Treatment with acetyl-l-carnitine during in vitro maturation of buffalo oocytes improves oocyte quality and subsequent embryonic development.

Oocyte quality is one of the important factors in female fertility, in vitro maturation (IVM), and subsequent embryonic development. In the present study, we assessed whether acetyl-l-carnitine (ALC) supplementation during in vitro maturation of buffalo oocytes could improve oocyte quality and subsequent embryonic development. To determine the optimal level of ALC supplementation, we matured cumulus-oocyte complexes in maturation medium supplemented with 0, 2.5, and 5 mM ALC. The oocytes with a polar body were selected for parthenogenetic activation (PA) and in vitro fertilization (IVF). We found that oocytes matured in 2.5 mM ALC had significantly higher PA blastocyst rate (P < 0.05) and blastocyst cell number than those of unsupplemented oocytes (P < 0.05) and a significantly higher IVF blastocyst rate than that of oocytes matured in 5 mM ALC (P < 0.05). In all further experiments, we supplemented the maturation medium with 2.5 mM ALC. We then tested whether ALC supplementation could improve various markers of oocytes and cumulus cells. We compared cell proliferation; concentrations of reactive oxygen species (ROS), intracellular ATP, estradiol, and progesterone; mitochondrial distribution; mitochondrial DNA copy number (mtDNA); and expression levels of four genes encoding oocyte-derived factors (GDF9, BMP15) and steroid hormones (StAR, P450scc) between the supplemented and unsupplemented oocytes and cumulus cells. Cumulus cells matured with ALC supplementation were more prolific than those matured without ALC supplementation (P < 0.05). Oocytes treated with ALC had lower concentrations of intracellular ROS (P < 0.05) and a higher rate of diffuse mitochondrial distributions (P < 0.05) than those of untreated oocytes. Additionally, the mtDNA was higher in the ALC-treated oocytes (P < 0.05) and cumulus cells (P < 0.05) than that in the untreated cells. The ALC-treated maturation medium had a higher postmaturation concentration of estradiol than that of the untreated medium (P < 0.05). Finally, the gene expression levels of P450scc and GDF9 were greater in ALC-treated oocytes and cumulus cells than those in untreated cells (P < 0.05). Therefore, in buffalo, our results suggest that ALC affects mitochondrial function, regulates oocyte-derived paracrine factors, and increases the production of steroid hormones, leading to increased quality of matured oocytes and improved embryonic development in vitro.

Xu HY ，Yang XG ，Lu SS ，Liang XW ，Lu YQ ，Zhang M ，Lu KH ... -  《-》

GDF8 activates p38 MAPK signaling during porcine oocyte maturation in vitro.

Growth Differentiation Factor 8 (GDF8) is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator. This factor is expressed as a paracrine factor in mural granulosa cells. To investigate the effects of GDF8 on the in vitro maturation (IVM) of porcine oocytes, we assessed the quality of matured oocytes as well as the specific gene transcription and protein activation levels in oocytes and cumulus cells (CCs) after IVM and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). Supplemental concentrations (0, 1, 10, and 100 ng/ml) of GDF8 were provided in IVM medium. Supplementation with GDF8 during IVM induced transcription of specific TGF-β receptor genes, such as ActRIIb and Alk4/5, and the recognition of the GDF8 by these receptors induced phosphorylation of p38 MAPK. Activated p38 MAPK signaling changed oocyte maturation and cumulus expansion-related gene transcription: Nrf2 and Bcl-2 in oocytes and PCNA, Nrf2, Has2, Ptx3, and TNFAIP6 in CCs. The altered gene expression pattern during IVM resulted in a 10% lower level of intracellular ROS in mature oocytes. The improved cytoplasmic maturation led to an increase in the fertilization efficiency and subsequent embryonic developmental competence. The embryonic development showed increases in the blastocyst formation rate and higher transcription levels of POU5F1 and BCL-2 in the blastocysts. The present study suggests that supplementation of GDF8 during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by reducing the intracellular ROS level in oocytes by altering the transcription of specific genes and increasing the phosphorylation of p38 MAPK during IVM. In conclusion, for the first time, our results demonstrate that GDF8 can act as a paracrine factor to modulate oocyte maturation by regulating p38 MAPK phosphorylation and intracellular ROS level during porcine IVM.

Yoon JD ，Hwang SU ，Kim E ，Jin M ，Kim S ，Hyun SH ... -  《-》

Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes.

Hot season is a major constraint to production and reproduction in buffaloes. The present work aimed to investigate the effect of season on ovarian function, developmental competence, and the relative abundance of gene expression in buffalo oocytes. Three experiments were conducted. In experiment 1, pairs of buffalo ovaries were collected during cold season (CS, autumn and winter) and hot season (HS, spring and summer), and the number of antral follicles was recorded. Cumulus oocyte complexes (COCs) were aspirated and evaluated according to their morphology into four Grades. In experiment 2, Grade A and B COCs collected during CS and HS were in vitro matured (IVM) for 24 hours under standard conditions at 38.5 °C in a humidified air of 5% CO2. After IVM, cumulus cells were removed and oocytes were fixed, stained with 1% aceto-orcein, and evaluated for nuclear configuration. In vitro matured buffalo oocytes harvested during CS or HS were in vitro fertilized (IVF) using frozen-thawed buffalo semen and cultured in vitro to the blastocyst stage. In experiment 3, buffalo COCs and in vitro matured oocytes were collected during CS and HS, and then snap frozen in liquid nitrogen for gene expression analysis. Total RNA was extracted from COCs and in vitro matured oocytes, and complementary DNA was synthesized; quantitative Reverse Transcription-Polymerase Chain Reaction was performed for eight candidate genes including GAPDH, ACTB, B2M, GDF9, BMP15, HSP70, and SOD2. The results indicated that HS significantly (P < 0.01) decreased the number of antral follicles and the number of COCs recovered per ovary. The number of Grade A, B, and C COCs was lower (P < 0.05) during HS than CS. In vitro maturation of buffalo oocytes during HS significantly (P < 0.01) reduced the number of oocytes reaching the metaphase II stage and increased the percentage of degenerated oocytes compared with CS. Oocytes collected during HS also showed signs of cytoplasmic degeneration. After IVF, cleavage rate was lower (P < 0.01) for oocytes collected during HS, and the percentage of oocytes arrested at the two-cell stage was higher (P < 0.01) than oocytes IVF during CS. Oocytes matured during CS showed a higher (P < 0.01) blastocyst rate than those matured during HS. Also, COCs recovered in HS showed significant (P < 0.05) upregulation of HSP70 mRNA expression compared with those recovered in CS. For in vitro matured oocytes, CS down regulated the transcript abundance of ACTB and upregulated GAPDH and HSP70 mRNA levels compared with HS condition. In conclusion, HS could impair buffalo fertility by reducing the number of antral follicles and oocyte quality. In vitro maturation of buffalo oocytes during HS impairs their nuclear and cytoplasmic maturation, fertilization, and subsequent embryo development to the morula and blastocyst stages. This could be in part because of the altered gene expression found in COCs and in vitro matured oocytes.

Abdoon AS ，Gabler C ，Holder C ，Kandil OM ，Einspanier R ... -  《-》

A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P < 0.05. Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 ± 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 ± 7.4% versus 53.3 ± 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 ± 9%, 37.2 ± 9.5% and 17.2 ± 8.3%, respectively), although fetal development did not differ. Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. N/A. This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.

Santiquet NW ，Greene AF ，Becker J ，Barfield JP ，Schoolcraft WB ，Krisher RL ... -  《-》

Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes.

In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions between follicular mural somatic cells and COCs. To restore the microenvironment, a coculture system was established using cumulus-derived somatic cells (CSCs) for IVM. The CSCs were cultured in Dulbecco's modified Eagle's medium for 48 hours with varying numbers of CSCs (0.0, 2.5 × 10(4), 5.0 × 10(4), and 10.0 × 10(4)) and then cultured in tissue culture medium 199 (TCM 199) for 4 hours before adding the oocytes. Cumulus-oocyte complexes from 3- to 6-mm follicles were matured in 500 μL of TCM 199 with eCG and hCG for 22 hours and then cultured in TCM 199 without hormones for 22 hours. After IVM, the group with 2.5 × 10(4) CSCs showed a significant increase in intracellular glutathione levels compared with the control group. In the evaluation of sperm penetration, efficient fertilization was increased in the groups with 2.5 × 10(4) and 5.0 × 10(4) CSCs compared with controls (44.9 and 46.5 vs. 32.1, respectively). The mRNA expression pattern analysis in matured COCs showed a significant upregulation of PCNA, COX-2, Has2, Ptx3, and Nrf2 in the 2.5 × 10(4) CSC group compared with controls. During COC maturation at 0, 11, 22, 33, and 44 hours, the 2.5 × 10(4) and 5.0 × 10(4) CSC groups showed a significantly altered mRNA expression of BMP15 and GDF9. The developmental competence of the matured oocytes in all groups was evaluated after IVF and parthenogenetic activation (PA). After IVF, the 2.5 × 10(4) CSC group showed significantly higher cleavage, blastocyst formation rate, and total cell numbers compared with controls (60.0%, 35.7%, and 127.3 vs. 43.2%, 21.1%, and 89.3, respectively). After PA, the 2.5 × 10(4) CSC group had significantly higher blastocyst formation rate and total cell number than the control group (52.0% and 120.4 vs. 35.4% and 90.9, respectively). In conclusion, these results suggest that the presence of a population of 2.5 × 10(4) CSCs during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by increasing the intracellular glutathione level via changing of a specific gene expression pattern during oocyte maturation.

Yoon JD ，Jeon Y ，Cai L ，Hwang SU ，Kim E ，Lee E ，Kim DY ，Hyun SH ... -  《-》