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.

Ultrastructure of human ovarian tissues and risk of cancer cells re-implantation after transplantation to chick embryo chorioallantois membrane (CAM) following vitrification or slow freezing.

Ovarian follicle depletion and premature ovarian failure are significant challenges in cancer patients subjected to radio- or chemotherapy. Ovarian tissue (OT) cryopreservation would be an option when other fertility preservation methods are not accessible. This study aimed to analyze the structure and ultrastructure of human OTs transplanted onto chick embryo chorioallantois membrane (CAM) after cryopreservation by vitrification or slow freezing. OTs from 10 cancer patients underwent cryopreservation. CAM transplantation was done on fresh and cryopreserved OTs, to assign samples to nine study groups as follows: 1) FI-FIII = fresh, 5- and 10-days post-CAM transplantation groups; 2) VI-VIII = vitrified, 5- and 10-days post-transplantation vitrified groups; 3) SFI-SFIII: slow frozen, 5- and 10-days post-transplantation slow freezing groups. Proliferation ability, folliculogenesis, and structural and ultrastructure were analyzed. The density of primordial follicles did not change after both freezing methods, but reduced after 5 (P ≥ 0.05) and 10 days (P ≤ 0.05) post-CAM transplantation. The follicular grade significantly decreased in all transplanted tissues (P ≤ 0.0). The proliferation marker increased after cryopreservation, but reduced after transplantation (P ≤ 0.05). TEM evaluation showed better follicular ultrastructure in the fresh group, after transplantation. Stromal ultrastructure appeared more preserved after vitrification compared with slow freezing. There was no sign of malignant cell contamination after transplantation. Some follicular TEM abnormalities were found in both methods of freezing, with a better transplantation rate after vitrification. Also, enhanced follicular activation resulted in faster follicular depletion in this method. The information regarding post grafting events would improve our knowledge for longer OTs' lifespans.

The detrimental effect of cell phone radiation on sperm biological characteristics in normozoospermic.

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.

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.

Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor).

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 (137 Cs) 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.

Efficacy of the in vitro splitting of human preimplantation embryos from ART programs

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 6­8-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. Furthermre, 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.

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.

Effective dosage of growth differentiation factor-9ß in folliculogenesis and angiogenesis in the sheep ovarian tissues grafted onto chick embryo chorioallantoic membrane.

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-9ß (GDF9ß), we decided to determine the most effective dose of GDF9ß 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 GDF9ß (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 GDF9ß 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 GDF9ß groups, the number of capillaries and the proliferative activity increased. The lower dose of GDF9ß (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 GDF9ß to the OT, grafted onto the CAM resulted in higher folliculogenesis and better transplantation features due to improvement in angiogenesis.

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.

Increased rounds of gonadotropin stimulation have side effects on mouse fallopian tubes and oocytes.

In this study, it was evaluated if increased rounds of gonadotropin stimulation could affect in mice: (i) expression levels of proteins regulating cell cycle and DNA repair in fallopian tubes and (ii) meiotic spindle morphology of ovulated oocytes. To this end, adult female mice were subjected or not (Control) to 6 or 8 rounds of gonadotropin stimulation. Ovulated oocytes were incubated with anti A/B tubulin to evaluate spindle morphology. Fallopian tubes were analyzed to detect Cyclin D1, phospho-p53/p53, phospho-AKT/AKT, phospho-GSK3B/GSK3B, SOX2, OCT3/4, phospho-B-catenin/B-catenin, phospho-CHK1 and phospho-H2A.X protein levels. After 6 rounds, Cyclin D1, p53 and phospho-p53 contents were higher than Control. After 8 rounds, the contents of phosphorylated AKT, GSK3B and p53 as well as of total p53, Cyclin D1 and OCT3/4 significantly increased in comparison with Control. Conversely, SOX2 and B-catenin were similarly expressed among all experimental groups. The finding that phospho-CHK1 and phospho-H2A.X protein levels were undetectable supported the absence of extensive DNA damage. Oocytes number and percentage of normal meiotic spindles drastically decreased from 6 rounds onward. Altogether, our results demonstrated that 6 and 8 cycles of gonadotropin stimulation reduce mouse reproductive performances by inducing over-expression and over-activation of proteins controlling cell cycle progression in fallopian tubes and by impairing oocyte spindle.

Effect of pre-in vitro maturation with cAMP modulators on the acquisition of oocyte developmental competence in cattle.

The administration of follicle-stimulating hormone (FSH) prior to oocyte retrieval improves oocyte developmental competence. During bovine embryo production in vitro, however, oocytes are typically derived from FSH-unprimed animals. In the current study, we examined the effect of pre-in vitro maturation (IVM) with cAMP modulators, also known as the second messengers of FSH, on the developmental competence of oocytes derived from small antral follicles (2-4 mm) of FSH-unprimed animals. Pre-IVM with N6,2'-O-dibutyryladenosine 3',5'-cyclicmonophosphate (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) for 2 h improved the blastocyst formation in oocytes stimulated by FSH or amphiregulin (AREG). Furthermore, pre-IVM enhanced the expression of the FSH- or AREG-stimulated extracellular matrix-related genes HAS2, TNFAIP6, and PTGS2, and epidermal growth factor (EGF)-like peptide-related genes AREG and EREG. Additionally, pre-IVM with dbcAMP and IBMX enhanced the expression of EGFR, and also increased and prolonged cumulus cell-oocyte gap junctional communication. The improved oocyte development observed using the pre-IVM protocol was ablated by an EGF receptor phosphorylation inhibitor. These results indicate that pre-IVM with cAMP modulators could contribute to the acquisition of developmental competence by bovine oocytes from small antral follicles through the modulation of EGF receptor signaling and oocyte-cumulus/cumulus-cumulus gap junctional communication.

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.

Does rescue in vitro maturation of germinal vesicle stage oocytes impair embryo morphokinetics development?

SummaryCurrently, 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.

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.