In Vitro Maturation Research Articles

Enhancing antioxidant levels and mitochondrial function in porcine oocyte maturation and embryonic development through notoginsenoside R1 supplementation.

This study examines the impact of Notoginsenoside R1 (NGR1), a compound from Panax notoginseng, on the maturation of porcine oocytes and their embryonic development, focusing on its effects on antioxidant levels and mitochondrial function. This study demonstrates that supplementing invitro maturation (IVM) medium with NGR1 significantly enhances several biochemical parameters. These include elevated levels of glutathione (GSH), nuclear factor erythrocyte 2-related factor 2 (NRF2) and mRNA expression of catalase (CAT) and GPX. Concurrently, we observed a decrease in reactive oxygen species (ROS) levels and an increase in JC-1 immunofluorescence, mitochondrial distribution, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and nuclear NRF2 mRNA levels. Additionally, there was an increase in ATP production and lipid droplets (LDs) immunofluorescence. These biochemical improvements correlate with enhanced embryonic outcomes, including a higher blastocyst rate, increased total cell count, enhanced proliferative capacity and elevated octamer-binding transcription factor 4 (Oct4) and superoxide dismutase 2 (Sod2) gene expression. Furthermore, NGR1 supplementation resulted in decreased apoptosis, reduced caspase 3 (Cas3) and BCL2-Associated X (Bax) mRNA levels and decreased glucose-regulated protein 78 kD (GRP78) immunofluorescence in porcine oocytes undergoing invitro maturation. These findings suggest that NGR1 plays a crucial role in promoting porcine oocyte maturation and subsequent embryonic development by providing antioxidant levels and mitochondrial protection.

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

Rescue in vitro maturation using ovarian support cells of human oocytes from conventional stimulation cycles yields oocytes with improved nuclear maturation and transcriptomic resemblance to in vivo matured oocytes

PurposeDetermine if the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes are bidirectionally influenced by co-culture during in vitro maturation (IVM).MethodsFertility patients aged 25 to 45 years old undergoing conventional ovarian stimulation donated denuded immature oocytes for research. Oocytes were randomly allocated to either OSC-IVM culture (intervention) or Media-IVM culture (control) for 24–28 h. The OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione, and doxycycline supplementation. The Media-IVM control lacked OSCs and contained the same supplementation. A limited set of in vivo matured MII oocytes were donated for comparative evaluation. Endpoints consisted of MII formation rate, morphological and spindle quality assessment, and gene expression analysis compared to in vitro and in vivo controls.ResultsOSC-IVM resulted in a statistically significant improvement in MII formation rate compared to the Media-IVM control, with no apparent effect on morphology or spindle assembly. OSC-IVM MII oocytes displayed a closer transcriptomic maturity signature to IVF-MII controls than Media-IVM control MII oocytes. The gene expression profile of OSCs was modulated in the presence of oocytes, displaying culture- and time-dependent differential gene expression during IVM.ConclusionThe OSC-IVM platform is a novel tool for rescue maturation of human oocytes, yielding oocytes with improved nuclear maturation and a closer transcriptomic resemblance to in vivo matured oocytes, indicating a potential enhancement in oocyte cytoplasmic maturation. These improvements on oocyte quality after OSC-IVM are possibly occurring through bidirectional crosstalk of cumulus-free oocytes and ovarian support cells.

Effect of Gentianella acuta (Michx.) Hulten against the arsenic-induced development hindrance of mouse oocytes.

The current study was designed to investigate the alleviative effect of Gentianella acuta (Michx.) Hulten (G. acuta) against the sodium arsenite (NaAsO2)-induced development hindrance of mouse oocytes. For this purpose, the in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs) was conducted in the presence of NaAsO2 and G. acuta, followed by the assessments of IVM efficiency including oocyte maturation, spindle organization, chromosome alignment, cytoskeleton assembly, cortical granule (CGs) dynamics, redox regulation, epigenetic modification, DNA damage, and apoptosis. Subsequently, the alleviative effect of G. acuta intervention on the fertilization impairments of NaAsO2-exposed oocytes was confirmed by the assessment of in vitro fertilization (IVF). The results showed that the G. acuta intervention effectively ameliorated the decreased maturation potentials and fertilization deficiency of NaAsO2-exposed oocytes but also significantly inhibited the DNA damages, apoptosis, and altered H3K27me3 expression level in the NaAsO2-exposed oocytes. The effective effects of G. acuta intervention against redox dysregulation including mitochondrial dysfunctions, accumulated reactive oxygen species (ROS) generation, glutathione (GSH) deficiency, and decreased adenosine triphosphate (ATP) further confirmed that the ameliorative effects of G. acuta intervention against the development hindrance of mouse oocytes were positively related to the antioxidant capacity of G. acuta. Evidenced by these abovementioned results, the present study provided fundamental bases for the ameliorative effect of G. acuta intervention against the meiotic defects caused by the NaAsO2 exposure, benefiting the future application potentials of G. acuta intervention in these nutritional and therapeutic research for attenuating the outcomes of arseniasis.

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus–oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

Expression pattern of ATG4C and its effect on early embryonic development of porcine oocytes

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0 % serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10 % serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0 % and 5 % serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P < 0.05) in the 0 % serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0 % serum group (P < 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P < 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P < 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

How to beat the bull: Lycopene as a tool to improve in vitro fertilization efficiency in bulls with high polyspermy

High levels of reactive oxygen species (ROS) derived from in vitro conditions compromise oocyte quality and subsequent polyspermy prevention by the zona and membrane block. Antioxidant supplementation, like lycopene, during in vitro maturation (IVM) mitigates ROS effects, yet, its efficacy in blocking polyspermy remains uncertain. This study aims to evaluate the effect of lycopene supplementation during IVM on oocyte maturation, fertilization, and developmental parameters. To this end, bovine oocytes were supplemented with 0.2 µM lycopene and fertilized with semen from three bulls. The three bulls showed different fertilization potential in vitro, with bull 1 showing the highest penetration and polyspermy rates and the lowest in vitro fertilization (IVF) efficiency. Interestingly, in bull 1, the treatment with lycopene improved IVF efficiency (p = 0.043) and reduced the polyspermy rate (p = 0.028). However, none of these effects were observed in bulls 2 and 3. Bulls with higher penetration rates exhibited better blastocyst rates although those rates did not seem to be associated with polyspermy or IVF efficiency. Oocyte mitochondrial distribution and activity and cortical granule migration and distribution were not influenced by lycopene. In conclusion, we demonstrated that lycopene addition during oocyte maturation had a positive impact on IVF efficiency by reducing polyspermy rates in a bull-dependent manner. The reduction in polyspermy rates was not caused by changes in cortical granule migration or oocyte mitochondrial distribution. Lycopene must therefore induce other changes in the oocyte that lower the in vitro penetration rates of specific bulls prone to polyspermy.

Accomplishment of canine cloning through in vitro matured oocytes: a pioneering milestone.

The in vitro maturation (IVM) rate of canine oocytes remains low compared to other mammals due to their unique reproductive characteristics. This study aimed to explore the effect of hormone supplementation during the IVM of canine immature oocytes on nuclear maturation and subsequently assess its potential application in canine somatic cell nuclear transfer (SCNT). Immature oocytes were collected and cultured in an IVM medium supplemented with hormones (follicle-stimulating hormone [FSH] and progesterone [P4]) or without hormones (control) for 24 hours. The maturation rates of oocytes in the hormone-treated group (94.92 ± 3.15%) were significantly higher than those in the control group (61.01 ± 4.23%). Both in vitro and in vivo matured oocytes underwent NT to evaluate their utility, and the fusion rates were higher in the in vitro matured group than those in the vivo matured group, not significant between in vivo and in vitro matured group (73.28% and 82.35%, respectively). As a result, 14 fused embryos from the in vitro matured group were transferred into two surrogates, with one surrogate achieving a successful pregnancy and delivering four puppies. Whereas in the in vivo matured group, 85 fused embryos were transferred to 8 surrogate mothers, leading to three surrogates becoming pregnant and delivering one, four, and two puppies. The pregnancy rates were not significant between both groups (50% and 37.50%), but the number of offspring exhibited a significant difference (28.57% and 8.23%). In conclusion, we achieved a remarkable milestone by successfully producing cloned puppies using in vitro matured oocytes, underscoring the feasibility of canine cloning from in vitro recovered oocytes. It is important to note that this study focused only on immature oocytes after ovulation and only during the estrus stage. Further research targeting other stages of the estrous cycle could potentially enhance canine cloning efficiency.

Cryopreservation of okapi (Okapia johnstoni) oocytes following in vitro maturation

Okapi (Okapia johnstoni) belong to the Giraffidae family and is the only representative of the Okapia genus. The species has been declared as endangered and due to its very elusive nature, little is known about its reproductive behavior. The wild population has continued to decrease over the last decades despite the different conservation programs developed worldwide. Captive breeding is difficult and the European population is not self-sustaining. An alternative tool to prevent the extinction of this charismatic species is the use of assisted reproductive technologies (ART). Specifically, we introduce a novel protocol for the in vitro maturation (IVM) and cryopreservation of matured oocytes obtained post-mortem. Out of sixteen cumulus-oocytes complexes isolated from one deceased individual, four matured metaphase II (MII) oocytes were successfully obtained following IVM. Furthermore, these matured oocytes demonstrated resilience during the vitrification/warming process and underwent sperm injection. Although no embryos were obtained, this study represents the first step towards the application of ART in okapi, providing a foundation for future reproductive interventions aimed at preserving self-sustaining captive populations.

Smart seq2 revealed distinct molecular responses during invitro porcine oocyte maturation before or after the addition of mogroside V.

Oocyte maturation involves both nuclear and cytoplasmic maturation. Mogroside V (MV) has been shown to enhance nuclear maturation, mitochondrial content, and developmental potential of porcine oocyte during invitro maturation (IVM). However, the impact of MV on cytoplasmic maturation and its underlying mechanisms are not understood. This study aimed to assess the effect of MV on cytoplasmic maturation. Germinal vesicle (GV) oocytes treated with MV exhibited a noticeable increase in cortical granules (CGs) formation. Additionally, MV enhanced the expression of NNAT and improved glucose uptake in mature oocytes. Further insights were gained through Smart-seq2 analysis of RNA isolated from 100 oocytes. A total of 11,274 and 11,185 transcripts were identified in oocytes treated with and without MV, respectively. Among quantified genes, 438 differentially expressed genes (DEGs) were identified for further analysis. Gene Ontology (GO) enrichment analysis indicated that these DEGs were primarily involved in DNA repair regulation, cellular response to DNA damage, intracellular components, and organelles. Furthermore, the DEGs were significantly enriched in three KEGG pathways: fatty acid synthesis, pyruvate metabolism, and WNT signalling. To validate the results, lipid droplets (LD) and triglyceride (TG) were examined. MV led to an increase in the accumulation of LD and TG production in mature oocytes. These findings suggest that MV enhances cytoplasmic maturation by promoting lipid droplet synthesis. Overall, this study provides valuable insights into the mechanisms through which MV improves oocyte quality during IVM. The results have significant implications for research in livestock reproduction and offer guidance for future studies in this field.

Impact of preovulatory follicle maturity on oocyte metabolism and embryo development.

Improved oocyte competence for embryo development and pregnancy was observed following ovulation of preovulatory follicles with greater physiological maturity, as indicated by estradiol production, prior to the gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) surge. It was hypothesized that follicular fluid from preovulatory follicles of greater maturity better supports the maturing oocyte's metabolic requirements and improves embryo development. The objective was to determine if differences in preovulatory follicular fluid due to follicle maturity influence oocyte metabolism during in vitro maturation (IVM) and affect embryo development. Bovine preovulatory follicular fluid was collected 18 h after a GnRH-induced LH surge. Serum estradiol concentration at GnRH administration categorized follicles as greater or lesser maturity. Immature bovine oocytes were submitted to 24 h IVM in medium supplemented with 20% follicular fluid from preovulatory follicles of greater or lesser maturity. Embryo development was recorded. Oocyte maturation media and media conditioned by developing embryos were submitted for metabolomics. A randomized block design was utilized to determine differences in embryo development and media metabolites (P ≤ 0.05). Blastocysts from oocytes matured in greater vs. lesser maturity follicular fluid had a more moderate rate of development (P = 0.01). At the conclusion of 24 h IVM, abundance of 66 metabolites differed between greater and lesser follicle maturity treatments. Nine metabolites differed in media conditioned by developing embryos. Metabolome results suggest improved amino acid, purine, and glucose metabolism, followed by a more efficient rate of embryo development, in oocytes matured in greater vs lesser maturity follicular fluid.

Follicular fluid-derived exosomal LncRNA LIPE-AS1 modulates steroid metabolism and survival of granulosa cells leading to oocyte maturation arrest in polycystic ovary syndrome.

Women who are of reproductive age can suffer from polycystic ovary syndrome (PCOS), an endocrine disorder. Anovulatory infertility is mostly caused by aberrant follicular development, which is seen in PCOS patients. Due to the dysfunction of reproductive and endocrine function in PCOS patients, assisted reproduction treatment is one of the main means to obtain clinical pregnancy for PCOS patients. Long non-coding RNA (lncRNA) as a group of functional RNA molecules have been found to participate in the regulation of oocyte function, hormone metabolism, and proliferation and apoptosis of granulosa cells. In this study, we investigated the role of lncRNAs in follicular fluid-derived exosomes and the underlying mechanism of lncRNA LIPE-AS1. We used RNA sequencing to analyze the lncRNA profiles of follicular fluid-derived exosomes in PCOS patients and controls. RT-qPCR was performed to detect the expression levels of these lncRNAs in control (n = 30) and PCOS (n = 30) FF exosome samples. Furthermore, we validated the performance of lncRNA LIPE-AS1 in oocyte maturation by in vitro maturation (IVM) experiments in mouse and steroid metabolism in granulosa cells. We found 501 lncRNAs were exclusively expressed in the control group and another 273 lncRNAs were found to be specifically expressed in the PCOS group. LncRNA LIPE-AS1, highly expressed in PCOS exosomes, was related to a poor oocyte maturation and embryo development in PCOS patients. Reduced number of MII oocytes were observed in the LIPE-AS1 group by in vitro maturation (IVM) experiments in mouse. LIPE-AS1 was also shown to modulate steroid metabolism and granulosa cell proliferation and apoptosis by LIPE-AS1/miR-4306/LHCGR axis. These findings suggested that the increased expression of LIPE-AS1, facilitated by follicular fluid exosomes, had a significant impact on both oocyte maturation and embryo development. We demonstrated the ceRNA mechanism involving LIPE-AS1, miR-4306, and LHCGR as a regulator of hormone production and metabolism. These findings indicate that LIPE-AS1 is essential in PCOS oocyte maturation and revealed a ceRNA network of LIPE-AS1 and provided new information on abnormal steroid metabolism and oocyte development in PCOS.

Nicotinamide Mononucleotide improves oocyte maturation of mice with type 1 diabetes

BackgroundThe number of patients with type 1 diabetes rises rapidly around the world in recent years. Maternal diabetes has a detrimental effect on reproductive outcomes due to decreased oocyte quality. However, the strategies to improve the oocyte quality and artificial reproductive technology (ART) efficiency of infertile females suffering from diabetes have not been fully studied. In this study, we aimed to examine the effects of nicotinamide mononucleotide (NMN) on oocyte maturation of mouse with type 1 diabetes mouse and explore the underlying mechanisms of NMN’s effect.MethodsStreptozotocin (STZ) was used to establish the mouse models with type 1 diabetes. The successful establishment of the models was confirmed by the results of body weight test, fasting blood glucose test and haematoxylin and eosin (H&E) staining. The in vitro maturation (IVM) rate of oocytes from diabetic mice was examined. Immunofluorescence staining (IF) was performed to examine the reactive oxygen species (ROS) level, spindle/chromosome structure, mitochondrial function, actin dynamics, DNA damage and histone modification of oocytes, which are potential factors affecting the oocyte quality. The quantitative reverse transcription PCR (RT-qPCR) was used to detect the mRNA levels of Sod1, Opa1, Mfn2, Drp1, Sirt1 and Sirt3 in oocytes.ResultsThe NMN supplementation increased the oocyte maturation rate of the mice with diabetes. Furthermore, NMN supplementation improved the oocyte quality by rescuing the actin dynamics, reversing meiotic defects, improving the mitochondrial function, reducing ROS level, suppressing DNA damage and restoring changes in histone modifications of oocytes collected from the mice with diabetes.ConclusionNMN could improve the maturation rate and quality of oocytes in STZ-induced diabetic mice, which provides a significant clue for the treatment of infertility of the patients with diabetes.

Mesenchymal stem cells promote ovarian reconstruction in mice

BackgroundStudies have shown that chemotherapy and radiotherapy can cause premature ovarian failure and loss of fertility in female cancer patients. Ovarian cortex cryopreservation is a good choice to preserve female fertility before cancer treatment. Following the remission of the disease, the thawed ovarian tissue can be transplanted back and restore fertility of the patient. However, there is a risk to reintroduce cancer cells in the body and leads to the recurrence of cancer. Given the low success rate of current in vitro culture techniques for obtaining mature oocytes from primordial follicles, an artificial ovary with primordial follicles may be a good way to solve this problem.MethodsIn the study, we established an artificial ovary model based on the participation of mesenchymal stem cells (MSCs) to evaluate the effect of MSCs on follicular development and oocyte maturation. P2.5 mouse ovaries were digested into single cell suspensions and mixed with bone marrow derived mesenchymal stem cells (BM-MSCs) at a 1:1 ratio. The reconstituted ovarian model was then generated by using phytohemagglutinin. The phenotype and mechanism studies were explored by follicle counting, immunohistochemistry, immunofluorescence, in vitro maturation (IVM), in vitro fertilization (IVF), real-time quantitative polymerase chain reaction (RT-PCR), and Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) assay.ResultsOur study found that the addition of BM-MSCs to the reconstituted ovary can enhance the survival of oocytes and promote the growth and development of follicles. After transplanting the reconstituted ovaries under kidney capsules of the recipient mice, we observed normal folliculogenesis and oocyte maturation. Interestingly, we found that BM-MSCs did not contribute to the formation of follicles in ovarian aggregation, nor did they undergo proliferation during follicle growth. Instead, the cells were found to be located around growing follicles in the reconstituted ovary. When theca cells were labeled with CYP17a1, we found some overlapped staining with green fluorescent protein(GFP)-labeled BM-MSCs. The results suggest that BM-MSCs may participate in directing the differentiation of theca layer in the reconstituted ovary.ConclusionsThe presence of BM-MSCs in the artificial ovary was found to promote the survival of ovarian cells, as well as facilitate follicle formation and development. Since the cells didn’t proliferate in the reconstituted ovary, this discovery suggests a potential new and safe method for the application of MSCs in clinical fertility preservation by enhancing the success rate of cryo-thawed ovarian tissues after transplantation.

Successful equine in vitro embryo production by ICSI – effect of season, mares’ age, breed, and phase of the estrous cycle on embryo production

This retrospective study aimed at identifying factors that contribute to the success of equine in vitro embryo production by intracytoplasmic sperm injection (ICSI). A total of 7993 ovum pick-up (OPU) sessions were performed, totaling 2540 donor mares and semen from 396 stallions. Oocytes were aspirated at multiple sites in Brazil and were sent to the laboratory, within 6 h from OPU, in pre-maturation medium where they were in vitro matured (IVM) followed by ICSI and in vitro embryo culture for 7–8 days. The number of recovered oocytes, matured oocytes, cleaved embryos and blastocysts were used to explore the effect of age and breed of the donor mare, time of year in which the mare was aspirated and phase of the estrous cycle on the day of follicular aspiration. Mares between 6 and 15 years old were superior to other age groups in most parameters evaluated, including the average number of blastocysts per OPU. The impact of age was similar when evaluated within two breeds, American Quarter Horse (AQHA) and Warmblood mares. We observed that breed (AQHA, Warmblood, Crioulo, Lusitano and Mangalarga) had an important effect on most of the parameter evaluated, including number of oocytes recovered, blastocysts produced per OPU, and blastocyst rates. The overall impact of season was less pronounced than age and breed, with the only statistically significant difference being a higher rate of oocyte maturation during the summer season. Finally, most of the parameters evaluated were superior in follicular phase mares, with or without dominant follicle than luteal phase mares. In conclusion, this retrospective study revealed that breed, age, season and stage of estrous at the time of OPU are all important parameters for the success of equine embryo production by ICSI. This technology enables producing embryos all-year-round from mares of different breeds and ages from OPU-derived oocytes collected at multiple sites.

Correction: Rose et al. The Effect of In Vitro Maturation (IVM) Protocol Changes on Measures of Oocyte/Embryo Competence. Reprod. Med. 2023, 4, 65–73

**Samuel E [...]

C-Phycocyanin improves the quality of goat oocytes after in vitro maturation and vitrification

In vitro maturation (IVM) and cryopreservation of goat oocytes are important for establishing a valuable genetic bank for domesticated female animals and improving livestock reproductive efficiency. C-Phycocyanin (PC) is a Spirulina extract with antioxidant, antiinflammatory, and radical scavenging properties. However, whether PC has positive effect on goat oocytes IVM or developmental competence after vitrification is still unknown. In this study, we found that first polar body extrusion (n = 293), cumulus expansion index (n = 269), and parthenogenetic blastocyst formation (n = 281) were facilitated by adding 30 μg/mL PC to the oocyte maturation medium when compared with the control groups and that supplemented with 3, 10, 100 or 300 μg/mL PC (P < 0.05). Although PC supplementation did not affect spindle formation or chromosome alignment (n = 115), it facilitated or improved cortical granules migration (n = 46, P < 0.05), mitochondria distribution (n = 39, P < 0.05), and mitochondrial membrane potential (n = 46, P < 10−4). Meanwhile, supplementation with 30 μg/mL PC in the maturation medium could significantly inhibit the reactive oxygen species accumulation (n = 65, P < 10−4), and cell apoptosis (n = 42, P < 0.05). In addition, PC increased the oocyte mRNA levels of GPX4 (P < 0.01), and decreased the mRNA and protein levels of BAX (P < 0.01). Next, we investigated the effect of PC supplementation in the vitrification solution on oocyte cryopreservation. When compared with the those equilibrate in the vitrification solution without PC, recovered oocytes in the 30 μg/mL PC group showed higher ratios of normal morphology (n = 85, P < 0.05), survival (n = 85, P < 0.05), first polar body extrusion (n = 62, P < 0.05), and parthenogenetic blastocyst formation (n = 107, P < 0.05). Meanwhile, PC supplementation of the vitrification solution increased oocyte mitochondrial membrane potential (n = 53, P < 0.05), decreased the reactive oxygen species accumulation (n = 73, P < 0.05), promoted mitochondria distribution (n = 58, P < 0.05), and inhibited apoptosis (n = 46, P < 10−3). Collectively, our findings suggest that PC improves goat oocyte IVM and vitrification by reducing oxidative stress and early apoptosis, which providing a novel strategy for livestock gamete preservation and utilization.

Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins.

Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3mprotein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. N/A.

Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function

Bone morphogenetic protein 15 (BMP15) plays a crucial role in the porcine follicular development. However, its exact functions in the in vitro maturation (IVM) of porcine oocytes remain largely unknown. Here, through cytoplasmic injection of a preassembled crRNA–tracrRNA–Cas9 ribonucleoprotein complex, we achieved BMP15 disruption in approximately 54 % of the cultured porcine oocytes. Editing BMP15 impaired the IVM of porcine oocytes, as indicated by the significantly increased abnormal spindle assembly and reduced first polar body (PB1) extrusion. The editing also impaired cytoplasmic maturation of porcine oocytes, as reflected by reduced abundant of Golgi apparatus and impaired functions of mitochondria. The impaired IVM of porcine oocytes by editing BMP15 possibly was associated with the attenuated SMAD1/5 and EGFR-ERK1/2 signaling in the cumulus granulosa cells (CGCs) and the inhibited MOS/ERK1/2 signaling in oocytes. The attenuated MOS/ERK1/2 signaling may contribute to the inactivation of maturation promoting factor (MPF) and the increased abnormal spindle assembly, leading to reduced PB1 extrusion. It also may contribute to reduced Golgi apparatus formation, and impaired functions of mitochondria. These findings expand our understanding of the regulatory role of BMP15 in the IVM of porcine oocytes and provide a basis for manipulation of porcine reproductive performance.