The determination of the relationship between p97/VCP, small VCP-interacting protein, two ERAD proteins and steroidogenesis in Leydig cell lines.

BACKGROUND: In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3ß-HSD in human and mouse. METHODS AND RESULTS: HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3ß-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3ß-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells. CONCLUSION: We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3ß-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.

Disruption of p97/VCP induces autophagosome accumulation, cell cycle arrest and apoptosis in human choriocarcinoma cells.

Gestational choriocarcinoma is aggressive trophoblastic disease. The development, progression and the cure of this disease is not well-established. p97/Valosin containing protein has been shown to play critical roles in many cellular processes. In various cancers, higher expression of p97/VCP has been reported and targeting of p97/VCP with its spesific inhibitors or siRNA's (siVCP) in cancer therapy was suggested. However, no study is avaible about the expression and function of p97/VCP in gestational choriocarcinoma. Hence, the aim of the study was to evaluate effects of p97/VCP inhibitor, DBeQ and siVCP on choriocarcinoma cells. We use human placental choriocarcinoma cell line (Jeg3) as model to find out the effects of DBeQ and VCP siRNA's (siVCP) on apoptotic and autophagic pathway by immunflouroscence staining, Western blotting, qPCR and flow-cytometry. p97/VCP siRNA's and DBeQ induced accumulation of autophagic proteins, LC3II and p62 in the cytoplasm of Jeg3 cells detected. Concurrently, Jeg3 cells treated with DBeQ and siVCP demonstrated G0/G1 cell cycle arrest, accompanied by accumulation of poly-ubiquitinated proteins. Moreover, disruption of p97/VCP by siRNA and DBeQ inhibited cancer cell growth managing the caspases-3 and -7. Our results show that inhibition of p97/VCP activity with DBeQ and depletion of p97/VCP expression with siRNA in Jeg3 cells induce caspase activation, inhibits cell proliferation and leads to a defect in autophagosome maturation, thus providing potential target for the prevention and treatment of choriocarcinoma.

Effects of regular whey protein consumption on rat thyroid functions

Background/aim: We aimed to investigate whether there was a significant difference in TSH, T3, T4 values and histopathologically evaluated thyroid tissues between rats that received isole hydrolyzed whey protein (IHWP) at different doses regularly and rats fed with only standard feed. Material & methods: Total 24 rats were randomly divided into three groups with 8 rats in each group. First group were fed with standard feed for 12 weeks. Second group were given standard feed + daily 0.3 g/kg IHWP and rats in the third group standard feed + 0.5 g/kg IHWP for 12 weeks. Blood samples were collected from all rats before and after IHWP administration. All rats were then sacrificed, and thyroid tissues were histopathologically examined. Results: Interfollicular connective tissue areas and TSH (0.35­4.90 µIU/L) were higher in the control group compared to 3 cc IHWP and 5 cc IHWP groups, while thyroid hormone T4 (0.7­1.48 ng/dL), and thyroid hormone synthesis parameters including intrafollicular colloid amount, follicular diameter, and epithelial height were significantly higher in 3 cc and 5 cc IHWP groups compared to the control. Conclusion: We think that regular daily use of IHWP may increase the synthesis of thyroid hormone due to its high amino acid content.

Evaluation of effects of repeated sevoflurane exposure on rat testicular tissue and reproductive hormones.

CONTEXT: Evaluation of inhalation anesthetics on sperm and reproductive hormones are extremely important. OBJECTIVE: Investigation of the effects of sevoflurane used as an inhalation anesthetic on sperm morphology and reproductive hormones in rat testes. MATERIALS AND METHODS: Forty Wistar-Albino male rats were divided into five groups of eight rats each. The control group received 2 L/min oxygen for seven days, 2 h/day while sevoflurane treatment S1 received 1 minimal alveolar concentration (MAC) sevoflurane + 2 L/min oxygen for seven days, 2 h/day, and sevoflurane S2 received 1 MAC sevoflurane + 2 L/min oxygen for seven days, 2 h/day followed by seven days of no treatment. Sevoflurane treatment S3 received 1 MAC sevoflurane + 2 L/min oxygen for 14 days, 2 h/day and sevoflurane treatment S4 received 1 MAC sevoflurane + 2 L/min oxygen for 14 days, 2 h/day, with no treatment for the following seven days. All rats were examined histologically after experimental procedures. Rat luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and inhibin levels were measured. RESULTS: Histological injury scores were significantly higher in S2, S3, and S4 receiving sevoflurane in comparison to the control group (p = 0.001, <0.001, and 0.001, respectively). Sperm motility and concentration decreased in S3 and S4 compared to the control group (p = 0.03 and 0.02, respectively). Significant differences were detected among all groups for serum LH, FSH, T, and inhibin serum concentrations (p < 0.05). CONCLUSION: Testicular and sperm morphology, and reproductive hormones were affected by chronic exposure to sevoflurane. However, more randomized, controlled, and well-designed clinical studies with larger population are needed to confirm of these results.

The apoptotic effect of a high dose of toluene on liver tissue during the acute phase: an experimental study.

The aim of this study is to investigate the acute toxic effects of high-dose toluene and its mechanisms on the liver tissue of toluene-treated rats. In this study, 16 adult male Wistar albino rats (200-220 g) were divided into two equal groups. Group I was used as a control group, while group II was exposed to high dose of toluene, 5200 mg/kg (6 ml/kg per gavage). After the 3-hour experimental period, blood samples and liver tissues were taken from the euthanized animals. Serum aspartate and alanine aminotransferase levels were assayed. Liver tissues were fixed in 10% neutral formalin, then embedded in paraffin and sectioned (5 µm thickness). Sections were stained with hematoxylin and eosin for histopathological examination. A terminal transferase dUTP nick end labeling assay was also done for the determination of apoptosis in liver tissues. For the determination of Bax and caspase-3 immunoreactivity, the sections were stained using avidin-biotin-peroxidase immunohistochemical method. The level of plasma transaminase was found to be increased in toluene administered rats. Additionally, slight degeneration of hepatocyte and mononuclear cell infiltration was observed in the liver tissue sections and a high (+++) immunoreactivity for Bax and caspase-3 protein was observed in the toluene group. This study showed that the high dose of toluene triggers apoptosis in the liver of rats via the mitochondrial pathway in acute period.

The immunoexpression of valosin-containing protein and small VCP-interacting protein in rat ovaries.

Numerous cellular processes are controlled by the ubiquitin-proteasome-mediated degradation pathway, involve the 97-kDa valosin-containing protein (p97/VCP). Small p97/VCP-interacting protein (SVIP) was first discovered as one of the novel androgen-responsive genes as well as one of the many cofactors controlling p97/VCP. The aim of the study was to investigate localization and immunoexpression of p97/VCP and SVIP in rat ovarian tissue. The histomorphological examination of rat ovarian tissue was performed by using haematoxylin-eosin (HE) staining. Using the immunohistochemical technique, cellular location and expression of p97/VCP and SVIP in rat ovarian tissue were examined. The nuclear and cytoplasmic immunoexpression of p97/VCP and SVIP was observed in the different stages of ovarian follicles and corpus luteum in the rat ovaries. The immunolocalization of SVIP and VCP in the rat ovaries suggest that they may be involved in the oogenesis. Further studies should be performed about the function of the VCP and SVIP in the female reproductive tract.

Investigation the Effect of Human Recombinant Epidermal Growth Factor on Rotator Cuff Healing: An Experimental Model.

Objective To investigate the effectiveness of human recombinant epidermal growth factor in the healing of rotator cuff tear in the rabbit shoulder. Methods Rotator cuff tears (RCTs) were experimentally created on both shoulders of 20 New Zealand rabbits. The rabbits were divided into the following groups: RCT (sham group; n = 5), RCT + EGF (EGF group; n = 5), RCT + transosseous repair (repair group; n = 5), and RCT + EGF + transosseous repair (combined repair + EGF group; n = 5). All rabbits were then observed for 3 weeks, and biopsies were taken from the right shoulders in the third week. After three more weeks of observation, all rabbits were sacrificed, and a biopsy removed from their left shoulders. All biopsy material was stained with haematoxylin & eosin (H&E) and vascularity, cellularity, the proportion of fibers and the number of fibrocartilage cells were evaluated under light microscope. Results The highest collagen amount and the most regular collagen sequence was detected in the combined repair + EGF group. The repair group and the EGF group showed higher fibroblastic activity and capillary formation when compared with the sham group, but the highest fibroblastic activity and capillary formation with highest vascularity was detected in the combined repair + EGF group ( p < 0.001). EGF seems to improve wound healing in the repair of RCT. The EGF application alone, even without repair surgery, seems to be beneficial to RCT healing. Conclusion In addition to rotator cuff tear repair, application of human recombinant epidermal growth factor has an effect on rotator cuff healing in rabbit shoulders.

Levetiracetam treatment in an experimental model of sciatic nerve injury: A randomized controlled trial.

AIM: This study examined whether levetiracetam contributes to improvements in the axon-nerve damage in an experimental rat model. MATERIALS AND METHODS: Forty-eight Wistar albino adult male rats weighing 250-300 gr were randomized into six groups having or not having sciatic nerve damages and receiving different (none, 300 and 600 mg/kg) levetiracetam doses, and control (non-levetiracetam). Functional gait analysis and tissue sample analysis with the aid of light microscopy and hematoxylin-eosin dye were evaluated between the groups. Additionally, scanning electron microscopy (SEM) was used for the detailed examination of sciatic nerves. S-100 (Schwann cell marker) immunoreactivities in sciatic nerve was detected by immunohistochemistry. RESULTS: Sciatic functional index of the injured rats receiving 300 mg/kg levetiracetam was -65.59 ± 29.48 and -47.13 ± 21.36 in the 2nd and 6th weeks, respectively (p < 0.001). Also, IMA and TOS levels were significantly higher in the control group compared to those receiving levetiracetam (p = 0.001 and p < 0.001, respectively).      The most significant nerve regeneration was in the group injured and treated with LEV 600 mg/kg (p < 0.05). CONCLUSION: There was a significant improvement in the sciatic functional index, histopathological findings, and parameters showing tissue oxidant status in rats with sciatic nerve injury receiving levetiracetam treatment. Further investigations should be performed to evaluate the contribution of levetiracetam as a treatment modality in sciatic nerve injuries.

Altered expression of COP9 signalosome proteins in preeclampsia.

OBJECTIVE: The presumptive factors that are released by the preeclamptic placenta to cause maternal disease are less well known. The constitutive photomorphogenic-9 (COP9) signalosome (CSN) complex, a multifunctional protein complex involved in modulating signal transduction, gene transcription, and protein stability in cells. Although the roles of most CSN components in early embryonic development have been studied, their role in preeclamptic human placentas is not known. Thus, this study was aimed to show the localization and the protein expression of CSN1 and CSN5 in normal and preeclamptic placenta. STUDY DESIGN: The distribution and the protein expression of CSN1 and CSN5 were analyzed in normal (n: 15) and preeclamptic (n: 15) human placenta by using immunohistochemistry (IHC) and Western blotting. RESULTS: CSN1 and CSN5 were mainly localized in the vascular endothelium, syncytiotrophoblast, stromal and Hofbauer cells in normal and preeclamptic placentas. However, a stronger immunoreactivity and protein expression for CSN1 and CSN5 were observed in preeclamptic placentas compared to normal term placentas. Western blotting of the tissue extracts confirmed the IHC results. CONCLUSIONS: Our results suggest that an increased level of CSN1 and CSN5 as an important part of the ubiquitin proteasome system (UPS) might be associated with the pathophysiology of preeclampsia.

Novel regulation mechanism of adrenal cortisol and DHEA biosynthesis via the endogen ERAD inhibitor small VCP-interacting protein.

Endoplasmic reticulum-associated degradation (ERAD) is a well-characterized mechanism of protein quality control by removal of misfolded or unfolded proteins. The tight regulation of ERAD is critical for protein homeostasis as well as lipid metabolism. Although the mechanism is complex, all ERAD branches converge on p97/VCP, a key protein in the retrotranslocation step. The multifunctionality of p97/VCP relies on its multiple binding partners, one of which is the endogenous ERAD inhibitor, SVIP (small VCP-interacting protein). As SVIP is a promising target for the regulation of ERAD, we aimed to assess its novel physiological roles. We revealed that SVIP is highly expressed in the rat adrenal gland, especially in the cortex region, at a consistently high level during postnatal development, unlike the gradual increase in expression seen in developing nerves. Steroidogenic stimulators caused a decrease in SVIP mRNA expression and increase in SVIP protein degradation in human adrenocortical H295R cells. Interestingly, silencing of SVIP diminished cortisol secretion along with downregulation of steroidogenic enzymes and proteins involved in cholesterol uptake and cholesterol biosynthesis. A certain degree of SVIP overexpression mainly increased the biosynthesis of cortisol as well as DHEA by enhancing the expression of key steroidogenic proteins, whereas exaggerated overexpression led to apoptosis, phosphorylation of eIF2α, and diminished adrenal steroid hormone biosynthesis. In conclusion, SVIP is a novel regulator of adrenal cortisol and DHEA biosynthesis, suggesting that alterations in SVIP expression levels may be involved in the deregulation of steroidogenic stimulator signaling and abnormal adrenal hormone secretion.

Developmental expression of p97/VCP (Valosin-containing protein) and Jab1/CSN5 in the rat testis and epididymis.

BACKGROUND: The ubiquitin proteasome system (UPS) is a key player in regulating many cellular processes via proteasomal degradation of ubiquitinated proteins. Recently published data show that Jab1/CSN5 interacts with p97/VCP and controls the ubiquitination status of proteins bound to p97/VCP in mouse and human cells. However, coexpression of p97/VCP and Jab1/CSN5 in the developing rat testis and epididymis has not previously been studied. METHODS: Testicular and epididymal tissues from 5-, 15-, 30-, and 60-day-old rats were examined by immunohistochemistry and Western blotting. Colocalisation of proteins was determined by immunofluorescence microscopy. RESULTS: In the 5-day-old rat testis, p97/VCP and Jab1/CSN5 were specifically expressed in gonocytes. The expression of p97/VCP and Jab1/CSN5 significantly increased at day 15 and was found in spermatogonia, Sertoli cells and spermatocytes. In 30- and 60-day-old rat testes, p97/VCP indicated moderate to strong expression in Sertoli cells, spermatogonia, round and elongating spermatids. However, moderate to weak expression was observed in spermatocytes. Jab1/CSN5 showed strong expression in spermatogonia and spermatocytes, while relatively moderate expression was observed in round and elongating spermatids in 30- and 60-day-old rat testes. In contrast, in the epididymis, the expression of both proteins gradually increased from 5 to 60 days of age. After rats reached 2 weeks of age, the expression of both proteins was mostly restricted to the basal and principal cells of the caput epididymis. CONCLUSIONS: Our study suggests that p97/VCP and Jab1/CSN5 could be an important part of the UPS in the developing rat testis and epididymis and that both proteins may be involved in the regulation of spermatogenesis and epididymal epithelial functions.

Effect of pioglitazone on the expression of ubiquitin proteasome system and autophagic proteins in rat pancreas with metabolic syndrome.

The metabolic syndrome (MetS) and pathologies associated with metabolic dysregulations a worldwide growing problem. Our previous study demonstrated that pioglitazone (PGZ) has beneficial effects on metabolic syndrome associated disturbances in the heart. However, mechanism mediating the molecular alterations of Ubiquitin proteasome system (UPS) and autophagy has not been investigated in rat pancreas with metabolic syndrome. For this reason, we first aimed to detect whether MetS effects on the expression of UPS (p97/VCP, SVIP, Ubiquitin) and autophagic (p62, LC3) proteins in rat pancreas. The second aim of the study was to find impact of pioglitazone on the expression of UPS and autophagic proteins in MetS rat pancreas. To answer these questions, metabolic syndrome induced rats were used as a model and treated with pioglitazone for 2 weeks. Pancreatic tissue injuries, fibrosis and lipid accumulation were evaluated histopathologically in control, MetS and MetS-PGZ groups. Apoptosis and cell proliferation of pancreatic islet cells were assessed in all groups. UPS and autophagic protein expressions of pancreas in all groups were detected by using immunohistochemistry, double-immunfluorescence and Western blotting. Compared with the controls, the rat fed with high sucrose exhibited signs of metabolic syndrome, such as higher body weight, insulin resistance, higher triglyceride level and hyperglycaemia. MetS rats showed pancreatic tissue degeneration, fibrosis and lipid accumulation when their pancreas were examined with Hematoxilen-eozin and Mallory trichrome staining. Metabolic, histopathologic parameters and cell proliferation showed greater improvement in MetS-PGZ rats and pioglitazone decreased apoptosis of islet cells. Moreover, SVIP, ubiquitin, LC3 and p62 expressions were significantly increased while only p97/VCP expression was significantly decreased in MetS-rat pancreas compared to control. PGZ treatment significantly decreased the MetS-induced increases in autophagy markers. Additionally, UPS and autophagy markers were found to colocalizated with insulin and glucagon. Colocalization ratio of UPS markers with insulin showed significant decrease in MetS rats and PGZ increased this ratio, whereas LC3-insulin colocalization displayed significant increase in MetS rats and PGZ reversed this effect. In conclusion, PGZ improved the pancreatic tissue degeneration by increasing the level of p97/VCP and decreasing autophagic proteins, SVIP and ubiquitin expressions in MetS-rats. Moreover, PGZ has an effect on the colocalization ratio of UPS and autophagy markers with insulin.

Ubiquitin proteasome system and autophagy associated proteins in human testicular tumors.

Ubiquitin proteasome sytem (UPS) and autophagy govern protein quality control by degradation and clearance of damaged proteins. Many proteins working in these pathways such as p97/VCP, Ubiquitin (Ub), Jab1/CSN5, p62, LC3B and Beclin 1 are known to be essential for different pathological conditions, especially in cancer, but their expression in human testicular tumors has not been characterized yet. In the present study, we aimed to investigate the expression of UPS (p97/VCP, Ubiquitin, Jab1/CSN5) and autophagic (p62, LC3B, Beclin 1) proteins in human testicular tumors and cancer adjacent normal testicular tissues. We used an immunohistochemical staining technique. 120 cases of testicular germ and non-germ cell tumors, which are 42 seminomas, 31 embryonal carcinomas, 11 yolk sac tumors, 25 intratubular germ cell neoplasms, 6 Leydig cell tumors, 5 Sertoli cell tumors, were collected and evaluated on tissue microarray. For the first time, the expression of p97/VCP, Ub, Jab1/CSN5, p62, LC3B and Beclin 1 in different type of human testicular tumors has been confirmed. We found that p97/VCP, Ub and Jab1/CSN5 were frequently expressed at higher levels in testicular tumours. In contrast to UPS markers, p62, LC3B and Beclin 1 showed significantly diminished expressions in testicular tumors. Accordingly, a negative correlation between p97/VCP and autophagic markers (p62 and LC3B) was found, suggesting a relationship between UPS and autophagy in different type of testicular tumors. The current results displayed elevated level of p97/VCP, Ub and Jab1/CSN5 expressions in contrast to the diminished expression of p62, LC3B and Beclin 1 in human testicular tumors, thereby supporting a correlation between p97/VCP and autophagic markers in testicular tumors.

Three dimensional nanofibrous and compressible poly(L-lactic acid) bone grafts loaded with platelet-rich plasma.

In this study, nanofibrous matrices of poly(L-lactic acid)-hydroxyapatite (PLLA-HAp) were successfully fabricated by three-dimensional (3D) electrospinning for use in the treatment of irregular bone damages. Compressibility analysis showed that 3D nanofibrous grafts occupied at least 2-fold more volume than their 2D form and they can easily take shape of the defect zone with irregular geometry. Moreover, the compression moduli of the PLLA and PLLA-HAp grafts were calculated as 8.0 ± 3.0 kPa and 11.8 ± 3.9 kPa, respectively, while the strain values of the same samples at the maximum load of 600 kPa were 164 ± 28% and 130 ± 20%, respectively. Treatment of the grafts with aqueous sodium hydroxide solution increased the surface roughness and thus the alloplastic graft materials (PLLA-HAp/M) protecting the fiber morphology were produced successfully. Then, platelet-rich plasma (PRP) was loaded into the surface modified grafts and activated with 10% calcium chloride. The efficiency of the activation was evaluated with flow cytometry and it was found that after activation the percentages of CD62 (P-selectin) and CD41/61 (glycoprotein IIb/IIIa) proteins increased approximately 4-fold. Surface hydrophilicity and biological activity of the PLLA-HAp grafts were enhanced by fibrin coating after PRP activation. Thein vitrocell culture studies which were carried out by using mouse pre-osteoblasts (MC3T3-E1) showed that graft materials supported by PRP increased cellular proliferation and osteogenic differentiation significantly. Thein vivoresults demonstrated that compared with bare PLLA-HAp/M grafts, the PRP loaded grafts (PRP-PLLA-HAp/M) induced significantly greater bone formation based on computed tomography, histological and immunohistochemical analyses. Our findings suggest that 3D PLLA nanofibrous matrices can be used as a graft material for irregular bone defects especially when combined with PRP as an osteogenic induction agent.

COP9 signalosome interacts ATP-dependently with p97/valosin-containing protein (VCP) and controls the ubiquitination status of proteins bound to p97/VCP.

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.

Inhibition of p97/VCP function leads to defective autophagosome maturation, cell cycle arrest and apoptosis in mouse Sertoli cells.

p97/valosin-containing protein (VCP) is expressed in many cells and plays critical functions in a broad range of diverse cellular processes. Because it is expressed in the mouse testes, predominantly in Sertoli cells, and is known to play a critical role in autophagy and apoptosis in different cell types, we set out to investigate its function in autophagosome maturation, apoptosis and cell cycle arrest in a mouse Sertoli cell line. To study the mechanism of p97/VCP action, p97/VCP siRNA and a specific p97/VCP inhibitor, N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), were used in the mouse 15P1 Sertoli cell line. Loss of p97/VCP activity due to DBeQ exposure and silencing of p97/VCP (siVCP) expression results in autophagosome (LC3 and p62) accumulation in the cytoplasm of Sertoli cells. The coexpression of autophagosomal and lysosomal markers (LAMP1 and LAMP2) was reduced in cells in which p97/VCP expression had been inactivated. To better understand in which step of autophagy p97/VCP functions, the interaction between autophagosomal and autolysosomal markers was studied by coimmunoprecipitation and colocalization experiments. The interaction between autophagosomal markers and lysosomal markers decreased in siVCP-expressing and DBeQ-exposed cells. Moreover, the expression of siVCP and DBeQ exposure caused cytoplasmic vacuolation, induced caspase 3-7-mediated cell death and decreased cell cycle progression in mouse Sertoli cells. Taken together, the results show that p97/VCP is essential for autophagosome maturation and cell survival in mouse Sertoli cells. When these functions are prevented, impaired autophagy and apoptosis may have a detrimental effect on germ cells and cause male infertility.

Analysis of the developmental expression of small VCP-interacting protein and its interaction with steroidogenic acute regulatory protein in Leydig cells.

Small VCP-interacting protein (SVIP) is a 9-kDa protein that is composed of 76 amino acids, and it plays a role in the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Recent studies have shown that SVIP is an androgen-responsive protein and its expression is regulated by androgens. Because no data are available regarding the cellular localization and expression of SVIP in the mouse testis, where androgens are highly expressed, immunohistochemistry and western blotting were performed. In the fetal testis, we found that moderate but consistent staining of SVIP is present in the cytoplasm of Leydig cells. In prepubertal and adult life, SVIP remains present in Leydig cells as well as in the cytoplasm of some peritubular and Sertoli cells. From postnatal day 15 onward, SVIP is strongly expressed in the cytoplasm of Leydig cells. Furthermore, TM3, MA-10 Leydig and Sertoli cell lines were also used to evaluate the expression of SVIP. To identify the interacting partners, such as steroidogenic acute regulatory (STAR) protein, colocalization studies were performed by fluorescence microscopy, showing that STAR colocalized with SVIP in the adult mouse testis. The expression changes of STAR were studied by using SVIP siRNAs in Leydig cell line cultures. Depletion of SVIP resulted in decreased expression of STAR. Additionally, the number and size of lipid droplets were significantly increased in SVIP-depleted Leydig cells. Taken together, our data identify SVIP as a marker of Leydig cell lineage and as a regulator of STAR protein expression and lipid droplet status in Leydig cells.

Surgical and Immunohistochemical Outcomes of Scleral Reconstruction with Autogenic, Allogenic and Xenogenic Grafts: An Experimental Rabbit Model.

Purpose: Choukroun's platelet-rich fibrin (PRF), a second-generation platelet concentrate, has unique morphological and chemical features and may be considered as a scaffold for scleral reinforcement and regeneration. The purpose of this study was to compare the use of xenogenic human-derived amniotic membrane (HAM), allogenic sclera, and autogenic PRF in rabbit lamellar scleral defect model with respect to both anatomical and immunohistochemical improvement. Methods: A total of 45 adult New Zealand rabbits were randomized into five groups: normal control; without surgical procedure, negative control; scleral defect model (SDM), xenogenic HAM; SDM+HAM graft, allogenic sclera; SDM+allogenic sclera graft, autogenic PRF; SDM+autogenic PRF graft. Clinical findings, Hematoxylin&Eozin (HE), Masson Trichrome, Verhoeff Acid Fuchsin, Transforming Growth Factor ß Receptor 1, Fibroblast Growth Factor, Bone Morphogenetic Protein 2, collagen type 1, aggrecan, and Matrix Metalloproteinase 2 were evaluated. Results: Ocular surface inflammation was significantly lower in normal control and autogenic PRF groups (p < .001). Graft was avascular and not integrated to scleral wound area in 25% rabbits of allogenic sclera group (p = .02), was out of the scleral wound in 33.3% rabbits of xenogenic HAM group (p > .05), all the grafts were at the normal location and viable in autogenic PRF group. The inflammation and vascularization in autogenic PRF group was significantly lower than negative control and xenogenic HAM groups in HE (p < .001). The collagen score of negative control and xenogenic HAM groups were significantly lower than normal control (p < .001) and autogenic PRF (p < .001) groups. There were insignificant differences between allogenic sclera and autogenic PRF groups (p > .05). For immunohistochemistry, the closest values to normal control group were detected in autogenic PRF group for all immunomarkers. Conclusion: Autogenic PRF showed superior features via its excellent anatomical and chemical composition for scleral regeneration when compared to single-layered xenogenic HAM and allogenic sclera grafts.

Is there a relationship between PCNA expression and diabetic placental development during pregnancy?

We aimed to investigate the distribution pattern of proliferating cell nuclear antigen (PCNA) by immunohistochemistry and Western blot in placentas of control and diabetic rats at different stages of pregnancy. It is still not clear how proliferation is coordinated and how this coordination is affected by diabetes in the placenta. Diabetes was induced by streptozocin on the first day of pregnancy. Animals were sacrificed on days 11, 13, 17 and 21 of pregnancy. In control placentas immunolabeling intensity of PCNA was the highest on days 11 and 13 of pregnancy and decreased with progression of pregnancy. In the diabetic groups immunolabeling was less intense on days 11 and 13 of pregnancy compared to controls. However, in parallel with placental weights, PCNA immunopositivity was more intense in diabetic groups than control groups on days 17 and 21 of pregnancy, and the difference was statistically significant on day 17. According to Western blot data, on days 11 and 13 of pregnancy the amount of PCNA was greater in control groups than in the diabetics, whereas it was greater in diabetic groups than the controls on days 17 and 21 of pregnancy. We conclude that PCNA may play a role in abnormal placenta formation resulting from diabetes.

Altered expression of p97/Valosin containing protein and impaired autophagy in preeclamptic human placenta.

INTRODUCTION: Autophagy increases in placenta-related obstetrical diseases such as preeclampsia and intrauterine growth retardation but the regulation of autophagy by ubiquitin proteasome pathway (UPP) proteins, p97/Valosin containing protein (VCP) and ubiquitin (Ub) have not been previuosly studied in preeclampsia. The objective of this study is to investigate the expression of UPP (p97/VCP and Ub), autophagosomal (p62 and LC3) and autolysosomal proteins (Lamp1 and Lamp2) in the normal and preeclamptic human placentas and to explore the regulatory mechanism of these proteins in autophagic pathway. MATERIAL AND METHODS: Different portions of normal term placentas (n = 20) and preeclamptic placentas (n = 10) were snap-frozen in liquid nitrogen for Western blotting and coimmunoprecipitation and others were fixed-embedded in paraffin for immunohistochemistry. Colocalization and coimmunoprecipitation experiments were done for the detection of interaction between p97/VCP and autophagic proteins. RESULTS: Compared with normal placentas, expression of p97/VCP was significantly reduced; however accumulation of ubiquitinlated proteins were significantly increased in preeclamptic placentas. The expression of autophagosomal proteins (LC3-II and p62) were significantly increased and no significant alterations of the expression of autolysosomal proteins were observed in preeclamptic placentas. Additionally, p97/VCP was found to colocalized and interact with autophagosomal and autolysosomal markers in normal and preeclamptic placentas. Autophagosome maturation diminished and autophagosomes had decreased localization with lysosomal markers in preeclamptic human placentas. CONCLUSION: Decreased expression of p97/VCP and increased expression of Ub in preeclampsia might be related to impaired autophagy and pathophysiology of preeclampsia. Therefore, our study highlights an important potential relationship between p97/VCP and autophagic proteins in preeclampsia.