Growth factor messenger ribonucleic acid expression during differentiation of porcine embryonic myogenic cells

The growth factors, IGF-I and II, their binding proteins, IGFBP, and members of the transforming growth factor (TGF) superfamily (myostatin and TGFbeta1) are known to regulate proliferation and differentiation of myogenic cells. We hypothesized that changes in the relative expression of members of the IGF and TGFbeta systems play a significant role in regulating myogenesis in porcine embryonic myogenic cell (PEMC) cultures. Therefore, determining the expression patterns of these factors during PEMC myogenesis is important. Consequently, we used real-time PCR to explore the pattern of IGF-I; IGF-II; IGFBP-2, -3, and -5; IGF-type-I receptor; myogenin; myostatin; and TGFbeta1 mRNA expression during PEMC myogenesis. The progression of differentiation was assessed using creatine kinase activity and myogenin mRNA expression. As anticipated, creatine kinase activity was low in PEMC cultures at 48 h and increased 20-fold (P < 0.0001) between 48 h and its peak at 144 h. Similarly, myogenin mRNA was low at 48 h and increased approximately 5-fold (P < 0.0001) as differentiation progressed, peaking at 120 h and decreasing at 144 h. The patterns of IGF-I and IGFBP-2 mRNA expression were similar and were relatively lower in 48-h PEMC cultures, increasing approximately 5-fold (P < 0.0001) to their greatest levels at 120 h. In contrast, IGF-II and IGFBP-5 mRNA levels were relatively high at 48 h, peaking at 72 h, and steadily decreasing by 60 and 80%, respectively (P < 0.001), at 144 h. The level of IGF-type-I receptor mRNA was relatively high until 96 h of culture, after which it decreased 40% (P < 0.01), reaching a low at 144 h. Levels of IGFBP-3 mRNA were relatively high at 48 h, dropped approximately 40% to their lowest level at 72 h (P < 0.001), and then increased approximately 60% (P < 0.001) to their greatest levels at 144 h. Levels of TGFbeta1 mRNA decreased approximately 30% (P < 0.0001) between 48 and 96 h, then quickly rebounded to a peak at 120 h, and by 144 h had dropped to the levels seen at 72 h. Myostatin mRNA was at its greatest level at 48 h and declined rapidly between 72 and 96 h, finally decreasing by approximately 80% at 144 h (P < 0.0001). Our data demonstrate that these factors are differentially regulated during PEMC myogenesis and provide new information about their pattern of mRNA expression in cultured porcine muscle cells.     

Low-density lipoprotein-related receptor protein 1 (LRP-1) is not required for insulin-like growth factor binding protein 3 (IGFBP-3) to suppress L6 myogenic cell proliferation

Insulin-like growth factor binding protein-3 (IGFBP-3) suppresses proliferation of numerous cell types, including myogenic cells, via both insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms; however, the mechanism of IGF-independent suppression of proliferation is not clearly defined. In nonmuscle cells, binding of IGFBP-3 to the low-density lipoprotein receptor-related protein-1 (LRP-1)/activated α(2)M receptor is reportedly required for IGFBP-3 to inhibit proliferation. These findings suggest that binding to this receptor also may be required for IGFBP-3 to suppress proliferation of cultured myogenic cells. To investigate the role of the LRP-1 receptor in suppression of myogenic cell proliferation by IGFBP-3, we have examined the effect of receptor-associated protein, an LRP-1 receptor antagonist, on recombinant porcine (rp)IGFBP-3 inhibition of L6 myogenic cell proliferation. Treatment with receptor-associated protein results in a 37% decrease (P < 0.05) in the ability of rpIGFBP-3 to inhibit L6-cell proliferation. In L6 cells subjected to LRP-1 small interfering RNA treatment for 48 h (LRP-1 silenced), LRP-1 mRNA levels were reduced by greater than 80% compared with control cultures treated with nonsense small interfering RNA (mock silenced). In addition, the 85-kDa transmembrane subunit of LRP-1 was undetectable in Western immunoblots of total protein lysates from LRP-1-silenced cells. Even though LRP-1 mRNA and protein levels were dramatically reduced in LRP-1-silenced L6 cells compared with mock-silenced controls, rpIGFPB-3 suppressed proliferation rate to the same extent in both LRP-1-silenced and mock-silenced cultures. Our results strongly suggest that, in contrast to data obtained for nonmuscle cell lines, the LRP-1 receptor is not required for IGFBP-3 to suppress proliferation of L6 myogenic cells.     

Effect of constitutive expression of porcine IGFBP-3 on proliferation and differentiation of L6 myogenic cells

We have previously shown that exogenous recombinant porcine IGFBP-3 (rpIGFBP-3) suppresses proliferation and differentiation of L6 myogenic cells in an IGF-I-dependent manner and suppresses proliferation of L6 myogenic cells via an IGF-I-independent mechanism. In order to assess the effects of endogenously produced IGFBP-3, we have transfected L6 myogenic cells with a pEF6/V5 vector containing pIGFBP-3 cDNA under the control of the human elongation factor 1alpha (hEF-1alpha) promoter and with the empty vector. We have isolated a cell population that constitutively produces porcine IGFBP-3 (tL6 cells) and a stable mock transfected cell population containing the empty vector (mtL6 cells). Constitutive expression of IGFBP-3 slightly reduced the expression of IGFBP-5 but had no effect on IGFBP-4 production by L6 myogenic cells. Immunoneutralization of IGFBP-3 increased both IGF-I- and Long-R3-IGF-I-stimulated proliferation of tL6 cells (58 and 33%, respectively) (P<0.01). These data indicate endogenous pIGFBP-3, like exogenous rpIGFBP-3, suppresses the proliferation of L6 myogenic cells via both IGF-I-dependent and -independent pathways. Immunoneutralization of IGFBP-3 also increased IGF-I-stimulated differentiation (21%, P<0.05) but had no effect on Long-R3-IGF-I stimulated differentiation of tL6 myogenic cells. Results indicate that exogenous and endogenous IGFBP-3 affect proliferation and differentiation of L6 myogenic cells in a similar way. Immunohistochemical localization data reveal that pre-incubation with anti-pIGFBP-3 dramatically reduces the level of intracellular IGFBP-3 in tL6 myogenic cells indicating that endogenously produced IGFBP-3 must first be secreted before it is internalized and that anti-pIGFBP-3 prevents internalization of IGFBP-3. TL6 and mtL6 cells provide a good system to further investigate the mechanisms by which IGFBP-3 affects proliferation and differentiation of myogenic cells.     

Effects of growth factors on insulin-like growth factor binding protein (IGFBP) secretion by primary porcine satellite cell cultures.

Insulin-like growth factor-binding proteins (IGFBP) regulate the biological functions of insulin-like growth factors (IGF) and may affect cell growth through IGF-independent actions. Growth factors and hormones have been shown to alter IGFBP production by target cells suggesting that the effects of these factors may be partially mediated by the local production of IGFBP. Growth factors, including IGF-I, transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) have potent effects on satellite cell proliferation and differentiation, and some of these factors have been shown to alter IGFBP production in various cell types. Consequently, some of their actions on muscle satellite cells may be mediated by the local production of IGFBP. In this study, we measured the effects of IGF-I, bFGF, and TGF-beta1 on IGFBP production by primary porcine satellite cell (PSC) cultures after first determining physiologically active concentrations of these growth factors to use according to [3H]thymidine incorporation dose responses. There is little information on the effects of these growth factors on IGFBP production in primary porcine myogenic cells due to the confounding affects of contaminating nonmuscle fibroblasts. Comparative studies show that primary porcine satellite cells produce IGFBP-3 and -5 whereas porcine muscle-derived nonfusing cells (FIB) produce IGFBP-2 and -4 but not IGFBP-3 or -5. Because of this, our investigations have focused on growth factor-induced production of IGFBP-3 and -5 in primary porcine satellite cells cultures. Both IGF-I and bFGF exhibited dose-dependent increases in [3H]thymidine incorporation with increasing concentration from 1 to 50 ng/mL (P < 0.05), whereas TGF-beta1 caused a dose-dependent decrease from 0.01 to 0.5 ng/mL (P < 0.05). When 20 ng/ mL of IGF-I was added to the media, IGFBP-3 was increased approximately 65% (P < 0.05) and IGFBP-5 was increased approximately twofold (P < 0.05). The addition of 0.5 ng/mL TGF-beta1 caused more than a two-fold increase in IGFBP-3 (P < 0.05) and approximately an 80% increase in IGFBP-5 (P < 0.05), whereas 50 ng/ mL of bFGF caused approximately 40% (P < 0.05) and 70% (P < 0.05) increases in IGFBP-3 and -5, respectively. Neither IGFBP-3 nor -5 was detectable in the conditioned media from fibroblasts whether or not IGF-I, TGF- beta1 or bFGF were present. These data suggest that the effects of IGF-I, TGF- beta1 and bFGF on porcine satellite cells may in part be through the autocrine/ paracrine production of IGFBP-3 and -5 by porcine satellite cells.     

Insulin-like growth factor (IGF)-I and IGF binding proteins-2, -3, -4, -5 in porcine corpora lutea during the estrous cycle; evidence for inhibitory actions of IGFBP-3

In this study we measured protein concentrations of insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBPs) 2-5 in porcine corpora lutea (CLs) throughout the estrous cycle (Experiment 1), and examined the effects of IGFBP-3 and IGFBP-3 antibody (AB) on luteal progesterone (P4) secretion in vitro (Experiment 2). For Experiment 1, (CLs) and serum were collected on days (D) 4, 7, 10, 13, 15 and 16 of the estrous cycle (n = 5 animals per day). IGF-I was extracted from CLs and sera, and measured by radioimmunoassay (RIA). IGFBPs were measured in CLs by ligand blots. For Experiment 2, CLs (from Experiment 1) were enzyme dissociated and luteal cells cultured (24 h) in Medium 199 (M199) containing (0-500 ng/ml) IGFBP-3 (+/-IGF-I; 100 ng/ml), or (0-10 microg/ml) IGFBP-3 AB. P4 in media was measured by RIA. In Experiment 1, luteal IGF-I concentrations (ng/g tissue) were maximal on day 4 and gradually decreased thereafter. Serum IGF-I concentrations (ng/ml) were highest on days 4 and 7, compared with days 10-15. Peak levels of luteal IGFBP-3 were also seen on days 4 and 7 of the cycle. Luteal IGFBP-2 concentrations showed a tendency to increase on day 16 (P < 0.05 versus day 10), but no significant changes in IGFBP-4 or -5 were seen. In Experiment 2, IGFBP-3 (w IGF) inhibited the steroidogenic actions of IGF-I, but had no significant actions alone (IGFBP-3 w/o IGF). Finally, IGFBP-3 AB stimulated P4 secretion on days 4 and 7, but not on days 10-16. We conclude that IGFBP-3 inhibits IGF-I actions in the porcine CL.     

Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.     

Effects of L-carnitine on fetal growth and the IGF system in pigs

The effects of L-carnitine on porcine fetal growth traits and the IGF system were determined. Fourth-parity sows were fed a gestation diet with either a 50-g top dress containing 0 (control, n = 6) or 100 mg of L-carnitine (n = 6). At midgestation, fetuses were removed for growth measurements, and porcine embryonic myoblasts (PEM) were isolated from semitendinosus. Real-time quantitative PCR was used to measure growth factor messenger RNA (mRNA) levels in the uterus, placenta, muscle, hepatic tissue, and cultured PEM. A treatment x day interaction (P = 0.02) was observed for maternal circulating total carnitine. Sows fed L-carnitine had a greater (P = 0.01) concentration of total carnitine at d 57 than control sows. Circulating IGF-I was not affected (P = 0.55) by treatment. Supplementing sows with L-carnitine resulted in larger (P = 0.02) litters (15.5 vs. 10.8 fetuses) without affecting litter weight (P = 0.07; 1,449.6 vs. 989.4 g) or individual fetal weight (P = 0.88) compared with controls. No treatment effect was found for muscle IGF-I (P = 0.36), IGF-II (P = 0.51), IGFBP-3 (P = 0.70), or IGFBP-5 (P = 0.51) mRNA abundance. The abundance of IGF-I (P = 0.72), IGF-II (P = 0.34), and IGFBP-3 (P = 0.99) in hepatic tissue was not influenced by treatment. Uterine IGF-I (P = 0.46), IGF-II (P = 0.40), IGFBP-3 (P = 0.29), and IGFBP-5 (P = 0.35) mRNA abundance did not differ between treatments. Placental IGF-I (P = 0.30), IGF-II (P = 0.18), IGFBP-3 (P = 0.94), and IGFBP-5 (P = 0.42) mRNA abundance did not differ between treatments. There was an effect of side of the uterus for IGF-I (P = 0.04) and IGF-II (P = 0.007) mRNA abundance; IGF-I mRNA abundance was greater in the left uterine horn than in the right uterine horn (0.14 and 0.07 relative units, respectively). Placental IGF-II mRNA abundance was greater (P = 0.007) in the left than in the right uterine horn (483.5 and 219.59, respectively). The abundance of IGFBP-3 was not affected by uterine horns in either uterine (P = 0.66) or placental (P = 0.13) tissue. There was no treatment difference for IGF-I (P = 0.31) or IGFBP-5 (P = 0.13) in PEM. The PEM isolated from sows fed L-carnitine had decreased IGF-II (P = 0.02), IGFBP-3 (P = 0.03), and myogenin (P = 0.04; 61, 59, and 67%, respectively) mRNA abundance compared with controls. These data suggest that L-carnitine supplemented to gestating sows altered the IGF system and may affect fetal growth and development.     

Sequential expression of myogenic regulatory factors in bovine skeletal muscle and the satellite cell culture

Myogenic regulatory factors (MRFs) are important in the control of skeletal muscle development. To understand myogenic regulation by MRFs in bovine adult muscle cells, their expressions, namely that of Myf5, MyoD, myogenin, and MRF4 in the biceps femoris muscle (BF) and in the satellite cell culture, were analyzed by RT-PCR. In the BF, all four MRFs were expressed and in particular, myogenin and MRF4 were strongly expressed, whereas Myf5 was faintly expressed. The satellite cells prepared from the BF expressed Myf5, but only a trace of MyoD, at day 9 of culture. During the growth of the cells to day 14, the MyoD and myogenin expressions gradually increased, and that of MyoD expression reached its maximum at the confluence of the culture. After induction of myogenic differentiation by a serum-free medium at day 14, Myf5 expression gradually decreased, and the up-regulated expression of MyoD was suppressed, whereas myogenin expression continued to increase sharply. Following the myogenin expression, MRF4 also drastically increased toward the myotube formation of the cells. When huge myotubes were formed at day 18, Myf5 was expressed at a low level, whereas the MyoD expression remained at a moderate level.     

Administration of estradiol, trenbolone acetate, and trenbolone acetate/estradiol implants alters adipogenic and myogenic gene expression in bovine skeletal muscle

Twenty crossbred yearling steers (421 kg) were used to evaluate the effects of implanting with trenbolone acetate (TBA; 120 mg), estradiol-17β (E(2); 25.7 mg), and a combination (120 mg of TBA and 24 mg of E(2)) on adipogenic and myogenic mRNA concentrations. Animals were blocked by BW and within each block were assigned to 1 of 4 treatments. Animals were housed and fed in individual pens with 5 animals per treatment. All animals were weighed weekly, and muscle biopsy samples were taken from the LM of each steer on d 0 (before implantation), 7, 14, and 28. Total RNA was isolated from each sample and real-time quantitative PCR was used to measure the quantity of C/EBPβ, PPARγ, stearoyl CoA desaturase (SCD), myogenin, and 3 isoforms of bovine myosin heavy chain (MHC) mRNA. Total BW gain from the 28-d period was adjusted to d 0 by use of covariant analysis, and steers in the implant groups tended (P = 0.09) to have increased BW gain compared with nonimplanted control steers. Analysis of the gene expression of MHC showed that neither implant nor day (P > 0.20) had a significant effect on the expression of type I or IIX MHC mRNA There was also no treatment effect (P > 0.20) on MHC-IIA and myogenin, but increasing days on feed increased (P = 0.05) the expression of MHC-IIA mRNA. Relative mRNA abundance of C/EBPβ, PPARγ, and SCD increased (P < 0.05) during days of feed but PPARγ decreased (P < 0.05) with the treatment of combined TBA/E(2) implant. Results of this study indicate that implanting with TBA, E(2), or both increased BW gain and decreased adipogenic gene expression of finishing steers without significantly affecting the concentration of type I, IIA, or IIX MHC mRNA. Increasing days on feed increased both MHC-IIA and adipogenic gene expression in bovine skeletal muscle biopsy samples. We conclude that administration of steroidal implants had no effect on the proportion of the 3 MHC mRNA isoforms but decreased C/EBPβ, PPARγ, and SCD mRNA in bovine skeletal muscle.     

Cloning and expression of equine insulin-like growth factor binding proteins in normal equine tendon

OBJECTIVES: To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons. ANIMALS: 7 horses. PROCEDURE: Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both nucleotide and predicted amino acid sequences were compared with those published for bovine, human, mouse, and ovine IGFBPs. Gene expression was quantitated by real-time PCR assay, and protein expression was evaluated by western ligand blot (WLB). RESULTS: Clones ranged in size from 262 to 522 bp and had high degrees of sequence homology with other mammalian species. Sequence homology was highest between bovine and equine IGFBPs (86% to 95%) and amongst the IGFBP-5 sequences from the various species (92% to 95%). Message for IGFBP-2 to -6, but not IGFBP-1, was expressed in normal tendon. Protein expression for IGFBP-2, -3, and -4 was detected byWLB in normal tendon and markedly increased in damaged tendons. CONCLUSIONS AND CLINICAL RELEVANCE: Results provide basic information and tools needed for further characterization of the role of the IGF system in tendon healing and may lead to the ability to potentiate the response of healing tendon to exogenous IGF-I via concurrent manipulation of IGFBPs.     

Melengestrol acetate alters muscle cell proliferation in heifers and steers

In vitro experiments were performed to investigate the effects of melengestrol acetate (MGA) or progesterone (P4) on bovine muscle satellite cells and C2C12 myoblasts. Addition of MGA at physiological and supraphysiological concentrations resulted in a dose-dependent decrease (P < 0.05) in DNA synthesis as measured by [3H]-thymidine incorporation (TI). Similarly, P4 addition (0.01 nM) reduced (P < 0.05) TI. Addition of MGA (10 nM) increased (P < 0.05) IGF-I mRNA abundance but did not affect myogenin mRNA. Progesterone addition (10 nM) increased myogenin mRNA abundance (P < 0.05). In C2C12 cultures, P4 addition resulted in a dose-dependent decrease in TI. The antiprogestin RU486, in combination with MGA or P4, also resulted in reduced (P < 0.05) TI. Treatment with RU486 alone had a negative effect (P < 0.05) on TI that was similar to the progestins. Treatment of C2C12 myoblasts with MGA (100 nM) resulted in an increase (P < 0.05) in myogenin mRNA. These studies suggest that progestins may reduce satellite cell proliferation, ultimately affecting carcass composition.     

二甲双胍对牛骨骼肌卫星细胞增殖与分化的影响

为探究二甲双胍对牛骨骼肌卫星细胞增殖和分化的影响，本研究将体外培养的牛骨骼肌卫星细胞分别用0（对照组）、1、2、4 mmol/L二甲双胍进行处理，采用CCK-8法筛选出二甲双胍作用于牛骨骼肌卫星细胞的最适浓度，接着通过EdU染色法检测二甲双胍处理牛骨骼肌卫星细胞后对其增殖的影响，然后对二甲双胍处理的牛骨骼肌卫星细胞进行体外成肌诱导分化，通过显微镜观察牛骨骼肌卫星细胞分化时期的细胞状态，然后利用Western blotting技术检测牛骨骼肌卫星细胞的分化标志因子肌球蛋白重链（MyHC）、肌细胞生成素（MyoG）在分化24、48和72 h的表达情况。结果表明，二甲双胍作用于牛骨骼肌卫星细胞的最适浓度为2 mmol/L。2 mmol/L二甲双胍处理牛骨骼肌卫星细胞后，其细胞增殖率显著降低（P<0.05），说明二甲双胍可以抑制牛骨骼肌卫星细胞的增殖；牛骨骼肌卫星细胞诱导分化后形成的肌管数量和直径均呈现减少趋势，牛骨骼肌卫星细胞成肌分化标志因子MyHC、MyoG在分化24、48和72 h的表达均显著低于0 mmol/L （对照）组（P<0.05），说明2 mmol/L二甲双胍能够抑制牛骨骼肌卫星细胞的成肌分化过程。研究结果表明，二甲双胍可以显著抑制牛骨骼肌卫星细胞的增殖及成肌分化过程。该研究为二甲双胍在肌肉发育调控及肌损伤修复方面的应用提供一定的理论依据。     

A method to establish co-cultures of myotubes and preadipocytes from collagenase digested neonatal pig semitendinosus muscles

The relationships between adipocyte and muscle cell development within muscle are important in the study of factors or agents that may improve meat quality. Neonatal porcine muscle has the potential to yield both cell types for cell culture because it contains developing adipocytes and a high number of muscle satellite cells. Therefore, we modified a conventional collagenase-based procedure to digest neonatal porcine muscle and subsequently cultured the resultant muscle stromal-vascular (SV) cells on several substrata in basal and dexamethasone (DEX)-containing media. Developing myotubes and preadipocytes were present in muscle SV cell cultures on laminin substrata following seeding and plating with fetal bovine serum (FBS) with or without DEX. Myotube number was much higher (P < 0.05) on laminin substrata compared with all other substrata, whereas preadipocyte number in muscle SV cell cultures was independent of substrata, as we have shown previously. This approach can be used to establish co-cultures of differentiating adipocytes and myotubes from collagenase-digested neonatal pig muscle. Because the comparison is within the same culture dish, this method allows for a direct comparison of the responses of adipogenic and myogenic cells to growth and differentiation factors. For example, DEX did not alter myogenesis (i.e., 11 +/- 3 vs. 11 +/- 4 myotubes per unit area for control and DEX-treated cultures, respectively), but it has been shown to markedly increase preadipocyte number in muscle SV cell cultures.     

Growth factor messenger RNA levels in muscle and liver of steroid-implanted and nonimplanted steers

Ribonuclease protection assays were used to measure steady-state semimembranosus muscle and/or hepatic levels of IGF-I, IGFBP-3, IGFBP-5, hepatocyte growth factor (HGF), and myostatin messenger RNA (mRNA) in steers implanted from 32 to 38 d with Revalor-S, a combined trenbolone acetate and estradiol implant. Insulin-like growth factor-ImRNA levels were 69% higher (P < 0.01, n = 7) in the livers of implanted steers than in the livers of nonimplanted steers. Similarly, IGF-I mRNA levels were 50% higher (P < 0.05, n = 7) in the semimembranosus muscles of implanted steers than in the same muscles from nonimplanted steers. Hepatic IGFBP-3 mRNA levels were 24% higher (P < 0.07, n = 7) in implanted steers than in nonimplanted steers. Hepatic HGF and IGFBP-5 mRNA levels did not differ between implanted and nonimplanted steers. Similarly, muscle IGFBP-3, IGFBP-5, HGF, and myostatin mRNA levels were not affected by treatment. Previous data from these same steers have shown that circulating IGF-I and IGFBP-3 concentrations were 30 to 40% higher (P < 0.01, n = 7) in implanted steers than in nonimplanted, control steers. Additionally, the number of actively proliferating satellite cells that could be isolated from the semimembranosus muscle was 45% higher (P < 0.01, n = 7) for implanted steers than for nonimplanted steers. Viewed together, these data suggest that increased muscle IGF-I levels stimulate increased satellite cell proliferation, resulting in the increased muscle growth observed in Revalor-S implanted steers.     

鹌鹑早期胚胎bcl-2基因表达的差异及发育性变化

采用RT-PCR方法,用Wpkci和β-actin引物进行多重PCR鉴定66～120 h鹌鹑胚胎样本性别后,选取不同时间点雌、雄胚胎各4枚,以β-actin为内标,测定bcl-2 mRNA的相对丰度;探讨bcl-2对鹌鹑早期胚胎发育的影响。结果表明：①雄性胚胎在66～90 h bcl-2 mRNA表达量一直维持在较低水平,96～102 h bcl-2 mRNA有所增加,但96与102 h间差异不显著（P＞0.05）,在108 h显著降低（P＜0.05）;与108 h相比,114 h显著升高,120 h维持在较高水平。雄性胚胎在66～108 h（72 h除外）bcl-2 mRNA表达均比雌性胚胎的表达量低,在114～120 h bcl-2 mRNA表达水平均比雌性胚胎的高。②雌性胚胎在66～90 h bcl-2 mRNA表达量一直维持在较低水平;与90 h相比,96 h显著升高,96～120 h逐渐降低,其中96～108 h下降,但三者间差异不显著（P＞0.05）;与108 h相比,114～120 h显著下降（P＜0.05）。③相同时间点雌、雄胚胎间的比较结果表明,84 h雌性胚胎bcl-2 mRNA表达显著高于雄性胚胎（P＜0.05）,90、108 h雌性胚胎bcl-2 mRNA表达极显著高于雄性胚胎（P＜0.01）;而在120 h雄性胚胎bcl-2 mRNA表达极显著高于雌性胚胎（P＜0.01）。鹌鹑胚胎发育早期,雌、雄样本bcl-2 mRNA表达存在一定的时序性,说明bcl-2对雌、雄鹌鹑早期分化及早期胚胎发育有重要影响。     

Exogenous somatotropin alters IGF axis in porcine endometrium and placenta

The aim of this study was to examine whether exogenous somatotropin (ST) can alter the insulin-like growth factor (IGF) axis in the porcine epitheliochorial placenta. Crossbred gilts were injected either 6 mg of recombinant porcine ST or vehicle from days 10 to 27 after artificial insemination (term day 116). Control and ST-treated gilts were euthanized on day 28 (8 control/5 treated), day 37 (4 control/6 treated), and day 62 (4 control/6 treated) of gestation. Endometrium and placental tissue samples were collected and subjected to mRNA analyses. In control gilts, somatotropin receptor (STR) and IGF-I mRNA abundance in the endometrium decreased with gestation. Conversely, the amounts of IGF-II mRNA and of IGF binding protein (BP)-2 and -3 mRNA, which were analyzed in endometrium and placental chorion, increased with gestation. The endometrium contained less IGF-II mRNA but more IGFBP-2 and-3 mRNA than the placental chorion. In response to pST treatment, the amounts of endometrial STR and IGF-I mRNA were lower at days 28 and 37, but higher at day 62 of gestation. The content of IGF-II mRNA was higher in the endometrium of pST-treated than control gilts on day 37. The amount of IGFBP-2 mRNA was increased on day 37 in endometrium and placenta of pST-treated gilts, whereas no changes in IGFBP-3 mRNA were observed. The IGF-II/IGFBP-2 ratio was higher in the placenta in response to pST on day 28 of gestation. Results show that pST treatment of pregnant gilts during early gestation alters IGF axis in maternal and fetal placental tissues and suggest pST may exert an effect on fetal growth by altering the relative amount of IGFBPs and IGFs at the fetal-maternal interface.     

m6A甲基化酶METTL3和WTAP在牦牛组织及脂肪细胞中的表达

本研究旨在探究m⁶A RNA甲基化酶METTL3和WTAP在牦牛(Bos grunniens)不同组织、前体脂肪细胞增殖与分化过程中的表达模式和前体脂肪细胞分化过程mRNA m⁶A的变化水平。采用qRT-PCR检测牦牛皮下脂肪、肌肉、心、肝、脾、肺、肾和皮下脂肪不同时期(18和30月龄)METTL3及WTAP的mRNA表达水平。应用I型胶原酶消化法获取牦牛前体脂肪细胞,油红O染色和脂肪分化标志基因的检测建立牦牛前体脂肪细胞分化模型,以及qRTPCR检测前体脂肪细胞增殖分化阶段METTL3和WTAP的mRNA表达水平。结果表明,肝脏组织中METTL3表达最高(P<0.05),皮下脂肪组织表达量最低(P<0.05);WTAP在皮下脂肪组织中的表达最为丰富,脾脏中的表达量最低(P<0.05)。30月龄皮下脂肪组织中METTL3和WTAP mRNA表达量高于18月龄。前体脂肪细胞诱导分化12 d时,细胞中出现多而密的脂环,脂肪细胞分化特异性标志基因FABP4、C/EBPα和PPARγ第12天的表达量显著高于第0天(P<0.05)。METTL3和WTAP的表达量在细胞增殖阶段(24、48和72 h)呈现“下降-上升”的表达趋势(P<0.05)。在牦牛前体脂肪细胞分化阶段(0、4、8和12 d),METTL3表达量呈现“上升-下降-上升”的趋势,WTAP呈现“上升-下降”的趋势。细胞分化阶段mRNA m⁶A水平呈现逐渐上升的趋势,在分化12 d时细胞内RNA的m⁶A丰度最高(P<0.05)。本研究获得METTL3和WTAP在牦牛不同组织和前体脂肪细胞增殖分化阶段的变化规律及细胞分化过程中mRNA m⁶A水平变化,初步揭示WTAP和METTL3对牦牛脂代谢具有重要的调控作用。     

Messenger ribonucleic acid expression of the MyoD gene family in muscle tissue at slaughter in relation to selection for porcine growth rate

Livestock meat production capacity is related to muscle fiber numbers and growth. Muscle fibers develop during early embryonic development from proliferating and differentiating myoblasts. Post-natal muscle growth requires satellite cell proliferation and differentiation. Myoblast and satellite cell proliferation and differentiation is regulated by the genes of the MyoD gene family (myogenin, myf-5, myf-6, and MyoD1). Our aim was to study the mRNA expression of these genes in postnatal muscle tissue in relation to porcine selection for growth rate or leanness. Five boars from a line selected for fast growth (F-line) and five boars from a line selected against backfat thickness (L-line) were slaughtered, and biopsies were taken from 12 muscles. Between-line effects, within-line effects in relation to the performance of the pigs, and muscle-specific effects were studied. Comparing the F-line with the L-line revealed significantly greater myogenin, myf-5, and MyoD1 mRNA expression in some muscles of the F-line. The expression of myf-6 showed a tendency for the opposite effect in some muscles. Muscles were ordered by their muscle-specific growth rate (b-value). Within-line evaluation of the data revealed a systematic muscle effect for the myf-6 expression level in the F-line because higher b-values correlated with increased myf-6 expression level. Backfat thickness was negatively related to myogenin expression in the F-line. A relationship was found between myogenin:MyoD1 mRNA expression ratio and meat color/muscle fiber type composition in the L-line. Furthermore, the myogenin:MyoD1 ratio was greater in muscles from F-line boars than in muscles from L-line boars, which relates to the difference between the lines in muscle fiber type. We conclude that the mRNA levels of the MyoD genes in porcine muscle tissue at slaughter showed different relationships to selection for growth rate when evaluated between selection lines and within selection lines.