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Metabolite supplementation during in vitro embryo development improves blastocyst quality, however, our understanding of the incorporation of metabolites during in vitro maturation (IVM) is limited. Two important metabolites, follistatin and choline, have beneficial impacts during in vitro culture; however, effects of supplementation during IVM are unknown. The objective of this study was to investigate combining choline and follistatin during IVM on bovine oocytes and subsequent early embryonic development. We hypothesized that supplementation of choline with follistatin would synergistically improve oocyte quality and subsequent early embryonic development. Small follicles were aspirated from slaughterhouse ovaries to obtain cumulus oocyte complexes for IVM with choline (0, 1.3 or 1.8 mM) and follistatin (0 or 10 ng/mL) supplementation in a 3 × 2 design. A subset of oocytes underwent transcriptomic analysis, the remaining oocytes were used for IVF and in vitro culture (IVC). Transcript abundance of CEPT1 tended to be reduced in oocytes supplemented with 1.8 mM choline and follistatin compared to control oocytes ( P = 0.07). Combination of follistatin with 1.8 mM choline supplementation during maturation, tended ( P = 0.08) to reduce CPEB4 in oocytes. In the blastocysts, HDCA8 , NANOG , SAV1 and SOX2 were increased with choline 1.8 mM supplementation without follistatin ( P < 0.05), while HDCA8 and SOX2 were increased when follistatin was incorporated ( P < 0.05). The combination of choline and follistatin during oocyte maturation may provide a beneficial impact on early embryonic development. Further research is warranted to investigate the interaction between these two metabolites during early embryonic development and long-term influence on fetal development.

We herein investigated the influence of temperature on the embryonic development (from fertilisation to hatching) of Mugil liza larvae. For this purpose, oocytes (>600 μm) and sperm were obtained from breeding stock at the laboratory of marine fish culture (LAPMAR). After fertilisation, 1200 eggs were distributed in 12 cylindrical experimental units of 400 mL under four different temperatures 18, 22, 26 and 30 ºC, all in triplicate. Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment. The eggs were visualized and photographed, and the classification of embryonic stages was performed. Temperature influenced the main events of the embryonic development of M. liza . More accelerated development was observed according to the increase in temperature until the gastrula phase. At temperatures of 22 and 26 °C, embryonic development occurred from fertilisation to hatching of the larvae. In the 18 °C treatment, it was verified that most of the embryos ceased development during the final phase of cleavage and the beginning of blastula formation, while in the 30 °C treatment patterns of embryo malformation were also verified, with erratic divisions of the blastomeres, resulting in irregular cells. Unlike what was observed at a temperature of 18 °C, none of the embryos incubated at 30 °C reached the blastopore closure phase, stopping in the gastrula. The larvae hatched in the treatments at 22 and 26 °C were viable and exhibited intense swimming, with a large amount of reserve material (yolk) and an evident drop of oil.

In vitro maturation of oocytes (IVM) represents an assisted reproductive technique that involves the minimal or absence of ovarian stimulation and is beneficial to specific groups of patients. These may include women with polycystic ovarian syndrome and/or patients who need a fertility preservation option before undergoing gonadotoxic treatment. However, when IVM is applied in cases where it is not recommended, it can be considered as an add-on technique, as described by the ESHRE Guideline Group on Female Fertility Preservation. Interestingly, IVM has not been proven yet to be as effective as conventional IVF in the laboratory, in terms of clinical pregnancy and live birth rates, while concerns have been raised for its long-term safety. As a result, both safety and efficacy of IVM remain still questionable and additional data are needed to draw conclusions.

The intra-ovarian presence of ootids, i.e. female gametes that have completed meiosis, is considered exceptional in the animal kingdom. The present study explores the first such case to be reported in a sea cucumber (Echinodermata: Holothuroidea). In the overwhelming majority of animals, including holothuroids, oocytes (i.e. immature female gametes) that are developing in the ovary undergo a primary arrest at the prophase stage of meiosis, which may last from days to decades. In free-spawning taxa, this arrest is normally lifted only during or shortly before transit in the gonoduct, when gamete release (spawning) is imminent. However, oocytes of the holothuroid Chiridota laevis were discovered to have resumed the second meiotic division including the completion of germinal vesicle breakdown and polar-body expulsion inside the ovary, effectively reaching the ootid stage concomitantly with ovulation (i.e. escape from follicle cells) prior to spawning. The potential drivers and significance of this exceptionally rare case of full intra-ovarian oogenic maturation are discussed.

There is no evidence, nor need, for a fast block to polyspermy in animal oocytes. The idea that oocytes have evolved a mechanism to allow the entry of one spermatozoon and repel all others has, however, gained consensus over the last century. The main culprit is the sea urchin, which has been used for over a century in in vitro studies of the fertilization process. Images of sea urchin oocytes with thousands of sperm attached to the surface are commonplace in textbooks and appeal to the nature of the reader implying an intriguing surface mechanism of sperm selection despite these oocytes being fixed for photography (Figure ). The abundance of gametes in this marine invertebrate and the ease of experimentation have given us the possibility to elucidate many aspects of the mechanism of fertilization, but has also led to ongoing controversies in reproductive biology, one being polyspermy prevention. Kinetic experiments by Rothschild and colleagues in the 1950s led to the hypothesis of a fast partial block to polyspermy in sea urchin oocytes that reduced the probability of a second spermatozoon from entering the oocyte by 1/20th. In the 1970s, Jaffe and colleagues suggested, with circumstantial evidence, that this partial block was due to the sperm-induced depolarization of the oocyte plasma membrane. However, the fate of supernumerary spermatozoa is determined well before the plasma membrane of the oocyte depolarizes. Transmembrane voltage does not serve to regulate sperm entry. Scholastic texts have inadvertently promulgated this concept across the animal kingdom with no logical correlation or experimentation and, as of today, a molecular mechanism to regulate sperm entry in oocytes has not been identified.

Oocytes with excessively large first polar bodies (PB1) often occur in assisted reproductive procedures. Many times these oocytes are discarded without insemination and, as a result, the application of this portion of oocytes has scarcely been reported to date. Few studies have examined large PB1 oocytes in infertile women and have virtually entirely studied genetic variations for large PB1 oocyte abnormalities. Here, we describe an unusual case of a live birth from a remarkably large PB1 oocyte in a frozen embryo transfer (FET) cycle. This is the first instance of a successful live birth resulting from a PB1 oocyte with an extremely large polar body measuring 80 μM × 40 μM in size. The large PB1 oocyte was performed by an early rescue intracytoplasmic sperm injection (r-ICSI) and was formed into a blastocyst on day 5. Following FET, a healthy boy baby weighing 3100 g was finally delivered by caesarean section at 37 weeks and 5 days after conception. Additionally, there were no complications throughout the antenatal period or the perinatal phase of this following full-term delivery. In this study, it is revealed for the first time that a huge PB1 oocyte can be fertilized, resulting in the growth of a blastocyst, a subsequent pregnancy, and a live birth. This new information prompts us to reconsider the use of large PB1 oocytes. More insightful talks should be given attention to prevent the waste of embryos because not all oocytes with aberrant morphology are unavailable.

The present study was conducted to elucidate (1) the influence of kisspeptin (KP) on the in vitro development of preantral follicles (PFs) and (2) evolution of KP receptor gene ( KISS1R ) expression during ovarian follicular development in sheep. Kisspeptin was supplemented (0-100 µg/ml) in the culture medium of PFs for 6 days. The cumulus-oocyte complexes (COCs) from cultured PFs were subsequently matured to metaphase II (MII) for an additional 24 h. The proportions of PFs exhibiting growth, antrum formation, average increase in diameter, and maturation of oocytes to MII stage were the indicators of follicular development in vitro . The expression of the kisspeptin receptor gene at each development stages of in vivo developed (preantral, early antral, antral, large antral and COCs from Graafian follicles) and in vitro cultured PFs supplemented with KP was assessed using a real-time polymerase chain reaction. The best development in all the parameters under study was elicited with 10 µg/ml of KP. Supplementation of KP (10 µg/ml) in a medium containing other growth factors (insulin-like growth factor-I) and hormones (growth hormone, thyroxine, follicle-stimulating hormone) resulted in better PF development. The KISS1R gene was expressed in follicular cells and oocytes at all the development stages of both in vivo developed and in vitro cultured follicles. Higher KISS1R gene expression was supported by culture medium containing KP along with other hormones and growth factors. Accordingly, it is suggested that one of the mechanisms through which KP and other growth factors and hormones influence the ovarian follicular development in mammals is through the upregulation of expression of the KP receptor gene.

Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.

Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo . However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.

The influence of the method of evaluating developmentally competent oocytes on their viability after cryopreservation still needs to be better understood. The objective of this study was to determine the cleavage and embryo developmental rates after parthenogenetic activation of cumulus-oocyte complexes (COCs) selected by different concentrations of brilliant cresyl blue (BCB) and cryopreservation. In the first experiment, COCs were separated into groups and incubated for 1 h in medium containing BCB (13 μM, 16 μM, or 20 μM). The control group was not exposed to BCB staining. In the second experiment, COCs were divided into four groups: 13 μM BCB(+), 13 μM BCB(-), fresh control (selected by morphologic observation and immediately in vitro matured) and vitrified control (selected by morphologic evaluation, vitrified, and in vitro matured). In the first experiment, the 13 μM BCB group displayed greater development rates at the morula stage (65.45%, 36/55) when compared with the other groups. In the second experiment, cleavage (47.05%, 72/153) and morula development (33.55%, 51/153) of the control group of fresh COCs were increased compared with the other groups. However, when comparing morula rates between vitrified COC control and BCB(+) groups, the BCB(+) group had better results (19.23%, 5/26 and 64.7%, 11/17, respectively). Our best result in rat COC selection by BCB staining was obtained using a concentration of 13 μM. This selection could be a valuable tool to improve vitrification outcomes, as observed by the BCB(+) group that demonstrated better results compared with the vitrified COC control.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

The production of in vitro embryos has sped up the dissemination of superior genetic material. However, the variation among the cattle response to oocyte and embryo production is a challenging factor. This variation is even higher in the Wagyu cattle as the breed has a small effective population size. The identification of an effective marker related to reproductive efficiency would allow the selection of more responsive females to reproductive protocols. The objective of this study was to evaluate the blood levels of anti-Müllerian hormone and associate it with oocyte recovery and blastocyst rate of embryos produced in vitro in Wagyu cows, as well as observe the hormone circulating levels in males. Serum samples from 29 females with seven follicular aspirations and four bulls were used. AMH measurements were performed using the bovine AMH ELISA kit. A positive correlation was identified between oocyte production and blastocyst rate ( r = 0.84, P = 9 × 10 -9 ), and AMH levels with oocyte ( r = 0.49, P = 0.006) and embryo ( r = 0.39, P = 0.03) production. The mean levels of AMH were different between animals with low (11.06 ± 3.01) and high (20.75 ± 4.46) oocyte production ( P = 0.01). Males showed high serological levels of AMH (3829 ± 2328 pg/ml) compared with other breeds. It is possible to use the serological measurement of AMH as a method to select Wagyu females with greater capacity for oocyte and embryo production. Further studies correlating AMH serological levels with Sertoli cell function in bulls are needed.

Pumilio3 ( Pum3 ), an evolutionarily distant homologue of the classical RNA-binding protein PUF (PUMILIO and FBF) family member, is also involved in the process of RNA metabolism through post-transcriptional regulation. However, the functions of Pum3 in mouse oocyte maturation and preimplantation embryonic development have not been elucidated. By comparing RNA levels in different tissues, we found that Pum3 was widely expressed in multiple tissues, but moderately predominant in the ovary. Histochemical staining suggested that the PUM3 protein exhibits positive signals in oocytes, granulosa cells and theca cells of different follicle stages. Oocyte immunofluorescence results showed a slightly higher level of PUM3 protein in metaphase II compared with the germinal vesicle (GV) stage. After knockdown of Pum3 in GV oocytes using siRNA injection (siPUM3), no obvious defect was observed in the processes of GV breakdown and polar body extrusion during in vitro maturation (IVM) for the siPum3 oocytes. Compared with the control group, the siPUM3 group displayed no significant abnormality in the cleavage and blastocyst formation rate of these fertilized oocytes. Therefore, we can conclude that depletion of Pum3 does not affect mouse oocyte maturation and early embryonic development in vitro .

Although ethanol treatment is widely used to activate oocytes, the underlying mechanisms are largely unclear. Roles of intracellular calcium stores and extracellular calcium in ethanol-induced activation (EIA) of oocytes remain to be verified, and whether calcium-sensing receptor (CaSR) is involved in EIA is unknown. This study showed that calcium-free ageing (CFA) in vitro significantly decreased intracellular stored calcium (sCa) and CaSR expression, and impaired EIA, spindle/chromosome morphology and developmental potential of mouse oocytes. Although EIA in oocytes with full sCa after ageing with calcium does not require calcium influx, calcium influx is essential for EIA of oocytes with reduced sCa after CFA. Furthermore, the extremely low EIA rate in oocytes with CFA-downregulated CaSR expression and the fact that inhibiting CaSR significantly decreased the EIA of oocytes with a full complement of CaSR suggest that CaSR played a significant role in the EIA of ageing oocytes. In conclusion, CFA impaired EIA and the developmental potential of mouse oocytes by decreasing sCa and downregulating CaSR expression. Because mouse oocytes routinely treated for activation (18 h post hCG) are equipped with a full sCa complement and CaSR, the present results suggest that, while calcium influx is not essential, CaSR is required for the EIA of oocytes.

Induction of puberty in cattle breeds that attain puberty in later stages, such as Gir, allows the earlier beginning of reproductive life and it might increase oocyte quality. Here, the ovulatory capacity of prepuberal Gir heifers was studied and its relationship to follicular growth, luteinizing hormone (LH) secretion and oocyte quality was evaluated. Peripubertal Gir heifers were treated with a progesterone-based protocol and according to ovulatory response were separated into groups: not-ovulated (N-OV) and ovulated (OV). Serial blood samples were taken 24 h after estradiol treatment on day 12 to evaluate LH secretion. Cumulus-oocyte complexes (COCs) were collected using ovum pick-up and assessed for brilliant cresyl blue (BCB) staining rate, IVF-grade oocytes rate, and mean oocyte diameter, in comparison with cow oocytes. Gene expression of developmental competence markers (ZAR1, MATER, and IGF2R) was also analyzed. The largest follicle diameters were similar between N-OV and OV groups on the day of estradiol treatment (d12) and the next day and decreased ( P = 0.04) in the N-OV group thereafter. LH pulse secretion was different between groups (N-OV = 3.61 ± 0.34 vs OV = 2.83 ± 0.21 ng/ ml; P = 0.04). COC assessment showed that the number of recovered oocytes, BCB+ rate, IVF-grade oocytes and oocyte size was similar ( P > 0.05) among groups, resembling adult cow patterns. ZAR1, MATER and IGF2R gene expression in oocytes were also similar ( P > 0.05) in N-OV and OV groups. In conclusion, our results demonstrate a lower LH secretion profile in peripubertal Gir heifers prone to ovulate after induction protocol, and that oocyte quality is not affected on a short-term basis by ovulation itself.

Oocyte-mediated somatic cell haploidization is a process in which a diploid cell halves its chromosomal content by segregating its homologue within the ooplasm. Replacing the donor oocyte nucleus with a patient's female diploid somatic nucleus can generate patient-genotyped oocytes. Insemination of these resulting constructs enables their activation and induces a reductive meiotic division, haploidizing the diploid female donor cell that can subsequently support syngamy with the male genome and create a zygote. So far, experimental data for this method have been limited and have not consistently proven the generation of chromosomally normal embryos. Overall, we achieved reconstruction of murine oocytes with a micromanipulation survival rate of 56.5%, and a correct haploidization and fertilization rate of 31.2%, resulting in a 12.7% blastocyst rate. Time-lapse analysis revealed that reconstructed embryos underwent a timely polar body extrusion and pronuclear appearance followed by a satisfactory embryonic cleavage, comparable with the control. Whole genome sequencing of the analyzed embryos indicated that 27.3% (6/22) were properly diploid. Our findings suggest that diploid cell haploidization may be a feasible technique for creating functional gametes in mammals.

This study investigated the effect of the flavonoid-based compound isorhamnetin (ISO) on maturation and developmental competence in oxidative stress-exposed porcine oocytes in vitro . Treatment with 2 μM ISO (2 ISO) increases the developmental rate of oxidative stress-exposed porcine oocytes during in vitro maturation (IVM). The glutathione level and mRNA expression of antioxidant-related genes ( NFE2L2 and SOD2 ) were increased in the 2 ISO-treated group, whereas the reactive oxygen species level was decreased. Treatment with 2 ISO increased mRNA expression of a cumulus cell expansion-related gene ( SHAS2 ) and improved chromosomal alignment. mRNA expression of maternal genes ( CCNB1, MOS, BMP15 and GDF9 ) and mitogen activated protein kinase (MAPK) activity were increased in the 2 ISO-treated group. The total cell number per blastocyst and percentage of apoptotic cells were increased and decreased in the 2 ISO-treated group, respectively. Treatment with 2 ISO increased mRNA expression of development-related genes ( SOX2, NANOG , and POU5F1 ) and anti-apoptotic genes ( BCL2L1 and BIRC5 ) and decreased that of pro-apoptotic genes ( CASP3 and FAS ). These results demonstrate that 2 ISO improves the quality of porcine oocytes by protecting them against oxidative stress during IVM and enhances subsequent embryo development in vitro . Therefore, we propose that ISO is a useful supplement for IVM of porcine oocytes.

Oocyte gene expression is a well controlled event that promotes gamete competence to undergo maturation, fertilization, and to support early embryo development, directly affecting reproductive outcomes. Considering that in vivo controlled ovarian stimulation or in vitro maturation (IVM) for the acquisition of mature oocytes has distinct implications for gene expression, we sought to evaluate the effects of these procedures on the expression of competence-related genes in single-cell oocytes. Healthy Nelore cows of reproductive age were synchronized to harvest in vivo matured oocytes; ovaries from slaughtered animals were used to obtain cumulus-oocyte complexes that were in vitro matured. Single-cell gene expression was performed using TaqMan Low-Density Arrays and 42 genes were evaluated. In silico analysis of protein interactions and Gene Ontology (GO) analysis was performed. Reduced gene expression was observed for 24 targets in IVM oocytes when compared with those of in vivo matured oocytes ( P < 0.05). Differences ranged from 1.5-fold to 4.8-fold higher in in vivo oocytes and the BMP15 (5.28), GDF9 (6.23), NOBOX (7.25), HSPA8 (7.85) and MSX1 (11.00) showed the greatest fold increases. The strongest score of functional interactions was observed between the CDC20 and CKS2 , with the differentially expressed gene CDC20 being the main marker behind GO enrichment. IVM negatively affected the expression of important genes related to oocyte competency, and showed higher expression levels in in vivo matured oocytes. In vivo controlled ovarian stimulation may be a better strategy to achieve proper oocyte competence and increase the success of assisted reproductive technologies.

The process of oocyte retrieval represents a key phase during the cycles of in vitro fertilization (IVF). It involves controlled ovarian stimulation to retrieve the highest number of oocytes possible. According to many previous studies, the higher the number of oocytes the higher the chances of obtaining embryos for multiple transfers. In this study, in total, 1987 patients were retrospectively reviewed to investigate the correlations between the number of retrieved oocytes and the subsequent IVF outcomes. Patients were divided into three groups according to the number of retrieved oocytes (Group 1: ≤5 oocytes; Group 2: 6-15 oocytes; Group 3: ≥15 oocytes). The results showed a significant negative correlation between oocyte number and maturation rate as well as fertilization rate. However, a significant positive correlation was found between oocyte number and the blastulation rate. The implantation rate after fresh embryo transfers was higher in group 2 (6-15 oocytes) compared with group 1 (≤5 oocytes). According to our findings, we conclude that oocyte numbers between 6 and 15 oocytes can result in the highest chances of positive IVF outcomes in terms of embryo quality and fresh embryo transfers with lower risks of ovarian hyperstimulation.

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.

Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower ( P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower ( P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased ( P < 0.05) the blastocysts rates. In the third experiment, the association of 2 μM 17AAG with HS for 12 h during IVM resulted in lower ( P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.

Recently, more attention has been raised towards fertility preservation in women with cancer. One option is in vitro maturation (IVM) of the immature oocytes as there is not enough time for induction of an ovarian stimulation protocol. The aim was to compare the IVM laboratory outcomes between stimulated and unstimulated (natural) in vitro fertilization (IVF) cycles. In total, 234 immature oocytes collected from 15 cancer patients who underwent an IVM programme (natural IVM) and 23 IVF cycles with a controlled ovarian hyperstimulation protocol (stimulated IVM) were analyzed. The oocyte morphology, zona pellucida (ZP), and meiotic spindle presence were measured using PolScope technology. Also, the rates of oocyte maturation and fertilization were assessed in both groups. The IVM rate was higher in the stimulated cycle ( P < 0.05), but the fertilization rate was insignificant in comparison with unstimulated cycles. There were no significant differences in the spindle visualization and ZP birefringence scoring between the groups ( P > 0.05). The oocyte normal morphology was better in the stimulated cycle compared with the natural cycle ( P < 0.05). In conclusion, IVM can be recommended for cancer patients as an alternative treatment when there is insufficient time for conventional IVF before chemotherapy initiation.

This study evaluated the effect of fibroblast growth factor-2 (FGF-2) on the morphology, primordial follicle activation and growth after in vitro culture of domestic cat ovarian tissue. Ovaries ( n = 12) from prepubertal domestic cats were collected and fragmented. One fragment was fixed for histological analysis (fresh control). The remaining fragments were incubated in control medium alone or with 10, 50 or 100 ng/ml FGF-2 for 7 days. After in vitro culture, the following endpoints were analyzed: morphology, activation by counting primordial and developing follicles, and growth (follicle and oocyte diameters). Treatment with 100 ng/ml FGF-2 maintained ( P > 0.05) the percentage of normal follicles similar to fresh control. Follicle survival was greater ( P < 0.05) after culture in 100 ng/ml FGF-2 than in 50 ng/ml FGF-2. The percentage of primordial follicles decreased ( P < 0.05) and the percentage of developing follicles increased ( P < 0.05) in all treatments compared with fresh tissue. The proportion of developing follicles increased ( P < 0.05) in tissues incubated with 100 ng/ml FGF-2 compared with control medium and other FGF-2 concentrations. Furthermore, culture in 10 or 100 ng/ml FGF-2 resulted in increased ( P < 0.05) follicle and oocyte diameters compared with fresh tissues and MEM + . In conclusion, FGF-2 at 100 ng/ml maintains follicle survival and promotes the in vitro activation and growth of cat primordial follicles.

Telomere shortening during oocyte growth and development is related to reproductive ageing and infertility. The main mechanism involved in the maintenance of telomeres is based on telomerase activity, a specialized enzyme complex, which is capable of adding TTAGGG repeats at the ends of the chromosomes. Mitochondrial dysfunction may cause progressive shortening of telomeres by promoting the generation of reactive oxygen species. Mitofusin-1 is a protein required for mitochondrial fusion. Mice with the mitofusin-1 ( Mfn1 ) deletion in the oocyte are characterized by accelerated follicular depletion and infertility, associated with defective oocyte maturation and follicular development. We hypothesized whether mitochondrial dysfunction in oocytes with targeted deletion of Mfn1 causes telomere shortening. We analyzed telomere length in oocyte and somatic cells in 3-, 6- and 9-month-old Mfn1 -/- and wild-type mice. Immunofluorescence in oocyte mice of TRF1 and H2A.X was assessed to evaluate the interplay between the end-protection functions and the response to DNA damage occurring inside the telomeric repeats. Mitochondrial dysfunction due to the deletion of Mfn1 does not seem to affect telomere length in mouse oocytes.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into S -adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs. As MTHFR controls the entry of one-carbon units into the methionine cycle, it is a candidate regulator of the SAM levels in oocytes and eggs. Mthfr transcripts are expressed in mouse oocytes and preimplantation embryos and MTHFR protein is present at each stage. In mature MII eggs, the apparent molecular weight of MTHFR was increased compared with GV oocytes, which we hypothesized was due to increased phosphorylation. The increase in apparent molecular weight was reversed by treatment with lambda protein phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. In contrast, LPP had no effect on MTHFR from GV oocytes, 2-cell embryos, or blastocysts. MTHFR was progressively phosphorylated after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing again after egg activation. As phosphorylation suppresses MTHFR activity, it is predicted that MTHFR becomes inactive during meiotic maturation and is minimally active in MII eggs, which is consistent with the reported changes in SAM levels during mouse oocyte maturation.

Over the last decades, extracellular vesicles (EVs) have been found to be implicated in a complex universal mechanism of communication between different cell types. EVs are nanostructures of lipid nature that have an exosomal or ectosomal biogenesis, responsible for the intercellular transport of proteins, lipids, carbohydrates, nucleic acids, ions, among other molecules. The content of EVs can vary due to various factors such as hormonal stimuli, non-physiological conditions, metabolic state, etc. Once EVs reach their target cell, they can modulate processes such as gene expression, metabolism, response to external factors, and can even be associated with the delivery of molecules involved in epigenetic inheritance processes in germ cells. In mammalian reproduction, EVs have been shown to play an important role, either in vivo or in vitro , modulating a variety of processes in sperm, oocytes and embryos, and in their respective environments. Moreover, EVs represent a biodegradable, harmless and specific vehicle, which makes them attractive allies to consider when improving assisted reproductive technologies (ARTs). Therefore, the present review aims to describe the content of the main EVs involved in mammalian reproduction and how they can vary due to different factors, as well as to detail how EVs modulate, directly or indirectly, different molecular processes in gametes and embryos. In addition, we will highlight the mechanisms that remain to be elucidated. We will also propose new perspectives according to the characteristics of each particular EV to improve the different ARTs.

Buffalo has many excellent economic traits and it is one of the greatest potential livestock. Compared with cattle, buffalo has poorer reproductivity, it is of great significance to improve the development potential of oocytes. Buffalo oocyte in vitro maturation (IVM) has been widely used in production, but the poor development ability of bovine oocytes IVM limits the development of buffalo reproductivity. Milrinone as a phosphodiesterase inhibitor could affect the maturation of oocytes in goat and mice, but there have been few reported studies in water buffalo. To optimize buffalo oocyte in vitro maturation systems, the effects of phosphodiesterase inhibitor (milrinone) on pre-maturation culture of buffalo oocytes were investigated in this study. Buffalo cumulus-oocyte complexes (COCs) were cultured in medium with different concentrations (0, 12, 25, 50 and 100 mol/l) of milrinone for different times (0, 4, 8, 12, 16, 22 and 24 h). The results showed that the buffalo COCs nuclear maturation process could be inhibited by milrinone (25-100 mol/l) in a dose-dependent manner. The inhibitory effect of milrinone on in vitro maturation of buffalo oocytes did not decrease with the extension of time. This indicated that milrinone can be used as a nuclear maturation inhibitor during the maturation process in buffalo oocytes. In addition, milrinone can inhibit the effect of follicle stimulating hormone (FSH)-induced IVM of buffalo oocytes, but with time FSH partially eliminated the inhibition. Therefore, inhibition of milrinone on the nuclear maturation of buffalo oocytes was reversible, and buffalo oocytes can mature normally after the inhibition is lessened.

This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

This study aimed to describe outcomes in four women aged 28-34 years with central cytoplasmic granulation (CCG) of the oocytes who underwent in vitro fertilization/intracytoplasmic sperm injection (ICSI) using gonadotrophin-releasing hormone (GnRH) agonist to replace human chorionic gonadotrophin (hCG) as a trigger of final oocyte maturation. The initial ICSI procedure showed that all four women had CCG of the ooplasm and poor quality embryos. Subsequent ICSI used an antagonist protocol with a GnRH agonist trigger replacing the agonist protocol, plus hCG triggered ovulation. Ooplasm and embryo quality were improved in all four patients. All four became pregnant and gave birth to live infants. This study provides GnRH agonist triggering that may improve ooplasm granularity and embryo quality.

The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

The process of embryonic development is crucial and radically influences preimplantation embryo competence. It involves oocyte maturation, fertilization, cell division and blastulation and is characterized by different key phases that have major influences on embryo quality. Each stage of the process of preimplantation embryonic development is led by important signalling pathways that include very many regulatory molecules, such as primary and secondary messengers. Many studies, both in vivo and in vitro, have shown the importance of the contribution of reactive oxygen species (ROS) as important second messengers in embryo development. ROS may originate from embryo metabolism and/or oocyte/embryo surroundings, and their effect on embryonic development is highly variable, depending on the needs of the embryo at each stage of development and on their environment (in vivo or under in vitro culture conditions). Other studies have also shown the deleterious effects of ROS in embryo development, when cellular tissue production overwhelms antioxidant production, leading to oxidative stress. This stress is known to be the cause of many cellular alterations, such as protein, lipid, and DNA damage. Considering that the same ROS level can have a deleterious effect on the fertilizing oocyte or embryo at certain stages, and a positive effect at another stage of the development process, further studies need to be carried out to determine the rate of ROS that benefits the embryo and from what rate it starts to be harmful, this measured at each key phase of embryonic development.

Tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are cytokines that are involved in the development, proliferation and apoptosis of ovarian follicular cells in domestic mammals. The expression of these cytokines in various follicular compartments, depending on the stage of follicle development, demonstrates their involvement in the control of primordial follicle growth up to the preovulatory stage. The mechanism of action of these factors depends on the presence of their receptors that transduce their biological actions. This review shows the expression sites of TNF-α, IL-1β and their receptors in ovarian follicles, and discusses the mechanism of action of these cytokines during follicle development, oocyte maturation and ovulation in domestic animals.

Infertility is the most common issue in the field of reproductive medicine. Many factors affect fertility status, including life-style choices such as cigarette smoking or alcohol use. The aim of this study was to investigate the effects of cigarette smoking on oocyte quality as well the quantity in young fertile women. In total, 56 young fertile women who were undergoing oocyte donation programmes were included in this study. The effects of cigarette smoking on antral follicle count, number of follicles and oocytes retrieved and morphology were assessed by an expert embryologist. The results of this study showed that cigarette smoking did not have a significant effect on the follicle count or the number of oocytes retrieved from young and fertile women. However, a significant difference was observed in the morphological assessment. In conclusion, although cigarette smoking does not seem to affect oocyte development, it had an effect on cytoplasmic anomalies and therefore may lower pregnancy chance. Therefore, it is crucial to give proper counselling to patients who are trying to become pregnant both naturally and by in vitro fertilization.

It is widely accepted that the oocyte plays a very active role in promoting the growth of the follicle by directing the differentiation of granulosa cells and secreting paracrine growth factors. In turn, granulosa cells regulate the development of the oocytes, establishing close bidirectional communication between germ and somatic cells. The presence of cortical cells with morphological characteristics, similar to primordial germ cells that express specific germline markers, stem cells and cell proliferation, known as adult cortical germ cells (ACGC) have been reported in phyllostomid bats. Using magnetic cell separation techniques, dissociation-cellular re-aggregation and organ culture, the behaviour of oocytes and ACGC was analyzed by interacting in vitro with mouse ovarian cells. Bat ACGC was mixed with disaggregated ovaries from a transgenic mouse that expressed green fluorescent protein. The in vitro reconstruction of the re-aggregates was evaluated. We examined the viability, integration, cellular interaction and ovarian morphogenesis by detecting the expression of Vasa, pH3, Cx43 and Laminin. Our results showed that the interaction between ovarian cells is carried out in the adult ovary of two species, without them losing their capacity to form follicular structures, even after having been enzymatically dissociated.

This study aims to develop an in vitro co-culture system of in situ goat preantral follicles with bone marrow-derived mesenchymal stem cells (BM-MSC), evaluating the influence of these cells on follicular growth, rate of activation and morphologically normal follicles. Fragments of ovarian cortex were cultured for 1 or 7 days in the presence of BM-MSC (BM-MSC+) and absence of BM-MSC (BM-MSC-). Histological sections of the fragments were analysed and data were obtained regarding morphological classification, survival rate of morphologically normal follicles and rate of follicular activation. Culture medium on days 1 and 7 was also sampled for nitrite concentration and reduced glutathione activity. There was a reduction (P < 0.05) in the percentage of morphologically normal follicles in the BM-MSC+ compared with the fresh control only on the seventh day of culture. When comparing treatments, on the seventh day of culture, a higher rate of morphologically normal preantral follicles was observed in BM-MSC+ (P < 0.05). In both treatments, primordial and developing follicle rates were similar to the fresh control (P > 0.05). When comparing treatments with each other, as well as with the fresh control, no differences were observed in follicular diameter (P > 0.05) or nitrite concentration (P > 0.05). The concentration of reduced glutathione was lower on the seventh day of co-culture in both treatments (P < 0.05). In conclusion, co-culture had no influence on follicular or oocyte development. However, it was critical to maintain the survival of preantral follicles during 7 days of culture.

This study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA-dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA-dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA-dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA-dLCr 1:25 and the controls. The DNA-dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA-dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA-dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

Activated pERK1/2 and pAKT are key players in supporting cell survival and proliferation pathways. Translocation of pERK1/2 into the nucleus, where it interacts with transcription factors and DNA itself, is instrumental in exerting an anti-apoptotic effect. In this study, pAKT levels, pERK1/2 nuclear localization and DNA fragmentation index (DFI) in cumulus cells of single cumulus-oocyte complexes of patients undergoing in vitro fertilization programmes were evaluated and correlated with the clinical outcome of the related embryos. For a positive clinical outcome of blastocyst development, pERK1/2 nuclear localization and DFI value had a significant inverse relationship, whereas the former and the intracellular accumulation of pAKT had a significant direct relationship. This relationship was not observed for the negative clinical outcome of the arrested embryos. Moreover, intracellular accumulation of pAKT and DFI value had a significant inverse relationship in all samples examined. The obtained data suggest that the intranuclear relocation of pERK1/2, along with an enhanced intracellular accumulation of pAKT, may exert a survival effect and increase cell viability, therefore providing a novel marker tool to choose the best oocyte to be fertilized and submitted to an intracytoplasmic sperm injection cycle.

The aim of this study was to investigate if there is an adverse effect of multiple controlled ovarian stimulation (COS) on the maturity of oocytes (MI and MII), fertilization rate, embryo developmental qualities and clinical pregnancy rates in donation cycles. In total, 65 patients undergoing oocyte donation cycles multiple times were included in this study. Patients were grouped as group A that consisted of donors with ≤2 stimulation cycles while B consisted of donors with ≥3 stimulation cycles; and group C included donors who had ≤15 oocytes, while group D had donors with ≥16 oocytes. Numbers of oocytes obtained, MI and MII oocytes, fertilization, embryo quality and clinical pregnancy outcomes were compared. Significant statistical differences were observed in total number of oocytes obtained, maturity of oocytes (MI and MII), fertilization rate, embryo qualities and clinical pregnancy outcomes of donors in groups A-D. Donors with ≤2 ovarian stimulation cycles had lower numbers of immature oocytes than donors with three or more stimulation cycles. However, donors with ≥3 stimulation cycles had higher numbers of mature oocytes, zygotes, with better day 3 embryo qualities and higher clinical pregnancy rates than donors with ≤2 stimulation cycles. Repeated COS does not seem to have any adverse effect on ovarian response to higher dose of artificial gonadotropin, as quality of oocytes collected and their embryological developmental potential were not affected by the number of successive stimulation cycles. The effect of multiple COS on the health of the oocyte donor needs to be assessed for future purpose.

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 μM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 μm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.

Around 60-80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus-oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

The present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus-oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 μM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.

SummaryDifferent parameters affect the success of assisted reproduction technology (ART) treatments. One of the advantages of using intracytoplasmic sperm injection (ICSI) is that it also enables the assessment of the oocyte morphology. To date there has not been a clear conclusion on aberrant oocyte morphology and its consequences on the success of ART treatments. Therefore, in this study, we aimed to investigate the fertilization, embryo development and pregnancy rates in patients who have oocytes with granular cytoplasm. Additionally, we investigated if there were more aneuploid embryos obtained from abnormal cytoplasmic morphology. In total, 5704 oocytes were collected and, of these, 4036 were metaphase II (MII) oocytes. The morphology of these oocytes was assessed following denudation and 970 oocytes were observed to have granular cytoplasm. There was no difference in the fertilization rates between the oocytes with normal cytoplasm (89%) and oocytes with granular cytoplasm (72%). Cleavage of embryos and the number of embryos that reached the blastocyst stage were also similar in these two groups. The aneuploidy rates between the two groups were also similar. However, clinical pregnancies were significantly lower in embryos obtained from oocytes with granular cytoplasm (37.5% vs 70%, P&lt;0.05). Therefore, the morphology of the oocyte is as important as morphology of the sperm. Even though normal fertilization and cleavage were achieved from oocytes with granular cytoplasm, their implantation potential was significantly compromised.

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P &lt;0.05). Blastocyst development was low in vitrified oocytes compared with controls (&lt;6% vs 38.9%, P &lt;0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P &lt;0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P &lt;0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P &lt;0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P &lt;0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

SummaryGerm plasm-related structures (GPRS) are known to accompany meiotic cell differentiation but their dynamics are still poorly understood. In this study, we analyzed the ultrastructural mechanisms of GPRS transformation during oogenesis and spermatogenesis of the bivalve mollusc Ruditapes philippinarum (Manila clam), exploring patterns of GPRS activity occurring at meiosis onset, sex-specific difference/similarity of such patterns, and the involvement of mitochondria during GPRS-assigned events. In the two sexes, the zygotene-pachytene stage of meiosis is anticipated by three shared steps. First, the dispersion of germ plasm granules containing the germ line determinant VASA occurs. Second, the VASA protein deriving from germ plasm granules enters neighbouring mitochondria and appears to induce mitochondrial matter release, as supported by cytochrome B localization outside the mitochondria. Third, intranuclear VASA entrance occurs and the protein appears involved in chromatin reorganization, as supported by VASA localization in synaptonemal complexes. In spermatogenesis, these three steps are sufficient for the normal course of meiosis. In oogenesis, these are followed by the action of 'germ plasm granule formation complex', a novel type of structure that appears alternative to the Balbiani body. The possibility of germ plasm involvement in reproductive technologies is also suggested.

SummaryThe aim of this study was to determine the capacity of the meiotic spindle (MS) to predict embryo implantation and live birth rates. For this purpose, we performed a broad systematic literature search. Of all publications retrieved, only those in which the implantation rates were related to some characteristics of the MS were evaluated. Despite the different methodology used in all the chosen studies, presence of the MS in oocytes was found to be positively associated with embryo implantation. Moreover, high retardance values, as well as strict criteria of normality in the MS structure, are significantly related to higher embryo implantation numbers and live birth rates.

SummaryIn this study we analyzed whether the in vivo storage of oocytes (time after ovulation until fertilization) affects the survival and the ploidy status of the yellowtail tetra Astyanax altiparanae. Fish were induced to spawn and, after ovulation, a small aliquot was stripped and immediately fertilized (positive control group). Subsequently, aliquots (~150 oocytes) were stripped and fertilized at various time points of 60, 120, 180 or 240 min. Developmental stages, abnormalities, survival and the ploidy status of the hatched larvae were examined. As expected, in the control group, 100% of the larvae were diploid. Conversely, triploid individuals were observed just at the 60 min treatment time point (0.6%). In vivo storage of oocytes also influenced the survival rates (P &lt; 0.05); the 180 and 240 min samples, respectively, presented lower survival rates at gastrula (50.10±6.26% and 40.92±5.32%), and somite (17.80±5.14% and 4.41±2.76%) stages and lower hatching rates (12.01±4.04% and 4.41±2.76%). A higher percentage (99.27±0.40%) of normal larvae and only a few abnormal larvae (0.73±0.40%) were observed in the control group (P = 0.0000). This observation did not differ from that observed at the 60 min treatment point (P = 0.9976). A significant increase in the percentage of abnormalities was observed in the other treatments, and, after 240 min, the highest percentage of abnormal larvae was seen (P=0.0024; 83.33±16.67%). In conclusion, we showed that oocyte ageing had a significant effect on survival and may affect the ploidy status in A. atiparanae.

SummaryTRIM28/KAP1/TIF1β was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (P&lt;0.05). We further show that TRIM28 is highly expressed during oocyte maturation and reaches peak levels at the 2-cell stage. In contrast, at this stage, TRIM28 expression in somatic cell nuclear transfer (SCNT) embryos decreased significantly (P&lt;0.05), suggesting that Trim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.

SummaryThe present study aimed to investigate the effect of quercetin as an alternative antioxidant to cysteamine on in vitro maturation. Oocytes were collected from goat ovaries, destined for in vitro maturation and distributed into three groups: CIS group, oocytes were immersed in MIV base medium; in Groups Q4 and Q8, oocytes were immersed in the medium of the CIS group, adding 4 μM or 8 μM of quercetin, respectively, and cultured for 24 h at 38.5°C with 5% CO2. The CIS and Q4 groups presented the same percentage of expanded cumulus cells, but the per cent in the Q8 group was significantly lower than that of the other groups (P&lt;0.05). The oocyte retraction rate in the Q8 group was higher (P&lt;0.05) than in the CIS and Q4 groups. Treatment with 8 μM of quercetin presented a lower proportion of expanded oocytes than the CIS group and 4 μM of quercetin (P&lt;0.05). The percentage of MII oocytes was higher in the Q4 group than in the CIS group (P&lt;0.05), but the percentages in the CIS and Q8 groups were similar. The rate of apoptosis was higher in the CIS group than in the other groups (P&lt;0.05). In addition, oocytes matured with 4 μM quercetin showed higher mitochondrial activity than matured oocytes in the CIS and Q8 groups (P&lt;0.05). In conclusion, 4 μM of quercetin can be used as an alternative to cysteamine in the in vitro maturation of goat oocytes.

SummaryThe high miscarriage rates that result following transfer of embryos derived from in vitro maturation (IVM) of oocytes necessitate improvements in the processes involved. This study aimed to improve the quality of in vitro matured oocytes using granulosa cell conditioned medium (GCCM) as the culture medium. In this work, germinal vesicle (GV)-stage oocytes from NMRI mice were collected and cultured using three types of culture medium: Base medium (BM) (control), 50% granulosa cell conditioned medium (GCCM50) and 100% GCCM (GCCM100). After IVM, the mitochondria activity potential and viability of metaphase II (MII) oocytes were evaluated by JC-1 and trypan blue staining, respectively. Maturational gene expression levels of CyclinB1, Cdk1 and Gdf9 in the control, GCCM50 and GCCM100 samples were analyzed using real-time polymerase chain reaction (PCR). The viability rate of in vitro matured oocytes was highest in the GCCM50 group. JC-1 staining showed that GCCM50 enhances mitochondrial activity more than the other groups (P < 0.05). Gene expression levels of Cdk1 and Gdf9 were higher in the group with GCCM50 treatment, than in the control and GCCM100 groups (P < 0.05), while the expression level of CyclinB1 did not differ among the groups. The results indicated that a 50% concentration of GCCM in combination with BM components enhanced MII and viability rates and mitochondria activity of mouse immature oocytes.

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.