To further develop defined conditions for in vitro fertilization (IVF) and in vitro culture (IVC) of in vitro-matured porcine oocytes, we evaluated the effects of theophylline, adenosine, and cysteine in a chemically defined medium during IVF. Viability to full term of in vitro-produced blastocysts after IVF and IVC in chemically defined medium was also investigated by embryo transfer to recipients. A chemically defined medium, porcine gamate medium (PGM), was modified from porcine zygote medium (PZM-4), which was previously established. PGM was used as a basal medium for IVF and PZM-4 was for the culture of presumptive zygotes. Addition of 2.5 mM theophylline to PGM significantly increased the percentage of male pronuclear formation compared with controls (no addition). Addition of 1 microM adenosine to PGM supplemented either with or without 2.5 mM theophylline significantly reduced the number of penetrated spermatozoa compared with controls (no addition of adenosine). Supplementation with 0.2 microM cysteine in PGM containing both 2.5 mM theophylline and 1 microM adenosine further increased the percentage of development to the blastocyst stage, compared with no supplementation of cysteine, but there was no difference in fertilization parameters, such as monospermy and pronuclear formation, regardless of presence or absence of theophylline and adenosine. When Day 5 blastocysts were transferred into four recipients (20-25 blastocysts per recipient), all recipients became pregnant and farrowed a total of 21 live piglets. The present results clearly demonstrate that porcine blastocysts can be produced by IVF and IVC in chemically defined media and that they can develop to full term after embryo transfer.

Background-Pharmacological and genetic studies indicate that the Na ϩ -H ϩ exchanger isoform 1 (NHE1) plays a critical role in myocardial ischemia and reperfusion (I/R) injury. We found that p90 ribosomal S6 kinase (RSK) phosphorylated serine 703 of NHE1, stimulating 14 -3-3 binding and NHE1 activity. Therefore, we hypothesized that inhibiting RSK in cardiomyocytes would prevent NHE1 activation and decrease I/R-mediated injury. Methods and Results-To examine the role of RSK in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative RSK (DN-RSK-TG

Peroxisome proliferator-activated receptors (PPAR) decrease the production of cytokine and inducible nitric-oxide synthase (iNOS) expression, which are associated with aging-related inflammation and insulin resistance. Recently, the involvement of the induction of heme oxygenase-1 (HO-1) in regulating inflammation has been suggested, but the exact mechanisms for reducing inflammation by HO-1 remains unclear. We found that overexpression of HO-1 and [Ru(CO) 3 Cl 2 ] 2 , a carbon monoxide (CO)-releasing compound, increased not only ERK5 kinase activity, but also its transcriptional activity measured by luciferase assay with the transfection of the Gal4-ERK5 reporter gene. This transcriptional activity is required for coactivation of PPAR␦ by ERK5 in C2C12 cells. [Ru(CO) 3 Cl 2 ] 2 activated PPAR␦ transcriptional activity via the MEK5/ERK5 signaling pathway. The inhibition of NF-B activity by ERK5 activation was reversed by a dominant negative form of PPAR␦ suggesting that ERK5/PPAR␦ activation is required for the anti-inflammatory effects of CO and HO-1. Based on these data, we propose a new mechanism by which CO and HO-1 mediate anti-inflammatory effects via activating ERK5/PPAR␦, and ERK5 mediates CO and HO-1-induced PPAR␦ activation via its interaction with PPAR␦.Muscle wasting is a major feature of the cachexia associated with diverse pathologies such as cancer, sepsis, diabetes, and aging (1). Several cytokines have been implicated in the pathogenesis of muscle wasting, most notably TNF-␣, 2 a pro-inflammatory cytokine that was originally called "cachectin" (1). In addition, aging-related chronic low grade inflammation by TNF-␣ plays an important role in insulin resistance (2). It has been proposed that chronic inflammation by TNF-␣-mediated NF-B activation and subsequent inducible nitric-oxide synthase (iNOS) induction relates to muscle wasting and insulin resistance as we will explain below. Cai et al. (3) have shown that activation of NF-B, through muscle-specific transgenic expression of activated IB kinase ␤ (MIKK), causes profound muscle wasting that resembles clinical cachexia. In contrast, no overt phenotype was seen upon muscle-specific inhibition of NF-B through expression of IB suppressor (MISR), and denervation and tumor-induced muscle loss were substantially reduced and survival rates improved by NF-B inhibition in MISR mice, which is consistent with a critical role for NF-B in the pathology of muscle wasting, especially in diabetes and during the process of aging (3). Recent studies suggest the involvement of iNOS in the pathogenesis of insulin resistance (4, 5). First, most inducers of insulin resistance, including obesity (6), free fatty acids (7), hyperglycemia (8, 9), TNF-␣, oxidative stress, and endotoxin, increase iNOS expression. Second, iNOS mediates the impaired insulin-stimulated glucose uptake by treatment with TNF-␣ and lipopolysaccharide in cultured muscle cells (10). iNOS expression is elevated in skeletal muscle of patients with type 2 diabetes (11, 12), and high fat diet-induced...

Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.

We investigated changes in peripheral concentrations of inhibin A, total inhibin, steroids, and gonadotropins throughout the intact estrous cycle of the sow in relation to ovarian changes determined by daily transrectal ultrasonography. All visible follicles of 3 mm or more in diameter were classified as small (R3 and !6 mm) or large (R6 mm). Follicular recruitment was identified in two periods of the cycle: one from the late luteal to the follicular phase, characterized by an increase in the number of small follicles followed by the appearance of large follicles; and another during the early luteal phase, consisting only of increased numbers of small follicles. Plasma concentrations of inhibin A increased (P!0.05), coinciding with the two periods of follicle emergence. Estradiol (E 2 ) levels increased (P!0.05) during the follicular phase, but not during the early luteal phase. An inverse relationship (P!0.01) between the patterns of inhibin and FSH concentrations was noted around the two periods of follicle emergence, but there was no relationship (PR0.1) between the patterns of plasma E 2 and FSH during the early luteal phase. In conclusion, measurement of plasma inhibin A levels combined with ultrasonographic examination of the ovaries revealed two periods of synchronous follicular growth during the sow's estrous cycle. The results strongly suggest that inhibin A functions as a negative feedback regulator of FSH secretion throughout the estrous cycle, whereas E 2 appears to influence FSH secretion only during the follicular phase.Reproduction (2010) 139 153-161