Maturation Of Cumulus-oocyte Complexes Research Articles

Effects of MEK1/2 inhibitor U0126 on FGF10-enhanced buffalo oocyte maturation in vitro

Fibroblast growth factor 10 (FGF10) plays critical roles in oocyte maturation and embryonic development; however, the specific pathway by which FGF10 promotes in vitro maturation of buffalo oocytes remains elusive. The present study was aimed at investigating the mechanism underlying effects of the FGF10-mediated extracellular regulated protein kinases (ERK) pathway on oocyte maturation and embryonic development in vitro. MEK1/2 (mitogen-activated protein kinase kinase) inhibitor U0126, alone or in combination with FGF10, was added to the maturation culture medium during maturation of the cumulus oocyte complex. Morphological observations, orcein staining, apoptosis detection, and quantitative real-time PCR were performed to evaluate oocyte maturation, embryonic development, and gene expression. U0126 affected oocyte maturation and embryonic development in vitro by substantially reducing the nuclear maturation of oocytes and expansion of the cumulus while increasing the apoptosis of cumulus cells. However, it did not have a considerable effect on glucose metabolism. These findings suggest that blocking the MEK/ERK pathway is detrimental to the maturation and embryonic development potential of buffalo oocytes. Overall, FGF10 may regulate the nuclear maturation of oocytes and cumulus cell expansion and apoptosis but not glucose metabolism through the MEK/ERK pathway. Our findings indicate that FGF10 regulates resumption of meiosis and expansion and survival of cumulus cells via MEK/ERK signaling during in vitro maturation of buffalo cumulus oocyte complexes. Elucidation of the mechanism of action of FGF10 and insights into oocyte maturation should advance buffalo breeding. Further studies should examine whether enhancement of MEK/ERK signaling improves embryonic development in buffalo.

Biochemical profiling of the follicular environment to predict oocyte competence in cattle.

To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.

Heavy Metals in Follicular Fluid Affect the Ultrastructure of the Human Mature Cumulus-Oocyte Complex.

It is known that exposure to heavy metal such as lead (Pb) and cadmium (Cd) has several adverse effects, particularly on the human reproductive system. Pb and Cd have been associated with infertility in both men and women. In pregnant women, they have been associated with spontaneous abortion, preterm birth, and impairment of the development of the fetus. Since these heavy metals come from both natural and anthropogenic activities and their harmful effects have been observed even at low levels of exposure, exposure to them remains a public health issue, especially for the reproductive system. Given this, the present study aimed to investigate the potential reproductive effects of Pb and Cd levels in the follicular fluid (FF) of infertile women and non-smokers exposed to heavy metals for professional reasons or as a result of living in rural areas near landfills and waste disposal areas in order to correlate the intrafollicular presence of these metals with possible alterations in the ultrastructure of human cumulus-oocyte complexes (COCs), which are probably responsible for infertility. Blood and FF metals were measured using atomic absorption spectrometry. COCs corresponding to each FF analyzed were subjected to ultrastructural analyses using transmission electron microscopy. We demonstrated for the first time that intrafollicular levels of Pb (0.66 µg/dL-0.85 µg/dL) and Cd (0.26 µg/L-0.41 µg/L) could be associated with morphological alterations of both the oocyte and cumulus cells' (CCs) ultrastructure. Since blood Cd levels (0.54 µg/L-1.87 µg/L) were above the current reference values established by the guidelines of the Agency for Toxic Substances and Disease Registry (ATSDR) and the Environmental Protection Agency (EPA) (0.4 µg/L), whereas blood Pb levels (1.28 µg/dL-3.98 µg/dL) were below the ATSDR reference values (≤5 µg/dL), we believe that these alterations could be due especially to Cd, even if we cannot exclude a possible additional effect of Pb. Our results highlighted that oocytes were affected in maturation and quality, whereas CCs showed scarcely active steroidogenic elements. Regressing CCs, with cytoplasmic alterations, were also numerous. According to Cd's endocrine-disrupting activity, the poor steroidogenic activity of CCs might correlate with delayed oocyte cytoplasmic maturation. So, we conclude that levels of heavy metals in the blood and the FF might negatively affect fertilization, embryo development, and pregnancy, compromising oocyte competence in fertilization both directly and indirectly, impairing CC steroidogenic activity, and inducing CC apoptosis.

Exploring the Impact of Controlled Ovarian Stimulation and Non-Invasive Oocyte Assessment in ART Treatments.

Invasive and noninvasive features are normally applied to select developmentally competent oocytes and embryos that can increase the take-home baby rates in assisted reproductive technology. The noninvasive approach mainly applied to determine oocyte and embryo competence has been, since the early days of IVF, the morphological evaluation of the mature cumulus-oocyte complex at the time of pickup, first polar body, zona pellucida thickness, perivitelline space and cytoplasm appearance. Morphological evaluation of oocyte quality is one of the options used to predict successful fertilization, early embryo development, uterine implantation and the capacity of an embryo to generate a healthy pregnancy to term. Thus, this paper aims to provide an analytical revision of the current literature relating to the correlation between ovarian stimulation procedures and oocyte/embryo quality. In detail, several aspects of oocyte quality such as morphological features, oocyte competence and its surrounding environment will be discussed. In addition, the main noninvasive features as well as novel approaches to biomechanical parameters of oocytes that might be correlated with the competence of embryos to produce a healthy pregnancy and live birth will be illustrated.

Effects of cyclic adenosine monophosphate modulating agents during oocyte pre-maturation and the role of melatonin on in vitro maturation of bovine cumulus-oocyte complexes

This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10−6, 10−7 or 10−8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10−8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10−7 or 10−8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10−6 or 10−8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10−8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10−8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.

FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. N/A.

Pre-maturation of oocytes for equine cloning using cAMP modulators

The asynchrony of cytoplasmic and nuclear maturation in cumulus oocyte complexes (COCs) due to decreased concentrations of cyclic adenosine phosphate (cAMP) has been shown to result in decreased oocyte developmental competence (Rose et al., Theriogenology. 2013;79: 142–148). The aim of this study was to evaluate the effect of pre-maturation treatment with cAMP modulators, previously shown to delay spontaneous nuclear maturation, on the developmental competence of oocytes used for equine cloning. Equine ovaries were obtained from a slaughterhouse and processed within 1-2h. The COCs were washed three times with H-SOF medium and transferred to cryovials filled with a 1:1 mixture of DMEM-F12 and TCM-199 containing 10% FCS and kept at 20-22°C. Groups of COCs were either untreated (control) or treated with 50 mM forskolin (FSK) and 100 mM 3-isobutyl-1-methylxanthine (IBMX) for 4 or 18h. Immature oocytes from all three groups were washed 3 times with H-SOF medium, then transferred to maturation medium, and incubated for 18h at 38.5°C in a humidified atmosphere of 5% CO2 in air. A total of 715 oocytes were collected in 6 replicates (191 oocytes inthe 4h FSK-IBMX group, 239 in the 18h FSK-IBMX group, and 285 oocytes in the control). The methods used for in vitro maturation (IVM), SCNT, and embryo culture were identical for all three groups. Six different fibroblast cell lines were used to provide the donor nuclei. Meiotic maturation, fusion, cleavage, and embryonic development in vitro were all evaluated. Data were analyzed using the Genstat 18th Edition statistical software package. While the meiotic maturation and cleavage rates were similar for the three groups, the proportion of fused couplets that developed to the blastocyst stage was lower for the 4 FSK-IBMX group (15.4 ± 5.7%) compared to the control group (25.6 ± 6.4%; P<0.05). Interestingly, the fusion rates for oocytes of the 4 and 18h FSK-IBMX groups (90.3 ± 4.7% and 94.3 ± 2.6%, respectively) were greater than that for oocytes of the control group (79.1 ± 2.1%; P<0.05). As a result, the pre-maturation treatment did not reduce the blastocyst yield from couplets reconstructed. Further studies are needed to determine the impact of such cAMP modulating treatments on embryos’ in vivo viability following transfer to recipient mares.

Ameliorating Effect of Sodium Selenite on Developmental and Molecular Response of Bovine Cumulus-Oocyte Complexes Matured in Vitro Under Heat Stress Condition.

Selenium (Se), an essential trace element, plays an important role in the antioxidative defense mechanism, and it has been proven to improve fertility and reproductive efficiency in dairy cattle. The present study evaluated the potential protective action of Se supplement of in vitro maturation (IVM) media on the maturation and subsequent development of bovine cumulus-oocyte complexes (COCs) exposed to heat stress (HS). The treatment with Se improved the viability of cumulus cells (CCs) and oocytes (P < 0.05). The proportion of oocytes reached metaphase II (MII) and those arrested at metaphase I (MI) was greater and lower in treatment than control respectively (P < 0.05). Supplementation with Se increased the percentage of cleaved embryos, total blastocysts, and blastocyst/cleavage ratio (P < 0.05). Moreover, the upregulation of CCND1, SEPP1, GPX-4, SOD, CAT, and downregulation of GRP78, CHOP, and BAX in both Se-treated CCs and oocytes were recorded. The upregulation of NRF2 was detected in Se-treated CCs other than in oocytes, which showed upregulation of IGF2R and SOX-2 as the markers of quality as well. Se supplement in IVM media improved the viability, maturation, and the level of transcripts related to antioxidant defense and quality of heat-treated oocytes, which coincided with greater subsequent development outcomes. Se ameliorated the viability of CCs along with upregulation of antioxidative candidate gene expression and downregulation of apoptosis-related ones to support their protective role on restoring the quality of oocytes against compromising effects of HS.

The Mature COC Promotes the Ampullary NPPC Required for Sperm Release from Porcine Oviduct Cells.

Porcine spermatozoa are stored in the oviductal isthmus after natural mating, and the number of spermatozoa is increased in the oviductal ampulla when the mature cumulus-oocyte complexes (COCs) are transferred into the ampulla. However, the mechanism is unclear. Herein, natriuretic peptide type C (NPPC) was mainly expressed in porcine ampullary epithelial cells, whereas its cognate receptor natriuretic peptide receptor 2 (NPR2) was located on the neck and the midpiece of porcine spermatozoa. NPPC increased sperm motility and intracellular Ca2+ levels, and induced sperm release from oviduct isthmic cell aggregates. These actions of NPPC were blocked by the cyclic guanosine monophosphate (cGMP)-sensitive cyclic nucleotide-gated (CNG) channel inhibitor l-cis-Diltiazem. Moreover, porcine COCs acquired the ability to promote NPPC expression in the ampullary epithelial cells when the immature COCs were induced to maturation by epidermal growth factor (EGF). Simultaneously, transforming growth factor-β ligand 1 (TGFB1) levels were dramatically increased in the cumulus cells of the mature COCs. The addition of TGFB1 promoted NPPC expression in the ampullary epithelial cells, and the mature COC-induced NPPC was blocked by the transforming growth factor-β type 1 receptor (TGFBR1) inhibitor SD208. Taken together, the mature COCs promote NPPC expression in the ampullae via TGF-β signaling, and NPPC is required for the release of porcine spermatozoa from the oviduct isthmic cells.

Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

How in vitro maturation changes the proteome of ovine cumulus-oocyte complexes?

The present study evaluated the effects of in vitro maturation (IVM) on the proteome of cumulus-oocyte complexes (COCs) from ewes. Extracted COC proteins were analyzed by LC-MS/MS. Differences in protein abundances (p &lt; 0.05) and functional enrichments in immature versusin vitro-matured COCs were evaluated using bioinformatics tools. There were 2550 proteins identified in the COCs, with 89 and 87 proteins exclusive to immature and mature COCs, respectively. IVM caused downregulation of 84 and upregulation of 34 proteins. Major upregulated proteins in mature COCs were dopey_N domain-containing protein, structural maintenance of chromosomes protein, ubiquitin-like modifier-activating enzyme 2. Main downregulated proteins in mature COCs were immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain 2, alpha-2-macroglobulin. Proteins exclusive to mature COCs and upregulated after IVM related to immune response, complement cascade, vesicle-mediated transport, cell cycle, and extracellular matrix organization. Proteins of immature COCs and downregulated after IVM were linked to metabolic processes, immune response, and complement cascade. KEGG pathways and miRNA-regulated genes attributed to downregulated and mature COC proteins related to complement and coagulation cascades, metabolism, humoral response, and B cell-mediated immunity. Thus, IVM influenced the ovine COC proteome. This knowledge supports the future development of efficient IVM protocols for Ovis aries.

Physiological and Functional Roles of Neurotrophin-4 During In Vitro Maturation of Porcine Cumulus-Oocyte Complexes.

Neurotrophin-4 (NT-4), a granulosa cell-derived factor and a member of the neurotrophin family, is known to promote follicular development and oocyte maturation in mammals. However, the physiological and functional roles of NT-4 in porcine ovarian development are not yet known. The aim of this study was to investigate the physiological role of NT-4-related signaling in the in vitro maturation (IVM) of porcine cumulus–oocyte complexes (COCs). The NT-4 protein and its receptors were detected in matured porcine COCs via immunofluorescence analysis. NT-4 was shown to promote the maturation of COCs by upregulating NFKB1 transcription via the neurotrophin/p75NTR signaling pathway. Notably, the mRNA expression levels of the oocyte-secreted factors GDF9 and BMP15, sperm–oocyte interaction regulator CD9, and DNA methylase DNMT3A were significantly upregulated in NT-4-treated than in untreated porcine oocytes. Concurrently, there were no significant differences in the levels of total and phosphorylated epidermal growth factor receptor and p38 mitogen-activated protein kinase between NT-4-treated and untreated cumulus cells (CCs); however, the level of phosphorylated ERK1/2 was significantly higher in NT-4-treated CCs. Both total and phosphorylated ERK1/2 levels were significantly higher in NT-4-treated than in untreated oocytes. In addition, NT-4 improved subsequent embryonic development after in vitro fertilization and somatic cell nuclear transfer. Therefore, the physiological and functional roles of NT-4 in porcine ovarian development include the promotion of oocyte maturation, CC expansion, and ERK1/2 phosphorylation in porcine COCs during IVM.

Increasing vitamin D levels to improve fertilization rates in cattle.

Recently, interest in supplementing vitamin D (Vit D) to improve aspects of health, mainly in human fertility, has emerged. Still, supplementation of Vit D above the minimum required levels has yet to be explored in cattle despite evidence for Vit D receptors in reproductive tissues. The objective of this study was to establish if a dose-response relationship exists between Vit D exposure and success of in vitro production (IVP) of embryos and, if acute supplementation of Vit D improves pregnancy rates during timed artificial insemination (TAI) of dairy cows. Cumulus-oocyte complexes (COCs) were obtained from ovaries acquired from a local abattoir and cultured in five different IVP treatments from three separate collections (Control, 50, 100, 150, and 200 ng/mL of 1,25(OH)2D3; n = 20-30 COCs/group). In Experiment 2, dairy breed cows (n = 100) were synchronized for TAI with the PresynchOvsynch protocol. Cows received 150,000 IU of Vit D (n = 48) or castor oil as control (n = 53) along with gonadotropin-releasing hormone (GnRH) 24 h before TAI. Serum samples were collected before and 24 h after treatment. A small cohort of cows (n = 4) received the same treatments in two separate cycles and follicular fluid (FF) was collected after 24 h for calcidiol (25OHD) analyses. Increased concentrations of Vit D resulted in decreased rates of maturation of COC (150 and 200 ng/mL vs. control and 50 ng/mL; P = 0.01). Supplementation with 50 ng/mL resulted in greater numbers of early blastocyst and blastocyst stage embryos (P < 0.009). Pregnancy at first breeding did not differ (P = 0.13) between groups, but serum 25OHD increased in treated females after 24 h (P = 0.002). The FF 25OHD levels were reflective of serum levels, however, the observed increase in the treatment cycle (P = 0.04) was parallel to an overall increase in serum 25OHD during the entire second cycle, likely due to increased environmental sunlight exposure (March, control vs. May, treatment). A similar increase in the serum 25OHD in the lactating commercial herd maintained in covered housing was not observed, although experiments were conducted during a similar timeframe. This herd had levels of 25OHD near the low end of sufficiency according to National Research Council (NRC) guidelines. We conclude mild Vitamin D supplementation with concentrations at the higher end of NRC guidelines can improve maturation rates of recovered COCs. However, longer term supplementation may be needed to appreciate any benefits on fertility.

Anti-Müllerian Hormone Inhibits FSH-Induced Cumulus Oocyte Complex In Vitro Maturation and Cumulus Expansion in Mice.

Simple SummaryAnti-Müllerian hormone (AMH) is a homodimeric glycoprotein composed of two identical subunits, which inhibits the recruitment of primordial follicles and the development of antral follicles in females. Anti-Müllerian hormone can be used as a diagnostic and prognostic marker for ovarian reserve, superovulation, embryo quality, and conception rate. However, few studies have focused on the effect of AMH on oocyte maturation. In the present study, we found Anti-Müllerian hormone has no effect on the nuclear maturation and cumulus expansion of cumulus oocyte complexes (COCs), whereas it has an inhibitory effect on follicle-stimulating hormone (FSH)-stimulated COCs nuclear maturation and cumulus expansion. These findings expand our knowledge of the functional role of AMH in modulating folliculogenesis.Anti-Müllerian hormone (AMH) is secreted by the ovaries of female animals and exerts its biological effects through the type II receptor (AMHR2). AMH regulates follicular growth by inhibiting the recruitment of primordial follicles and reducing the sensitivity of antral follicles to FSH. Despite the considerable research on the actions of AMH in granulosa cells, the effect of AMH on the in vitro maturation of oocytes remains largely unknown. In the current study, we showed that AMH is only expressed in cumulus cells, while AMHR2 is produced in both cumulus cells and oocytes. AMH had no significant effect on COCs nuclear maturation, whereas it inhibited the stimulatory effects of FSH on COCs maturation and cumulus expansion. Moreover, AMH treatment effectively inhibited the positive effect of FSH on the mRNA expressions of Hyaluronan synthase 2 (Has2), Pentraxin 3 (Ptx3), and TNF-alpha-induced protein 6 (Tnfaip 6) genes in COCs. In addition, AMH significantly decreased the FSH-stimulated progesterone production, but did not change estradiol levels. Taken together, our results suggest that AMH may inhibit the effects of FSH-induced COCs in vitro maturation and cumulus expansion. These findings increase our knowledge of the functional role of AMH in regulating folliculogenesis.

Melatonin improves meiosis maturation against diazinon exposure in mouse oocytes

AimsOrganophosphorus pesticide diazinon (DZN) has adverse effects on animals and humans by direct contact or the spread of food chain. The antioxidant melatonin has protective effects on female reproduction. This study aimed to explore the effects of DZN on meiosis maturation in mouse cumulus oocyte complexes (COCs) and the effects of melatonin. Main methodsDifferent concentrations of DZN and melatonin were added during the in vitro maturation of COCs. Then we detected the extrusion rate of the first polar body, the number of sperms binding to oocyte, mitochondrial membrane potential, reactive oxygen species (ROS), early apoptosis. Subsequently, the expression of Juno, CX37, CX43 and ERK1/2 were detected by immunofluorescence staining and Western blotting. Key findingsDZN exposure results in the failure of nuclear and cytoplasmic maturation of oocyte meiosis. Destruction of repositioning and function of mitochondria increases the levels of ROS and early apoptosis. The DZN-exposed oocytes express less Juno resulting to bind less sperms than normal. The loss of gap junctions and failure to activate ERK1/2 also contribute to the failure of cytoplasmic maturation. All these ultimately lead to the poor oocyte quality and low fertility. Appropriate melatonin can effectively restore all these defects. SignificanceUnder DZN exposure, melatonin can significantly improve the quality of oocytes, and melatonin promotes oocyte maturation by protecting gap junction and restoring ERK1/2 pathway, which is a new breakthrough for improving female fertility.

Predictors of the ovarian superstimulatory response and oocyte collection in prepubertal heifers

The objectives were to investigate the relationships between antral follicle counts and plasma AMH and FSH at the time of follicular wave emergence in prepubertal calves, and to determine the effects of age and duration of gonadotropin treatment on the ovarian superstimulatory response in pre- and post-pubertal heifers. Hereford crossbred prepubertal (Replicate 1 and 2, n = 20) and post-pubertal heifers (Replicates 1, n = 8; Replicate 2, n = 8) were assigned randomly to 2 treatment groups and given FSH for either 4 or 7 d (25 mg pFSH im at 12-h intervals). Prepubertal heifers were first treated at 4 mo and again at 7 mo of age. Blood samples were collected immediately before the first FSH administration, that was initiated 36 h after follicular ablation. An LH treatment (12.5 mg im) was given 12 h after the last FSH injection. Follicular fluid and cumulus-oocyte complexes (COC) were collected 24 h after LH treatment. At wave emergence, the number of follicles ≥1 mm (AFC, 31.1 ± 4.0 vs 16.2 ± 1.8; P < 0.001) and the plasma concentrations of AMH (606.4 ± 90.5 vs 279.6 ± 28.3 pg/mL; P = 0.001) were higher at 4 than at 7 mo of age, while plasma FSH concentrations did not differ between ages. At oocyte collection, a higher number of follicles ≥6 mm were observed in prepubertal calves at 4 mo of age and post-pubertal heifers than in calves at 7 mo of age (32.4 ± 5.4 and 22.0 ± 2.3 vs 14.9 ± 2.0, respectively; P = 0.003). Intrafollicular concentrations of estradiol were lower (23.7 ± 4.5 vs 144.0 ± 29.5 ng/mL; P < 0.0001) and of progesterone tended to be higher (217.5 ± 29.3 vs 157.0 ± 33.9 ng/mL; P = 0.07) in the 7- than in the 4-d groups. A greater number of COC was collected from calves at 4 mo of age and heifers than the 7-mo-old calves (13.4 ± 2.6 and 6.0 ± 1.0 vs 5.8 ± 1.1, respectively; P = 0.008). Overall, the 7-d FSH treatment tended to result in a greater proportion of expanded COC than the 4-d treatment in calves (50.1 ± 7.7 vs 31.9 ± 6.8%; P = 0.07). In summary, there was a positive relationship between AFC and plasma AMH concentrations at the time of wave emergence. A higher AFC was observed in calves at 4- than 7-mo of age, which resulted in greater ovarian response to gonadotropin treatment. Following an exogenous LH stimulus, COC maturation rates were greater in the 7-d than in the 4-d FSH treatment groups, resulting in collection of a higher proportion of fully expanded COC.

Sequential IVM by CNP preincubation and cooperating of PGE2 with AREG enhances developmental competence of SCNT reconstructs in goat

Developmental competence of in vitro matured cumulus oocyte complexes (COCs) in conventional IVM (C.IVM) is lower than in vivo maturated COCs and is related to unsynchronized nuclear and cytoplasmic maturation. To overcome this dearth, COCs can be exposed to granulosa secreted factors in a two-step system. Therefore, in the first experiment, 1000 nM of C-type natriuretic peptide for 8 h was determined (CAPA), as the best time and concentration to retain oocytes in germinal vesicle stage. This condition, also reduces lipid droplets and increases the expression of ATGL and PLIN2 involved in lipolysis and lipogenesis, respectively. In the second experiment, maturation was stimulated with prostaglandin E2 and amphiregulin for 18 h (CAPA-IVM), and their optimal concentrations based on blastocyst formation rates through in vitro fertilization (IVF) were determined as 1 and 600 nM, respectively. In the third experiment, the in vitro and in vivo developmental competency of SCNT embryos in CAPA-IVM group were determined. Despite similar blastocyst formation rates in IVF and SCNT between CAPA-IVM and C.IVM, the quality of blastocysts were quality was higher in CAPA-IVM, which reflected itself, as higher ICM/TE ratio and also expression of NANOG in SCNT blastocysts. Pregnancy rate, live births rate and SCNT efficiency were not significant between CAPA-IVM and C.IVM groups. Therefore, CAPA-IVM can improve the developmental competency of SCNT derived embryos.

THE RESEARCH OF TLR4 INVOLVEMENT IN SIGNALING PATHWAYS ACTIVATED IN MURINE OVARIAN CELLS UNDER THE INFLUENCE OF LIPOPOLYSACCHARIDE

Toll-like receptor 4 (TLR4) is a widely described transmembrane protein involved in the inflammatory process. Bacterial infection is one of the main factors influencing the change in TLR4 expression. At the same time, the level of TLR4 expression directly correlates with the severity of the process, which in some cases allows considering these receptors as an early markers of infection. Early it was shown disruption of ovarian cell functions under the condi-tions of experimental endotoxemia in mice. Pathological changes of follicular environment of oocytes resulted in impairment of oocyte meiotic maturation. However, involvement of TLR4 in activation of signaling pathways in ovar-ian cells under the influence of lipopolysaccharide (LPS), requires careful study. In the present work we have investi-gated pharmacological effect of TAK242 (inhibitor of TLR4) on oocyte meiotic maturation in vitro under the influ-ence of LPS. The study was done on cumulus oocyte complexes (COCs) and oocytes, denuded from cumulus cells, which were isolated from ovaries of Albino mice. It was shown that LPS inhibited meiotic maturation in vitro of COCs and cumulus-denuded oocytes. However, 15 min pretreatment with TLR4 inhibitor (ТАК242) with the subsequent ad-dition of LPS to the culture medium, improved meiotic maturation of both COCs and cumulus-denuded oocytes. More-over, simultaneous exposure to ТАК242 and LPS did not show any significant differences in meiotic maturation of both COCs and cumulus-denuded oocytes compared with 15 min pretreatment with ТАК242. The results obtained may indicate the presence of TLR4 on murine oocytes and cumulus cells. Also the results can serve as the basis for the re-search of feasibility of therapeutic use of TLR4 inhibitors in diseases in the course of which interaction between TLR4 and its ligands (in particular LPS) occur.

Effect of oestrogen on mouse follicle growth and meiotic resumption.

Many studies have shown that oestrogen affects late follicular development, but whether oestrogen is involved in other aspects of folliculogenesis remains unclear. In this study, two antagonists of oestrogen, tamoxifen and G15, were used to determine the effects of oestrogen on folliculogenesis. Mouse preantral follicles and cumulus-oocyte complexes (COCs) were cultured in vitro. The results showed that follicle growth stimulated using pregnant mare serum gonadotrophin (PMSG) was inhibited using tamoxifen, whether in vivo or in vitro. The average diameters, the maximum diameters of follicles and the numbers of follicles with a diameter of more than 300 μm decreased significantly following a 4-day culture with tamoxifen. G15, the antagonist of oestrogen via the membrane receptor, did not change follicular growth stimulated by PMSG in vitro. Results of in vitro maturation of COCs showed that germinal vesicle breakdown (GVBD) occurred spontaneously (95.1%) after 2 h in culture, and the GVBD ratio changed little with the addition of either oestrogen or 10 μM G15. However, first polar body (PBI) extrusion was driven by oestrogen markedly and supplementation with 10 μM G15 inhibited PBI extrusion (82.4% vs 55.0%) significantly. These results demonstrated that oestrogen promotes follicle growth through the nuclear receptor during follicle growth and then triggers the transition of metaphase to anaphase through the membrane receptor during meiotic resumption. So oestrogen plays a progressive role in the two phases of follicle growth and oocyte meiotic resumption.

DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

BackgroundThe evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development.ResultsThe DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length (CTL) and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69%, Toxicity; 66.7 ± 4.57%, Vitrification) and cumulus cells viability (32.7 ± 5.85%, Toxicity; 7.7 ± 2.21%, Vitrification) compared to control (95.5 ± 4.04%, oocytes; 89 ± 4.24%, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65%, 22.6 ± 3.05%, respectively) and vitrification (32.3 ± 9.29%, 20 ± 1%, respectively). It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in embryo development to the blastocyst stage was observed after CPAs exposure (1.66 ± 0.57%) or vitrification (2 ± 1%) compared to control (22.3 ± 2.51%). This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells.ConclusionThis study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells.