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Simple Summary: In 2011, the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident resulted in the release of vast quantities of radioactive substances. In this study, we performed an ultrastructural analysis of the testis of the wild large Japanese field mouse (Apodemus speciosus), inhabiting the area near the ex-evacuation zones of the FDNPP and chronically exposed to various levels of low-dose-rate (LDR) radiation, to evaluate the effects of ionizing radiation on the male reproductive system and the possible consequences for future generations. Our results showed a preserved morphology of seminiferous tubules. However, ultrastructural changes, such as wide intercellular spaces, cytoplasmic vacuolization, vacuolated mitochondria, and increased lipid droplet clusters, were found, which could be linked to the mechanisms related to spermatogenesis. Long-term chronic LDR radiation exposure associated with the FDNPP accident had no pronounced adverse effect on spermatogenesis in A. speciosus, even if testis presented some ultrastructural changes. Our findings could be used in future studies to evaluate the effects of radiation on reproductive health in wild animals. 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. [ABSTRACT FROM AUTHOR]

Highlights: What are the main findings? For the first time, the in vitro effects of simulated microgravity using a Random Positioning Machine on the ultrastructural features of human metaphase II oocytes were investigated by transmission electron microscopy. Microgravity might compromise oocyte quality affecting the ultrastructural morphology of mitochondria, endoplasmic reticulum, and cortical granules due to a possible alteration of the cytoskeleton. What is the implication of the main finding? Microgravity-induced organelle modifications potentially affect the oocyte's ability to fully mature and fertilize and develop into a viable embryo. The Gravity Force to which living beings are subjected on Earth rules the functionality of most biological processes in many tissues. It has been reported that a situation of Microgravity (such as that occurring in space) causes negative effects on living beings. Astronauts returning from space shuttle missions or from the International Space Station have been diagnosed with various health problems, such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance, including impaired visual acuity, altered metabolic and nutritional status, and immune system dysregulation. Microgravity has profound effects also on reproductive functions. Female astronauts, in fact, suppress their cycles during space travels, and effects at the cellular level in the early embryo development and on female gamete maturation have also been observed. The opportunities to use space flights to study the effects of gravity variations are limited because of the high costs and lack of repeatability of the experiments. For these reasons, the use of microgravity simulators for studying, at the cellular level, the effects, such as those, obtained during/after a spatial trip, are developed to confirm that these models can be used in the study of body responses under conditions different from those found in a unitary Gravity environment (1 g). In view of this, this study aimed to investigate in vitro the effects of simulated microgravity on the ultrastructural features of human metaphase II oocytes using a Random Positioning Machine (RPM). We demonstrated for the first time, by Transmission Electron Microscopy analysis, that microgravity might compromise oocyte quality by affecting not only the localization of mitochondria and cortical granules due to a possible alteration of the cytoskeleton but also the function of mitochondria and endoplasmic reticulum since in RPM oocytes we observed a switch in the morphology of smooth endoplasmic reticulum (SER) and associated mitochondria from mitochondria-SER aggregates to mitochondria–vesicle complexes. We concluded that microgravity might negatively affect oocyte quality by interfering in vitro with the normal sequence of morphodynamic events essential for acquiring and maintaining a proper competence to fertilization in human oocytes. [ABSTRACT FROM AUTHOR]

Simple Summary: Exposure to endocrine-disrupting pollutants, such as the fungicide mancozeb, is linked to various environmental health hazards, including female fertility. Although the dithiocarbamate mancozeb has low reported toxicity in mammals, it impairs female reproductive functions in exposed animals and humans. The specific mechanism of action of mancozeb and the damage to cell structures in the female reproductive system are still unclear. This study aims to describe the ultrastructure of mouse oocytes exposed in vitro to increasing concentrations of mancozeb (0.001–1 μg/mL) by light and transmission electron microscopy and to perform a morphometric analysis over significant organelles. While from 0.001 to 0.1 μg/mL, oocyte ultrastructure was comparable to controls, at the highest concentration (1 µg/mL), a decrease in the numerical density of mitochondria and cortical granules, an altered organelle distribution, and flattening of microvilli were observed. These results could be responsible for the adverse effect of this fungicide on mammalian reproductive performance. 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. [ABSTRACT FROM AUTHOR]

(1) Background: Polycystic ovarian syndrome (PCOS) is a common and multifactorial disease affecting reproductive-age women. Although PCOS ovarian and metabolic features have received extensive research, uterine dysfunction has been poorly investigated. This research aims to investigate morphological and molecular alterations in the PCOS uterus and search for modulating effects of different carnitine formulations. (2) Methods: CD1 mice were administered or not with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) for 20 days, alone or with 0.40 mg L-carnitine (LC) and 0.20 mg acetyl-L-carnitine (ALC) in the presence or absence of 0.08 mg propionyl-L-carnitine (PLC). Uterine horns from the four groups were subjected to histology, immunohistochemistry and immunoblotting analyses to evaluate their morphology, collagen deposition, autophagy and steroidogenesis. Oxidative-/methylglyoxal (MG)-dependent damage was investigated along with the effects on the mitochondria, SIRT1, SOD2, RAGE and GLO1 proteins. (3) Results: The PCOS uterus suffers from tissue and oxidative alterations associated with MG-AGE accumulation. LC-ALC administration alleviated PCOS uterine tissue alterations and molecular damage. The presence of PLC prevented fibrosis and maintained mitochondria content. (4) Conclusions: The present results provide evidence for oxidative and glycative damage as the main factors contributing to PCOS uterine alterations and include the uterus in the spectrum of action of carnitines on the PCOS phenotype. [ABSTRACT FROM AUTHOR]

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After its discovery in 1825 by the physiologist J.E. Purkinje, the human germinal vesicle (GV) attracted the interest of scientists. Discarded after laparotomy or laparoscopic ovum pick up from the pool of retrieved mature oocytes, the leftover GV was mainly used for research purposes. After the discovery of Assisted Reproductive Technologies (ARTs) such as in vitro maturation (IVM), in vitro fertilization and embryo transfer (IVF-ET) and intracytoplasmic sperm injection (ICSI), its developing potential was explored, and recognized as an important source of germ cells, especially in the case of scarce availability of mature oocytes for pathological/clinical conditions or in the case of previous recurrent implantation failure. We here review the ultrastructural data available on GV-stage human oocytes and their application to ARTs. [ABSTRACT FROM AUTHOR]

Radiofrequency electromagnetic radiation emitted from cell phone has harmful effects on some organs of the body, such as the brain, heart, and testes. This study aimed to assess the effects of cell phones on sperm parameters, DNA fragmentation, and apoptosis in normozoospermic. Normal sperm samples were divided into two groups of control and case. The samples from the case were placed for 60 min at a distance of approximately 2.5 cm from the cell phone set in the active antenna position. Control samples were exposed to cell phones without active antennas. All specimens were analysed by World Health Organization criteria. Sperm viability, sperm with chromatin abnormality and maturity, DNA fragmentation, and apoptosis were examined. Viability and motility in the case were significantly lower than the control (p <.001, p =.004 respectively). The percentage of apoptotic sperms and DNA fragmentation were significantly higher in the case when compared with the control (p =.031, p <.001 respectively). The other parameters studied such as morphology, chromatin abnormality, and maturity showed no significant difference between the case and control groups. Cell phone waves had a detrimental effect on human sperm's biological features. Therefore, it is recommended to keep the cell phone away from the pelvis as much as possible. [ABSTRACT FROM AUTHOR]

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex‐evacuation zone. Wildlife within the alert zone have been exposed to low‐dose‐rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex‐evacuation zone of the FDNPP accident. The radioactivity of caesium‐137 (137Cs) was measured by gamma‐ray spectrometry, and the measured radioactivity concentration was 300–6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze‐thawing sperm penetration ability was confirmed using the interspecific zona pellucida‐free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons. [ABSTRACT FROM AUTHOR]

Background/aim: The aim of this study was to evaluate the efficiency of in vitro embryo splitting (IES) procedures. We also assessed the quality of the blastocysts developed from embryos obtained from different sources. Materials and methods: Good quality embryos at 6--8-cell stages were categorized according to their fertilization sources: 1) frozenwarmed donated embryos, 2) chromosomally abnormal embryos, 3) parthenogenetic embryos, and 4) embryos derived from fertilization of in vitro matured oocytes (rescue IVM). After IES, splitting and developmental efficiency was assessed. Furthermore, the quality of the developed blastocysts was evaluated by Hoechst and propidium iodide (PI) staining. Results: The data showed a high rate of both splitting and developmental efficiency in the frozen-warmed embryos after IES (140% and 71.7%, respectively), followed by chromosomally abnormal embryos (96.8% and 52.5%, respectively). Results of the Hoechst and PI staining showed that the mean ± SD cell numbers of the control group were higher (113.11 ± 16.01) than that of twins A (donor blastomeres embryos, 58 ± 12.2) and B (recipient blastomeres embryos, 50.4 ± 8.5), respectively. Conclusion: Chromosomally normal embryos enrolled in IES are more potent to develop into viable blastocysts. For research purposes, 1PN and 3PN embryos are the best options for splitting procedures, regardless of the poor quality of developed blastocysts. [ABSTRACT FROM AUTHOR]

Over the last forty years, many trials have been performed using mammalian embryo cultures with reduced oxygen tension (O2) to encourage proper embryo development and increase the success rate for in vitro fertilization (IVF) outcome. Even if the use of atmospheric O2 (20%) affects invitro embryo development and intracellular redox balance, the use of low (5% O2, physiologic) and ultra-low (close or less to 5% O2) O2 applied to invitro embryo culture is still under debate. Numerous studies in various mammalian species have shown that embryo development improves when culturing embryos under low O2, although culture conditions are not the only factors involved in the success of IVF. This article reviews the literature data of the last four decades and discusses the current evidence on the use of low and ultra-low O2 in embryo culture, and examines the impact of multiple factors on IVF outcomes. O2: oxygen tension; IVF: invitro fertilization; IVC: invitro culture; ET: embryo transfer; ROS: reactive oxygen species; ARTs: assisted reproductive technologies. [ABSTRACT FROM AUTHOR]

Aim: Scientists have tried to culture and transplant the ovarian tissues (OT), to preserve fertility in cancer patients. However, one of the main limitations to the applicability of this technique is the folliculogenesis disruption after transplantation. Due to the roles exerted by growth differentiation factor‐9β (GDF9β), we decided to determine the most effective dose of GDF9β on promotion of folliculogenesis and angiogenesis in sheep OT grafted onto the chick chorioallantoic membrane (CAM). Methods: Fresh sheep OT were grafted onto the CAM for 5 days, and divided into four groups based on the addition of increasing doses of GDF9β (0, 150, 200 and 250 ng/mL). Following culture, histological (hematoxylin and eosin [H&E] staining) and immunohistological studies (Ki‐67) were done. Fibrotic and necrotic regions were measured using MICROVISIBLE software. For comparing the follicle development rates between the groups as well as differences in the Ki‐67‐positive follicles, analysis of variance was applied. Results: In both 200 and 250 ng/mL GDF9β groups, significantly higher rates of intermediary and primary follicles were observed, also the numbers of good quality follicles increased in the aforementioned groups and the rates of fibrotic and necrotic areas decreased. Moreover, in the 200 and 250 ng/mL GDF9β groups, the number of capillaries and the proliferative activity increased. The lower dose of GDF9β (150 ng/mL) neither activated the primordial follicles nor lead to an increase in the number of growing follicles. Conclusion: Addition of high dosages of GDF9β to the OT, grafted onto the CAM resulted in higher folliculogenesis and better transplantation features due to improvement in angiogenesis. [ABSTRACT FROM AUTHOR]

Advanced glycation end-products (AGEs) are involved in the pathogenesis and consequences of polycystic ovary syndrome (PCOS), a complex metabolic disorder associated with female infertility. The most powerful AGE precursor is methylglyoxal (MG), a byproduct of glycolysis, that is detoxified by the glyoxalase system. By using a PCOS mouse model induced by administration of dehydroepiandrosterone (DHEA), we investigated whether MG-dependent glycative stress contributes to ovarian PCOS phenotype and explored changes in the Sirtuin 1 (SIRT1) functional network regulating mitochondrial functions and cell survival. In addition to anovulation and reduced oocyte quality, DHEAovaries revealed altered collagen deposition, increased vascularization, lipid droplets accumulation and altered steroidogenesis. Here we observed increased intraovarian MG-AGE levels in association with enhanced expression of receptor for AGEs (RAGEs) and deregulation of the glyoxalase system, hallmarks of glycative stress. Moreover, DHEA mice exhibited enhanced ovarian expression of SIRT1 along with increased protein levels of SIRT3 and superoxide dismutase 2 (SOD2), and decreased peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC1α), mitochondrial transcriptional factor A (mtTFA) and translocase of outer mitochondrial membrane 20 (TOMM20). Finally, the presence of autophagy protein markers and increased AMP-activated protein kinase (AMPK) suggested the involvement of SIRT1/AMPK axis in autophagy activation. Overall, present findings demonstrate that MG-dependent glycative stress is involved in ovarian dysfunctions associated to PCOS and support the hypothesis of a SIRT1-dependent adaptive response. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Currently, rescue in vitro maturation (IVM) is not a routine method in assisted reproductive treatment (ART) programmes but is a promising procedure for ART to improve IVM. The aim of this study was to compare embryo morphokinetics of germinal vesicles (GV) with metaphase II (MII) oocytes from controlled ovarian hyperstimulation (COH) cycles by time-lapse photography monitoring (TLM). Morphokinetics of the same number of embryos derived from the in vivo (group I) and rescue of in vitro matured oocytes (group II) from 310 patients were analyzed and compared retrospectively. The time to form second PB extrusion (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf) and time of two to eight discrete cells (t2–t8) were assessed. Abnormal cleavage patterns such as uneven blastomeres at the two-cell stage, cell fusion (Fu), trichotomous mitoses (TM), and the rates of embryo arrest were assessed. These data showed that tPB2, tPNa, tPNf, t2, t3 and t4 stages took place later in group II compared with group I (P <0.001, P =0.017, P <0.001, P <0.001, P <0.001, P <0.001, respectively). The rates of uneven blastomeres, Fu, TM, and embryo arrest were increased significantly in group II compared with group I (P =0.001, P <0.001, P =0.003, P <0.001, respectively). Based on the exact annotation of timing parameters and cleavage patterns, the present data agreed with the concept that rescue IVM of oocytes negatively influences embryo morphokinetics. Therefore, cautious use of embryos derived from rescue IVM of GV oocytes should be made. [ABSTRACT FROM AUTHOR]