ABSTRACT
Inside eukaryotic cells, membrane contact sites (MCSs), regions where two membrane-bound organelles are apposed at less than 30 nm, generate regions of important lipid and calcium exchange. This review principally focuses on the structure and the function of MCSs between the endoplasmic reticulum (ER) and the plasma membrane (PM). Here we describe how tethering structures form and maintain these junctions and, in some instances, participate in their function. We then discuss recent insights into the mechanisms by which specific classes of proteins mediate nonvesicular lipid exchange between the ER and PM and how such phenomena, already known to be crucial for maintaining organelle identity, are also emerging as regulators of cell growth and development.
Subject(s)
Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Animals , Humans , Models, BiologicalABSTRACT
RESEARCH QUESTION: Is ovarian stimulation with levonorgestrel intrauterine system (LNG-IUS) in situ and co-treatment with letrozole safe and effective in patients undergoing fertility-sparing combined treatment for atypical endometrial hyperplasia (AEH) or early endometrial cancer limited to the endometrium? DESIGN: Retrospective case-control study recruiting women who had undergone fertility-sparing 'combined' treatment and ovarian stimulation with letrozole and LNG-IUS in situ. The 'three steps' hysteroscopic technique was used. Once complete response was achieved, the ovaries were stimulated, and mature oocytes cryopreserved. The LNG-IUS was removed, and embryos transferred. A comparative analysis was conducted between the two control groups of the initial outcomes of ART (number of oocytes and MII oocytes retrieved): healthy infertile women undergoing ovarian stimulation for IVF/ICSI (control group A); and patients diagnosed with breast cancer who underwent ovarian stimulation with letrozole (control group B). RESULTS: Of the 75 patients analysed, 15 underwent oocyte cryopreservation after achieving a complete response to fertility-sparing treatment (study group); 30 patients in control group A and B, respectively. No statistically significant differences were observed in retrieved oocytes and mature oocytes between the study and control groups. In the nine patients who underwent embryo transfer, clinical pregnancy (55.6%), cumulative live birth (44.4%) and miscarriage (20%) rates were reported. In three patients with AEH, recurrence occurred (12%) at 3, 6 and 16 months after removing the LNG-IUS to attempt embryo transfer, respectively. CONCLUSION: Fertility-sparing hysteroscopic combined treatment and subsequent ovarian stimulation with letrozole and LNG-IUS in situ could be suggested to women with AEH or early endometrial cancer who ask for future fertility preservation.
Subject(s)
Endometrial Neoplasms , Fertility Preservation , Letrozole , Levonorgestrel , Ovulation Induction , Humans , Female , Levonorgestrel/administration & dosage , Levonorgestrel/therapeutic use , Letrozole/therapeutic use , Letrozole/administration & dosage , Retrospective Studies , Adult , Ovulation Induction/methods , Case-Control Studies , Fertility Preservation/methods , Pregnancy , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/complications , Cryopreservation , Endometrial Hyperplasia/drug therapy , Intrauterine Devices, Medicated , Pregnancy RateABSTRACT
OBJECTIVES: To assess the effectiveness of a topical treatment for cervical ectropion with vaginal ovules containing aqueous extract of Triticum vulgare . METHODS: Prospective observational cohort study conducted between November 2020 and November 2022 at the colposcopy clinics of 2 different hospitals. Sexually active patients with a colposcopic diagnosis of cervical ectropion, awaiting ablative procedure, were included in the study and prescribed medical therapy with vaginal T. vulgare ovules (group 1) or wait-and-see therapy (group 2). At both enrollment and 2-month follow-up, a symptomatologic evaluation recording the intensity of postcoital bleeding, leucorrhea, and dyspareunia was performed with a Visual Analog Scale (VAS) questionnaire, and a colposcopic examination was performed, calculating the size of the ectropion area. RESULTS: A total of 116 patients (58 in group 1 and 58 in group 2) were included. Topical treatment with vaginal ovules containing aqueous extract of T. vulgare for 2 months resulted in significant decrease of symptoms related to cervical ectropion and significant reduction of the size of the ectropion area. CONCLUSIONS: Our results support the efficacy of topical treatment with vaginal ovules containing aqueous extract of T. vulgare for symptomatic cervical ectropion; our study also suggests that expectant management, despite being widely practiced, seems to lead to a worsening of the natural history of ectropion.
Subject(s)
Ectropion , Plant Extracts , Triticum , Humans , Female , Prospective Studies , Adult , Plant Extracts/therapeutic use , Plant Extracts/administration & dosage , Middle Aged , Treatment Outcome , Ectropion/drug therapy , Young Adult , Colposcopy , Administration, TopicalABSTRACT
Uterine Tumors Resembling Ovarian Sex Cord Tumors (UTROSCTs) are rare uterine mesenchymal neoplasms with uncertain biological potential. These tumors, which affect both premenopausal and postmenopausal women, usually have a benign clinical course. Nevertheless, local recurrences and distant metastases have been described. By analyzing 511 cases retrieved from individual reports and cases series, we provide here the most comprehensive overview of UTROSCT cases available in the literature, supplemented by two new cases of UTROSCTs. Case 1 was an asymptomatic 31-year-old woman who underwent a laparoscopic resection of a presumed leiomyoma. Case 2 was a 58-year-old postmenopausal woman with abnormal vaginal bleeding who underwent an outpatient hysteroscopic biopsy of a suspicious endometrial area. In both cases, immunohistochemical positivity for Calretinin and Inhibin was noted, typical for a sex cord differentiation. In both cases, total laparoscopic hysterectomy with bilateral salpingo-oophorectomy was performed. In light of the available literature, no pathognomonic clinical or imaging finding can be attributed to UTROSCT. Patients usually present with abnormal uterine bleeding or pelvic discomfort, but 20% of them are asymptomatic. In most cases, a simple hysterectomy appears to be the appropriate treatment, but for women who wish to become pregnant, uterus-preserving approaches should be discussed after excluding risk factors. Age, tumor size, lymphovascular space invasion, nuclear atypia, and cervical involvement are not reliable prognostic factors in UTROSCT. The current research suggests that aggressive cases (with extrauterine spread or recurrence) can be identified based on a distinct genetic and immunohistochemical phenotype. For instance, UTROSCTs characterized by GREB1::NCOA1-3 fusions and PD-L1 molecule expression appear to be predisposed to more aggressive behaviors and recurrence, with GREB1::NCOA2 being the most common gene fusion in recurrent tumors. Hence, redefining the criteria for UTROSCTs may allow a better selection of women suitable for fertility-sparing treatments or requiring more aggressive treatments in the future.
Subject(s)
Leiomyoma , Ovarian Neoplasms , Sex Cord-Gonadal Stromal Tumors , Uterine Neoplasms , Humans , Female , Adult , Middle Aged , Neoplasm Recurrence, Local , Uterine Neoplasms/diagnosis , Uterine Neoplasms/surgery , Uterus , Hysterectomy , Leiomyoma/surgery , Sex Cord-Gonadal Stromal Tumors/diagnosis , Sex Cord-Gonadal Stromal Tumors/surgery , Sex Cord-Gonadal Stromal Tumors/genetics , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/surgery , Ovarian Neoplasms/pathologyABSTRACT
Fertility-sparing treatments have become important for young women with atypical endometrial hyperplasia (AEH) or endometrial carcinoma (EC) who wish to preserve their reproductive potential. Evidence indicates a strong relationship between weight and EC and the effect of weight loss on reducing the risk of EC. We report the case of a young obese woman with a body mass index (BMI) of 46.6 kg/m2, diagnosed with grade 2 endometrial endometrioid adenocarcinoma, who underwent a combined fertility-sparing treatment with hysteroscopic resection followed by insertion of a levonorgestrel intrauterine system. After twelve months of failure to achieve a complete response, bariatric surgery was proposed to lose weight and improve the response to treatment. Histologic regression was achieved three months after surgery, with a weight loss of 30 kg and fifteen months after combined treatment of endometrial cancer. We reviewed the literature to summarize the evidence on the role of bariatric surgery and weight loss in modifying the oncologic and reproductive outcomes of women undergoing fertility-sparing treatment for atypical endometrial lesions.
Subject(s)
Endometrial Neoplasms , Fertility Preservation , Pregnancy , Female , Humans , Hysteroscopy , Endometrial Neoplasms/surgery , Levonorgestrel , Weight LossABSTRACT
STUDY OBJECTIVE: To demonstrate our hysteroscopic technique using the mini-resectoscope for the treatment of complete uterine septum with or without cervical anomalies. DESIGN: A step-by-step video demonstration of the technique with the use of an educational video. SETTINGS: We present 3 patients diagnosed as having complete uterine septum (U2b according to the ESHRE/ESGE classification) with or without cervical anomalies (C0, normal cervix; C1, septate cervix; C2, double "normal" cervix"), 2 of them with a longitudinal vaginal septum (V1). The first case is a 33-year-old woman with history of primary infertility diagnosed as having a complete uterine septum with normal cervix (class U2bC0V0 according to the ESHRE/ESGE classification). Case 2 is a 34-year-old woman with infertility and abnormal uterine bleeding, diagnosed as having complete uterine and cervical septum and a partial nonobstructive vaginal septum (class U2bC1V1). Case 3 is a 28-year-old woman with infertility and dyspareunia, diagnosed as having a complete uterine septum, double "normal" cervix, and nonobstructive longitudinal vaginal septum (class U2bC2V1) Still 3. The procedures were performed in a tertiary care university hospital. INTERVENTION: The 3 procedures were performed in the operative room using a 15 Fr continuous flow mini-resectoscope and bipolar energy with the patient under general anesthesia Still 1 and Still 2. No complications were encountered in any of the 3 cases. After all procedures, a gel based on hyaluronic acid was applied to minimize postoperative adhesion formation. Patients were discharged home the same day of the procedure after a short period of observation. CONCLUSION: Hysteroscopic treatment of patients with uterine septa associated or not with cervical anomalies using miniaturized instruments is a feasible and effective option for the management of patients with these complex müllerian anomalies.
Subject(s)
Infertility , Septate Uterus , Uterine Cervical Diseases , Pregnancy , Female , Humans , Adult , Hysteroscopy/methods , Uterus/surgery , Uterus/abnormalities , Uterine Cervical Diseases/complications , Uterine Cervical Diseases/surgeryABSTRACT
Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b-Stx1 SNARE complexes at endoplasmic reticulum (ER)-PM contacts. Here, we show that Sec22b interacts with members of the extended synaptotagmin (E-Syt) family of ER lipid transfer proteins (LTPs), and this interaction depends on the longin domain of Sec22b. Overexpression of E-Syts stabilizes Sec22b-Stx1 association, whereas silencing of E-Syts has the opposite effect. Overexpression of wild-type E-Syt2, but not mutants unable to transfer lipids or attach to the ER, increase the formation of axonal filopodia and ramification of neurites in developing neurons. This effect is inhibited by a clostridial neurotoxin cleaving Stx1, and expression of the Sec22b longin domain and a Sec22b mutant with an extended linker between the SNARE and transmembrane domains. We conclude that Sec22b-Stx1 ER-PM contact sites contribute to PM expansion by interacting with LTPs, such as E-Syts.This article has an associated First Person interview with the first author of the paper.
Subject(s)
Endoplasmic Reticulum , Neurites , Cell Membrane/metabolism , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Humans , Neurites/metabolism , SNARE Proteins/metabolism , Synaptotagmins/geneticsABSTRACT
BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is predominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.
Subject(s)
Embryonic Development , Oocytes , Animals , Spectrometry, Fluorescence , Oocytes/metabolism , Sea Urchins , Cryopreservation/methodsABSTRACT
Voltage-gated ion channels are plasma membrane proteins that generate electrical signals following a change in the membrane voltage. Since they are involved in several physiological processes, their dysfunction may be responsible for a series of diseases and pain states particularly related to neuronal and muscular systems. It is well established for decades that bioactive peptides isolated from venoms of marine mollusks belonging to the Conus genus, collectively known as conotoxins, can target different types and isoforms of these channels exerting therapeutic effects and pain relief. For this reason, conotoxins are widely used for either therapeutic purposes or studies on ion channel mechanisms of action disclosure. In addition their positive property, however, conotoxins may generate pathological states through similar ion channel modulation. In this narrative review, we provide pieces of evidence on the pathophysiological impacts that different members of conotoxin families exert by targeting the three most important voltage-gated channels, such as sodium, calcium, and potassium, involved in cellular processes.
Subject(s)
Conotoxins , Ion Channels , Animals , Conotoxins/chemistry , Conus Snail , Humans , Ion Channels/drug effects , Pain/drug therapyABSTRACT
Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disease. The hypothesis that alterations in the microbiome are involved in the genesis of PCOS has been postulated. Aim of this review is to summarize the available literature data about the relationship between microbiome and PCOS. A search on PubMed and Medline databases was performed from inception to November 20Most of evidence has focused on the connection of intestinal bacteria with sex hormones and insulin-resistance: while in the first case, a relationship with hyperandrogenism has been described, although it is still unclear, in the second one, chronic low-grade inflammation by activating the immune system, with increased production of proinflammatory cytokines which interfere with insulin receptor function, causing IR (Insulin Resistance)/hyperinsulinemia has been described, as well as the role of gastrointestinal hormones like Ghrelin and peptide YY (PYY), bile acids, interleukin-22 and Bacteroides vulgatus have been highlighted. The lower genital tract microbiome would be affected by changes in PCOS patients too. The therapeutic opportunities include probiotic, prebiotics and synbiotics, as well as fecal microbiota transplantation and the use of IL-22, to date only in animal models, as a possible future drug. Current evidence has shown the involvement of the gut microbiome in PCOS, seen how humanized mice receiving a fecal transplant from women with PCOS develop ovarian dysfunction, immune changes and insulin resistance and how it is capable of disrupting the secondary bile acid biosynthesis. A future therapeutic approach for PCOS may involve the human administration of IL-22 and bile acid glycodeoxycholic acid.
Subject(s)
Gastrointestinal Microbiome , Polycystic Ovary Syndrome/microbiology , Animals , Diet , Female , Genitalia, Female/microbiology , Hormones/metabolism , Humans , Insulin Resistance , Polycystic Ovary Syndrome/therapyABSTRACT
Hepatocyte Nuclear Factor 1 beta (HNF1ß) is a transcription factor which plays an important role during early organogenesis, especially of the pancreato-biliary and urogenital tract. Furthermore, HNF1ß is an established marker in the differential diagnosis of ovarian cancer and shows a distinct nuclear expression in the clear cell carcinoma subtype. Recently, it has been described in yolk sac tumor, which represents a common component in many non-seminomatous germ cell tumors. Due to its broad histologic diversity, the diagnosis may be challenging and additional tools are very helpful in the workup of germ cell tumors. Immunohistochemistry was used to study HNF1ß expression in a tissue microarray (TMA) of 601 testicular germ cell tumors including seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma, germ cell neoplasia in situ (GCNIS), and normal tissue. The expression pattern was compared to glypican 3 (GPC3) and α-fetoprotein (AFP), two markers currently in use for the detection of yolk sac tumor. HNF1ß showed a distinct nuclear staining in comparison to the cytoplasmic pattern of GPC3 and AFP. The sensitivity and specificity of HNF1ß were 85.4% and 96.5%, of GPC3 83.3% and 90.7%, of AFP 62.5% and 97.7%. We conclude that HNF1ß allows a reliable distinction of yolk sac tumor from other germ cell tumor components. Therefore, we propose HNF1ß as a novel and robust marker in the immunohistochemical workup of testicular germ cell tumors.
Subject(s)
Biomarkers, Tumor/metabolism , Endodermal Sinus Tumor/diagnosis , Hepatocyte Nuclear Factor 1-beta/metabolism , Testicular Neoplasms/diagnosis , Adult , Carcinoma, Embryonal/diagnosis , Carcinoma, Embryonal/metabolism , Carcinoma, Embryonal/pathology , Choriocarcinoma/diagnosis , Choriocarcinoma/metabolism , Choriocarcinoma/pathology , Diagnosis, Differential , Endodermal Sinus Tumor/metabolism , Endodermal Sinus Tumor/pathology , Humans , Immunohistochemistry , Male , Seminoma/diagnosis , Seminoma/metabolism , Seminoma/pathology , Sensitivity and Specificity , Teratoma/diagnosis , Teratoma/metabolism , Teratoma/pathology , Testicular Neoplasms/metabolism , Testicular Neoplasms/pathology , Testis/metabolism , Testis/pathology , Tissue Array AnalysisABSTRACT
Intradermal therapy, known as mesotherapy, is a technique used to inject a drug into the surface layer of the skin. In particular, it involves the use of a short needle to deposit the drug in the dermis. The intradermal microdeposit modulates the drug's kinetics, slowing absorption and prolonging the local mechanism of action. It is successfully applied in the treatment of some forms of localized pain syndromes and other local clinical conditions. It could be suggested when a systemic drug-sparing effect is useful, when other therapies have failed (or cannot be used), and when it can synergize with other pharmacological or nonpharmacological therapies. Despite the lack of randomized clinical trials in some fields of application, a general consensus is also reached in nonpharmacological mechanism of action, the technique execution modalities, the scientific rationale to apply it in some indications, and the usefulness of the informed consent. The Italian Mesotherapy Society proposes this position paper to apply intradermal therapy based on scientific evidence and no longer on personal bias.
Subject(s)
Analgesics/administration & dosage , Dermis/metabolism , Mesotherapy/methods , Pain/prevention & control , Skin Absorption , Analgesics/pharmacokinetics , Animals , Forecasting , Humans , Injections, Intradermal , Italy , Mesotherapy/instrumentation , Mesotherapy/trends , Practice Guidelines as Topic , Treatment OutcomeABSTRACT
The present study investigates whether leuprolide acetate (Lupron) adds to the efficacy of traditional sex offender treatment. A group of sex offenders receiving both Lupron and cognitive behavioral therapy (CBT; n = 25) were compared with a group of sex offenders receiving only CBT (n = 22). Treated subjects were compared with norms available with reference to the Static-99R, as well as compared with a sample of untreated, nonsexual violent offenders (n = 81), to provide baseline data regarding risk of violent recidivism. Results indicated that subjects receiving Lupron were at significantly higher risk of recidivism and significantly more likely to be diagnosed with a paraphilia than subjects receiving only CBT, a priori. Both treated groups of sexual offenders recidivated at substantially lower rates than predicted by the Static-99R. Currently, this study represents the only, long-term outcome study on Lupron administration using officially recorded recidivism as the primary dependent measure.
Subject(s)
Cognitive Behavioral Therapy , Criminals , Leuprolide/therapeutic use , Paraphilic Disorders/therapy , Recidivism/prevention & control , Sex Offenses , Adult , Aged , Aged, 80 and over , Humans , Male , Middle Aged , Paraphilic Disorders/drug therapy , Paraphilic Disorders/psychologyABSTRACT
BACKGROUND: Mitochondria play a role in type 1 diabetes (T1D) particularly in the treatment and prevention of disorder consequences. Due to their demonstrated role in diabetes pathology, mitochondrial proteins can be an interesting starting point to study candidate antigens in T1D. We investigated the role of relevant post-translational modifications (PTM) on a synthetic mitochondrial peptide as putative antigen. METHODS: The antibody response in T1D was evaluated by solid phase-ELISA using a collection of synthetic peptides bearing different PTMs. We investigated the role of lipoylation, phosphorylation, and glycosylation. The PTMs were introduced at position 173 of the mitochondrial pyruvate dehydrogenase E2 complex peptide PDC-E2(167-184) and at position 7 of a structure-based designed ß-turn peptide as an irrelevant sequence to investigate the role of the specific PDC-E2 peptide sequence. RESULTS: IgM titres in 31 T1D patients were higher than IgGs to all the synthetic PTM peptides. Results demonstrated the crucial role of lysine lipoamide, serine O-phosphorylation, and O-glycosylation into the PDC-E2(167-184) peptide sequence for IgM antibody recognition. CONCLUSIONS: Results highlight the importance of immune dysregulation in T1D, furthermore, if confirmed in a large number of patients, they will contribute to add novel diagnostic markers for the understanding the physiopathology of the disease.
Subject(s)
Antibodies/immunology , Diabetes Mellitus, Type 1/immunology , Mitochondrial Proteins/chemistry , Peptide Fragments/immunology , Peptide Fragments/metabolism , Protein Processing, Post-Translational , Adult , Diabetes Mellitus, Type 1/metabolism , Female , Glycosylation , Humans , Male , Phosphorylation , Stereoisomerism , Thioctic Acid/analogs & derivatives , Thioctic Acid/chemistry , Thioctic Acid/metabolismABSTRACT
Duchenne muscular dystrophy (DMD) is a lethal disease, determined by lack of dystrophin (Dp427), a muscular cytoskeletal protein also expressed by selected neuronal populations. Consequently, besides muscular wasting, both human patients and DMD animal models suffer several neural disorders. In previous studies on the superior cervical ganglion (SCG) of wild type and dystrophic mdx mice (Lombardi et al. 2008), we hypothesized that Dp427 could play some role in NGF-dependent axonal growth, both during development and adulthood. To address this issue, we first analyzed axon regeneration potentials of SCG neurons of both genotypes after axotomy in vivo. While noradrenergic innervation of mdx mouse submandibular gland, main source of nerve growth factor (NGF), recovered similarly to wild type, iris innervation (muscular target) never did. We, therefore, evaluated whether dystrophic SCG neurons were poorly responsive to NGF, especially at low concentration. Following in vitro axotomy in the presence of either 10 or 50ng/ml NGF, the number of regenerated axons in mdx mouse neuron cultures was indeed reduced, compared to wild type, at the lower concentration. Neurite growth parameters (i.e. number, length), growth cone dynamics and NGF/TrkA receptor signaling in differentiating neurons (not injured) were also significantly reduced when cultured with 10ng/ml NGF, but also with higher NGF concentrations. In conclusion, we propose a role for Dp427 in NGF-dependent cytoskeletal dynamics associated to growth cone advancement, possibly through indirect stabilization of TrkA receptors. Considering NGF activity in nervous system development/remodeling, this aspect could concur in some of the described DMD-associated neural dysfunctions.
Subject(s)
Axons/drug effects , Dystrophin/genetics , Nerve Growth Factor/pharmacology , Nerve Regeneration/drug effects , Neurons/drug effects , Superior Cervical Ganglion/cytology , Animals , Animals, Newborn , Axons/ultrastructure , Axotomy , Caspase 3/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Dystroglycans/metabolism , Dystrophin/metabolism , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Iris/innervation , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Nerve Fibers/metabolism , Nerve Fibers/pathology , Nerve Fibers/ultrastructure , Nerve Tissue Proteins/metabolism , Neurons/ultrastructure , Receptor, trkB/metabolism , Tubulin/metabolism , Tyrosine 3-Monooxygenase/metabolismABSTRACT
The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.
Subject(s)
Aquatic Organisms/cytology , Environmental Monitoring/methods , Fluorescent Dyes/chemistry , Invertebrates/cytology , Spectrometry, Fluorescence/methods , Spermatozoa/drug effects , Water Pollution/adverse effects , Animals , Aquatic Organisms/drug effects , Cell Survival/drug effects , DNA/chemistry , Invertebrates/drug effects , Male , Microscopy, Confocal , Organic Chemicals/chemistry , Propidium/chemistry , Species Specificity , Spermatozoa/cytology , Staining and LabelingABSTRACT
In marine animals with external fertilization, gametes are released into seawater where fertilization and embryo development occur. Consequently, pollutants introduced into the marine environment by human activities may affect gametes and embryos. These xenobiotics can alter cell physiology with consequent reduction of fertilization success. Here the adverse effects on the reproductive processes of the marine invertebrate Ciona intestinalis (ascidian) of different xenobiotics: lead, zinc, an organic tin compound and a phenylurea herbicide were evaluated. By using the electrophysiological technique of whole-cell voltage clamping, the effects of these compounds on the mature oocyte plasma membrane electrical properties and the electrical events of fertilization were tested by calculating the concentration that induced 50% normal larval formation (EC50). The results demonstrated that sodium currents in mature oocytes were reduced in a concentration-dependent manner by all tested xenobiotics, with the lowest EC50 value for lead. In contrast, fertilization current frequencies were differently affected by zinc and organic tin compound. Toxicity tests on gametes demonstrated that sperm fertilizing capability and fertilization oocyte competence were not altered by xenobiotics, whereas fertilization was inhibited in zinc solution and underwent a reduction in organic tin compound solution (EC50 value of 1.7 µM). Furthermore, fertilized oocytes resulted in a low percentage of normal larvae with an EC50 value of 0.90 µM. This study shows that reproductive processes of ascidians are highly sensitive to xenobiotics suggesting that they may be considered a reliable biomarker and that ascidians are suitable model organisms to assess marine environmental quality.