Growth differentiation factor 8 regulates SMAD2/3 signaling and improves oocyte quality during porcine oocyte maturation in vitro†.

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 μM were added as control, SB, SB + GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.

Yoon JD ，Hwang SU ，Kim M ，Jeon Y ，Hyun SH ... -  《-》

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 ... -  《-》

Disruption of bidirectional oocyte-cumulus paracrine signaling during in vitro maturation reduces subsequent mouse oocyte developmental competence.

Yeo CX ，Gilchrist RB ，Lane M 《-》

GDF8 enhances SOX2 expression and blastocyst total cell number in porcine IVF embryo development.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.

Yoon JD ，Hwang SU ，Kim M ，Lee G ，Jeon Y ，Hyun SH ... -  《-》

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 ... -  《-》