RIPK4 regulates cell-cell adhesion in epidermal development and homeostasis.

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer.

Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

Ultrastructure of mitochondria of human oocytes in different clinical conditions during assisted reproduction.

Infertility affects around 8% of couples with a slight change in percentage in the last years. Despite the significant efforts made in Assisted Reproductive Technologies (ARTs) in handling this disorder, oocyte quality remains a crucial factor for a positive outcome. A better understanding of the dynamics underlying oocyte maturation, fertilization, and embryo development remains one of the main areas for progress in the ARTs field. Mitochondria are believed to play an essential role in these processes. Mitochondria have a crucial part in producing energy for oocyte maturation and embryo development throughout precise cellular functions comprising Ca2+ homeostasis regulation, glycolysis, amino acid and fatty acid metabolism, and regulation of apoptosis. Recent studies suggest that mitochondrial structure, content, and function may be related to oocyte competence, embryo viability, and implantation success during ARTs. Their defects could lead to low fertilization rates and embryonic development failure. This review aimed to provide an overview of the available literature data surrounding the correlation between changes at ultrastructural level of mitochondria or correlated-mitochondrial aggregates and oocyte quality and ARTs treatments. Our reported data demonstrated that oocyte mitochondrial ultrastructural alterations could be partial or complete recovery during the early embryo stages. However, these changes could persist as quiescent during the pre-implantation embryo development, causing abnormalities that become evident only during fetal and postnatal life. These factors led to consider the mitochondria as a crucial marker of oocyte and embryo quality, as well as a strategic target for further prospective therapeutical approaches.

Does in vitro application of pentoxifylline have beneficial effects in assisted male reproduction?

Application of nonspecific phosphodiesterases inhibitors, such as pentoxifylline (PTX), is a strategy utilised to aid sperm selection from immotile sperm samples prior to ICSI. No extensive studies have yet been performed to verify the safety of the clinical outcomes of ICSI after PTX administration. In this article, we summarise the data reported in the literature that assess the implication of in vitro usage of PTX on sperm parameters, as well as clinical outcomes during assisted male reproduction programme.

Type of protein supplement in cryopreservation solutions impacts on the degree of ultrastructural damage in frozen-thawed human oocytes.

Protein sources used as supplements of IVF culture media are known to have several implications for the function and stability of embryo culture environment. In fact, they i) transport biologically active molecules ii) chelate heavy metals, iii) regulate media pH, iii) scavenge reactive oxygen species (ROS) and iv) attenuate osmotic stress to which cells are exposed in sub-optimal culture conditions. Instead, their specific relevance to the formulation of cryopreservation solutions used for gamete and embryo cryopreservation remains uncertain. In the present work, we tested the hypothesis that different protein supplements present in cryopreservation solutions, serum or plasma protein solution (PPS), or different concentrations of the same supplement (serum), are associated with different types and/or magnitude of cryopreservation-derived cell damage. To this end, using cryopreservation solutions containing serum or PPS, donated supernumerary human mature oocytes were frozen-thawed by slow freezing and compared with fresh controls. Ultrastructural markers of oocyte quality were adopted as objective measure to assess possible damage from cryopreservation. The study results indicate that the adoption of serum minimises cell damage induced by cryopreservation. Indeed, typical hallmarks of cryodamage in human oocytes, i.e. loss of cortical granules, zona pellucida hardening and above all vacuolization, were largely reduced in oocytes cryopreserved with solutions containing serum, especially if used a higher concentration. This suggest that oocyte cryopreservation still has significant margins of improvement that may derive also from composition of cryopreservation media.

Repeated hyperstimulation affects the ultrastructure of mouse fallopian tube epithelium.

Controlled ovarian hyperstimulation (COH) is routinary used in assisted reproductive technologies (ARTs) to increase the yields of mature oocytes. The possibility that patients with a history of failures or poor-responders may develop side-effects following these treatments is still debated. Epidemiological studies reported controversial results about pregnancy outcome and the risk of developing gynecological cancers. By using a mouse model, here we compared the ultrastructural features of fallopian tubes (FTs) obtained from mice undergoing or not (control, CTR) four (4R) and eight (8R) rounds of gonadotropin stimulation. Although the morphological characteristics of oviductal layers seemed unaffected by repeated treatments, dose-response ultrastructural alterations in the ampulla appeared in the 4R group and even more in the 8R group. The targets were oviductal ciliated (CCs) and non-ciliated (NCCs) cells, which showed damaged mitochondria and glycogen accumulations in the cytoplasm. The drastic reduction of CCs, evident after 4R, was supported by the absence of cilia. After 8R, glycogen granules were significantly reduced and massive degeneration of mitochondria, which appeared swollen and/or vacuolated, occurred in NCCs. Moreover, disintegrated mitochondria were found at the periphery of mitophagic vacuoles with evident signs of cristolysis. The morphometric analysis evidenced a significant increase in the density and frequency of damaged mitochondria after 4R and 8R. The absence of cilia, necessary to sustain oviductal transport of oocytes, spermatozoa and embryos, may originate from either mitochondrial dysfunction or glycogen consumption. These results suggest that repeated COH treatments could induce alterations impairing fertilization and embryo transport toward the uterus.

Oxygen concentration alters mitochondrial structure and function in in vitro fertilized preimplantation mouse embryos.

STUDY QUESTION: Does the oxygen concentration in the culture medium [either physiologic (5%) or atmospheric (20%)] affect mitochondrial ultrastructure and function in preimplantation mouse embryos generated by IVF? SUMMARY ANSWER: Embryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen. WHAT IS KNOWN ALREADY: ART are widely used and have resulted in the birth of more than 8 million children. A variety of media and oxygen concentrations are used to culture embryos. Embryos cultured under physiological O2 tension (5%) reach the blastocyst stage faster and have fewer alterations in gene expression when compared with embryos cultured under atmospheric oxygen conditions (20%). The mechanisms by which oxygen tension affects preimplantation development remain unclear, but mitochondria are believed to play an important role. The aim of this study was to evaluate how mitochondrial ultrastructure and function in IVF embryos were affected by culture under physiologic (5%) or atmospheric (20%) oxygen concentrations. STUDY DESIGN, SIZE, DURATION: Zygotes, 2-cell, 4-cell, morula and blastocyst were flushed out of the uterus after natural fertilization and used as controls. IVF was performed in CF1 x B6D2F1 mice and embryos were cultured in Potassium simplex optimized medium (KSOM) with amino acids (KAA) under 5% and 20% O2 until the blastocyst stage. Embryo development with the addition of antioxidants was also tested. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mitochondrial function was assessed by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, ATP levels, and the expression of selected genes involved in mitochondrial function. Mitochondria ultrastructure was evaluated by transmission electron microscopy (TEM). MAIN RESULTS AND THE ROLE OF CHANCE: Embryos cultured under 20% O2 had fewer mitochondria and more vacuoles and hooded (abnormal) mitochondria compared to the other groups (P < 0.05). At the blastocyst stage the mitochondria of IVF embryos cultured in 20% O2 had lower mtDNA copy number, a denser matrix and more lamellar cristae than controls. Overall IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, together with changes in the expression of selected mitochondrial genes (P < 0.05). ATP levels were significantly lower than controls only under 5% O2, with the 20% O2 IVF group having intermediate levels. Unexpectedly, adding antioxidant to the culture medium did not improve development. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Findings in mice embryos might be different from human embryos. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that changes in the mitochondria may be part of the mechanism by which lower oxygen concentration leads to better embryo development and further emphasize the importance of mitochondria as a locus of reprogramming. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by R01 HD 082039 to PFR, the Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy (RIA 2016-2018) and the Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Italy (University grants 2016-2017). The authors declare no competing interests.

The impact of sperm DNA fragmentation on ICSI outcome in cases of donated oocytes.

PURPOSE: The aim of this study is to evaluate the sperm DNA fragmentation index (DFI) in oocyte donation cycles and correlate it with the sperm parameters, the male characteristics, the embryo quality and the outcome of intracytoplasmic sperm injection (ICSI). METHODS: A total of 150 couples participating in an oocyte donation program were included in the study. Sperm samples were assessed by conventional sperm analysis. DFI was evaluated using the Halosperm kit, a sperm chromatin dispersion test (SCD). RESULTS: The relations between DNA damage and epidemiological male factors (age, height, weight), standard semen parameters (concentration, total and forward motility, and morphology), and embryological and clinical parameters (fertilization rate, total blastocyst number, number of good quality blastocyst, clinical pregnancy) were analyzed. DFI was positively correlated with advanced male age (r = 0.23, p < 0.05) and negatively correlated with total sperm and forward motility (r = - 0.29, r = - 0.27, respectively; p < 0.05). DFI was not significantly correlated with pregnancy outcome in oocyte donation cycles (r = - 0.05, p > 0.05). When good quality blastocysts were chosen, a trend toward the development of good quality embryos was detected in the presence of a low DFI (r = - 0.20, p = 0.08). CONCLUSIONS: DFI does not significantly affect the outcome of ICSI in oocyte donation cycles. Even in cases of advanced paternal age that a high DFI resulted sperm DNA fragmentation seems not to adversely affect the final outcome.

Mancozeb impairs the ultrastructure of mouse granulosa cells in a dose-dependent manner.

Mancozeb, an ethylene bis-dithiocarbamate, is widely used as a fungicide and exerts reproductive toxicity in vivo and in vitro in mouse oocytes by altering spindle morphology and impairing the ability to fertilize. Mancozeb also induces a premalignant status in mouse granulosa cells (GCs) cultured in vitro, as indicated by decreased p53 expression and tenuous oxidative stress. However, the presence and extent of ultrastructural alterations induced by mancozeb on GCs in vitro have not yet been reported. Using an in vitro model of reproductive toxicity, comprising parietal GCs from mouse antral follicles cultured with increasing concentrations of mancozeb (0.001-1 µg/ml), we sought to ascertain the in vitro ultrastructural cell toxicity by means of transmission (TEM) and scanning (SEM) electron microscopy. The results showed a dose-dependent toxicity of mancozeb on mouse GCs. Ultrastructural data showed intercellular contact alterations, nuclear membrane irregularities, and chromatin marginalization at lower concentrations, and showed chromatin condensation, membrane blebbing, and cytoplasmic vacuolization at higher concentrations. Morphometric analysis evidenced a reduction of mitochondrial length in GCs exposed to mancozeb 0.01-1 µg/ml and a dose-dependent increase of vacuole dimension. In conclusion, mancozeb induced dose-dependent toxicity against GCs in vitro, including ultrastructural signs of cell degeneration compatible with apoptosis, likely due to the toxic breakdown product ethylenethiourea. These alterations may represent a major cause of reduced/delayed/missed oocyte maturation in cases of infertility associated with exposure to pesticides.

Human cumulus cell sensitivity to vitrification, an ultrastructural study.

SummaryCumulus cells (CCs) play an important role in the regulation of female gamete development, meiotic maturation, oocyte-sperm interaction, capacitation and acrosome reaction. However, their role in maintaining oocyte competence after vitrification is unclear as controversial data on their protecting action against oocyte cryoinjuries are available. Here we described the effects of vitrification on the ultrastructure of human CCs collected from women undergoing assisted reproductive technologies (ARTs). In total, 50 patches of CCs, sampled from high-quality human cumulus-oocyte complexes, were randomly allocated into two groups after patient informed consent: 1, fresh CCs (controls, n = 25); 2, vitrified CCs (n = 25). Samples were then prepared and observed by transmission electron microscopy. In fresh CCs, in which small cell clusters were visible, cell membranes were joined by focal gap junctions. Microvilli were rare and short. Nuclei, mitochondria, smooth endoplasmic reticulum (SER), Golgi apparatus and lipid droplets appeared well preserved; vacuoles were scarce. After vitrification, we observed two populations of CCs: light CCs, with a smooth appearance and few short microvilli; and dark CCs, with numerous and long microvilli. In both, most of the organelles appeared similar to those of fresh CCs. Lipid droplets were denser and more numerous, with respect to fresh CCs. They were mainly located in the peri-nuclear and sub-plasmalemmal regions. Numerous packed electron-negative vacuoles were visible. The vitrification procedure did not cause alterations in the fine structure of major organelles, except for an increased amount of lipid droplets and vacuoles. This specific sensitivity of human CCs to vitrification should be considered during ARTs.

Vitrification of human immature oocytes before and after in vitro maturation: a review.

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.

EGF-FSH supplementation reduces apoptosis of pig granulosa cells in co-culture with cumulus-oocyte complexes.

In cattle breeding, co-culture with granulosa cells (GCs) is one of the strategies to improve oocyte maturation and fertilization potential, but yields are still suboptimal due to GC apoptosis. We previously set up an in vitro co-culture system of cumulus-oocyte-complexes (COCs) anchored to GC multilayers adhering to the basal lamina (COCGs), in which GC apoptosis was inhibited by FSH supplementation. Here, we assessed the antiapoptotic effect of EGF (5 ng/ml-EGF5) alone or in synergism to FSH (50mU/ml-FSH50) on pig COCGs. COCG morphology, apoptotic rate, procaspase-8 and-9 expression levels and surface ultrastructure were determined. Results showed an increased % of apoptotic GCs in control and EGF5 (≈80%) respect to sampling (≈3%) and caspase-8 and -9 activation. In contrast, apoptotic cells were significantly reduced by FSH50 (≈35%) supplementation, with inactive Procaspase-8 and -9 highly expressed. The pro-survival effect of FSH was strengthened by EGF (EGF5+FSH50), as evidenced by a significant reduction of apoptosis (≈15%) and high expression levels of Procaspase-8 and -9. Ultrastructural analysis revealed that GC multilayers were characterized by round-to-ovoid cells connected each other and to the basal lamina by cytoplasmic projections. Microvilli shortening/thickening/reduction, cytoplasmic projection rarefaction, blebbing of apoptotic bodies and degenerating/atresic GCs were observed in control and EGF5 groups. FSH50 induced the formation of an abundant mucinous matrix, due to granulosa expansion. Blebs and atresic areas were rarely observed. In EGF5+FSH50 group, GCs were well-preserved, richly covered by microvilli and connected by numerous cytoplasmic projections. Degenerative phenomena were rarely observed. In conclusion, EGF in synergism with FSH seems to better counteract GC apoptosis in a co-culture of pig GC multilayers.

Ultrastructure of human oocytes after in vitro maturation.

STUDY HYPOTHESIS: How does the ultrastructure of human oocytes matured in vitro compare with oocytes collected from women after full hormonal stimulation? STUDY FINDING: The ultrastructure of human oocytes matured in vitro is largely, but not entirely, similar to those matured in vivo. WHAT IS KNOWN ALREADY: Embryos derived from in vitro-matured oocytes often have limited developmental potential, possibly as an effect of inappropriate in vitro maturation (IVM) conditions. Transmission electron microscopy (TEM) is a valuable research tool to compare in vivo and in vitro matured oocytes. However, previous studies on the ultrastructure of human IVM oocytes were done with inadequate material or inappropriate IVM conditions, and have limited significance. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Immature cumulus cell-enclosed oocytes, retrieved from mid-sized antral follicles of women requiring IVM treatment, were matured in vitro for 30 h. No leftover germinal vesicle-stage oocytes collected from fully stimulated cycles were used. Control in vivo matured oocytes were obtained from age-matched women undergoing full ovarian stimulation. In vitro and in vivo matured oocytes were analysed by TEM and compared according to previously established morphometric criteria of oocyte quality. MAIN RESULTS AND THE ROLE OF CHANCE: All oocytes had normal ooplasm showing uniform distribution of organelles. Mitochondrial morphology appeared similar between the maturation conditions. Cortical granules were found typically stratified in a single, mostly continuous row just beneath the ooplasm in all oocytes. Microvilli were well preserved after IVM. Vacuoles were only occasionally found in all oocytes and, if present, they were frequently associated with lysosomes. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and mitochondria-vesicles (MV) complexes were commonly found in in vivo matured oocytes. However, large MV complexes partially replaced M-SER aggregates in IVM oocytes. LIMITATIONS, REASONS FOR CAUTION: As a note of caution it should be noticed that, being laborious and technically demanding, TEM cannot be applied to a large number of samples in a single investigation. Therefore, our data require further independent confirmation. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggests the notion that TEM remains a valuable research tool that can also offer quantitative data if associated with morphometric criteria of evaluation. Therefore, it can be adopted to test pre-clinically the performance of novel in vitro systems that are demanded to make oocytes IVM more successful in the human. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was independently funded by Biogenesi Reproductive Medicine Centre, Monza, Italy. All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes.

PURPOSE: Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration. METHODS: Samples were studied by light and transmission electron microscopy. RESULTS: We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed. CONCLUSIONS: This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism.

Ultrastructure of immature and mature human oocytes after cryotop vitrification.

In vitro maturation of vitrified immature germinal vesicle (GV) oocytes is a promising fertility preservation option. We analyzed the ultrastructure of human GV oocytes after Cryotop vitrification (GVv) and compared it with fresh GV (GVc), fresh mature metaphase II (MIIc) and Cryotop-vitrified mature (MIIv) oocytes. By phase contrast microscopy and light microscopy, the oolemmal and cytoplasmic organization of fresh and vitrified oocytes did not show significant changes. GVv oocytes showed significant ultrastructural alterations of the microvilli in 40% of the samples; small vacuoles and occasional large/isolated vacuoles were abnormally present in the ooplasm periphery of 50% of samples. The ultrastructure of nuclei and mitochondria-vesicle (MV) complexes, as well as the distribution and characteristics of cortical granules (CGs), were comparable with those of GVc oocytes. MIIv oocytes showed an abnormal ultrastructure of microvilli in 30% of the samples and isolated large vacuoles in 70% of the samples. MV complexes were normal, but mitochondria-smooth endoplasmic reticulum aggregates appeared to be of reduced size. CGs were normally located under the oolemma but presented abnormalities in distribution and matrix electron density. In conclusion, Cryotop vitrification preserved main oocyte characteristics in the GV and MII stages, even if peculiar ultrastructural alterations appeared in both stages. This study also showed that the GV stage appears more suitable for vitrification than the MII stage, as indicated by the good ultrastructural preservation of important structures that are present only in immature oocytes, like the nucleus and migrating CGs.

Repeated ovarian stimulation does not affect the expression level of proteins involved in cell cycle control in mouse ovaries and fallopian tubes.

PURPOSE: To understand if repeated cycles (2-4 rounds) of gonadotropin stimulation could affect intracellular localization/content of proteins controlling cell cycle progression in mouse fallopian tubes (FT) and ovaries. METHODS: FT and ovaries of estrous mice (control) and of stimulated mice were analyzed to detect Oct-3/4, Sox-2, p53, ß-catenin, pAKT and cyclin D1 localization/content. Spindles and chromosome alignment were analyzed in ovulated oocytes. RESULTS: After round 4, FT and ovaries of control and stimulated groups showed no differences in Oct-3/4, Sox-2 and ß-catenin localization nor in Oct-3/4, Sox-2, p53, ß-catenin and pAKT contents. Cyclin D1 level increased significantly in FT of treated mice. Oocytes number decreased meanwhile frequency of abnormal meiotic spindles increased with treatments. CONCLUSIONS: Repetitive stimulations affected oocyte spindle morphology but did not induce changes in a set of proteins involved in cell cycle progression, usually altered in ovarian cancer. The significant increase of cyclin D1 in the FT requires further investigation.

Ultrastructural Analysis of Large Japanese Field Mouse (Apodemus speciosus) Testes Exposed to Low-Dose-Rate (LDR) Radiation after the Fukushima Nuclear Power Plant Accident.

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, great attention has been paid to the impact of chronic low-dose-rate (LDR) radiation exposure on biological systems. The reproductive system is sensitive to radiation, with implications connected to infertility. We investigated the testis ultrastructure of the wild large Japanese field mouse (Apodemus speciosus) from three areas contaminated after the FDNPP accident, with different levels of LDR radiation (0.29 µSv/h, 5.11 µSv/h, and 11.80 µSv/h). Results showed good preservation of the seminiferous tubules, comparable to the unexposed animals (controls), except for some ultrastructural modifications. Increases in the numerical density of lipid droplet clusters in spermatogenic cells were found at high levels of LDR radiation, indicating an antioxidant activity rising due to radiation recovery. In all groups, wide intercellular spaces were found between spermatogenic cells, and cytoplasmic vacuolization increased at intermediate and high levels and vacuolated mitochondria at the high-level. However, these findings were also related to the physiological dynamics of spermatogenesis. In conclusion, the testes of A. speciosus exposed to LDR radiation associated with the FDNPP accident showed a normal spermatogenesis, with some ultrastructural changes. These outcomes may add information on the reproductive potential of mammals chronically exposed to LDR radiation.

A New Strong-Acid Free Route to Produce Xanthan Gum-PANI Composite Scaffold Supporting Bioelectricity.

Conductive hybrid xanthan gum (XG)-polyaniline (PANI) biocomposites forming 3D structures able to mimic electrical biological functions are synthesized by a strong-acid free medium. In situ aniline oxidative chemical polymerizations are performed in XG water dispersions to produce stable XG-PANI pseudoplastic fluids. XG-PANI composites with 3D architectures are obtained by subsequent freeze-drying processes. The morphological investigation highlights the formation of porous structures; UV-vis and Raman spectroscopy characterizations assess the chemical structure of the produced composites. I-V measurements evidence electrical conductivity of the samples, while electrochemical analyses point out their capability to respond to electric stimuli with electron and ion exchanges in physiological-like environment. Trial tests on prostate cancer cells evaluate biocompatibility of the XG-PANI composite. Obtained results demonstrate that a strong acid-free route produces an electrically conductive and electrochemically active XG-PANI polymer composite. The investigation of charge transport and transfer, as well as of biocompatibility properties of composite materials produced in aqueous environments, brings new perspective for exploitation of such materials in biomedical applications. In particular, the developed strategy can be used to realize biomaterials working as scaffolds that require electrical stimulations for inducing cell growth and communication or for biosignals monitoring and analysis.

Ultrastructural Evaluation of Mouse Oocytes Exposed In Vitro to Different Concentrations of the Fungicide Mancozeb.

Mancozeb is a widely used fungicide, considered to be an endocrine disruptor. In vivo and in vitro studies evidenced its reproductive toxicity on mouse oocytes by altering spindle morphology, impairing oocyte maturation, fertilization, and embryo implantation. Mancozeb also induces dose-dependent toxicity on the ultrastructure of mouse granulosa cells, including chromatin condensation, membrane blebbing, and vacuolization. We evaluated the effects on the ultrastructure of mouse oocytes isolated from cumulus-oocyte complexes (COCs), exposed in vitro to increasing concentrations of mancozeb. COCs were matured in vitro with or without (control) low fungicide concentrations (0.001-1 µg/mL). All mature oocytes were collected and prepared for light and transmission electron microscopy. Results showed a preserved ultrastructure at the lowest doses (0.001-0.01 µg/mL), with evident clusters of round-to-ovoid mitochondria, visible electron-dense round cortical granules, and thin microvilli. Mancozeb concentration of 1 µg/mL affected organelle density concerning controls, with a reduction of mitochondria, appearing moderately vacuolated, cortical granules, and microvilli, short and less abundant. In summary, ultrastructural data revealed changes mainly at the highest concentration of mancozeb on mouse oocytes. This could be responsible for the previously described impaired capability in oocyte maturation, fertilization, and embryo implantation, demonstrating its impact on the reproductive health and fertility.