Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development

Morphological behavior of human periodontal ligament fibroblasts towards the exposition to dentinal derivates biomaterial.

The histological composition of the dentinal tissue lead researchers to explore its use as autologous grafting materials for regeneration interventions in oral and maxillofacial surgeries. Due to the novelty of the use of this material, few reported cases and few in vitro stud- ies are available in literature. The aim of the study was to evaluate the morphological behaviour of human periodontal ligament fibro- blasts (HPLF) towards different types of dentine derivates grafting material. The study design included the evaluation of mineralized dentine (SG), of deproteinized and demineralized dentine (DDP) and demineralized dentine (TT) as test materials, and the evaluation of deproteinized bovine bone (BIOS) as positive control material in contact with the HPLF cell line after 24 h, 72 h and 7 days of in vitro cul- ture. The evaluated outcomes were the morphological characteristics such as cellular shape and surface by light microscopy (LM) and scanning electron microscopy (SEM), and the adhesion using confocal microscopy (CLSM). The LM observations showed the pres- ence of densely packed cells, whilst the SEM observations showed how fibroblasts exposed to DDP and TT presented cytoplasmatic ex- tensions, while SG and BIOS presented, in addition to digitations and cytoplasmatic extensions, the thickening of the cellular mem- brane. The CLMS observations showed the expression of cyto- skeletal elements (vinculin, actin and integrin) involved in the adhesion process. Overall, the experimental materials induced a positive response of the HPLFs in terms of proliferations and adhesion. The knowledge of the effects of the degree of mineralization of the dentine on the cellular behavior will help clinician in the choice of the type of dentine derivates material according to the required clinical situation

Pre-Implantation Mouse Embryos Cultured In Vitro under Different Oxygen Concentrations Show Altered Ultrastructures.

Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O2) concentrations to culture human embryos. Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2

Association between female reproductive health and mancozeb: systematic review of experimental models

Abstract: Mancozeb is a widely used fungicide approved for use in agriculture in many countries with long persistence in the environment and consequent bioaccumulation in tissues and biological fluids. Despite the large amount of studies published in recent years, the relationship between mancozeb exposure and female reproductive health is not fully elucidated. In order to summarize current evidence on mancozeb exposure and female reproductive disease, we performed a systematic review of literature. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to make this review. An adapted version of the National Toxicology Program’s Office of Health and Assessment and Translation (OHAT) framework was used to evaluate the risk of bias. Electronic search on two databases (PubMed and Scopus) was used to find experimental studies (in vitro and in vivo) on mancozeb exposure. The database search identified 250 scientific articles, 20 of which met our inclusion criteria. Selected data were then reviewed and summarized in tables. Overall, mancozeb represents a hazard for female reproductive health, with different mechanisms of action. Undoubtedly more experimental and epidemiological studies are required to definitively validate mancozeb as reproductive toxicant

Ultrastructure of isolated mouse ovarian follicles cultured in vitro

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth <it>in vitro </it>have been developed and many studies exist on factors influencing the development of <it>in vitro </it>grown oocytes. However, a very few reports concern the ultrastructural morphology of <it>in vitro </it>grown follicles.</p> <p>Methods</p> <p>The present study was designed to evaluate, by transmission and scanning electron microscopy, the ultrastructural features of isolated mouse preantral follicles cultured <it>in vitro </it>for 6 days in a standard medium containing fetal calf serum (FCS). The culture was supplemented or not with FSH.</p> <p>Results</p> <p>The follicles cultured in FCS alone, without FSH supplementation (FCS follicles), did not form the antral cavity. They displayed low differentiation (juxta-nuclear aggregates of organelles in the ooplasm, a variable amount of microvilli on the oolemma, numerous granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Eighty (80)% of FSH-treated follicles formed the antral cavity (FSH antral follicles). These follicles showed various ultrastructural markers of maturity (spreading of organelles in ooplasm, abundant microvilli on the oolemma, scarce granulosa cell-oolemma contacts, granulosa cell proliferation). Areas of detachment of the innermost granulosa cell layer from the oocyte were also found, along with a diffuse granulosa cell loosening compatible with the antral formation. Theca cells showed an immature morphology for the stage reached. Twenty (20)% of FSH-treated follicles did not develop the antral cavity (FSH non-antral follicles) and displayed morphological differentiation features intermediate between those shown by FCS and FSH antral follicles (spreading of organelles in the ooplasm, variable amount of microvilli, scattered granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment).</p> <p>Conclusions</p> <p>It is concluded that FSH supports the <it>in vitro </it>growth of follicles, but the presence of a diffuse structural granulosa cell-oocyte uncoupling and the absence of theca development unveil the incomplete efficiency of the system. The present study contributes to explain, from a morphological point of view, the effects of culture conditions on the development of mouse <it>in vitro </it>grown follicles and to highlight the necessity of maintaining efficient intercellular communications to obtain large numbers of fully-grown mature germ cells.</p

Streptococcus spp. and Fusobacterium nucleatum in tongue dorsum biofilm from halitosis patients: a fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) study

The present study involved a qualitative and quantitative evaluation of tongue dorsum biofilms sampled from halitosis patients and healthy volunteers. The aim of the study was to quantify the distribution of Streptococcus spp. and Fusobacterium nucleatum within the oral halitosis biofilm in order to highlight the role of these bacterial members in halitosis. Tongue plaque samples from four halitosis-diagnosed patients and four healthy volunteers were analyzed and compared. The visualization and quantification of the tongue dorsum biofilm was performed combining fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Eubacteria, Streptococcus spp. and Fusobacterium nucleatum were stained using specific fluorescent probes. For a comparison of the two tested biofilm groups the Wilcoxon rank-sum test was used. Morphological analysis by CLSM illustrated the distribution of the species which were tracked. Streptococcus spp. appeared to be enclosed within the samples and always associated to F. nucleatum. Furthermore, compared to the control group the biofilm within the halitosis group contained significantly higher proportions of F. nucleatum and Streptococcus spp., as revealed by the FISH and CLSM-analysis. The total microbial load and relative proportions of F. nucleatum and Streptococcus spp. can be considered as causative factors of halitosis and thus, as potential treatment targets