Effect of milking frequency on lactation persistency and mammary gland remodeling in mid-lactation cows.

The objective of this study was to evaluate the effect of milking frequency on milk production and composition, mammary cell proliferation, apoptosis, and gene expression. For this investigation, 10 Holstein cows that were being milked twice a day in mid lactation were selected. To study the effect of differential milking, 2 quarters were milked once daily and the other 2 were milked thrice daily for 8wk. After that period, twice-daily milking was resumed for all quarters, and data were collected for an additional 6wk. Mammary gland biopsies were taken 1wk before differential milking (wk -1) and after 4 and 8wk of differential milking. Milk samples were collected weekly throughout the experiment. Once-daily milking resulted in an immediate reduction in milk yield, whereas thrice-daily milking resulted in an increase in milk yield. During differential milking, the daily milk yield of the quarters milked once daily declined by 0.54kg/wk, on average, but remained constant in the quarters milked thrice daily. Part of the difference in milk yield between the glands pairs persisted after twice-daily milking was reinitiated. In the quarters milked once daily, milk BSA concentration increased, indicating an increase in tight junction leakiness, and zymographic analysis of milk enzymes showed increased activity of several proteases. Reducing the milking frequency also increased mammary cell apoptosis and, surprisingly, mammary cell proliferation. Interestingly, milk concentrations of stanniocalcin-1 and insulin-like growth factor-I and mammary gland expression of several genes were also modulated by milking frequency. For example, expression of insulin-like growth factor I receptor was downregulated during once-daily milking. Last, expression of the short and long isoforms of the prolactin receptor and of CSN2 (beta-casein) were upregulated during thrice-daily milking. Taken together, these data suggest that milking frequency not only affects mammary gland remodeling and the expression of paracrine factors but also modulates hormone sensitivity.

Local control of mammary involution: is stanniocalcin-1 involved?

There is considerable evidence to indicate the existence of local control of mammary gland involution, but the exact nature of this control has yet to be defined. Stanniocalcin-1 (STC-1) is a newly discovered mammalian hormone that seems involved in the lactation process and may be implicated in the control of involution. As a first step in investigating this hypothesis, the change in STC-1 levels in milk and serum was measured during drying off. Nine Holstein cows in late lactation were milked twice daily on half the gland, while the other half was left unmilked for a 14-d period. Milk and blood samples and mammary biopsies were taken on d -7, 1, 2, 7, and 14 relative to the onset of the nonmilking period. The concentrations of STC-1 in serum and milk were determined by RIA. The albumin concentration and proteinase activity of the milk were determined. Apoptosis of the mammary epithelium was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Finally, the effects of milk on cellular activity and apoptosis were tested in vitro on mammary epithelial cells by measuring the turnover of tetrazolium salts and by counting the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells. The drying off of 2 quarters increased the milk production of the quarters that were milked by 30%. Milk proteinase activity and BSA and STC-1 concentrations increased in the nonmilked quarters, but remained unchanged in the milked quarters. Moreover, at d 2, the apoptotic rate of the mammary cells was higher in the nonmilked quarters than in the milked quarters (0.22 +/- 0.04 vs. 0.07 +/- 0.04%, respectively). Finally, in vitro experimentation demonstrated that mammary epithelial cells cultured in the presence of milk from involuting quarters had 3-fold more apoptotic cells as compared with those cultured in milk from the milked quarters at d 14. The metabolic rate was reduced by 14.6% for milk from d 7 and 23.6% for milk from d 14. Interestingly, the metabolic rate was negatively correlated with the STC-1 concentration in milk (r = -0.65). This study shows for the first time that STC-1 in milk is increased following milk stasis, although its exact role in the involution process remains to be clarified.

Serum levels of stanniocalcin-1 in Holstein heifers and cows.

Stanniocalcin-1 is a hormone that possesses both paracrine and endocrine functions and has recently been identified in mammals. While paracrine functions have been determined for several organs, the role of circulating stanniocalcin-1 in cattle is still unclear but, observations in mice and cows suggest that stanniocalcin-1 plays a role in both gestation and lactation. The changes in serum stanniocalcin-1 content in different physiological states have never been evaluated in ruminants. We measured stanniocalcin-1 levels in sera from cattle ranging in age from post-weaned calves to 17-month-old heifers and in sera from cows during lactation and pregnancy. Our results indicate that the blood concentration of stanniocalcin-1 is increased by pregnancy, but not by lactation. The highest levels of stanniocalcin-1 were found in young calves and during the non-lactating period preceding calving. This suggests that stanniocalcin-1 is important for gestation and the preparation of the mammary gland for lactation.

Effect of stage of lactation and parity on mammary gland cell renewal.

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8 +/- 1.3 and 25.2 +/- 0.8 kg/d) and 50 DIM (38.0 +/- 1.2 and 29.8 +/- 1.1 kg/d), but it was the same for both groups at 250 DIM (23.9 +/- 1.5 and 23.8 +/- 1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, beta-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.

Short communication: suppression of estrous cycles in lactating cows has no effect on milk production.

The decline in milk yield observed after peak production in dairy animals results from apoptotic death of mammary epithelial cells. In cows, this decrease in milk yield can be accelerated by injection of 17beta-estradiol, thus evoking a possible role of estrogens in the regulation of bovine mammary gland involution. In nonpregnant cows, mammary involution could be induced or enhanced by the return of estrous cycles and the accompanying cyclic peaks of estrogen concentration in the serum of lactating animals. To test this hypothesis, we inserted implants of a GnRH agonist, deslorelin, in an ear of each cow (n = 10) on d 10 and 100 of lactation, to temporarily suppress the return of ovarian cycles. Cows were studied from calving to d 210 of lactation. Deslorelin had no impact on feed intake or animal health. Deslorelin significantly reduced serum concentrations of 17beta-estradiol and progesterone as compared with untreated cows (n = 10). Deslorelin had no effect on milk fat and protein, whereas milk lactose content was lower in treated cows than in control cows on d 100 of lactation. Finally, there was no difference in milk production between the 2 groups of cows. These results are consistent with previous observations that showed that delaying estrous cycles after calving had no effect on milk yield and they extend those observations to late lactation. Based on milk production data, the estrogen profiles associated with recurring estrous cycles apparently do not cause bovine mammary tissue to undergo gradual involution.

Antioxidants to prevent bovine neutrophil-induced mammary epithelial cell damage.

Activated neutrophils are able to produce a large quantity of bactericidal molecules such as reactive oxygen species that have been associated with tissue damage in several inflammation models. The protective effects of antioxidants in a context of neutrophil-induced damage to mammary epithelial cells were first evaluated in vitro using a coculture model of activated bovine neutrophils and a bovine mammary epithelial cell line (MAC-T cells). Cell damage was determined by quantifying the release of lactate dehydrogenase by MAC-T cells in culture medium. Morphological observation of cells stained with acridine orange was used to visualize the extent of cell damage. When incubated with neutrophils activated by lipopolysaccharides and phorbol 12-myristate 13-acetate, MAC-T cells released large amounts of lactate dehydrogenase indicating significant cell damage. The addition of dimethylthiourea or bathocuproine disulfonic acid did not reduce the damage whereas catechin, deferoxamine or glutathione ethyl ester significantly reduced neutrophil-induced cytotoxicity in a dose-dependent manner. The effect of deferoxamine, an iron chelator, on the growth of Escherichia coli and the ability of bovine neutrophils to phagocytose these bacteria were then assessed in vitro. Our data showed that deferoxamine did not interfere with the phagocytic activity of neutrophils but inhibited growth of the bacteria. Overall, our results suggest that antioxidants may be effective tools for protecting mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.

Milk fatty acid composition and mammary lipid metabolism in Holstein cows fed protected or unprotected canola seeds.

Six midlactation Holstein cows were fed a total mixed ration supplemented with either 4.8% canola meal, 3.3% unprotected canola seeds plus 1.5% canola meal, or 4.8% formaldehyde-protected canola seeds, according to a double 3 x 3 Latin square design. Each period lasted 3 wk; experimental analyses were restricted to the last week of each period. Mammary biopsies were taken the last day of each period for gene expression measurements. Milk production and milk protein percentage were reduced by canola seeds, whether protected or unprotected. Protected canola seeds also decreased dry matter intake. Feeding canola seeds reduced the content of C8 to C16 fatty acids in milk and increased the content of oleic acid (C18:1c9). Unprotected canola seeds elevated the concentrations of C18:0. Protected canola seeds increased the C18:2 and C18:3 content, and reduced the C18d:0/C18:1c9 ratio. Similar results were obtained for plasma fatty acids, with some specific features, such as an increased C16:0/C16:1 ratio with protected canola seeds. Canola seeds had no significant effects on insulin, triglycerides, or cholesterol present in serum, but increased the concentration of nonesterified fatty acids; a greater increase was obtained with protected canola seeds. Expression levels of acetyl-CoA carboxylase and delta 9-stearoyl-CoA desaturase genes measured in the mammary gland did not differ significantly between diets. Therefore, the reduced C18s:0/C18:1c9 ratio observed in milk with protected canola seeds was not due to an enhanced expression of the delta-9 desaturase in the mammary gland.

Matrix metalloproteinases and their inhibitors in human pituitary tumors.

OBJECTIVE: To determine the expression of matrix metalloproteinases (MMP)-1, -2, and -3 and the tissue inhibitors of metalloproteinases (TIMP)-1, -2, and -3 in 12 tissue samples from normal pituitary glands and in 28 human pituitary tumors ranging from Grade 0 to Grade IV, and to establish a correlation between the level of expression of MMPs and TIMPs and the tumor grade. METHODS: The expression of MMPs and TIMPs was determined by Western blotting. MMP activity was detected by gelatin zymography. RESULTS: MMPs were expressed in the majority of tumors, and their levels of expression were unrelated to tumor grade or to their invasive phenotype. Some correlation was observed between MMP activity detected by zymography and tumor grade. TIMP-2 and TIMP-3 were poorly expressed in high-grade tumors and strongly expressed in normal pituitary glands and in the majority of low-grade tumors. CONCLUSION: No correlation could be established between the invasive potential of tumors and MMP-1, -2, and -3 expression levels. Some correlation was observed between MMP activity detected by zymography and tumor grade. A good inverse correlation was observed between TIMP-2 and TIMP-3 expression levels and tumor grade. These data suggest that monitoring the expression of TIMP-2 and TIMP-3 or gelatinolytic activity could be of prognostic value.

Expression of matrix metalloproteinases and their inhibitors in human brain tumors.

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy.

Expression of matrix metalloproteinases and their inhibitors in human brain tumors.

Sixty human brain tumors, including grade I meningiomas, schwannomas, and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas and oligodendrogliomas, and grade IV glioblastomas and lung and melanoma metastases were analyzed for expression of four matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs), and MMP activity. No marked correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. All 60 tumors showed a similar pattern of activity in zymography, MMP-2 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were low in tumors of grade III but significantly higher in tumors of grade I, particularly schwannomas. Altogether, these data suggest that: (1) the balance between MMP-2 and TIMP-2 is important in human brain tumors; and (2) TIMP expression may be a valuable marker for tumor malignancy.

Rescue of polyomavirus DNA after co-transfection of recombinant plasmids with viral DNA fragments.

Plasmid DNA bearing a single copy of the mouse polyomavirus (Py) genome (template A) was transfected into murine cells together with another DNA (template B) carrying intact the viral sequence interrupted in template A. Rescue of unit-length Py DNA including markers from both templates was observed as long as the viral DNA in B overlapped that split in A by one kbp or more. Such rescue was not detectably enhanced by linearizing either or both template(s), and occurred in the absence of template replication. These findings are suggestive of an intermolecular recombination process taking place soon after transfection and starting with homologous pairing between A and B. Such pairing would facilitate removal of vector DNA from one template (A), followed by closure of the resulting break or gap through recombination with the other template (B). Since B may consist of a PCR-synthesized DNA fragment, these observations could conceivably serve as the basis for a method of generating mutant viral genomes.

p53-mediated DNA renaturation can mimic strand exchange.

The process of strand exchange is considered to be the hallmark of DNA recombination. Proteins known to carry out such exchange are believed to operate via one or the other of two mechanisms. RecA-like proteins promote the formation of a three-stranded or triplex synaptic intermediate in which strand exchange occurs, whereas other proteins would allow the coordinated exonucleolytic degradation of one strand in the duplex DNA and its replacement by an invading strand of similar sequence and polarity. In view of properties ascribed to it, we have attempted to determine whether p53 belongs to one or the other of these groups of proteins. The in vitro assay used relies on a double-stranded (ds) oligonucleotide (oligo 1+2) and a single-stranded (ss) oligonucleotide (oligo 3), part of which is complementary to oligo 1. The data collected suggest that, under the conditions of the assay, oligo 1+2 undergoes partial denaturation; p53 then catalyzes renaturation of oligo 1 with oligo 3, rather than true strand exchange. Since p53 is not known for being able to 'melt' DNA, it would seem unlikely that this protein would effect strand exchange in vivo without assistance from another, denaturing, protein.

An enhancer of recombination in polyomavirus DNA.

Previous work from this laboratory has indicated that intramolecular homologous recombination of polyomavirus (Py) DNA is dependent upon promoter structure or function. In this report, we demonstrate that Py DNA contains not two but three binding sites for transcription factor YY1, all located on the late side of viral origin of replication (ori) and the third well within the VP1 coding sequence. This third site (Y3), which may or may not play a role in transcription regulation, is immediately adjacent to a previously described recombination hot spot (S1/S2). We found that Py replicons carrying an altered Y3 site recombined in a manner suggesting partial inactivation of the S1/S hot spot. Point mutations precluding the binding of YY1 to Y3 in vitro depressed hot spot activity in vivo; however, of the two reciprocal products reflecting recombination at this spot, only that carrying the mutated Y3 site arose at a reduced rate. These results are interpreted in light of a model assuming that recombination occurs within a transcriptionally active viral chromatin tethered to the nuclear matrix by YY1.

Topoisomerase activity associated with polyoma virus large tumor antigen.

Polyomavirus (Py) large tumor antigen (LT) was produced in mammalian or insect cells infected with a suitable viral expression vector, and purified by a procedure combining immunoprecipitation with ion-exchange chromatography. Fractions containing the bulk of LT displayed a DNA-relaxing activity (LT-topo) which could be attributed neither to topoisomerase II (topo II) nor to topoisomerase I (topo I) encoded by the cell or the viral vector. On the one hand, LT-topo relaxed pBR322 DNA in a reaction which, unlike that characteristic of topo II, was ATP-independent and inhibited by camptothecin. On the other hand, serum from scleroderma patients which strongly inhibited calf thymus topo I had no effect on LT-topo, which absolutely required Mg2+ ions to relax DNA. Thus, LT-topo is either inherent to LT or belongs to a LT-bound enzyme similar to, but distinct from, topo I.

Intramolecular recombination in polyomavirus DNA is a nonconservative process directed from the viral intergenic region.

Previously, we have studied intramolecular homologous recombination in polyomavirus replicons under conditions allowing only one amplifiable recombination product to be generated from a single precursor molecule. In order to detect putative reciprocal product(s), we have now constructed precursor polyomavirus replicons which contain two copies, instead of one copy, of the viral intergenic region, including the origin of replication as well as both promoters. Upon transfection of mouse cells, constructs containing directly repeated intergenic regions yielded distinct amplifiable products, in number depending upon the functional integrity of both intergenic regions. Our data indicate that of two possible reciprocal products, a given precursor molecule would yield either one or the other but never both at the same time. Most striking, however, is the observation that promoter function is required for recombination, while the origin of replication function may be needed only for amplification of the recombination product once it has been formed. The data reported here confirm and extend previous data suggesting that (i) transcription is instrumental in recombination between direct repeats and (ii) nonconservative recombination involving direct repeats relies upon two promoters of opposing polarities.

Intramolecular recombination in polyomavirus DNA is controlled by promoter elements.

We show here that intramolecular homologous recombination in polyomavirus (Py) DNA depends upon discrete sequence elements of the viral regulatory region which are believed to regulate transcription initiation and exert little or no cis-control over replication. Either deleting the viral early promoter (EP) or inverting the viral late promoter (LP) strongly impairs viral DNA recombination under conditions allowing viral DNA replication to proceed undisturbed. These findings suggest that bi-directional transcription proceeding from the intergenic region favors intramolecular recombination.

Topoisomerase activity associated with SV40 large tumor antigen.

Purified preparations of simian virus 40 (SV40) large tumor antigen (LT) from three different sources, including LT expressed from a recombinant baculovirus, were found to relax negatively supercoiled cyclic DNA molecules, whether or not they contained SV40 sequences. Relaxation was stimulated by MgCl2 but not by ATP, and inhibited by camptothecin, suggesting the involvement of an enzymatic activity similar to that of topoisomerase I (topo I). However, the pH requirements for relaxation by respectively LT and topo I are different. Also, antibodies reacting with LT inhibited relaxation by preparations of LT but not topo I, whereas antibodies inhibiting relaxation by topo I had no effect on relaxation by LT. Reconstruction experiments suggested that both procedures used to purify LT, immunoaffinity chromatography and DEAE-Sepharose chromatography, separate topo I from LT. Finally, relaxing activity was found in over 40 preparations of LT, and in the few instances where activity could not be found, it probably had been lost during storage, rather than absent from the start. Whereas these results seem to exclude that the activity being detected is that of a contaminant of LT, they would be consistent with this activity being that of a stable topo-LT complex, or else intrinsic to LT itself.

Crossover site selection during recombination of polyomavirus replicons.

Two hybrid replicons containing polyomavirus (Py) genomes with large duplications of the viral late coding sequences were transfected into various permissive mouse cell lines. In all cell lines, either replicon yielded the sole amplifiable product expected from intramolecular homologous recombination, unit-length Py DNA (P155). In normal and in Py-transformed cells, such recombination was highly effective and involved sequences previously found to act as recombination hot spots (S repeats). In cells transformed by simian virus 40, however, these hot spots were inoperative in the generation of P155, which occurred with a reduced efficiency. These data confirm and extend earlier data indicating that the nature of products arising from recombination in Py replicons is tightly controlled by both cis- and trans-acting factors.

DNA nicking favors PCR recombination.

We attempted to use the polymerase chain reaction (PCR) to monitor in vitro recombination in a plasmid containing directly repeated sequences. Some of the plasmid preparations which had not been exposed to recombination conditions were however found to behave in the PCR test as if they had undergone homologous recombination. We show here that such false positives are attributable to a small degree of nicking and/or breaking of the DNA template. Presumably, such damage allows the formation of hybrid parental duplexes containing at least one truncated strand, the 3' end of which maps within the homology; extension of this 3' end by the polymerase then results in a linkage of sequences identical to that arising from homologous recombination.

The same mammalian replicon yields distinct recombination products in different cell lines.

We have observed previously that some chimeric replicons inclusive of a partly duplicated polyomavirus (Py) genome yield unit-length Py DNA (P155) at high frequency when transfected into normal or Py-transformed mouse cells. We demonstrate here that one such replicon generates either P155 or illegitimate recombination products in other mouse cells, transformed by simian virus 40. Use of the polymerase chain reaction indicates that each of the illegitimate products carried a different deletion, but that all deletions mapped within a rather well defined portion of the precursor replicon. Thus, these products were organized as if two hotspots for recombination existed in the Py late-coding region, one being located within or near one of the duplicated sequences characteristic of the chimeric replicon. Since this particular hotspot has already been shown to be involved in the generation of P155, the data reported here could indicate that a single recombination mechanism can yield either homologous (P155) or illegitimate products. How the DNA interacts with certain proteins, such as papovavirus large tumor antigen, could explain why one or the other type of product is formed.