonia mitosis are regulated by distinct factors, they both undergo differentiation in response to retinoic acid. In contrast to postpubertal spermatogenesis, the early steps of germ cell development have only recently attracted attention, unveiling genes and pathways regulating SSC self-renewal and proliferation. Yet, less is known on the mechanisms regulating differentiation. The processes leading from gonocytes to spermatogonia have been seldom investigated. While the formation of abnormal gonocytes or SSCs could lead to infertility, defective gonocyte differentiation might be at the origin of testicular germ cell tumors. Thus, it is important to better understand the molecular mechanisms regulating these processes. This review summarizes and compares the present knowledge on the mechanisms regulating mammalian gonocyte and spermatogonial differentiation.

Description of Bursaphelenchus piceae sp. n. (Nematoda: Parasitaphelenchinae) from larval galleries of the six-toothed spruce bark beetle, Pityogenes chalcographus (L.) (Coleoptera: Curculionidae: Scolytinae), in Norway spruce, Picea abies (L.) Karsten

Bursaphelenchus piceae sp. n. is described from the bark of Norway spruce, Picea abies. Adults and propagative juveniles of the nematode are found in larval galleries of the six-toothed spruce bark beetle, Pityogenes chalcographus, while its dauer juveniles aggregate in Malpighian tubules of older larvae, pupae and adult beetles. The new species is characterised by the body length of 719 (530-945) μm in female and 632 (474-808) μm in male, its moderately slender body (a = 41.4 (35.4-50.2) and 42.6 (35.1-47.2) in female and male, respectively), and small spicules (16.0 (14.0-18.3) μm along arc and 13.7 (12.2-15.5) μm along chord). The extended anterior vulval lip in female, lateral fields with four incisures, long post-uterine sac extending for 67-91% of vulva-anus distance, and number (7) and arrangement of male caudal papillae suggest that B. piceae sp. n. is closely related to the xylophilus-group yet it differs by the relatively small, claw-like spicules with no cucullus at the tip. The close relation of B. piceae sp. n. to the xylophilus-group has been confirmed by DNA sequencing and phylogenetic analysis. Morphologically B. piceae sp. n. most closely resembles B. tokyoensis and B. fagi but can be separated from both by the unique shape of the spicules. The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). The presence of dauer juveniles of B. piceae sp. n. in Malpighian tubules of adult P. chalcographus may lead to extensive damage to this organ as shown by local expansion of the tubule basement membrane and degradation of its cellular epithelium. Detailed phylogenetic analysis revealed that B. piceae sp. n. together with five other bark beetle-associated Bursaphelenchus species, namely B. trypophloei, B. masseyi, B. tiliae, B. tokyoensis and B. fagi, constitutes a small phylogenetic clade that is most closely located to, but separate from, the xylophilus-group.

324 TESTIS-SPECIFIC EXPRESSION OF Oct4-EGFP TRANSGENE IN PIG

Oct4 is a transcription factor essential for establishment and maintenance of pluripotency in mammalian stem cells. Oct4 expression was found in early embryos and germ cells throughout fetal development. In male mice, Oct4 expression is found in mitotically arrested prospermatogonia until birth. After onset of spermatogenesis, expression is maintained in type A spermatogonia, but is downregulated in type B spermatogonia and in spermatocytes (Pesce et al. 1998 Mech. Dev). Previously, we successfully generated Oct4-EGFP reporter pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev.). This animal model is unique because it allows in vivo and in vitro visualisation of Oct4-positive cells. Germ line specific Oct4-EGFP expression was analysed in testis isolated from young (<1 week) and adult (>7 months) pigs. Squash preparation of testicular tissue isolated from adult transgenic boars revealed high amounts of EGFP-positive cells compared to young piglets. We confirmed Oct4 and EGFP expression in the testis from young and adult transgenic animals using Northern blot analysis. Specific expression of Oct4 and EGFP in testis could be observed in blots as a single band of 1.5 kb. As a loading control, the blot was rehybridized with a β-actin probe. Mammalian testes contain different cell types, including germ cells, Sertoli cells, Leydig cells, and peritubular cells. To define the cellular origin of EGFP-expressing cells, we isolated these cells from adult transgenic testis using fluorescence-activated cell sorting (FACS)-based techniques. Analysis of isolated EGFP positive cells with qRT-PCR demonstrated the presence of marker genes specific for undifferentiated (Oct4, UTF1, FGFR3, PGP 9.5, THY-1, SALL4, and GFRα1) and differentiated (BOLL and PRM2) germ cells. Markers specific for Sertoli cells (vimentin) and Leydig cells (LHCGR) were not observed. To verify the localization of EGFP-positive cells in seminiferous tubules, we performed immunohistochemical detection of GFP in adult pig testis. Unlike the Oct4-EGFP reporter mouse model, GFP protein was not found in spermatogonia attached to the basement membrane of seminiferous tubules, but instead were found in differentiated germ cells, including spermatocytes and spermatids. These results show that the Oct4-EGFP expression in testis differs between mouse and porcine Oct4-EGFP transgenic models. To verify that the EGFP expression driven by the mouse Oct4 promoter in porcine testis reflects the endogenous Oct4 expression profile, Western blot and histochemical analyses are currently underway.

Profiling of Cxcl12 receptors, Cxcr4 and Cxcr7 in murine testis development and a spermatogenic depletion model indicates a role for Cxcr7 in controlling Cxcl12 activity.

In mice the chemokine Cxcl12 and its receptor Cxcr4 participate in maintenance of the spermatogonial population during postnatal development. More complexity arises since Cxcl12 also binds to the non-classical/atypical chemokine receptor Cxcr7. We explored the expression pattern of Cxcl12, Cxcr4 and Cxcr7 during postnatal development in mouse testes and investigated the response of Cxcl12, Cxcr4, Cxcr7 and SSC-niche associated factors to busulfan-induced germ cell depletion and subsequent recovery by RNA expression analysis and localization of the proteins. In neonatal testes transcript levels of Cxcl12, Cxcr4 and Cxcr7 were relatively low and protein expression of Cxcr7 was restricted to gonocytes and spermatogonia. During development, RNA expression of Cxcl12 remained stable but that of Cxcr4 and Cxcr7 increased. Cxcr7 was expressed in germ cells located at the basement membrane of the seminiferous tubules. In adult testes, transcript levels of Cxcl12 were highest while the localization of Cxcr7 did not change. Following germ cell depletion, a significantly increased expression of Cxcl12 and a decreased expression of Cxcr7 were observed. Germ cells repopulating the seminiferous tubules were immunopositive for Cxcr7. We conclude that Cxcr7 expression to be restricted to premeiotic germ cells throughout postnatal testicular development and during testicular recovery. Hence, the spermatogonial population may not only be simply controlled by interaction of Cxcl12 with Cxcr4 but may also involve Cxcr7 as an important player.

The KIT is a putative marker for differentiating spermatogonia in stallions

Putative markers have been discovered and are used to identify and separate certain lineage of spermatogonia. The KIT is a marker for differentiating spermatogonial stem cells in several species including mice and goats. The objectives of this study were (1) to investigate reproductive stage-dependent KIT expression patterns in stallions and (2) to identify spermatogonia subpopulations expressing KIT in stallion testes. To achieve these objectives, testicular samples were obtained during routine field castration of stallions. The reproductive stage of the stallions was classified as pre-pubertal (<1 year, n=3), pubertal (1–1.5 year, n=4), post-pubertal (2–3 year, n=6), or adult (4–8 year, n=6). For immunohistochemistry, KIT was used at a dilution of 1:200. In the pre-pubertal and pubertal stage, most germ cells were immunolabeled with KIT. In the post-pubertal and adult stages, immunolabeling of KIT was evident in the germ cells attached to the basement membrane of the seminiferous tubules with exception of some spermatogonia. Co-immunolabeling with KIT and deleted in azoospermia like (DAZL) showed different co-staining patterns, including KIT only, both KIT and DAZL), or DAZL positive germ cell populations alone. The KIT was not immunolabeled in Sertoli or Leydig cells at any reproductive stages. The result of Western blot analysis verified the cross-activity of the KIT antibody with horse testes tissue. In conclusion, KIT appears to be expressed in differentiating spermatogonia, and may be used to identify and isolate differentiating germ cells from stallions.

Potential spermatogenesis recovery with bone marrow mesenchymal stem cells in an azoospermic rat model.

Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90α, integrinβ1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy.

Stage-dependent DAZL localization in stallion germ cells

Deleted in azoospermia-like (DAZL) is used as a germ cell marker in several species, including mice, rats, pigs, rhesus monkeys, bulls, and humans. Our objectives with this study were to investigate DAZL expression in stallion germ cells by using immunofluorescence, immunocytochemistry, and western blotting, and to determine the effects of reproductive stage and breeding season on the DAZL-positive cell population in seminiferous tubule cross sections. Testes were obtained during routine castration procedures at a large animal clinic and routine field service castration. The reproductive stage of the stallions was classified as pre-pubertal (<1 yr), pubertal (1–1.5 yr), post-pubertal (2–3 yr), or adult (4–8 yr). Using immunofluorescent staining, we showed that DAZL is localized to the cytoplasm of some, but not all, spermatogonia in pre-pubertal and pubertal horses. In the post-pubertal and adult testes, DAZL immunostaining was observed in spermatogonia proximal to the basement membrane of seminiferous tubules; however, few spermatogonia attached to the basement membrane were not immunolabeled. DAZL immunostaining was also observed in primary spermatocytes, but not in secondary spermatocytes, spermatids, or spermatozoa. DAZL protein was not detected in Leydig, Sertoli, or myoid cells of the testes at any reproductive stage. The immunocytochemistry analysis showed that DAZL immunolabeling was also localized to the cytoplasm of isolated germ cells such as spermatogonia or primary spermatocytes. We conclude that DAZL can be used as a marker of pre-meiotic germ cells in stallions.

Chronic Stress Influences Sexual Motivation and Causes Damage to Testicular Cells in Male Rats

The suppressing effects of chronic stress on sexual desire have long been noted. Yet the biological mechanisms underlying such effects, especially at the level of cellular biology of testicular cells, have not been fully investigated. In the present study, we used a chronic unpredictable mild stress model to examine the association between chronic stress and structural alterations in the male reproductive system. The main outcome measures were the structural changes in sperm cells and Leydig cells of male rats. We used Agmo and Ellingsen's procedure to study partner preference behavior and observed the morphology of Leydig cells and germ cells in the control and stress groups. Our methods included histology, electron microscopy, and animal behavior tests. The results showed that after 5 weeks of chronic stress exposure, partner preference behavior was impaired, the total surface area of Leydig cells and the number and diameter of seminiferous tubules decreased significantly, and the number and size of Leydig cells, as well as the number and the short-axis diameter of spermatogenic cells, also decreased. At the ultrastructural level, transmission electron microscopy revealed that the basement membranes of seminiferous tubules in stressed rats was far thinner, had a low density, and was uneven in thickness compared with the normal group, with enhanced apoptosis in germ cells. We conclude that chronic stress can trigger organic damage to testicular cells in male rats.

Tubulointerstitial nephritis antigen expression in chronic kidney disease and its clinical significance

To explore tubulointerstitial nephritis antigen (TIN-ag) expression of chronic kidney disease (CKD) patients' renal tissue and the correlation to clinical phenotype. Through digital drawing lots, a total of 77 CKD patients from October 2012 to February 2013 at our department were randomly selected. All of them underwent biopsy. Based upon their pathological findings, they were divided into 2 groups of minimal change disease (MCD) and non-minimal change disease (NMCD). The stains of hematoxylin and eosin and Masson were used to observe renal pathological changes and immunofluorescence for detecting the TIN-ag expression of kidney tissue. The serum levels of creatinine, blood urea nitrogen, estimated glomerular filtration rate (eGFR), 24-hour urine output, 24-hour urine protein, α1-microglobulin, β2-microglobulin, pathological casts, N-acetyl-beta-glucosaminidase (NAG), specific gravity and other clinical parameters were monitored to examine their relationship between renal tissue TIN-ag expression. TIN-ag expression was distinct in renal tubular basement membrane of MCD patients while weak in primary focal segmental glomerulosclerosis (FSGS)(n = 16), IgA nephropathy (n = 23), MN (n = 14) and LN (n = 15) renal tissue. Immunofluorescence quantitative analysis showed that tubular TIN-ag fluorescence intensity of NMCD group was significantly lower than that of MCD group (4.84(3.02, 10.73) vs 20.79(8.19, 37.00), P < 0.01). In addition, TIN-ag expression in renal interstitial collagen area deposition of 0 grade group was higher than that of collagen area deposition 1-3 grades group (all P < 0.05). Serum α1-microglobulin and pathological urine cast, 24-hour urine protein of CKD patients were negatively correlated with kidney tubules TIN-ag expression (r = -0.312, -0.298, -0.214, all P < 0.05). Serum creatinine, blood urea nitrogen, serum β2-microglobulin and eGFR of CKD patients had no significant correlations with TIN-ag expression (P > 0.05). TIN-ag expression of CKD patients with lower expression levels of NAG was significantly higher than that of normal levels of NAG expression. TIN-ag expression of low urine specific gravity group was lower than that of normal urine specific gravity group (P < 0.05). TIN-ag expression of renal tissue tubule basement membrane in NMCD group is significantly lower than that in MCD group. TIN-ag expression is negatively correlated with renal tissue fibrosis. Expression of serum α1-microglobulin and concentrations of urinary pathology tube, 24-hour urine protein, NAG expression and urine specific gravity are negatively correlated with renal tissue TIN-ag expression in CKD patients.

Making gametes from pluripotent stem cells--a promising role for very small embryonic-like stem cells.

The urge to have one’s own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo – existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.

Bursaphelenchus tiliae sp. n. (Nematoda: Parasitaphelenchidae), a nematode associate of the bark beetle Ernoporus tiliae (Panz.) (Coleoptera: Curculionidae, Scolytinae), in small-leaved lime, Tilia cordata Mill.

Bursaphelenchus tiliae sp. n. is described from the bark of dead branches of the small-leaved lime, Tilia cordata, infested with the lime bark beetle, Ernoporus tiliae. The nematode adults and propagative juveniles were present in larval galleries, while dispersal dauer juveniles colonised the Malpighian tubules of adult beetles. Bursaphelenchus tiliae sp. n. is characterised by the body length of 664 (568-817) μm in female and 622 (515-754) μm in male, moderately slender body (a = 41.0 (34.7-47.9) and 43.1 (37.8-51.9) in female and male, respectively), and spicules 20.0 (18.1-21.8) μm long. The extended anterior vulval lip in female, lateral fields with four incisures, long post-uterine sac, and number (7) and arrangement of male caudal papillae, may indicate that B. tiliae sp. n. is closely related to the xylophilus group. It differs from this group by smaller, arcuate spicules with relatively short rostrum, narrow capitulum (5.2-6.9 μm), and indistinct condylus in an almost continuous line with dorsal lamina. Distal third of spicule dorsal contour is usually almost straight. The spicules have a distinct, disk-like cucullus at the terminus. The status of the new species is confirmed by the unique molecular profile of the ITS region (ITS-RFLP) and its close relation to the xylophilus group by DNA sequencing and phylogenetic analysis of the 28S rDNA region. The presence of B. tiliae sp. n. dauer juveniles in Malpighian tubules of their vector bark beetle, E. tiliae, caused limited pathogenicity expressed as a local expansion of the tubule basement membrane and partial degradation of the cellular epithelium.

Histopathological Changes in Testis after Partial Caput Epididymectomy of Teaser Rams

Three healthy adult local breed rams were subjected to partial caput epididymectomy to evaluate histopathological changes. The skin in caudal aspect of scrotal raphe pouch was transected longitudinally. The first testis was forced toward the other, the tunica vaginalis was incised at the upper of the epididymis body and a piece of one cm in length was removed from it with ligation. The same procedure was repeated for the other testis. Time of sexual desire was significantly longer (p&lt;0.05) after one and two weeks of the operation than three and four weeks for control, while it was longest after one weeks among all other periods. Sperm concentration was significantly lower (p&lt;0.05) after 2, 3 and 4 weeks than the first week for control, while it was significantly lower after first week than control. The histopathological changes in testicular tissue were evaluated after one, three and six months of the operation. The results revealed thickening of the basement membrane of seminiferous tubules and degeneration, necrosis of spermatogonia and sertoli cells. Some seminiferous tubules appeared only with covering basement membrane. The Leydig cells were proliferated at the first period, while there was decrease in their number at the last period.

Gonocyte transformation to spermatogonial stem cells occurs earlier in patients with undervirilisation syndromes

AimFertility post-orchidopexy is dependent on transformation of neonatal gonocytes (G) into adult dark spermatogonia at about 3months, the same time as gonadotrophins stimulate androgen secretion. We examined how androgen blockade affects transformation of gonocytes to spermatogonial stem cells (SSC) during this period in patients with undervirilisation syndromes. MethodsPatients with undervirilisation syndromes (n=30, 1.5weeks–16years) underwent review of medical records, pathology reports, and H&E slides of testes (ethics HREC32164). Fluorescent immunohistochemistry against anti-Mullerian hormone (AMH, Sertoli cells), mouse VASA homologue (MVH, germ cells) and DAPI (nuclei) allowed the number of MVH-positive gonocytes/spermatogonial stem cells per seminiferous tubular cross-section (G/T or SSC/T) to be counted. ResultsGonocytes (MVH-positive cells in the tubular lumen) were present in 15/16 patients under 2 years old. SSC (MVH-positive cells on the tubule basement membrane) were present in 25/30 patients. With increasing age, the mean number of SSC/T decreased from ~4 to 0, and G/T decreased from ~1.5 to 0. SSC were present in CAIS and PAIS patients at 1.5 and 3.5weeks old, respectively. ConclusionsGonocytes transform into SSC earlier than expected in patients with undervirilisation syndromes. Lack of androgens may stimulate non-androgenic regulators to trigger transformation. Understanding how gonocytes transform may enable optimization of spermatogonial development to preserve fertility post-orchidopexy.