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Objectives were to evaluate the role of canonical WNT signaling in development of the preimplantation embryo. Signaling was activated with 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and inhibited with Dickkopf-related protein 1 (DKK1). Treatment of bovine embryos with AMBMP at day 5 after insemination decreased development to the blastocyst stage at day 7 and reduced numbers of trophectoderm and inner cell mass cells. At high concentrations, AMBMP caused disorganization of the inner cell mass. DKK1 blocked actions of AMBMP but did not affect development in the absence of AMBMP. Examination of gene expression in day 6 morulae by microarray revealed expression of 16 WNT genes and other genes involved in WNT signaling; differences in relative expression were confirmed by PCR for 7 genes. In conclusion, the preimplantation embryo possesses a functional WNT signaling system and activation of the canonical pathway can inhibit embryonic development.

. Age-associated decrease in contraction-induced activation of downstream targets of Akt/mTor signaling in skeletal muscle. Am J Physiol Regul Integr Comp Physiol 290: R1080 -R1086, 2006. First published November 23, 2005 doi:10.1152/ajpregu.00277.2005In this study, we investigated the effect of age on the association of eukaryotic initiation factor 4E (eIF4E) with eukaryotic initiation factor 4G (eIF4G), as well as the activity of its binding protein (4E-BP1) and the activity of glycogen synthase kinase-3 (GSK-3) after a single bout of rat hindlimb muscle contractile activity elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Tibialis anterior (TA) and plantaris (Pla) muscles from adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 ϫ Brown Norway rats were collected immediately or 6 h after HFES. eIF4E-eIF4G association was elevated at 6 h of recovery in TA (1.9 Ϯ 0.2-fold, P Ͻ 0.05) and immediately and 6 h after exercise in Pla (2.1 Ϯ 0.3-and 2.1 Ϯ 0.7-fold, P Ͻ 0.05) in Y rats. No significant increase was observed in O rats. An increase in 4E-BP1 phosphorylation was observed only 6 h after HFES in TA (5.0 Ϯ 2.0-fold, P Ͻ 0.05) in Y rats. Phosphorylation of GSK-3␣ was increased immediately and 6 h after contraction in TA (1.6 Ϯ 0.3-and 4.1 Ϯ 0.8-fold, P Ͻ 0.05) and Pla (1.7 Ϯ 0.2-and 2.1 Ϯ 0.4-fold, P Ͻ 0.05) in Y rats and remained unaffected in O rats. Phosphorylation of GSK-3␤ was observed only immediately after HFES in TA (1.5 Ϯ 0.2-fold, P Ͻ 0.05) in Y rats. Overall, eIF4E-eIF4G association and phosphorylation of 4E-BP1 and GSK-3 are increased after HFES in adult, but not in aged, animals. These observations suggest that the anabolic response to muscle stimulation is attenuated with aging and may contribute to the limited capacity of hypertrophy in aged animals. sarcopenia; exercise; signaling; hypertrophy AGING IS ASSOCIATED WITH SKELETAL muscle atrophy (sarcopenia), characterized by decreased strength, functional limitations, and physical disability (4,5,15). Although resistance training can increase muscle size and strength, the myogenic response to exercise and the capacity for muscle hypertrophy in older animals and humans appear to be limited (7, 53). The cellular mechanisms responsible for the age-associated decline in response to exercise are not well understood.The mammalian target of rapamycin (mTOR) signaling kinase, which can be activated by Akt/protein kinase B, has emerged as a crucial regulator of skeletal muscle hypertrophy (8, 41). Acute and chronic effects of contractile activity have been described to increase the phosphorylation of mTOR and its downstream target, the 70-kDa ribosomal protein p70 S6K1 (8,10,37,40). p70 S6K1 is pivotal in the control of translation, inasmuch as it regulates a subset of mRNAs containing 5Ј-terminal polypyrimidine tracts (TOP sequences), which encode ribosomal proteins and factors essential to the translational machinery (49). mTOR also phosphorylates eukaryotic initiation factor (eIF) 4E (eIF4E) binding protein (4E-BP1) (16...

INTRODUCTION Space flight and bed rest (BR) lead to muscle atrophy. This study assessed the effect of essential amino acid supplementation (EAA) and resistance training with decreased energy intake on molecular changes in skeletal muscle after 28d BR and 14d recovery. METHODS Thirty-one men (31–55yr) subjected to an 8±6% energy deficit were randomized to receive EAA without resistance training (AA, n=7), EAA 3 h after (RT, n=12), or 5 min before (AART, n=12) resistance training. RESULTS During BR, myostatin transcript levels increased 2-fold in the AA group. During recovery, IGF1 mRNA increased in all groups while Pax7, MyoD, myogenin and MRF4 transcripts increased in AA only (all p<0.05). MAFbx transcripts decreased 2-fold with AA and RT. Satellite cells did not change during BR or recovery. DISCUSSION This suggests that EAA alone is the least protective countermeasure to muscle loss, and several molecular mechanisms are proposed by which exercise attenuates muscle atrophy during bed rest with energy deficit.

Because caspase-3 is considered a primary executioner of apoptosis and has been implicated as a mediator of luteal regression, we hypothesized that corpora lutea (CL) derived from caspase-3 null mice would exhibit a delayed onset of apoptosis during luteal regression, when compared with CL derived from wild-type (WT) mice. To test this hypothesis, ovulation was synchronized in immature (postpartum d 24-27) WT and caspase-3-deficient female littermates by exogenous gonadotropins. Individual CL were isolated by manual dissection, 30 h after ovulation, and placed in organ culture dishes in the absence of serum and growth factors. At the time of isolation (0 h) and after 24, 48, and 72 h in culture, the CL were removed and assessed for the presence of processed (active) caspase-3 enzyme and for apoptosis by multiple criteria. There was no evidence of active caspase-3 enzyme or apoptosis in either WT or caspase-3-deficient CL before culture. However, CL derived from the WT mice exhibited a time-dependent increase in the level of active caspase-3 and apoptosis during culture. By comparison, CL derived from caspase-3-deficient mice, cultured in parallel, failed to exhibit any detectable active caspase-3 and showed attenuated rates of apoptosis. To extend these findings derived from ex vivo culture experiments, ovaries were collected from WT and caspase-3 null female littermates at 2, 4, or 6 d post ovulation, and the occurrence of apoptosis within the CL was analyzed. Whereas ovaries of WT mice had only residual luteal tissue at d 6 post ovulation, ovaries collected from caspase-3-deficient mice retained many CL, at d 6 post ovulation, that were similar in size to those observed in the early luteal phase of WT mice. Importantly, there was no dramatic increase in apoptosis in CL of caspase-3-deficient mice at any time point examined post ovulation, indicating that the involution process had indeed been delayed. In contrast, the levels of progesterone declined regardless of genotype. These data provide the first direct evidence that caspase-3 is functionally required for apoptosis to proceed normally during luteal regression. However, caspase-3 is not a direct mediator of the decrease in steroidogenesis associated with luteolysis.

Because caspase-3 is considered a primary executioner of apoptosis and has been implicated as a mediator of luteal regression, we hypothesized that corpora lutea (CL) derived from caspase-3 null mice would exhibit a delayed onset of apoptosis during luteal regression, when compared with CL derived from wild-type (WT) mice. To test this hypothesis, ovulation was synchronized in immature (postpartum d 24-27) WT and caspase-3-deficient female littermates by exogenous gonadotropins. Individual CL were isolated by manual dissection, 30 h after ovulation, and placed in organ culture dishes in the absence of serum and growth factors. At the time of isolation (0 h) and after 24, 48, and 72 h in culture, the CL were removed and assessed for the presence of processed (active) caspase-3 enzyme and for apoptosis by multiple criteria. There was no evidence of active caspase-3 enzyme or apoptosis in either WT or caspase-3-deficient CL before culture. However, CL derived from the WT mice exhibited a time-dependent increase in the level of active caspase-3 and apoptosis during culture. By comparison, CL derived from caspase-3-deficient mice, cultured in parallel, failed to exhibit any detectable active caspase-3 and showed attenuated rates of apoptosis. To extend these findings derived from ex vivo culture experiments, ovaries were collected from WT and caspase-3 null female littermates at 2, 4, or 6 d post ovulation, and the occurrence of apoptosis within the CL was analyzed. Whereas ovaries of WT mice had only residual luteal tissue at d 6 post ovulation, ovaries collected from caspase-3-deficient mice retained many CL, at d 6 post ovulation, that were similar in size to those observed in the early luteal phase of WT mice. Importantly, there was no dramatic increase in apoptosis in CL of caspase-3-deficient mice at any time point examined post ovulation, indicating that the involution process had indeed been delayed. In contrast, the levels of progesterone declined regardless of genotype. These data provide the first direct evidence that caspase-3 is functionally required for apoptosis to proceed normally during luteal regression. However, caspase-3 is not a direct mediator of the decrease in steroidogenesis associated with luteolysis.

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Exposure of bovine conceptuses to colony-stimulating factor 2 (CSF2) from days 5 to 7 of development can increase the percentage of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10 ng/ml CSF2 from days 5 to 7 after insemination, transferred into cows, and flushed from the uterus at day 15 of pregnancy. There was a tendency (PZ0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2-treated conceptus (35% for control versus 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-t (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (PZ0.07). In addition, CSF2 increased mRNA for IFNT2 (PZ0.08) and keratin 18 (P!0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at day 15 of pregnancy.