Expression and localisation of extracellular matrix degrading proteases and their inhibitors during the oestrous cycle and after induced luteolysis in the bovine corpus luteum

The corpus luteum (CL) offers the opportunity to study high proliferative processes during its development and degradation processes during its regression. We examined the mRNA expression of matrix metalloproteases (MMP)-1, MMP-2, MMP-9, MMP-14, MMP-19, tissue inhibitor of MMP (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), uPA-receptor (uPAR), PA-inhibitors (PAI)-1, PAI-2 in follicles 20 h after GnRH application, CLs during days 1-2, 3-4, 5-7 and 8-12 of the oestrous cycle as well as after induced luteolysis. Cows in the mid-luteal phase were injected with Cloprostenol and the CLs were collected at 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2alpha injection. Real-time RT-PCR determined mRNA expressions. Expression from 20 h after GnRH to day 12: MMP-1, MMP-2, MMP-14 and tPA showed a clear expression, but no regulation. TIMP-1 and uPAR mRNA increased when compared with the follicular phase. TIMP-2, MMP-9, MMP-19 and uPA increased from the follicular phase to days 8-12. PAI-1 and PAI-2 expression increased from days 1-7 and decreased to days 8-12. Induced luteolysis: MMP-1, MMP-2, MMP-9, MMP-14, MMP-19 and TIMP-1 all increased at different time points and intensities, whereas TIMP-2 was constantly decreased from 24 to 64 h. The plasminogen activator system and their inhibitors were up-regulated from 2 to 64 h, tPA was already increased after 0.5 h. Immunohistochemistry for MMP-1, MMP-2, MMP-14: an increased staining for MMP-1 and MMP-14 was seen in large luteal cells beginning 24 h after PGF2alpha application. MMP-2 showed a strong increase in staining in endothelial cells at 48 h.     

Localization and temporal regulation of tissue inhibitors of metalloproteinases 3 and 4 in bovine preovulatory follicles

The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are potential regulators of the focalized extracellular matrix degradation required for ovulation. The objectives of the present study were to determine localization and temporal regulation of TIMP-3 and TIMP-4 mRNA and protein in bovine preovulatory follicles. Ovaries containing preovulatory follicles were collected at 0, 12 and 20 h after GnRH injection for real-time PCR quantification of TIMP-3 and TIMP-4 mRNAs and immunohistochemical localization studies. Additional samples collected at 0, 6, 12, 18 and 24 h post GnRH injection were subjected to Western analysis to determine temporal changes in TIMP-3 and TIMP-4 proteins in the apex and base of preovulatory follicles. Results indicate the gonadotropin surge regulates TIMP-3 and TIMP-4 expression. TIMP-3 and TIMP-4 mRNAs increased within 12 h after GnRH injection. TIMP-3 protein was localized to granulosal and thecal layers of preovulatory follicles and adjacent ovarian stroma, whereas TIMP-4 immunoreactivity was localized to granulosal and thecal cells and ovarian blood vessels. Amounts of TIMP-3 and TIMP-4 proteins in the follicular apex peaked within 12 h post GnRH injection and subsequently declined by 24 h. However, amounts of TIMP-3 and TIMP-4 proteins in the base were not elevated after GnRH administration. Results demonstrate that mRNA and protein for both TIMP-3 and TIMP-4 are increased in bovine preovulatory follicles following the gonadotropin surge. Coordinate expression of TIMPs and MMPs may help regulate the extracellular matrix remodeling characteristic of the ovulatory process.     

Expression and localization of extracellular matrix-degrading proteinases and their inhibitors in the bovine mammary gland during development, function, and involution

In degrading the extracellular matrix, matrix metalloproteinases (MMP) and the plasminogen activator (PA) system may play a critical role in extensive remodeling that occurs in the bovine mammary gland during development, lactation, and involution. Therefore, the aim of our study was to investigate the mRNA expression of MMP-1, MMP-2, MMP-14, MMP-19, tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, urokinase-type PA, tissue-type PA, urokinase-type PA receptor, and PA inhibitor-1 by quantitative PCR and to localize with immunohistochemistry MMP-1, MMP-2, MMP-14, and TIMP-2 proteins in the bovine mammary gland during pubertal mammogenesis, lactogenesis, galactopoiesis, and involution. Expression of mRNA for each of the studied factors was relatively lower during galactopoiesis and early involution but was markedly increased during mammogenesis and late involution, 2 stages in which tissue remodeling is especially pronounced. The localization of proteins for MMP-1, MMP-14, and TIMP-2 showed a similar trend with strong staining intensity in cytoplasm of mammary duct and alveolar epithelial cells during pubertal mammogenesis and late involution. Interestingly, MMP-2 protein was localized only in the cytoplasm of endothelial cells during late involution. Our study demonstrated clearly that expression of extracellular matrix-degrading proteinases coincides with a concomitant expression of their inhibitors. High expression levels of MMP, TIMP, and PA family members seem to be a typical feature of the nonlactating mammary gland.     

Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture

During ovarian folliculogenesis, ascorbic acid may be involved in collagen biosynthesis, steroidogenesis and apoptosis. The aims of this study were to determine the effects of ascorbic acid on bovine follicle development in vitro. Preantral follicles were cultured for 12 days in serum-free medium containing ascorbic acid (50 microg ml(-1)). Half of the medium was replaced every 2 days, and conditioned medium was analysed for oestradiol and matrix metalloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was assessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absence of serum, there was significant (P < 0.05) follicle growth and oestradiol secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle growth (P < 0.05), but compromised follicle integrity. By day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions (P < 0.05), and significantly (P < 0.01) less granulosa and theca cell death was observed in these follicles than in control follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. In conclusion, this culture system was capable of supporting follicle differentiation over the 12 day culture period. Furthermore, ascorbic acid maintains bovine follicle health and basement membrane remodelling in vitro.     

采后茉莉酸甲酯处理对桃果实青霉病及细胞壁降解酶的影响

以桃果实为试材,研究了采后不同浓度茉莉酸甲酯（Me JA）处理对损伤接种桃果实扩展青霉（Penicillium expansum）病斑扩展的影响,并分析了最佳浓度Me JA处理后桃果实细胞壁降解酶活性的变化。同时研究离体条件下Me JA对P.expansum菌丝生长和孢子萌发的影响。结果表明,100μmol/L Me JA对桃果实P.expansum的病斑抑制效果最好,并且显著（p≤0.05）抑制了菌丝生长和孢子萌发。100μmol/L Me JA处理明显抑制了桃果实多聚半乳糖醛酸酶（PG）、果胶甲酯酶（PME）、果胶甲基反式消除酶（PMTE）、多聚半乳糖醛酸反式消除酶（PGTE）和β-葡萄糖苷酶活性。由此表明,Me JA抑制桃果实青霉病的发生与延缓果实软化有关。      

Effect of high intensity pulsed electric fields on enzymes and vitamins in bovine raw milk

Pulsed electric fields (PEF) were applied to fresh bovine raw milk using a laboratory-scale continuous PEF system to study the impact on selected native enzyme activities and on vitamin degradation. The activities of protease, lipase, alkaline phosphatase and lactoperoxidase following PEF treatment at electric field strengths ranging from 15 to 35 kV/cm for treatment times of 12.5 µs to 75 µs were examined. Reductions in enzyme activity, even under the most severe conditions employed, were quite modest, amounting to 14%, 37% and 29% for lipase, protease and alkaline phosphatase, respectively, while the activity of lactoperoxidase remained unchanged. The PEF treatment had no effect on the levels of thiamin, riboflavin, retinol and α-tocopherol in the milk .     

Effect of pH,salt and chemical rinses on bacterial attachment to extracellular matrix proteins

Microbial contamination of carcass surfaces occurs during slaughter and post-slaughter processing steps, therefore interventions are needed to enhance meat safety and quality. Although many studies have been done at the macro-level, little is known about specific processes that influence bacterial attachment to carcass surfaces, particularly the role of extracellular matrix (ECM) proteins. In the present study, the effect of pH and salt (NaCl, KCl and CaCl₂) on attachment of Escherichia coli and Salmonella isolates to dominant ECM proteins: collagen I, fibronectin, collagen IV and laminin were assessed. Also, the effects of three chemical rinses commonly used in abattoirs (2% acetic acid, 2% lactic acid and 10% trisodium phosphate (TSP)) were tested. Within a pH range of 5–9, there was no significant effect on attachment to ECM proteins, whereas the effect of salt type and concentration varied depending on combination of strain and ECM protein. A concentration-dependant effect was observed with NaCl and KCl (0.1–0.85%) on attachment of E. coli M23Sr, but only to collagen I. One-tenth percent CaCl₂ produced the highest level of attachment to ECM proteins for E. coli M23Sr and EC614. In contrast, higher concentrations of CaCl₂ increased attachment of E. coli EC473 to collagen IV. Rinses containing TSP produced >95% reduction in attachment to all ECM proteins. These observations will assist in the design of targeted interventions to prevent or disrupt contamination of meat surfaces, thus improving meat safety and quality.     

Effect of high-pressure treatment at various temperatures on indigenous proteolytic enzymes and whey protein denaturation in bovine milk

The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55 degrees C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20 degrees C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16-18%) and PA (38-62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40 degrees C and at the pressures 450 and 650 MPa at 55 degrees C. Synergistic negative effects on the amount of native beta-lactoglobulin were observed at 450 and 650 MPa and on the amount of native alpha-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native beta-lactoglobulin and alpha-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage.     

Effects of whole cottonseed diet and recombinant bovine somatotropin on ovarian follicles in lactating dairy cows

The effects of whole cottonseed (WCS) in the diet and the administration of bovine somatotropin (bST) on ovarian follicular dynamics and plasma progesterone (P4) concentrations were examined in cows during a period of synchronized follicular growth. Lactating Holstein cows (n = 28) were randomly assigned to treatments in a 2 x 2 factorial arrangement. Diets consisted of WCS (15% of dry matter) or no WCS, and bST at a dose of 0 or 208 mg/14 d. Dietary treatments began within 24 h of calving and bST treatments began within 7 d postpartum. Cows received GnRH at 65 +/- 3 d postpartum (d 0), PGF2alpha, (d 7), a second dose of GnRH (d 9), and were inseminated 16 h later (d 10). Ovarian changes were monitored daily by ultrasonography from d 0 to 9. On d 9,93% of cows had a preovulatory follicle and 86% ovulated. For Class 2 (6 to 9 mm) follicles, a diet x bST interaction was detected, with bST stimulating Class 2 follicles in cows fed WCS, but not in cows on the control diet. Neither diet nor bST affected numbers of Class 1 (2 to 5 mm) or Class 3 (> or = 10 mm) follicles or sizes of the subordinate and dominant follicles. During the luteal phase of the cycle, lactating cows fed WCS tended to have elevated concentrations of plasma P4, whereas bST was without effect. Plasma concentrations of high-density lipoprotein cholesterol were increased in cows fed WCS. Number and diameter of corpora lutea did not differ among treatments.     

Effect of cold storage and freezing of bovine and porcine liver on activity and subcellular distribution of mitochondrial enzymes

The subcellular distribution of the mitochondrial enzymes lipoamide dehydrogenase (LIPDH), citrate synthase (CS), and beta-hydroxyacyl-CoA-dehydrogenase (HADH) in bovine and porcine liver tissue was studied by measuring the enzyme activities in a phosphate buffer extract of tissue (total activity) and in liver press-juice (cell plasma). In slaughter-fresh liver most of the activity was located in the mitochondria. During storage of liver under refrigeration (+2 degrees C) for several days a large decrease in total LIPDH activity and a lesser decrease in HADH activity, but no change in CS activity were observed. There was no or only little release of the three enzymes into the cell plasma during storage; this indicates that storage of liver at +2 degrees C was not accompanied by a marked damage of mitochondria. Freezing (-20 degrees C) and thawing of bovine and porcine liver caused some losses of the total activity of HADH and particularly of LIPDH but not changes in CS activity. There was a considerable increase in the activities of LIPDH, CS, and HADH in the press juice after freezing and thawing of liver tissue. Apparently freezing of liver results in damage to the mitochondria and, therefore, in a partial release of the three enzymes from the inner membrane of the mitochondrion into the cell plasma. By storage of liver under refrigeration the mitochondria became more sensitive to freezing and thawing. Prolonged frozen-storage of liver resulted in an increased release of LIPDH, CS, and HADH into the cell plasma.     

Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures

Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145-151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4 h with enzyme inhibitor and then challenged with progesterone for 1 h. Cell viability was unaffected by inhibitor treatment and averaged 84 ± 1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5 ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.     

Influence of stage of cycle, corpus luteum location, follicle size, and number of large follicles on estradiol-17 beta concentrations in bovine follicles

Concentrations of estradiol-17 beta in follicular fluid were correlated to follicular size, stage of estrous cycle, location of corpus luteum, and presence of large follicles. Paired ovaries were obtained from 481 nonpregnant cows at slaughter and follicles were classified as ipsilateral or contralateral to the corpus luteum. Follicular fluid estradiol-17 beta concentrations from 2494 small, 1485 medium, and 396 large follicles were quantified by radioimmunoassay. Stage of estrous cycle was estimated by visual examination of the corpus luteum. Follicles in stage 1 of the estrous cycle (d 1 to 4) had the highest estradiol-17 beta concentration and the smallest mean follicular diameter. Location of follicles relative to the corpus luteum had no influence on estradiol-17 beta concentrations. As follicular size increased, concentration of estradiol-17 beta also increased. The presence of a single large follicle did not affect the concentration of estradiol-17 beta in medium or small follicles. In contrast, if multiple large follicles occurred in the same cow, concentrations of estradiol-17 beta were significantly lower in medium but not small follicles.     

Gene expression for LH receptor, 17 alpha-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17 alpha-hydroxylase (17 alpha OH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n = 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated by in situ hybridization. LH-R and 17 alpha OH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17 alpha OH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteal-like cells expressing StAR. In summary, our study suggests that in spite of the presence of 17 alpha OH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein. During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.     

Role of transforming growth factor-beta1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.     

Effect of indomethacin administration on glucose homeostasis and pancreatic hormones in lactating goats

To determine glucogenic and hormonal responses in ruminants to indomethacin, a prostaglandin synthetase inhibitor, four lactating Toggenburg goats were used in a crossover design. Fifteen milligrams of indomethacin or 3 ml of saline were injected subcutaneously twice daily with propionate infused intrajugularly at a rate of 2 mM/min for 75 min as a challenge. Plasma glucose concentrations were increased by indomethacin injections. Plasma concentrations of insulin, glucagon, and 6-keto-prostaglandin F1 alpha were not affected by indomethacin injections. Propionate infusion elevated plasma glucose, insulin, and glucagon concentrations. A decreased insulin release in response to propionate challenge was observed with indomethacin injections.     

Effect of long-term infusion with recombinant growth hormone-releasing factor and recombinant bovine somatotropin on development and function of dominant follicles and corpora lutea in Holstein cows.

The objective of this study was to evaluate the effects of recombinant bovine growth hormone-releasing factor (rGRF) or recombinant bovine somatotropin (rbST) on growth and function of the first-wave dominant follicle and corpus luteum. Primiparous Holstein cows (117 d postpartum) were infused with 12 mg/d of rGRF (n = 10) or 29 mg/d of rbST (n = 10) for 63 d, and non-infused cows (n = 10) were controls. At slaughter on d 5 of an estrous cycle, blood and ovaries were collected and data from cows with a corpus luteum were analyzed (control, n = 8; rGRF, n = 5; rbST, n = 6). Treatment with rGRF or rbST increased somatotropin (ST) and IGF-I in serum similarly compared with controls. In contrast, rbST-treated cows had higher concentrations of ST in follicular fluid (FF) compared with rGRF-treated and control cows. In addition, rbST, but not rGRF, increased the number and decreased the size of estrogen-active follicles (EA; estradiol > progesterone concentrations in FF), increased the abundance of IGF binding proteins-2, -3, and -4 in FF from EA follicles, and increased the number but decreased the size of corpora lutea and decreased concentration of progesterone in serum compared with controls. Based on these results, we concluded that long-term infusion of rbST alters growth and function of the first-wave dominant follicle and the corpus luteum in cattle.     

Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymes and extracellular proteases, and prevention of cell lysis

The Bacillus subtilis spo0A mutant is an adequate host for extracellular protein production (e.g., alpha-amylase). However the mutant was prone to cell lysis. SDS-PAGE and zymography of cell wall lytic proteins indicated that the spo0A mutant contained high amounts of two major autolysins (LytC [CwlB] and LytD [CwlG]) and two minor cell wall lytic enzymes (LytE [CwlF] and LytF [CwlE]). On the other hand, the expression of eight extracellular protease genes was very poor or absent in the spo0A mutant. An eight-extracellular-protease-deficient mutant (Dpr8 strain) was constructed and the strain also exhibited cell lysis. The autolysins from the spo0A mutant were degraded by the supernatant of the wild type but not degraded by that of the Dpr8 mutant. These results suggest that the extensive cell lysis of the spo0A mutant was partially caused by the stability of autolysins via the decrease of the extracellular proteases. The introduction of a major autolysin and/or SigD mutations into the spo0A mutant was effective for preventing cell lysis.