A specific adenylyl cyclase inhibitor (DDA) and a cyclic AMP-dependent protein kinase inhibitor (H-89) block the action of equine growth hormone on in vitro maturation of equine oocytes.

The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus-oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein kinase (H-89; 10-9, 10-11 or 10-15 M concentration) and a specific adenylate cyclase inhibitor, 2',3'-dideoxyadenosine (DDA; 10-8, 10-10 or 10-14 M concentration) were used to observe whether they could block the eGH effect.

Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells.

The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis.

Selection of developmentally competent immature equine oocytes with brilliant cresyl blue stain prior to in vitro maturation with equine growth hormone.

Immature oocytes synthesize a variety of proteins that include the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Brilliant cresyl blue (BCB) is a vital blue dye that assesses intracellular activity of G6PDH, an indirect measure of oocyte maturation. The objective was to evaluate the BCB test as a criterion to assess developmental competence of equine oocytes and to determine if equine growth hormone (eGH) enhanced in vitro maturation (IVM) of equine oocyte.

The effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on in vitro maturation of equine oocytes.

The objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus-oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.

Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species.

Activation of AMP-activated protein kinase may not be involved in AICAR- and metformin-mediated meiotic arrest in bovine denuded and cumulus-enclosed oocytes in vitro.

The adenosine monophosphate-activated protein kinase (AMPK) activators, 5'-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) and metformin (MET), inhibit resumption of meiosis in bovine cumulus-enclosed oocytes (CEO) and denuded oocytes (DO). The objectives of this study were to: (1) examine the effects of AMPK inhibitors on bovine oocyte meiosis in vitro; and (2) determine if AICAR or MET activates oocyte and/or cumulus cell AMPK. The AMPK inhibitor compound C (CC; 0.

Effects of manipulating the nitric oxide/cyclic GMP pathway on bovine oocyte meiotic resumption in vitro.

The objective of this study was to examine the effects of manipulating the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on bovine oocyte nuclear maturation in vitro. Cumulus-enclosed oocytes (CEO) were recovered from abattoir-derived ovaries and cultured in M199+FCS for 7 or 21h in the presence of various molecules affecting the NO/cGMP pathway, and then fixed and stained for evaluation of the stage of nuclear maturation. Cyclic GMP levels were also measured in cumulus-oocyte complexes after 3 and 6 h of culture.

Effects of adenosine monophosphate-activated kinase activators on bovine oocyte nuclear maturation in vitro.

The purpose of this study was to examine the effects of an activator of AMPK (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR)) on bovine oocyte nuclear maturation in vitro. After 7 hr of culture, AICAR (1 mM) significantly increased the percentages of cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) remaining at the germinal vesicle stage. After 22 hr of culture, AICAR significantly reduced the percentage of CEO reaching metaphase II (MII).

Effects of culture media and energy sources on the inhibition of nuclear maturation in bovine oocytes.

The influence of the culture medium and energy sources on spontaneous nuclear maturation and inhibition of maturation in bovine cumulus-enclosed oocytes (CEO) was examined. CEO were cultured in Medium 199, minimum essential medium, M16, or synthetic oviduct fluid (SOF), all containing 3 mg/mL bovine serum albumin (BSA), and SOF without BSA, alone or supplemented with hypoxanthine (HYPO, 4 mM) or forskolin (FSK, 100 microM) for 21 h. More CEO remained at the GV stage in M16 compared to other media (P < 0.

Effects of phosphodiesterase inhibitors on spontaneous nuclear maturation and cAMP concentrations in bovine oocytes.

It was previously demonstrated that inhibition of cAMP degradation with phosphodiesterase type 3 (PDE3) inhibitors resulted in the maintenance of bovine cumulus-oocyte complexes (COC) and denuded oocytes (DO) in meiotic arrest, while a PDE4 inhibitor was without effect. In this study, different inhibitors of PDE3 and PDE4 were tested for their effects on bovine oocyte nuclear maturation. Bovine COC and DO were cultured in TCM-199+10% fetal bovine serum (FBS) with or without different concentrations of the PDE inhibitors.

Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate.

The objectives of this study were to compare different methods of evaluating sperm plasmalemma and to determine their relationship with in vitro fertilization rate. A single batch of frozen semen from each of eight beef bulls was used for assessment of sperm viability and for in vitro fertilization. Conventional viability tests included sperm morphology, motility, acrosome integrity, and abnormal DNA condensation.

Effect of oocyte quality on the relative abundance of specific gene transcripts in bovine mature oocytes and 16-cell embryos.

Although the developmental potential of oocytes is related to oocyte quality, whether the expression of specific genes is altered in oocytes of different quality and in resulting embryos is not known. Semi-quantitativereverse transcription-polymerase chain reaction was used to compare the relative abundance of 2 transcripts for housekeeping proteins (beta-actin and ribosomal protein L30) and 3 transcripts for growth factor ligand or receptors (platelet derived growth factor receptor alpha (PDGFRalpha), basic fibroblast growth factor (bFGF)), in mature bovine oocytes of high versus low developmental potential. The transcripts for L30, PDGFRalpha, and bFGF in 16-cell embryos originating from these oocytes were also examined.

Effects of oocyte quality on development and transcriptional activity in early bovine embryos.

The objective of this study was to evaluate the effects of oocyte quality on in vitro development and the level of transcriptional activity in early bovine embryos. Cumulus-oocyte complexes (COC) were divided into six classes based on their cumulus investment and on the texture of the ooplasm. Embryos originating from oocytes with more than five layers of cumulus cells and with slight expansion of the cumulus and/or granulation in the ooplasm (class II) developed to the blastocyst stage as frequently as embryos originating from oocytes of class I which showed no signs of atresia (13.