Maturation Medium Research Articles

TGF-β-Mediated Modulation of Cell-Cell Interactions in Postconfluent Maturing Corneal Endothelial Cells.

PurposeTransforming growth factor-beta (TGF-β) is known to influence many cell functions. In the corneal endothelium, TGF-β1 exerts contextual effects, promoting endothelial–mesenchymal transition in proliferating cells and enhancing barrier integrity in early confluent maturing cells. Herein, we studied how TGF-β isoforms participate in the formation of corneal endothelial intercellular junctions.MethodsCorneal endothelial cells (CECs) were cultured using a two-phase media approach. When CECs reached confluence, the proliferation medium was replaced with maturation medium, which was supplemented or not with TGF-β isoforms. The cell morphology (circularity index), intercellular junction protein expression, trans-endothelial electrical resistance (TEER), and permeability of 7-day postconfluent CECs were assessed. Gene transcription and signaling pathways that were activated following maturation in the presence of TGF-β2 were also studied. The beneficial effect of TGF-β2 on CEC maturation was evaluated using ex vivo corneas mounted on a corneal bioreactor.ResultsThe results showed increases in circularity index, membrane localization of junction-related proteins, and TEER when TGF-β isoforms were individually added during the maturation phase, and TGF-β2 was the most effective isoform. Gene profiling revealed an increase in extracellular matrix-related gene expression. In ex vivo cell adhesion experiments, CECs that were matured in the presence of TGF-β2 had a higher circularity index and cell density and exhibited cell membrane-localized junction-related protein expression at earlier time points.ConclusionsThese results suggest that TGF-β2 can strengthen cell–cell and cell–substrate adhesion, which accelerates barrier integrity establishment and thus enhances CEC functionality.

Quercetin protects mouse oocytes against chromium-induced damage in vitro and in vivo

BackgroundChromium (Cr) is a naturally-occurring element that is used in various fields of industry. Humans may be exposed to hexavalent chromium [Cr(VI)], which is one of the stable valence states of the chromium through contaminated soil, air, and water. Exposure to Cr(VI) through contaminated drinking water, soil and air causes various cancers and also fertility problems in animals and humans. Quercetin (QCT), a common flavonoid compound, has numerous biological effects as an antioxidant and free radical scavenger, but its function and mechanisms in reproductive processes in various species remain unclear. This study aims to determine the chromium effects on mice oocyte quality and the ameliorative effect of QCT in both in vitro and in vivo experimental models. MethodsFor the in vitro experiment, oocytes were collected and divided into the control, sham, QCT-treated, Cr(VI) (potassium dichromate), and treatment [Cr(VI)+QCT] groups. Collected oocytes were cultured in maturation medium with or without 10 µM quercetin and 10 µM Cr(VI) for 14 h based on the defined experimental design. For the in vivo experiment, the mice were randomly divided into the control, sham, QCT-treated, Cr(VI), and Cr(VI) + QCT groups. Control and sham mice received regular drinking water and diet. Cr(VI) group received Cr(VI) (50 ppm in drinking water) and Cr(VI) + QCT group received 50 ppm Cr(VI) with QCT (20 mg/kg body wt, through i.p) for a period of 21 days and then oocytes were collected and cultured for 14 h for in vitro maturation. For both experiments, at the end of the culture period, we examined the ameliorative effect of QCT on oocyte maturation, spindle formation, ROS production, mitochondrial function, and apoptosis. ResultsOur in vitro and in vivo results showed that Cr(VI) disrupt the oocyte maturation and spindle formation (P < 0.001). Furthermore, we found that exposure to Cr(VI) significantly increased ROS levels and decreased mitochondrial membrane potential (P < 0.001). In addition, exposure to Cr(VI) induced early apoptosis and downregulated the Bcl-2 mRNA expression and upregulated the Caspase-3 and Bax mRNAs expression (P < 0.01). Finally, quercetin significantly restored the detrimental effects of Cr(VI). ConclusionThe results indicated that quercetin protects the oocytes against Cr(VI) toxicity through the suppression of oxidative stress and apoptosis. The conclusions drawn from our study's findings suggest that quercetin might be useful agent for oocyte maturation in case of possible exposure to toxic substances such as chromium.

THE EFFECTS OF L-CARNITINE AND CONDITIONED MEDIUM OF MESENCHYMAL STEM CELL SUPPLEMENTATION ON MATURATION, FERTILIZATION, AND EMBRYO QUALITY OF OOCYTES OBTAINED FROM ENDOMETRIOSIS MICE

To investigate the comparative effects of L-carnitine and conditioned medium of mesenchymal stem cell (BMSCCM) inclusion into in vitro maturation (IVM) medium on maturation, fertilization, and developmental competence of oocytes derived from endometriosis (EMS) mice models.

VL Mandua 380: A medium maturing, high yielding and blast tolerant finger millet cultivar for rainfed organic agro-ecology of hills

The medium duration blast tolerant finger millet variety VL Mandua 380 was developed at ICAR-Vivekananda Parvatiya Krishi Anusandhan Sansthan, Almora from the cross between GEC-440 (blast resistant core collection germplasm line) and VL 149 ((blast resistant cultivars adapted to hills) followed by a pedigree method of selection in the segregating generations. Under rainfed organic conditions, it has recorded an average grain yield of 1,877 kg/ha, which is 34.1 and 46.9 per cent higher yield over the check varieties VL Mandua 324 (1,400 kg/ha) and PRM-1 (1278 kg/ha), respectively in SVT trial conducted over 3 years from 2014-16. It is a medium duration variety with a duration of 113-118 days. The grains are nutritious with a higher concentration of calcium (322.5 mg/100 g) and zinc (4.3 mg/100 g) in comparison to the check variety VL Mandua 324 (294.0 mg /100 g and 2.8 mg/100 g, respectively). It exhibited resistant reaction to leaf blast and moderate reaction to finger and neck blast in the multilocation trials conducted over nine locations. Based on its superior performance for grain yield, grain quality and blast tolerance VL Mandua 380 was released by State Varietal Release Committee, Uttarakhand on 5th, February 2018. Subsequently, it was notified by the Central Sub-Committee on Crops Standards, Notification and Release of Varieties for Agricultural Crops vide notification number S.O.1498 (E) dated April 1, 2019. The specific area of adaptation of this variety is the rainfed organic condition of Uttarakhand hills. Keywords: Finger millet, VL Mandua 380, medium maturity, high calcium content, blast tolerance

Effect of Supplementation of Cryoprotectant Solution with Hydroxypropyl Cellulose for Vitrification of Bovine Oocytes.

Simple SummaryThis study investigated the effects of the addition of hydroxypropyl cellulose (HPC) to a vitrification solution of bovine oocytes. For the vitrification, bovine metaphase II oocytes were pretreated with a solution containing 10% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 5 min, then exposed to a solution containing 30% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 30 sec, and then directly plunged into liquid nitrogen. The survival rate of the oocytes was significantly higher in the 50 HPC group than in the 0, 10, and 100 HPC groups. The reactive oxygen species (ROS) levels of the non-vitrified-thawed (VT) and the 50 HPC groups were significantly lower than the 0, 10, and 100 HPC groups. The mRNA expression levels of the genes related to proapoptotic (Bax) was higher in the 0 and 100 HPC groups than in the 50 and non-VT groups. The mRNA levels of antiapoptotic genes (BCl2) were higher in the non-VT than in the other groups. The development rates of the embryos (day 8) obtained via parthenogenetic activation (PA) were determined in the non-VT, 0 HPC, and 50 HPC groups. The development rate was significantly higher in the non-VT and 50 HPC groups than in the 0 HPC group. Conclusion: Supplementation of vitrification solution with HPC improves the survival of VT bovine oocytes and the development capacity of embryos derived from these oocytes via PA.The technology of successful cryopreservation is a very important factor in research and commercial applications. However, the survival and development of the vitrified-thawed (VT) oocytes are lower than those of non-vitrified-thawed (non-VT) oocytes. This study investigated the effect of the addition of hydroxypropyl cellulose (HPC) to a vitrification solution of bovine oocytes. For the vitrification, bovine metaphase II oocytes were pretreated with a solution containing 10% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 5 min, then exposed to a solution containing 30% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 30 sec, and then directly plunged into liquid nitrogen. Oocytes exposed to 0, 10, 50, and 100 µg/mL HPC were named the 0, 10, 50, and 100 HPC groups, respectively. Samples were thawed via sequential incubation in Dulbecco’s phosphate-buffered saline (D-BPS) supplemented with 10% fetal bovine serum and decreasing concentrations of sucrose (1, 0.5, 0.25, and 0.125 M) for 1 min each time. After thawing, VT oocytes were treated at 0.05% hyaluronidase, and cumulus cells were removed by mechanical pipetting. The oocytes were washed with HEPES-buffered Tyrode’s medium and incubated in a droplet of previously cultured in vitro maturation medium for 1 h to recover. The survival rate of the oocytes was significantly higher in the 50 HPC group (84.2%) than in the 0 (75.4%), 10 (80.4%), and 100 (75.5%) HPC groups. The reactive oxygen species (ROS) levels of the non-VT and 50 HPC groups were lower than the 0, 10, and 100 HPC groups. The mRNA levels of proapoptotic genes (Bax) were lower in the non-VT, 0, and 50 HPC groups than in the other groups. The mRNA expression levels of antiapoptotic genes (BCl2) was higher in the non-VT than in the other groups. The mRNA level of a stress-related gene (Hsp70) was lower in the 50 HPC than in the other groups. At day 8, the developmental capacity of embryos obtained via parthenogenetic activation (PA) was determined in the non-VT, 0 HPC, and 50 HPC groups. The cleavage rate of the non-VT group was significantly higher, but the blastocyst development rate and total cell number per blastocyst did not significantly differ between the non-VT and 50 HPC groups. The mRNA levels of proapoptotic genes (Bax and Caspase-3) and a stress-related gene (Hsp70) were higher in the 0 HPC group than in the non-VT and 50 HPC groups. In conclusion, supplementation of vitrification solution with HPC improves the survival rate of VT bovine oocytes and the development capacity of embryos derived from these oocytes via PA.

Neuroregenerative Potential of Stem-Cells-from-Apical-Papilla–Derived Neuronal Cell Spheroids Regulated by Stem Cells from Apical Papillae Under Various Microenvironments in a Pulp-On-Chip System

IntroductionStem cells from apical papillae (SCAPs) display potent neuroregenerative potential by secreting neurotrophic factors and differentiating into neuronal-like cells, which are regulated by local niches. This study aimed to explore the interactions between SCAP-derived neuronal cell spheroids and SCAPs under various microenvironments in a pulp-on-a-chip system. MethodsSCAPs were induced into neuronal cells by a cocktail of chemical molecules. The expression of neuronal markers was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. SCAP-derived neuronal cell spheroids were generated with micromolded agarose gels and embedded in collagen hydrogel. After characterization, the SCAP-derived neuronal cell spheroids were cocultured with SCAPs in a pulp-on-chip system under various microenvironments: α-Minimum Essential Medium (MEM)/neuronal maturation medium (NMM) (neurosphere only); undifferentiated SCAPs with α-MEM/NMM (neurosphere + SCAP); osteogenic-induced SCAPs (Oi-SCAPs) with osteogenic medium/NMM containing 0.02% hydroxyapatite nanoparticles (neurosphere + Oi-SCAP/hydroxyapatite nanoparticle); and Oi-SCAPs with α-MEM/NMM (neurosphere + Oi-SCAP). Neurite outgrowth and MAP2 and TUJ1 expression in SCAP-derived neuronal cell spheroids were assessed by immunostaining. ResultsSCAPs were efficiently chemically induced into neuronal-like cells. The expression levels of MAP2 and TUJ1 were upregulated in SCAP-derived neuronal cell spheroids when cocultured with undifferentiated SCAPs, followed by Oi-SCAPs with α-MEM and neurospheres cultured alone, while coculturing with Oi-SACPs in osteogenic medium/NMM with hydroxyapatite nanoparticles displayed the lowest expression. Neurite spreading was more prominent in the spheroids cocultured with undifferentiated SCAPs than in those of the other 3 groups. ConclusionThis work revealed that local microenvironments critically regulate the neuroregenerative potential of SCAP-derived neuronal cell spheroids.

Diverging olfactory sensitivities to yeast volatiles reflect resource partitioning of tephritids and drosophilids

As pests of fruits and vegetables, ovipositing tephritid fruit flies are infamous for their frugivory. Yet, adult tephritids have remained saprophytic in their feeding behavior, as they require decomposing, protein rich media for sexual maturation and oogenesis. Drosophilid fruit flies, in contrast, are saprophytic both during oviposition and feeding. Here we compared the sensory and behavioral responses of two tephritid (Bactrocera dorsalisandCeratitis capitata) and two drosophilid species (Drosophila melanogasterandDrosophila suzukii) to differentially aged cultures of the yeastSaccharomyces cerevisiae. We assessed convergence and divergence in the detection of and behavioral response to these attractive substrates, and how these might be linked to the roles of the substrates for the different taxa. The headspace shifted substantially as broth cultures transitioned from active (1-day) to inactive (8- and 15-days). Interestingly,Drosophilaflies were significantly attracted to actively fermenting 1-day old yeast cultures, whereas the preference shifted to older cultures for the tephritids.Bactrocera dorsalisflies preferred inactive, lysing cultures (8- and 15-days old). We identified compounds from the 1- to 8-days old broth cultures that elicited antennal responses in each species. Synthetic blends composed of antennally active compounds evoked similar behavioral responses as broth cultures. Similarly, the attractiveness of less attractive broth cultures (1- and 8-days old for drosophilids and tephritids, respectively) could be augmented by adding volatiles of the more attractive cultures. The results show that the volatile profiles of fermenting substrates evolve quantitatively and qualitatively, and that fly species key into volatile blends that indicate suitability of the substrates for their purposes. For drosophilids early arrival at fermenting substrates confers a competitive advantage to offspring. In contrast, for tephritid the concentration and availability of protein is facilitated by older, lysed yeast cultures. The data from this comparative study are also instrumental in the development of novel lures for these pests.

Longwan 5: A Semi-Leafless Sugar Snap Pea Cultivar Resistant to Powdery Mildew

Garden pea (Pisum sativum L.) is an important legume crop, which is widely planted in Yunnan-Guizhou Plateau and Sichuan Basin of southwest China. It has developed rapidly in spring planting agroecological zone in Northwest China in recent years. The major constraints to its cultivation are lodging and infection of powdery mildew. Breeding of high yielding cultivars resistant to powdery mildew is of great significance for the sustainability of pea production, because few local garden pea cultivars are resistant to the disease. Varietal diversification is needed to develop pea cultivars with resistance to lodging and powdery mildew. Breeding work was initiated to develop a high-yielding garden pea cultivar with medium maturity, double podding and resistance to powdery mildew. Longwan 5 (X9002) is high yielding, superior quality, multiple resistance, and climate resilient garden pea cultivar developed by hybridization between Shuanghua 101 and Baofeng 3. It is a semi-leafless pea variety with superiority over existing approved varieties Qizhen 76 and Xucai 1 in terms of green pod yield, medium maturity, and double podding. Longwan 5 gave a significantly higher average green pod yield (12,376 kg/ha) than check varieties Qizhen 76 (11,132 kg/ha) and Xucai 1 (11,649 kg/ha) across five locations and three years, which was 11.2% and 6.3% higher than control varieties, respectively. This variety is tolerant to lodging, powdery mildew disease, and wide climate resilient for spring cultivation as well as for autumn cultivation in irrigated conditions or rain-fed agricultural areas with annual precipitation of 450–650 mm in China. Cultivation of this variety on large scale will surely increase the production of peas in China and will also prove beneficial for farmers increasing their income.

Co-culturing or conditioned medium of Sertoli cells: Which one supports in vitro maturation of bovine oocytes and developmental competency of resulting embryos?

Sertoli cells (SCs) as supportive cells in the seminiferous tubule play an essential role in the nutrition and development of adjacent cells by secreting several beneficial growth factors, stimulators and cytokines which can be conceived to improve the developmental competency of oocyte or embryo in the co-culture system. This study aimed to improve the maturation of bovine oocytes and consequently the development of resulting embryos in co-culture with SCs and their conditioned medium (CM). The retrieved cumulus-oocyte complexes (COCs) from the abattoir-derived ovaries were matured in maturation medium alone (control group), in co-culture with ovine SCs (co-culture group), and in presence of 10% CM prepared in 33°C and 39°C (CM33 and CM39 groups). The nuclear maturation competency and subsequent embryo development rate of cultured COCs in all groups were evaluated. The results of this study showed that SCs and CM increased meiosis resumption from GV to the MII compared to the control group, significantly (p < 0.05). Besides, the degenerated oocytes in the co-culture group were significantly higher than those in the control, CM33 and CM39 groups (p < 0.05), and the lowest cleavage rate belonged to the co-culture group (p < 0.05). The blastocyst rate was also lower in the co-culture group than other groups and there was a significant difference between the control and two CM groups (p < 0.05). The Sertoli cells can be suitable for co-culturing with oocytes during IVM but detrimental for subsequent embryo development. In turn, Sertoli cell-derived conditioned medium (SC-CM) can provide sufficient bioactive materials for COCs to enhancing oocyte competence and embryo development.

Bovine ICSI: limiting factors, strategies to improve its efficiency and alternative approaches

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique mainly used to overcome severe infertility problems associated with the male factor, but in cattle its efficiency is far from optimal. Artificial activation treatments combining ionomycin (Io) with 6-dimethylaminopurine after piezo-ICSI or anisomycin after conventional ICSI have recently increased the blastocyst rate obtained. Compounds to capacitate bovine spermatozoa, such as heparin and methyl-β-cyclodextrin and compounds to destabilize sperm membranes such as NaOH, lysolecithin and Triton X-100, have been assessed, although they have failed to substantially improve post-ICSI embryonic development. Disulfide bond reducing agents, such as dithiothreitol (DTT), dithiobutylamine and reduced glutathione, have been assessed to decondense the hypercondensed head of bovine spermatozoa, the two latter being more efficient than DTT and less harmful. Although piezo-directed ICSI without external activation has generated high fertilization rates and modest rates of early embryo development, other studies have required exogenous activation to improve the results. This manuscript thoroughly reviews the different strategies used in bovine ICSI to improve its efficiency and proposes some alternative approaches, such as the use of extracellular vesicles (EVs) as 'biological methods of oocyte activation' or the incorporation of EVs in the in vitro maturation and/or culture medium as antioxidant defence agents to improve the competence of the ooplasm, as well as a preincubation of the spermatozoa in estrous oviductal fluid to induce physiological capacitation and acrosome reaction before ICSI, and the use of hyaluronate in the sperm immobilization medium.

Copper deficiency affects the developmental competence of porcine oocytes matured in vitro.

The trace element Cu is required for the activity of various enzymes essential for physiological processes. In this study, we elucidated the copper transport system in porcine follicular cells and investigated the effect of Cu chelation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). Cu chelation was induced by adding tetraethylenepentamine (TEPA) to the maturation media (TCM199-PVA). First, we identified the localization and relative levels of the copper transporter CTR1 in follicular cells. The level of CTR1 protein was the highest in mature cumulus cells; moreover, CTR1 was mainly localized in the cytoplasmic vesicular compartment in oocytes, whereas it was evenly distributed in the cytoplasm in cumulus cells. A total of 42 h after IVM, the TEPA-treated group showed reduced maturation rates compared to those of the control (p < 0.05). This negative effect of TEPA disappeared when it was added to the media with Cu (Cu + TEPA group). The TEPA treatment during IVM significantly increased the mRNA levels of the Has2 gene, which is related to cumulus expansion (p < 0.05). Both Cu supplementation and chelation significantly increased the reactive oxygen species (ROS) levels in porcine oocytes (p < 0.05). When we analyzed the transcript levels of folliculogenesis-related genes in Cu chelation conditions, only the expression of MAPK3 in cumulus cells significantly increased compared to that of the control. We also evaluated the subsequent embryonic development of PA embryos. TEPA-treated oocytes showed significantly decreased blastocyst formation rates compared to those of the control. The TEPA-induced toxic effect was alleviated when Cu was added with TEPA. Our findings suggest that the Cu transport system plays an important role in the porcine follicular development process and that the Cu deficiency negatively affects porcine oocyte maturation, as well as their subsequent developmental competence.

Influence of different environments on oocyte maturation and development of bovine embryos in vitro

According to the International Embryo Technology Society, the number of bovine embryos produced by in vitro fertilization technology is increasing every year. However, despite the large volumes of their production, the effectiveness of this method is still low and needs to be improved. Therefore, the purpose of this study was to compare the effectiveness of two commercial media – Origio Sequential Series (Origio, Denmark) and a panel of products manufactured by Minitube (Germany) in terms of oocyte maturation and development of bovine embryos in vitro. At the first stage of the study, a comparative evaluation of oocyte maturation media was performed: based on TCM 199 (Minitube) and Universal (Origio) culture media. At the second stage, the protocols for culturing bovine embryos were compared: Minitube and the two-stage Origio culture protocol with changing media. Therewith, it was found that the use of TCM 199 medium for oocyte maturation is more effective compared to Universal. Thus, at 48 hours of cultivation (the initial stage of embryo development), 64.3 ± 1.0 and 60.3 ± 1.4% of 2-8 cell embryos were obtained, and on Day 8 – 25.3 ± 1.0 and 20.0 ± 0.6% of blastocysts, respectively. The results of a comparison of bovine embryo culture protocols showed that when using both Minitube and Origio media, the percentage of division and the percentage of resulting embryos corresponded to their known values. It was found that the Minitube cultivation protocol is more effective than Origio. At 48 hours, the number of embryos obtained using the Minitube culture protocol was 1.3% higher compared to Origio, on Day 6 – by 7.8%, and on Day 8 – by 3.8%. The results obtained are a necessary component of the development of successful processes to produce bovine embryos in vitro with further implementation in the ruminant reproduction biotechnology

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Effect of pre-IVF incubation in maturation medium on oocyte maturity, fertilization, embryonic development, and clinical outcomes following embryo transfer

Objective: Collected human cumulus-oocyte complexes (COCs) are usually inseminated after 4 to 6 hours in in vitro fertilization (IVF) laboratories. The purpose of this study was to determine the effect of short-term pre-IVF incubation in culture medium on subsequent oocyte maturation, fertilization, and embryonic development, as well as clinical outcomes. Methods: Sixty patients were divided randomly into 2 groups, pre-IVF incubation for 5 hours: 1) with (+) the designed oocyte maturation medium; 2) without (−) the designed oocyte maturation medium (transferred directly to fertilization medium for 5 hours before insemination). Oocyte maturation and fertilization were assessed, and the rate of cleavage and good quality embryos were evaluated between the 2 groups on days 2 and 3, respectively. Blastocyst development was based on the remaining number of embryos on day 3, continuously cultured to day 5 after embryo transfer or frozen on day 3, and was compared between the 2 groups. Clinical pregnancy, implantation, and miscarriage rates were also compared. Results: Oocyte maturation rates did not differ between groups (85.8 ± 14.1% vs. 90.7 ± 9.1%). However, the range of oocyte maturation rates (58.3%–100.0%) for each patient was significantly higher in the (−) group than in the (+) pre-incubation group (71.4%–100.0%). There were no differences in fertilization rates (89.9 ± 10.0% vs. 86.5 ± 12.2%) and good quality embryos (70.8 ± 19.1% vs. 62.1 ± 23.7%) between groups; however, the blastocyst development rates were significantly different between groups (73.1 ± 20.1% vs. 58.8 ± 18.2%, P &lt;0.05). Nevertheless, clinical pregnancy (62.5% vs. 61.1%) and implantation (46.9% vs. 47.2%) rates did not differ between groups. Conclusions: These results indicate that a short pre-IVF incubation time in the designed culture medium promotes oocyte maturation and embryonic development, suggesting that short pre-IVF incubation of COCs in the designed culture medium may be important for subsequent final oocyte maturation and early embryonic development.

L–carnitine improves developmental competence of buffalo oocytes in vitro

Objective: To investigate the effect of L-carnitine on in vitro maturation and subsequent in vitro embryo production of buffalo oocytes. Methods: Cumulus oocyte complexes (COCs) were aspirated from ovaries of slaughtered buffaloes. COCs were classified into good and fair qualities based on morphological observation of numbers and integrity of cumulus cells surrounding the oocyte. Both categories of COCs were placed in in vitro maturation medium with supplementation of different concentrations (0, 0.250, 0.375 or 0.500 mg/mL) of L-carnitine. Oocytes from both qualities were in vitro fertilized and in vitro cultured for 7 days, to examine the developmental competence. Results: Supplementation of L-carnitine to in vitro maturation medium increased the cumulus cell expansion rate of COCs to grade A, and reduced the cumulus cell expansion of COCs to grade B and grade C in both good and fair quality oocytes. Similarly, L-carnitine induced the in vitro meiotic progression of buffalo oocytes to metaphase Π in both good and fair quality oocytes. Additionally, L-carnitine reduced the rate of oocyte degeneration in both good and fair quality oocytes. L-carnitine increased the rate of cleaved formation at day 2 and blastocyst formation at day 7 during in vitro culture in both qualities of oocytes. Moreover, a higher rate of blastocyst production was observed in L-carnitine-treated fair quality oocytes, which was higher than the results in the untreated good quality oocytes. Conclusions: L-carnitine enhances meiotic maturation and subsequent embryo development from both good and fair quality buffalo oocytes.

Fragile X Syndrome Patient–Derived Neurons Developing in the Mouse Brain Show FMR1-Dependent Phenotypes

BackgroundFragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. MethodsTo mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient–derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. ResultsThe transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1–positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons. ConclusionsThis transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context.

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development. In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment. At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated. From 0.1 to 10 μmol L–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal. When the LPA concentration was 15 μmol L–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst. Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2 and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol L–1. The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L–1 to LPA-10 μmol L–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L–1 LPA-treated embryos compared with LPA-10 μmol L–1 group (P<0.05). The inner cell mass in 15 μmol L–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal. Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells. The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells. They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L–1, while 15 μmol L–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells. The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L–1 group and LPA-10 μmol L–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L–1 group compared with LPA-10 μmol L–1 group (P<0.05). Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L–1 concentration. This study references the IVF embryo production and embryonic stem cell research of domestic animals.

Effects of follicular fluid exosomes on in vitro maturation of porcine oocytes

Exosomes are related to effective communication between cells. In this study we aimed to investigate the effect of porcine follicular fluid exosomes (FF-Exo) on cumulus expansion, oocyte mitochondrial membrane potential, and maturation in in vitro culture. We used different concentrations of FF-Exo (Exo-0, Exo-1, Exo-10, Exo-20, and Exo-40) and added them to an oocyte maturation medium. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot (WB) showed that the isolated samples were exosomes. Immunofluorescence showed that exosomes could be taken up by cumulus cells. Compared with the Exo-0 group, there was no significant difference in oocyte maturation rate in the Exo-1 group (p > 0.05), while the Exo-10 group (p < 0.05), Exo-20 group (p < 0.01) and Exo-40 group (p < 0.01) significantly increased. The maturation rate of the Exo-20 and Exo-40 groups was the highest, and there was no significant difference between the two groups (p > 0.05). However, different concentrations of treatment could not effectively induce cumulus expansion and the results of JC1 showed that it had no significant effect on mitochondrial membrane potential (p > 0.05). In conclusion, the results suggest that porcine FF-Exo are involved in oocyte nuclear maturation.

Importance of Antioxidant Supplementation during In Vitro Maturation of Mammalian Oocytes.

Simple SummaryIn vitro embryo production (IVEP) is a technology that is widely used in the field of reproductive biology. The first and most important step of IVEP is termed in vitro maturation or IVM, in which the oocyte is allowed to mature in a synthetic medium under controlled laboratory settings. The quality of in vitro matured oocytes is still low compared to that of those matured inside an animal’s body. One of the key elements that was found to affect oocyte quality is the accumulation of large amounts of reactive oxygen species (ROS) within oocytes during IVM. The addition of antioxidants to oocyte IVM media is one of the most effective methods for preventing ROS accumulation. In this article, we highlight the latest events showing how ROS harms mammalian oocytes. We also discuss the possible impacts of antioxidant supplementation on the IVM rate and oocyte quality. The in vitro embryo production (IVEP) technique is widely used in the field of reproductive biology. In vitro maturation (IVM) is the first and most critical step of IVEP, during which, the oocyte is matured in an artificial maturation medium under strict laboratory conditions. Despite all of the progress in the field of IVEP, the quality of in vitro matured oocytes remains inferior to that of those matured in vivo. The accumulation of substantial amounts of reactive oxygen species (ROS) within oocytes during IVM has been regarded as one of the main factors altering oocyte quality. One of the most promising approaches to overcome ROS accumulation within oocytes is the supplementation of oocyte IVM medium with antioxidants. In this article, we discuss recent advancements depicting the adverse effects of ROS on mammalian oocytes. We also discuss the potential use of antioxidants and their effect on both oocyte quality and IVM rate.

Rescue IVM of Denuded GV- and MI-Stage Oocytes of Premenopausal Rats with Oncostatin M, Insulin-like Growth Factor I, and Growth Hormone.

Immature oocytes are retrieved and matured through in vitro maturation (IVM). Maturation, fertilization rates, and embryo development via IVM are all lower than those found in vitro fertilization (IVF) cycles. We investigated the effects of oncostatin M (OSM), insulin-like growth factor-1 (IGF-I), and growth hormone (GH) in rescue IVM. A total of 111 germinal vesicle (GV) and 17 metaphase I (MI) oocytes were obtained after conventional IVF from 28 female Wistar albino rats. Denuded immature oocytes were cultured in maturation medium supplemented with OSM, IGF-1, or GH. The quantities of metaphase II (MII) oocytes matured from the GV stage were 17 of 30 (56.6%), 15 of 28 (53.5%), 10 of 30 (33.3%), and 7 of 23 (30.3%), in control, OSM, IGF-I, and GH groups, respectively. Maturation rates in control and OSM groups were higher than those in IGF-I and GH groups (p = 0.001). The quantities of MII oocytes matured from MI stage were 7 of 7 (100%), 4 of 4 (100%), 1 of 1 (100%), and 1 of 5 (20%) in control, OSM, IGF-I, and GH groups, respectively. Maturation rates from MI to MII stages in control, OSM, and IGF-I groups were higher than those in the GH group (p = 0.004). Acceptable maturation rates are observed with OSM in rat oocytes in rescue IVM.