RESUMO
Fertilization is essential for species survival. Although Izumo1 and Juno are critical for initial interaction between gametes, additional molecules necessary for sperm:egg fusion on both the sperm and the oocyte remain to be defined. Here, we show that phosphatidylserine (PtdSer) is exposed on the head region of viable and motile sperm, with PtdSer exposure progressively increasing during sperm transit through the epididymis. Functionally, masking phosphatidylserine on sperm via three different approaches inhibits fertilization. On the oocyte, phosphatidylserine recognition receptors BAI1, CD36, Tim-4, and Mer-TK contribute to fertilization. Further, oocytes lacking the cytoplasmic ELMO1, or functional disruption of RAC1 (both of which signal downstream of BAI1/BAI3), also affect sperm entry into oocytes. Intriguingly, mammalian sperm could fuse with skeletal myoblasts, requiring PtdSer on sperm and BAI1/3, ELMO2, RAC1 in myoblasts. Collectively, these data identify phosphatidylserine on viable sperm and PtdSer recognition receptors on oocytes as key players in sperm:egg fusion.
Assuntos
Oócitos/metabolismo , Fagócitos/metabolismo , Fosfatidilserinas/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Angiogênicas/metabolismo , Animais , Antígenos CD36/metabolismo , Proteínas do Citoesqueleto/metabolismo , Epididimo , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Mioblastos Esqueléticos , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Fosfatidilserinas/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , c-Mer Tirosina Quinase/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Neuronal and endothelial nitric oxide synthases (nNOS and eNOS respectively) play major roles in generating the nitric oxide bioactivity necessary for erectile function. S-nitrosylation has been shown to regulate NOS activity. The presence of S-nitrosylated NOS in the penis and the impact of NOS S-nitrosylation/denitrosylation on erectile function were examined. S-nitrosylated forms of NOS were identified by biotin-switch assay followed by western blot analysis. Erectile function in S-nitrosoglutathione reductase deficient (GSNO+/-) and null (GSNO-/-) mice were assessed by continuous cavernous nerve electrical stimulation (CCNES). Glutathione ethyl ester (GSHee) was used to manipulate S-nitrosylated NOS levels. Immunohistological and immunofluorescence analyses were used to identify the location of eNOS and GSNO-R in corporal tissue. eNOS and nNOS were S-nitrosylated in unstimulated penises of the mice. CCNES resulted in a time-dependent increase in eNOS S-nitrosylation with peak eNOS S-nitrosylation observed during detumescence. S-nitrosylated nNOS levels were unchanged. Intracorporal injection of GSHee reduced S-nitrosylated eNOS levels, enhancing time to maximum intracorporal pressure (ICP). eNOS and GSNO-R co-localize to the endothelium of the corpus cavernosum in the mouse and the human. ICP measurements obtained during CCNES demonstrate GSNO-R+/- and GSNO-R-/- animals cannot maintain an elevated ICP. Results suggest eNOS S-nitrosylation/denitrosylation is an important mechanism regulating eNOS activity during erectile function. GSNO-R is a key enzyme involved in the eNOS denitrosylation. The increase in eNOS S-nitrosylation (inactivation) observed with tumescence may begin a cycle leading to detumescence. Clinically this may indicate that alterations in the balance of S-nitrosylation/denitrosylation either directly or indirectly contribute to erectile dysfunction.
Assuntos
Aldeído Oxirredutases/metabolismo , Disfunção Erétil/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ereção Peniana/fisiologia , Aldeído Oxirredutases/genética , Animais , Endotélio Vascular/metabolismo , Disfunção Erétil/genética , Masculino , Camundongos , Camundongos Knockout , Pênis/metabolismoRESUMO
AIMS: The present study aims to investigate the role of Akt in the regulation of urinary bladder organ hypertrophy caused by partial bladder outlet obstruction (pBOO). MAIN METHODS: Male rats were surgically induced for pBOO. Real-time PCR and western blot were used to examine the levels of mRNA and protein. A phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was used to inhibit the activity of endogenous Akt. KEY FINDINGS: The urinary bladder developed hypertrophy at 2â¯weeks of pBOO. The protein but not mRNA levels of type I collagen and α-smooth muscle actin (αSMA) were increased in pBOO bladder when compared to sham control. The phosphorylation (activation) levels of Akt1 (p-Ser473), mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and 4E-BP1 were also increased in pBOO bladder. LY294002 treatment reduced the phosphorylation levels of Akt1 and 4E-BP1, and the protein levels of type I collagen and αSMA in pBOO bladder. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) were increased in pBOO bladder, and PCNA up-regulation occurred in urothelial not muscular layer. LY294002 treatment had no effect on the mRNA and protein levels of PCNA in pBOO bladder. LY294002 treatment partially reduced the bladder weight caused by pBOO. SIGNIFICANCE: pBOO-induced urinary bladder hypertrophy is attributable to fibrosis, smooth muscle cellular hypertrophy, and urothelium cell hyper-proliferation. Akt1-mediated protein synthesis in pBOO bladder contributes to type I collagen and αSMA but not PCNA up-regulation. Target of Akt1 is necessary but not sufficient in treatment of urinary bladder hypertrophy following pBOO.
Assuntos
Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/genética , Bexiga Urinária/patologia , Animais , Vias Biossintéticas/genética , Cromonas/farmacologia , Inibidores Enzimáticos , Fibrose , Hipertrofia , Masculino , Morfolinas/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Obstrução do Colo da Bexiga Urinária/patologia , Urotélio/patologiaRESUMO
Specialized phagocytes are a newly appreciated classification of phagocyte that currently encompasses Sertoli cells (SCs) of the testes and the retinal pigment epithelial cells (RPE) of the retina. While these cells support very different tissues, they have a striking degree of similarity both as phagocytes and in ways that go beyond cell clearance. The clearance of apoptotic germ cells, cell debris, and used photoreceptor outer segments are critical functions of these cells, and the unique nature of their clearance events make specialized phagocytes uniquely suited for studying the larger implications of cell clearance in vivo. The shared functions of specialized phagocytes could provide novel insights into how phagocytosis impacts tissue homeostasis and immune modulation. In this review, we examine the remarkable similarities between SCs and RPE as specialized phagocytes and the physiological effects of cell clearance within a tissue.
Assuntos
Fagócitos/fisiologia , Fagocitose/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Células de Sertoli/fisiologia , Animais , Homeostase/imunologia , Homeostase/fisiologia , Humanos , Masculino , Fagócitos/citologia , Fagócitos/imunologia , Fagocitose/imunologia , Retina/citologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/imunologia , Células de Sertoli/citologia , Células de Sertoli/imunologia , Testículo/citologiaRESUMO
Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk -/- Bai1 Tg ) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk -/- mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk +/+ and Mertk -/- mice. Prior to the onset of photoreceptor degeneration, Mertk -/- mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.
Assuntos
Apoptose , Células Germinativas/metabolismo , Fagocitose , Epitélio Pigmentado da Retina/metabolismo , Células de Sertoli/metabolismo , c-Mer Tirosina Quinase/metabolismo , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Células Germinativas/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Epitélio Pigmentado da Retina/patologia , Células de Sertoli/patologia , c-Mer Tirosina Quinase/genéticaRESUMO
Few apoptotic corpses are seen even in tissues with high cellular turnover, leading to the notion that the capacity for engulfment in vivo is vast. Whether corpse clearance can be enhanced in vivo for potential benefit is not known. In a colonic inflammation model, we noted that the expression of the phagocytic receptor Bai1 was progressively downmodulated. Consistent with this, BAI1-deficient mice had more pronounced colitis and lower survival, with many uncleared apoptotic corpses and inflammatory cytokines within the colonic epithelium. When we engineered and tested transgenic mice overexpressing BAI1, these had fewer apoptotic cells, reduced inflammation, and attenuated disease. Boosting BAI1-mediated uptake by intestinal epithelial cells (rather than myeloid cells) was important in attenuating inflammation. A signaling-deficient BAI1 transgene could not provide a similar benefit. Collectively, these complementary genetic approaches showed that cell clearance could be boosted in vivo, with potential to regulate tissue inflammation in specific contexts.
Assuntos
Proteínas Angiogênicas/genética , Apoptose/imunologia , Colite/imunologia , Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , Animais , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colo/imunologia , Colo/patologia , Citocinas/imunologia , Sulfato de Dextrana , Células HCT116 , Humanos , Inflamação/imunologia , Mucosa Intestinal/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/imunologiaRESUMO
INTRODUCTION: MicroRNAs (miRs) are noncoding, endogenous RNA molecules that regulate gene expression and play roles in response to vascular injury. AIM: The aim of this study was to identify miRs expressed in corporal tissue (CT) and to determine whether miRs demonstrate differential expression in a mouse model of diet-induced erectile dysfunction (ED). METHODS: RNA was isolated from the CT from control mice and mice with diet-induced ED. A quantifiable miR profiling technique (NanoString) was used to determine the expression of over 600 miRs. MAIN OUTCOME MEASURES: Differential expression analysis was performed using a negative binomial regression model for count-based data. Mean expression levels, fold change, and false discovery-corrected P values were determined. Candidate miRs were validated via quantitative polymerase chain reaction (Q-PCR). RESULTS: In control mice, NanoString analysis revealed that 181 miRs were expressed above background levels and 5 miRs were expressed at high levels. Diet-induced ED resulted in the up-regulation of 6 miRs and the down-regulation of 65 miRs in the CT compared with mice on control diet. Focusing on the upregulated miRs, we chose five for Q-PCR validation. Of these five, two (miR-151-5p and miR-1937c) demonstrated significance via Q-PCR, whereas the other three (miR-720, miR-1937a, miR-205) trended in the correct direction. CONCLUSIONS: MiRs may play a significant role in mRNA regulation in CT and specific miRs may be involved in diet-induced vasculogenic ED. Future studies are aimed at determining the mRNA targets of these miRs.
Assuntos
Impotência Vasculogênica/genética , RNA Mensageiro/biossíntese , Animais , Dieta , Modelos Animais de Doenças , Regulação para Baixo , Masculino , Camundongos , MicroRNAs , Pênis/fisiopatologia , Reação em Cadeia da Polimerase , Regulação para CimaRESUMO
INTRODUCTION: During female sexual arousal, clitoral blood flow is controlled by endothelial nitric oxide synthase (eNOS) and its product, nitric oxide (NO). The mechanisms regulating eNOS activity and NO bioavailability in the clitoris are largely unknown. AIM: To identify proteins involved in regulation of eNOS activity within the clitoris and to evaluate the effects of S-nitrosoglutathione reductase (GSNO-R) and eNOS nitrosylation/denitrosylation on clitoral blood flow. METHODS: Immunohistochemistry for eNOS, caveolin-1 (Cav1), heat shock protein-90 (Hsp90), phosphodiesterase type 5 (PDE5), GSNO-R, and soluble guanylate cyclase (sGC) was performed on human and murine clitoral tissue. Western blot analysis was performed for eNOS, phosphorylated eNOS (phospho-eNOS, Ser1177), Cav1, Hsp90, sGC, PDE5, phosphoinositide 3-kinase (PI3K), Akt (protein kinase B), and GSNO-R on protein from human clitoral tissue. A biotin switch assay was used to analyze the S-nitrosylation of eNOS, nNOS, and GSNO-R. Clitoral blood flow was measured in wild-type and GSNO-R(-/-) mice at baseline and during cavernous nerve electrical stimulation (CNES). MAIN OUTCOME MEASURES: Localization of eNOS regulatory proteins and clitoral blood flow. RESULTS: eNOS and GSNO-R co-localized to the vascular endothelium and sinusoids of human clitoral tissue. Immunohistochemistry also localized Cav1 and Hsp90 to the endothelium and PDE5 and sGC to the trabecular smooth muscle. Expression of S-nitrosylated (SNO)-eNOS and SNO-GSNO-R was detected by biotin switch assays. Wild-type control mice exhibited increased clitoral blood flow with CNES whereas GSNO-R(-/-) animals failed to show an increase in blood flow. CONCLUSIONS: Several key eNOS regulatory proteins are present in the clitoral tissue in a cellular specific pattern. S-nitrosylation of eNOS may also represent a key regulatory mechanism governing eNOS activation/deactivation since mice deficient in GSNO-R failed to increase clitoral blood flow. Additional studies are necessary to define the role of S-nitrosylation in the genital vascular response and its subsequent impact on female sexual function.
Assuntos
Clitóris/enzimologia , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico/fisiologia , Aldeído Oxirredutases/fisiologia , Animais , Caveolina 1/metabolismo , Clitóris/irrigação sanguínea , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Endotélio/metabolismo , Endotélio Vascular/metabolismo , Feminino , Guanilato Ciclase/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Guanilil Ciclase SolúvelRESUMO
PURPOSE: Bilateral cavernous nerve injury results in up-regulation of ROCK signaling in the penis. This is linked to erectile dysfunction in an animal model of post-prostatectomy erectile dysfunction. We evaluated whether daily treatment with the ROCK inhibitor Y-27632 (Tocris Bioscience, Ellisville, Missouri) would prevent erectile dysfunction in a rat model of bilateral cavernous nerve injury. MATERIALS AND METHODS: Sprague-Dawley® rats underwent surgery to create sham (14) or bilateral (27) cavernous nerve injury. In the injury group 13 rats received treatment with Y-27632 (5 mg/kg twice daily) and 14 received vehicle. At 14 days after injury, rats underwent cavernous nerve stimulation to determine erectile function. Penes were assessed for neuronal and nitric oxide synthase membrane-endothelial nitric oxide synthase. ROCK2 was assessed by Western blot. Cyclic guanosine monophosphate was determined by enzyme-linked immunosorbent assay. Cavernous homogenates were tested for ROCK and protein kinase G enzymatic activity. Penile apoptosis was evaluated using the Apostain technique (Alexis, San Diego, California). Data were analyzed on ROCK using ANOVA and the t test. RESULTS: While erectile function was decreased in rats with bilateral cavernous nerve injury, daily administration of Y-27632 improved erectile responses. Injury decreased neuronal and nitric oxide synthase membrane-endothelial nitric oxide synthase but ROCK2 was significantly increased. Y-27632 treatment restored neuronal nitric oxide synthase, nitric oxide synthase membrane-endothelial nitric oxide synthase and cyclic guanosine monophosphate levels, and protein kinase G activity. Treatment significantly decreased ROCK2 protein and ROCK activity. There were significantly fewer apoptotic cells after treatment than in injured controls. CONCLUSIONS: These results provide evidence for up-regulation of the RhoA/ROCK signaling pathway with detrimental effects on erectile function after bilateral cavernous nerve injury. ROCK inhibition improved erectile dysfunction associated with bilateral cavernous nerve injury by preserving penile nitric oxide bioavailability and decreasing penile apoptosis.
Assuntos
Amidas/farmacologia , Apoptose/efeitos dos fármacos , Disfunção Erétil/enzimologia , Óxido Nítrico/metabolismo , Ereção Peniana/efeitos dos fármacos , Pênis/inervação , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Disfunção Erétil/etiologia , Masculino , Pênis/enzimologia , Pênis/lesões , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacosRESUMO
INTRODUCTION: Diabetes mellitus (DM) is a major risk factor for developing erectile dysfunction (ED) and men with DM are often less responsive to phosphodiesterase type 5 (PDE5) inhibitors than ED due to other causes. AIMS: The aim of this study was to explore potential mechanisms whereby PDE5 inhibitors may have reduced efficacy in type 2 DM. METHODS: At 4 weeks of age, mice were either fed a high-fat diet (HFD) for 22-36 weeks or fed regular chow (control). An additional group of mice in the same genetic background had a genetic form of type 1 DM. MAIN OUTCOME MEASURES: Glucose tolerance testing, intracorporal pressures (ICPs), oxidative stress (OS), apoptotic cell death (active caspase-3 and apostain), PDE5, p53, and cyclic guanosine monophosphate (cGMP) levels, and histological examination of inflow arteries were performed in mice fed a HFD and control mice. A group of mice with type 1 DM were studied for PDE5 expression levels. RESULTS: All mice fed a HFD had impaired glucose tolerance compared with the age-matched mice fed on standard chow diet (control). HFD fed mice had reduced maximum ICPs following in vivo cavernous nerve electrical stimulation and increased apoptotic cell death, OS, and p53 levels in the corporal tissue. Interestingly, PDE5 levels were increased and cGMP levels were decreased. In contrast, mice with type 1 DM did not have increases in PDE5. CONCLUSIONS: Taken together, our results suggest that type 2 DM-induced ED is associated with findings that could lead to reduced cGMP and may account for reduced efficacy of PDE5 inhibitors.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/sangue , Diabetes Mellitus Experimental/enzimologia , Disfunção Erétil/enzimologia , Inibidores da Fosfodiesterase 5/farmacologia , Animais , Apoptose/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica , Disfunção Erétil/patologia , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Endogâmicos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pênis/irrigação sanguínea , Pênis/patologiaRESUMO
AIMS: To determine whether cavernous nerve injury (CNI) alters lower urinary tract function, we assessed bladder and urethral function over time in a mouse model of CNI. METHODS: Twelve-week-old male C57BL/6 mice were divided into three groups: unoperated (UO; n = 6), sham-operated (SO; n = 18), and bilateral CNI (n = 30) group. At 1, 2, 4, 6, 8, 10 days bladder and urethral function were evaluated in these three groups using cystometry (CMG) and leak point pressure (LPP) recording under anesthesia. RESULTS: There was no significant difference in maximum detrusor pressure between groups at all times. Compared with the UO group, bladder compliance, and capacity in the CNI group were significantly decreased at Days 1, 2, 4 (P < 0.05) and recovered gradually from Day 6 to Day 10. In the SO group, they were decreased at Day 1, however, recovered more rapidly than the CNI group. Non-voiding contractions (NVC) developed in the CNI group at all times. Intercontraction interval were significantly decreased in SO and CNI groups and recovered more rapidly in SO group. In the SO group NVC were observed only at Days 1 and 2. LPP in the CNI group was decreased significantly at Days 1 and 2 (P < 0.05) and rapidly recovered with time compared with the UO and SO groups. CONCLUSION: In a mouse model of CNI, a transient decrease in bladder compliance, capacity, LPP and increased NVC was observed. These changes gradually recovered from Day 6 after CNI. Our findings suggest that CNI may affect bladder and urethral function, but alterations are reversible.
Assuntos
Traumatismos dos Nervos Periféricos/fisiopatologia , Uretra/inervação , Bexiga Urinária/inervação , Incontinência Urinária/fisiopatologia , Animais , Complacência (Medida de Distensibilidade) , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pressão , Recuperação de Função Fisiológica , Fatores de Tempo , Micção , UrodinâmicaRESUMO
PURPOSE: Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. MATERIALS AND METHODS: After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. RESULTS: Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. CONCLUSIONS: Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate.
Assuntos
Microdissecção/métodos , Recuperação Espermática , Testículo/citologia , Animais , Azoospermia/induzido quimicamente , Bussulfano/efeitos adversos , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Túbulos Seminíferos/metabolismo , Coleta de Tecidos e Órgãos/métodosRESUMO
Rapid and efficient removal of apoptotic cells by phagocytes is important during development, tissue homeostasis and in immune responses. Efficient clearance depends on the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the corpse-derived cellular material. However, the factors that influence continued clearance by phagocytes are not known. Here we show that the mitochondrial membrane potential of the phagocyte critically controls engulfment capacity, with lower potential enhancing engulfment and vice versa. The mitochondrial membrane protein Ucp2, which acts to lower the mitochondrial membrane potential, was upregulated in phagocytes engulfing apoptotic cells. Loss of Ucp2 reduced phagocytic capacity, whereas Ucp2 overexpression enhanced engulfment. Mutational and pharmacological studies indicated a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice were impaired in phagocytosis in vitro, and Ucp2-deficient mice showed profound in vivo defects in clearing dying cells in the thymus and testes. Collectively, these data indicate that mitochondrial membrane potential and Ucp2 are key molecular determinants of apoptotic cell clearance. As Ucp2 is linked to metabolic diseases and atherosclerosis, this newly discovered role for Ucp2 in apoptotic cell clearance has implications for the complex aetiology and pathogenesis of these diseases.
Assuntos
Apoptose , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Fagócitos/citologia , Fagócitos/metabolismo , Fagocitose/fisiologia , Animais , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Canais Iônicos/deficiência , Canais Iônicos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Fagócitos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Timo/citologia , Proteína Desacopladora 2RESUMO
OBJECTIVES: To determine cytokine responses in rat epididymal tissues after retrograde Escherichia coli inoculation of the cauda epididymidis via the intact and obstructed vas deferens. METHODS: Adult male Sprague-Dawley rats were divided into 3 groups: bilateral sham vasectomy followed by unilateral sham retrograde inoculation in the vas deferens (group A), bilateral sham vasectomy followed by unilateral retrograde inoculation of E. coli (group B), and bilateral vasectomy followed by left-sided inoculation of E. coli (group C). Three days later, the cauda epididymides and proximal vasa were subjected to histologic examination and assay for 9 cytokines. RESULTS: Groups A and C showed no histologic evidence of epididymal inflammation. Group B had leukocyte infiltrates in the inoculated tissue. Cytokine levels in the injected cauda epididymides were low in groups A and C; however, interleukin (IL)-1α, IL-1ß, and IL-4 were significantly increased in group B. The 6 other cytokines showed no significant change after E. coli infection though tumor necrosis factor-α, and IL-6 did show strong trends for increase. Contralateral epididymides never showed an inflammatory response. CONCLUSIONS: Experimental epididymitis induced by retrograde movement of bacteria in the vas deferens results in different responses by different cytokines. The cytokine responses and the histologically evident inflammation are prevented by vasectomy.
Assuntos
Citocinas/biossíntese , Epididimite/microbiologia , Escherichia coli/metabolismo , Vasectomia/métodos , Animais , Citocinas/metabolismo , Epididimo/microbiologia , Regulação da Expressão Gênica , Humanos , Inflamação , Masculino , Ratos , Ratos Sprague-Dawley , Ducto Deferente/microbiologiaRESUMO
Apoptosis and the subsequent clearance of dying cells occurs throughout development and adult life in many tissues. Failure to promptly clear apoptotic cells has been linked to many diseases. ELMO1 is an evolutionarily conserved cytoplasmic engulfment protein that functions downstream of the phosphatidylserine receptor BAI1, and, along with DOCK1 and the GTPase RAC1, promotes internalization of the dying cells. Here we report the generation of ELMO1-deficient mice, which we found to be unexpectedly viable and grossly normal. However, they had a striking testicular pathology, with disrupted seminiferous epithelium, multinucleated giant cells, uncleared apoptotic germ cells and decreased sperm output. Subsequent in vitro and in vivo analyses revealed a crucial role for ELMO1 in the phagocytic clearance of apoptotic germ cells by Sertoli cells lining the seminiferous epithelium. The engulfment receptor BAI1 and RAC1 (upstream and downstream of ELMO1, respectively) were also important for Sertoli-cell-mediated engulfment. Collectively, these findings uncover a selective requirement for ELMO1 in Sertoli-cell-mediated removal of apoptotic germ cells and make a compelling case for a relationship between engulfment and tissue homeostasis in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Fagocitose/fisiologia , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermatozoides/citologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Angiogênicas/metabolismo , Animais , Linhagem Celular , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Fosfatidilserinas/metabolismo , Epitélio Seminífero/citologia , Epitélio Seminífero/patologia , Células de Sertoli/patologia , Transdução de Sinais , Espermatozoides/patologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
INTRODUCTION: Over the past 15 years, significant advances have been made in the treatment of erectile dysfunction (ED). The most significant of these advances has been pharmacological treatment of ED with phosphodiesterase type 5 (PDE5) inhibitors. This therapy greatly increased the awareness of ED and has helped stimulate research into the underlying causes of ED. While treatment with PDE5 inhibitors continues to be the current therapy of choice, approximately 40% of men treated with PDE5 inhibitors fail to have significant improvement in erectile function and PDE5 inhibitors do not reverse the vasculopathic processes associated with ED. With this in mind, new therapies must be developed. The treatment with angiogenic growth factors such as vascular endothelial cell growth factor (VEGF) may be one such therapy. AIM: This review will focus on defining key terms in the angiogenic process, angiogenic growth factors, and different delivery methods, and summarize results from angiogenic therapies for the treatment of ED. METHODS: A review of the literature was performed on all angiogenic therapies for the treatment of ED. A brief review on the angiogenic factors was also performed. RESULTS: Angiogenic therapies for the treatment of ED are possible and promising; however, further investigation is needed to advance clinically. CONCLUSIONS: Although numerous studies have now employed angiogenic factors for the possible treatment of ED in several animal models, we are still not at the point to begin human investigations. Future studies need to examine proper dosage of the angiogenic agent, route of delivery, time course for delivery, and combination therapies.
Assuntos
Indutores da Angiogênese/uso terapêutico , Impotência Vasculogênica/tratamento farmacológico , Pênis/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/uso terapêutico , Animais , Fator 2 de Crescimento de Fibroblastos/fisiologia , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Humanos , Masculino , Pênis/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/fisiologia , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y(2) as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y(2)-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y(2)(-/-) (also known as P2ry2(-/-)) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y(2)(-/-) mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y(2)-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.
Assuntos
Trifosfato de Adenosina/metabolismo , Apoptose/fisiologia , Fagócitos/citologia , Fagocitose/fisiologia , Transdução de Sinais , Timo/citologia , Uridina Trifosfato/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Fatores Quimiotáticos/metabolismo , Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Humanos , Células Jurkat , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fagócitos/efeitos dos fármacos , Fagócitos/metabolismo , Fagocitose/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Transdução de Sinais/efeitos dos fármacos , Uridina Trifosfato/farmacologiaRESUMO
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that plays an essential role in oxygen homeostasis. HIF-1alpha is constitutively made in cells; however, it is ubiquitinated and degraded under normoxic conditions. Hypoxia prevents the ubiquitination of HIF-1alpha, resulting in stabilization of the protein and activation of target genes. Because of its vascular arrangement and the high metabolic demand of spermatogenesis, the testis has been described previously as functioning on the brink of hypoxia; thus, we have hypothesized that HIF-1alpha is constitutively expressed and stabilized in the testis, where it could play a role in testicular homeostasis. Western blot analysis using nuclear proteins from liver, kidney, and testis revealed the presence of HIF-1alpha only in the testis. Immunohistochemistry confirmed this result and revealed that HIF-1alpha was specifically located in interstitial Leydig cells. Electromobility shift assays employing nuclear extracts from the TM3 Leydig cell line revealed that these cells express HIF-1alpha that is capable of binding DNA under normoxic conditions. Furthermore, we found that protein levels can be increased further when the TM3 cells are cultured under hypoxic conditions. Finally, transient transfections of TM3 Leydig cells revealed that the promoter of the mouse 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1) gene, which encodes a key enzyme in testosterone production, is a potential target of HIF-1alpha. In conclusion, HIF-1alpha is constitutively present in the Leydig cells of the murine testis, where it potentially regulates Hsd3b1 transcription, and thus male reproductive function.
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17-Hidroxiesteroide Desidrogenases/genética , Regulação da Expressão Gênica/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Intersticiais do Testículo/metabolismo , 17-Hidroxiesteroide Desidrogenases/biossíntese , Animais , Western Blotting , Hipóxia Celular/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Imunofluorescência , Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Oxidative stress results from the production of oxygen radicals in excess of the antioxidant capacity of the stressed tissue. Many conditions or events associated with male infertility are inducers of oxidative stress. X-irradiation, for example, or exposure to environmental toxicants and the physical conditions of varicocele and cryptorchidism have been demonstrated to increase testicular oxidative stress, which leads to an increase in germ cell apoptosis and subsequent hypospermatogenesis. Such stress conditions can cause changes in the dynamics of testicular microvascular blood flow, endocrine signaling, and germ cell apoptosis. Testicular oxidative stress appears to be a common feature in much of what underlies male infertility, which suggests that there may be benefits to developing better antioxidant therapies for relevant cases of hypospermatogenesis.
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Estresse Oxidativo , Testículo/fisiopatologia , Antioxidantes/metabolismo , Sistema Endócrino/metabolismo , Humanos , Infertilidade Masculina/etiologia , Masculino , Espécies Reativas de Oxigênio/metabolismo , Doenças Testiculares/metabolismo , Doenças Testiculares/fisiopatologia , Testículo/irrigação sanguíneaRESUMO
PURPOSE: Despite techniques to preserve the cavernous nerves during radical prostatectomy erectile dysfunction remains a complication. We determined whether bilateral cavernous nerve resection induces apoptosis in the penis. We also determined whether treatment with the phosphodiesterase-5 inhibitor tadalafil prevents apoptosis as well as the specific mechanisms involved. MATERIALS AND METHODS: Mice were subjected to cavernous nerve resection or sham surgery. Penises were processed for the identification of apoptotic cells, changes in phosphorylation of several protein kinases and immunolocalization of specific kinases. Mice were also placed on tadalafil or vehicle after cavernous nerve resection and the penises were processed as described. Statistical analysis was performed with the Mann-Whitney U test for comparisons among groups or Student's t test. RESULTS: An increase in apoptotic cavernous smooth muscle and endothelial cells was evident by 2 weeks, which further increased 4 and 6 weeks after cavernous nerve resection. Apoptosis coincided with an increase in the phosphorylation of c-jun N-terminal kinase and p38 mitogen activated protein kinase. Phospho-c-jun N-terminal kinase was immunolocalized to endothelial and smooth muscle cells. Treatment with tadalafil decreased the number of apoptotic cells and increased the phosphorylation of the 2 survival associated kinases Akt and extracellular signal-regulated kinase 1/2. CONCLUSIONS: These results provide a rationale for the early use of phosphodiesterase-5 inhibition following radical prostatectomy or extensive pelvic surgery, during which there may be injury to the cavernous nerves, to aid in the return of erectile function.