Endocrine disruption of uterine insulin-like growth factor expression in the pregnant gilt

Early exposure of pregnant gilts to oestrogen, prior to the normal period of porcine conceptus oestrogen secretion, disrupts the uterine environment resulting in complete embryonic mortality during the period of placental attachment to the uterine surface. The current study evaluates the uterine insulin-like growth factor (IGF) system following endocrine disruption of early pregnancy in gilts through exposure to exogenous oestrogen on Days 9 and 10 of gestation. Endometrial IGF gene and protein expression, IGF-I receptor (IGF-IR) gene expression, and uterine lumenal content of IGF binding proteins (IGFBPs) were evaluated in control and oestrogen-treated gilts on Days 10, 12, 13, 15 and 17 of gestation. Oestrogen treatment altered endometrial IGF-I and IGF-IR gene expression on Days 12 and 13 of gestation. Uterine content of IGF-I and IGF-II in control gilts was greatest on Days 10, 12, and 13 followed by a four- to sixfold decrease on Day 15 of gestation. Oestrogen treatment caused a premature proteolysis of IGFBPs within the pregnant pig uterus on Day 10 of gestation, and an earlier decline in uterine lumenal IGF-I content. Results demonstrate that early exposure of pregnant gilts to oestrogen causes premature loss of uterine IGFs during the period of conceptus elongation. Timing for the release of uterine IGFs during early porcine conceptus development may play an important function in the ability of the conceptus to attach and survive during the establishment of pregnancy.     

Porcine endometrial and conceptus tissue kallikrein 1, 4, 11, and 14 gene expression

Previous studies have suggested that the porcine endometrium may express several tissue kallikreins during the estrous cycle and early pregnancy. The present study investigated porcine endometrial and conceptus tissue kallikrein 1, 4, 11, and 14 mRNA expression during the estrous cycle and early pregnancy. Tissue kallikrein (KLK) gene expression was evaluated using quantitative RT-PCR and in situ hybridization. KLK1 expression was similar across the estrous cycle and early pregnancy, and localized to the endometrial luminal (L) and glandular (G) epithelium. KLK4 endometrial mRNA expression was greatest on days 0, 5, and 10 when compared with days 12, 15, and 17 of the estrous cycle and greater in cyclic compared with pregnant gilts. Expression of KLK4 was more intense in the stroma and uterine epithelium from days 0 to 10 of the estrous cycle. Endometrial KLK11 mRNA was not different between cyclic and pregnant gilts but the expression was greatest on days 10 and 12 compared with all other days evaluated. There was an increased intensity of KLK11 gene expression in the stratum compactum on day 10 of the estrous cycle and early pregnancy. Endometrial KLK14 mRNA expression was not detectable on days 5 and 10 but was expressed on days 0, 12, 15, and 17 of the estrous cycle and pregnancy. KLK14 expression was localized in the uterine L and G epithelium, and stroma throughout the endometrium after day 10. Conceptus KLK1 mRNA did not change from days 10 to 17 of gestation. However, conceptus KLK4, and 14 mRNA expression was greatest on day 10 with expression declining after day 14 of gestation. Expression of the various tissue kallikreins in the endometrium and conceptus during the estrous cycle and early pregnancy in the pig can serve in the activation of growth factors and tissue remodeling during the establishment of pregnancy.     

Prostaglandin E(2) and F(2 alpha) production by equine conceptuses and concentrations in conceptus fluids and uterine flushings recovered from early pregnant and dioestrous mares

A growing equine conceptus must suppress the cyclical release of PGF(2 alpha) from the endometrium to effect maternal recognition of its presence in the uterus. Paradoxically, the conceptus itself secretes PGF(2 alpha), together with other prostaglandins. In this study, the PGF(2 alpha) and PGE(2) content of, and production in vitro by, day 10-32 equine conceptuses were measured and the influence of pregnancy on the concentrations of these prostaglandins in the uterine lumen was examined. In vitro, the release of both prostaglandins per mg conceptus tissue was very high on day 10 after ovulation and lower thereafter. However, while PGF(2 alpha) production decreased further after day 18 of gestation, PGE(2) production remained high until day 32. Prostaglandin concentrations in yolk sac fluid were unaffected by gestational age and PGE(2) concentrations in this compartment were two to five times higher than PGF(2a) concentrations. PGF(2 alpha) concentrations reached high values in uterine flushings recovered from cyclic mares during days 14-16 after ovulation, the expected time of luteolysis, but were negligible in flushings recovered from pregnant mares at this time. Beyond day 18 of gestation, PGF(2 alpha) concentrations in uterine flushings were high and strikingly similar to those recorded during cyclical luteolysis. It is concluded that the equine conceptus effects maternal recognition of pregnancy primarily by inhibiting the ability of the endometrium to release PGF(2 alpha) during days 12-16 after ovulation. However, the conceptus appears to delay, rather than prevent, the development of the uterine PGF(2 alpha) release pathway and an alternative mechanism must prevent luteolysis from being triggered during days 18-32 of gestation.     

Consequences of conceptus exposure to colony-stimulating factor 2 on survival, elongation, interferon-τ secretion, and gene expression

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 (P=0.07) 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-τ (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 (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.08) 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.     

Effect of different nutritional regimens before ovulation on plasma concentrations of metabolic and reproductive hormones and oocyte maturation in gilts

This study examined the effect of feeding either a maintenance (1.35 kg day(-1)) or high (3.5 kg day(-1)) plane of nutrition for 19 days after oestrus on oocyte maturity and both reproductive and metabolic hormone concentrations in gilts. Blood samples were collected each day from oestrus until slaughter on day 19 and during two pulse bleeds (15 min samples for 8 h) conducted on day 12 and day 18. After slaughter, oocytes were recovered from the presumed ovulatory population of follicles, matured in vitro for 46+/-2 h with 10% of their own follicular fluid, and then fixed and stained to determine the stage of nuclear maturation of the oocyte. Gilts fed the high diet had a higher proportion of oocytes that reached metaphase II than gilts fed the maintenance diet (88.3+/-2.71% versus 68.2+/-6.48%; P=0.013). Circulating concentrations of insulin-like growth hormone I (IGF-I) and the number of LH pulses were lower (P<0.05) in gilts fed the maintenance diet compared with gilts fed the high diet on day 12 and day 18. Mean oestradiol and progesterone concentrations were higher (P<0.05) for gilts fed the maintenance diet compared with gilts fed the high diet. Leptin concentrations were also higher on day 19 in gilts fed the high diet (2.16+/-0.26 ng ml(-1) (n=9) versus 3.20+/-0.32 (n=11), P=0.025). The results of this study indicate that improved oocyte quality (increased proportion of oocytes that reached metaphase II) is associated with a number of changes in reproductive and metabolic hormones. Further studies are required to indicate which hormonal mechanism may, in turn, lead to increased embryo survival and eventual litter size.     

Increased ovulation rate in gilts treated with dihydrotestosterone

Treatment with testosterone increases ovulation rate in pigs. The present study was conducted to examine the effects of 5alpha-dihydrotestosterone (DHT), a non-aromatizable androgen receptor ligand, on ovulation rate and amounts of androgen receptor and FSH receptor mRNAs in postpubertal gilts. In Expt 1, ovulation rate in response to daily i.m. injections of 0, 6, 60 or 600 microg DHT kg(-1) body weight from day 13 of the oestrous cycle (day 0 = day 1 of oestrus) to the following oestrus increased with each dose of DHT (P < 0.05). The mean increase in number of corpora lutea ranged from approximately three to 17 over the three dosages of DHT. In Expt 2, gilts treated daily with 60 microg DHT kg(-1) body weight during the early follicular phase (from day 13 to day 16), coincident with follicular recruitment, or the late follicular phase (day 17 to oestrus), had higher (P < 0.05) rates of ovulation compared with gilts that received vehicle, and were not different from gilts treated with DHT from day 13 to oestrus. Percentage recovery of day 3 embryos was not altered when gilts were treated from day 13 to day 16 or from day 17 to oestrus; however, treatment of gilts with DHT from day 13 to oestrus decreased recovery of day 3 (Expt 1) or day 11 (Expt 2) conceptuses. Daily administration of 6 microg DHT kg(-1) body weight to gilts from day 13 of the oestrous cycle to the following oestrus (Expt 3) did not affect the relative amounts of androgen receptor mRNA, but increased (P < 0.05) the amounts of FSH receptor mRNA in preovulatory follicles as determined by RT-PCR. The results of these experiments indicate that androgens may regulate ovulation rate in gilts. One of the roles of androgens might be regulation of the amounts of FSH receptor mRNA in ovarian follicles.     

Seminal plasma regulates endometrial cytokine expression, leukocyte recruitment and embryo development in the pig

In pigs, uterine exposure to the constituents of semen is known to increase litter size but the underlying physiological mechanisms remain undefined. Studies in rodents and humans implicate immune modulating moieties in seminal plasma as likely candidates, acting through enhancing the receptivity of the female tract. In this study, the acute and longer term effects of seminal plasma on cytokine expression and leukocyte abundance in the pig endometrium during early pregnancy have been characterised. The reproductive tracts of gonadotrophin-primed pre-pubertal gilts treated with intrauterine infusions of either pooled seminal plasma or phosphate-buffered saline (PBS) were retrieved at 34 h, or on day 5 and day 9 after treatment. Seminal plasma elicited an endometrial inflammatory infiltrate comprised of predominantly macrophages and major histocompatibility complex class II+-activated macrophages and dendritic cells. The abundance of these cells was greatest at the pre-ovulatory (34 h) time-point and their increase relative to PBS-treated tissues was maintained until day 9 after seminal plasma treatment. Seminal plasma induced the expression of the cytokines, granulocyte macrophage colony-stimulating factor, interleukin-6 and monocyte chemoattractant protein-1, and the eicosanoid-synthesising enzyme cyclo-oxygenase-2. Expression was maximal 34 h after treatment but altered expression patterns as a consequence of seminal plasma induction persisted through early pregnancy. These changes were accompanied by altered dynamics in pre-implantation embryo development with an increase in the number of embryos and in their viability after seminal plasma treatment. Together, these findings implicate factors in seminal plasma in programming the trajectory of uterine cytokine expression and leukocyte trafficking during early pregnancy and in regulating pre-implantation embryo development in the pig.     

Changes in major proteins in the embryonic capsule during immobilization (fixation) of the conceptus in the third week of pregnancy in the mare

During the third week of pregnancy, the equine conceptus is enclosed within a capsule, the glycan composition of which changes at around day 16 (ovulation = day 0) when the conceptus becomes immobilized (fixed) in the uterine lumen. Our objective was to characterize the process of fixation by identifying changes in major capsule-associated proteins. Individual equine conceptuses (n = 55) were collected transcervically by uterine lavage between days 13.5 and 26.5. Major proteins extracted from capsules were compared with those in fluids from the uterus and yolk sac by SDS-PAGE. Until day 14, a major capsule-associated protein that migrated at approximately 10 kDa was identified by N-terminal sequencing as equine beta2 microglobulin (beta2M). During fixation, beta2M in the capsule underwent limited proteolysis to an approximately 8 kDa form lacking nine amino acids from the N terminus, and was subsequently degraded. Expression of beta2M mRNA was detected in the yolk-sac wall tissues and endometrium between days 13.5 and 17.5. During this period, beta2M in the capsule was evidently not part of a complex with major histocompatibility complex class 1 heavy alpha chain bands because these were undetectable in the capsule and uterine lavage. Uterocalin (p19) was detected in uterine lavage and capsule throughout fixation, but in yolk-sac fluid only before fixation. These studies indicate that intact beta2M is a major protein associated with the embryonic capsule before fixation, after which it undergoes limited proteolysis to a truncated approximately 8 kDa form that remains in the capsule after the conceptus is immobilized.     

Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows: I. Ovarian, conceptus, and growth hormone-insulin-like growth factor system responses

The objective was to examine effects of bovine somatotropin (bST), pregnancy, and dietary fatty acids on reproductive responses in lactating dairy cows. Beginning at approximately 17 d in milk (DIM), a comparison was made of isoenergetic diets comprising supplementary lipids of whole cottonseed vs. calcium salts of fish oil enriched lipid (FO). Ovulation was synchronized in cows with a presynchronization plus Ovsynch protocol, and cows were inseminated artificially by appointment or not inseminated (d 0 = time of synchronized ovulation; 77 ± 12 DIM). On d 0 and 11, cows received bST (500 mg) or no bST. All cows were slaughtered on d 17. Number of cows in each group was as follows: control diet had 5 bST-treated cyclic (bST-C), 5 non-bST-treated cyclic (no bST-C), 4 bST-treated pregnant (bST-P), and 5 non-bST-treated pregnant (no bST-P) cows; and cyclic cows fed FO diet had 4 bST-treated (bST-FO) and 5 non-bST-treated cyclic (no bST-FO-C) cows. Feeding FO increased milk production, number of class 1 follicles (2 to 5 mm), and decreased insulin during the period before d 0 compared with control-fed cows. The bST increased milk production, pregnancy rate [83% (5/6) vs. 40% (4/10)], conceptus length (45 vs. 34 cm), and interferon-τ in the uterine luminal flushings (9.4 vs. 5.3 μg) with no effect on interferon-τ mRNA concentration in the conceptus. Treatment with bST increased plasma growth hormone (GH) and insulin-like growth factor (IGF)-I. Among control-fed cows (cyclic and pregnant), bST decreased progesterone concentration in plasma. Cows fed FO had less plasma insulin than control-fed cyclic cows, and FO altered the plasma GH (bST-FO > bST-C) and IGF-I (bST-C > bST-FO-C) responses to bST injections. Endometrial IGF-I mRNA was reduced in pregnant cows and tended to decrease in those fed FO. The IGF-II mRNA was increased in the endometrium of pregnant and bST-treated cows fed the control diet. Cows fed FO had increased concentrations of IGF-II mRNA, when bST was not injected. The insulin-like growth factor binding protein-2 (IGFBP-2) mRNA was increased in bST-P cows, whereas bST decreased the IGFBP-2 mRNA in all cyclic cows. In summary, bST and FO seemed to modulate reproductive responses that may be beneficial to the developing conceptus and pregnancy rate.     

Concentrations of progesterone in milk of cows administered an intravaginal progesterone insert

Milk from pregnant cows contains concentrations of progesterone (P4) considered safe for human consumption. The objective of this study was to determine if concentrations of P4 in milk during administration of an intravaginal progesterone insert (CIDR insert) are less than concentrations of P4 in milk associated with pregnancy. Results have implications for human use of milk from cows receiving CIDR inserts. Holstein cows (N = 64; > 40 and < 150 d after calving) were administered 25 mg of PGF2alpha i.m. (study d 0) and 20 cows detected in estrus from 2 to 4 d later were assigned randomly to either control (N = 10; no further treatment) or CIDR insert (N = 10; 1.38 g of P4) inserted on study d 17 (14 +/- 1 d after estrus) and removed 7 d later. Composite milk samples were collected contemporaneously from each of the 20 estrous cycling cows and from 10 pregnant cows (> or = 60 and < or = 220 d of gestation) twice daily from study d 17 to 27. Concentrations of P4 in defatted milk samples were quantified using a validated radioimmunoassay. Mean logs of areas under the curve of concentrations of P4 from the afternoon on study d 17 through the afternoon on study d 27 were 3.05 ng day/ml for control, 3.33 ng day/ml for CIDR insert, and 3.81 ng day/ml for pregnant cows. Therefore, increased P4 due to pregnancy was 0.76 ng day/ml (3.81-3.05), whereas the increase in P4 due to CIDR insert was only 0.28 ng day/ml (3.33-3.05). Applying a 95% confidence interval to 0.28 ng day/ml provided an upper value of 0.70 ng day/ml, lower than the increase due to pregnancy. Because milk from pregnant cows is considered safe for human consumption, it follows that milk from cows administered CIDR inserts should also be considered safe, based on concentrations of P4.     

Dihydrotestosterone influenced numbers of healthy follicles and follicular amounts of LH receptor mRNA during the follicular phase of the estrous cycle in gilts

The present experiments were conducted to determine androgenic effects on numbers, health, and amounts of gonadotropin receptor mRNA in late developing follicles of gilts. Gilts (n=5 per group) received daily injections of one of the following treatments on days 13-16 or days 13-18 of the estrous cycle: corn oil, 5alpha-dihydrotestosterone (DHT, 10 mg), flutamide (1.5 g, an androgen receptor inhibitor), DHT (10 mg) plus flutamide (1.5 g), testosterone (10 mg), and testosterone (10 mg) plus flutamide (1.5 g). Ovarian follicles > or =5 mm in diameter were evaluated on day 17 or 19, 24 h after receiving the last treatment dose. Follicles were classified as healthy (H), moderately atretic (MA), or very atretic (VA). Treatment with DHT increased (P<0.05) the numbers of H follicles relative to control gilts on days 17 and 19. DHT administration from days 13 to 16 diminished (P<0.05) the amounts of LH receptor (LHR) mRNA in H follicles from day 17 (relative amounts: 1.45+/-0.33 and 2.72+/-0.33 for DHT- and vehicle-treated gilts respectively). The effects of DHT on numbers of H follicles and LHR mRNA were not observed in gilts receiving DHT plus flutamide. Androgens did not influence numbers of MA, VA, and total follicles, or follicular estradiol-17beta concentrations and amounts of FSHR mRNA. Treating gilts with DHT during follicular recruitment and selection did not induce changes in the numbers of total follicles > or =5 mm, but rather increased the numbers of healthy follicles in this follicular population in association with decreased amounts of LHR mRNA.     

Postmortem changes in bovine troponin T isoforms on two-dimensional electrophoretic gel analyzed using mass spectrometry and western blotting: The limited fragmentation into basic polypeptides

To comprehend postmortem changes in troponin T (TnT), whole beef proteins were developed on a two-dimensional electrophoretic gel. Multiple TnT-related spots were identified by both western blotting and MALDI-TOF MS utilizing bovine TnT isoform mRNA sequences. More than 10 TnT fast-type isoform spots (pI 5.7-9.6<) and the two slow-type isoform spots (pI 5.6-5.7) were observed at slaughter. All the isoforms were degraded exclusively into basic spots (pI 9.6<) at day 14 postmortem. Some TnT-related phosphorylated spots present at slaughter had disappeared by day 14, suggesting that the phosphorylated N-terminal region was cut off during beef aging. The intact isoforms and the fragments were identified by the MS with sequence coverage of 20.8-62.7%, and two of the fragments included the cutting site peptide of a conventional 30kDa or of a slow TnT-derived fragment. These results revealed that all of the TnT isoforms are cut exclusively in the glutamic acid-rich amino-terminal region during postmortem aging.     

Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation

Endometrial glands are necessary for conceptus implantation and growth. In the ovine uterine gland knockout (UGKO) model, blastocysts hatch normally but fail to survive or elongate. This peri-implantation defect in UGKO ewes may be due to the absence of endometrial glands or, alternatively, to the lack of certain epithelial adhesion molecules or the inability of the endometrium to respond to signals from the conceptus. Two studies were performed to examine these hypotheses. In study one, normal (n = 8) and UGKO (n = 12) ewes were mated at oestrus (day 0) with intact rams and their uteri were flushed 14 days after oestrus. Normal ewes (n = 4) were also flushed on 14 days after oestrus. Uterine flushes from bred normal ewes contained filamentous conceptuses (n = 7 of 8), whereas those from UGKO ewes contained no conceptus (n = 5 of 12), a growth-retarded, tubular conceptus (n = 6 of 12), or a fragmented, filamentous conceptus (n = 1 of 12). In all groups, expression of mucin 1 and integrin alpha(v), alpha(5), beta(3) and beta(5) was localized at the apical surface of the endometrial luminal epithelium with no detectable differences between normal and UGKO ewes. Uterine flushes from pregnant ewes, but not cyclic or UGKO ewes, contained abundant immunoreactive interferon tau and the cell adhesion proteins, osteopontin and glycosylation-dependent cell adhesion molecule one. In study two, UGKO ewes were fitted with uterine catheters 5 days after oestrus, infused with recombinant ovine interferon tau or control proteins from 11 to 15 days after oestrus, and underwent hysterectomy 16 days after oestrus. Expression of several interferon tau-stimulated genes (ISG17, STAT1, STAT2 and IRF-1) was increased in the endometrium from interferon tau-infused UGKO ewes. These results support the hypothesis that the defects in conceptus elongation and survival in UGKO ewes are due to the absence of endometrial glands and their secretions rather than to alterations in expression of anti-adhesive or adhesive molecules on the endometrial luminal epithelium or to the responsiveness of the endometrium to the conceptus pregnancy recognition signal.     

Uterotrophic effects of relaxin related to age and estrogen receptor activation in neonatal pigs

While uterotrophic effects of relaxin are well documented, the mechanism through which relaxin promotes uterine growth is incompletely understood. Studies in rats suggest that relaxin-stimulated uterine edema depends on estrogen receptor (ER) activation. Here, neonatal pigs were used to investigate the interaction between relaxin and ER signaling pathways. Gilts were treated either at birth (postnatal day (PND) 0) (study 1) before the onset of endometrial ERalpha expression, or on PND 12 (study 2) after the onset of ERalpha expression. In study 1, gilts were treated with estradiol-17beta or porcine relaxin for two days and uteri were collected on PND 2. In study 2, PND 12 gilts were treated with a single injection of the ER antagonist ICI 182,780 (ICI) or vehicle. Two hours later, gilts were given either estradiol-17beta or porcine relaxin for two days. When administered for two days from birth (study 1), neither estradiol-17beta nor relaxin affected uterine weight or protein content. However, uterine luminal epithelial height was greater in relaxin- than in vehicle-treated gilts. In contrast, in study 2, both estradiol and relaxin increased uterine weight, protein content and uterine luminal epithelial height on PND 14. These effects were inhibited by pre-treatment with ICI in both estradiol- and relaxin-treated gilts. The results indicate that uterotrophic effects of relaxin in the neonatal pig are related to age and to both the relative presence and state of activation of the ER system in developing uterine tissues between birth and PND 14.     

Beneficial effects of a high fibre diet on oocyte maturity and embryo survival in gilts

The present study examined the effects of feeding gilts a high fibre diet from the third post-pubertal oestrus until either day 19 of the same cycle or insemination at the following oestrus on oocyte maturity, embryo survival and associated changes in reproductive hormone concentrations. Gilts fed with the high fibre diet had lower circulating oestradiol concentrations on days 17, 18 and 19 of the cycle and increased LH pulse frequency on day 18. More oocytes recovered on day 19 from gilts receiving the high fibre diet were at metaphase II after 46-h culture in medium containing 10% of their own follicular fluid, despite fewer large (>7 mm) follicles in these gilts when compared with control animals. There was no effect of diet on ovulation rate, corpora lutea size or progesterone concentrations on days 10-12 after insemination, but embryo survival on days 27-29 after insemination was higher in gilts that received the high fibre diet. This study demonstrates that a high fibre diet that increases embryo survival also improves oocyte maturity and provides information on endocrine correlates that may shed light on underlying mechanisms.     

Fibroblast growth factors activate mitogen-activated protein kinase pathways to promote migration in ovine trophoblast cells

Fibroblast growth factors (FGFs) 2 and FGF10 are uterine- and conceptus-derived factors that mediate trophoblast activities in cattle and sheep. To extend our understanding of how FGFs may control peri-implantation development in ruminants, we determined whether FGF2 and FGF10 impact trophoblast cell migration. Transwell inserts containing 8 μm pores were used to examine whether FGF2 or FGF10 supplementation increased oTr1 cell migration. Supplementation with 0.5 ng/ml FGF2 or FGF10 did not affect oTr1 cell migration number, but exposure to 5 or 50 ng/ml FGF2 or FGF10 increased (P<0.05) oTr1 cell migration when compared with controls. The involvement of specific MAP kinase (MAPK) cascades in mediating this FGF response was examined by using pharmacological inhibitors of specific MAPKs. Western blot analysis indicated that FGF2 and FGF10 increased phosphorylation status of MAPKs 1, 3, 8, 9, and 14. Exposure to specific inhibitors blocked FGF induction of each MAPK. Exposure to inhibitors before supplementation with FGF2 or FGF10 prevented FGF induction of cell migration, indicating that each of these signaling molecules was required for FGF effects. A final series of studies examined whether FGF2 and FGF10 also mediated the migration of a bovine trophoblast line (CT1 cell). Increases in migration were detected in each cell line by supplementing 5 or 50 ng/ml FGF2 or FGF10 (P<0.05). In summary, FGF2 and FGF10 regulate migratory activity of ovine trophoblast cells through MAPK-dependent pathways. These outcomes provide further evidence that FGFs function as mediators of peri-implantation conceptus development in cattle and sheep.     

腐乳白斑的化学组成及其抑制方法的研究

为探寻腐乳白斑的抑制手段,揭示白斑的物质基础,以腐乳白斑和不同品牌、不同品种腐乳为供试材料,测定溶解性、燃烧性、氨基酸组成,粗蛋白和钙离子含量,并通过毛霉菌株诱变、加入抗氧化剂处理研究腐乳白斑的抑制方法。结果表明:腐乳白斑易溶于甲酸溶液,粗蛋白含量为38.45%,钙含量为0.05 g/100 g,表明白斑的构成是以游离酪氨酸为主的氨基酸等结晶混合物。不同品牌、不同品种腐乳的氨基酸含量比较发现,白斑的形成与腐乳发酵工艺有关。毛霉菌株诱变后的白腐乳氨基酸含量有差异并出现白斑,说明物理诱变毛霉菌并不能彻底解决腐乳白斑问题。调节酒精度数发现,酒精度为13%的腐乳白斑形成率最低。在后期发酵向配料中加入抗氧化剂大豆异黄酮和茶多酚,在室温下后发酵半年,无白斑出现,表明抗氧化剂的添加有明显抑制腐乳白斑形成的作用。