Vanessa Lollivier

Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows

In most mammals, prolactin (PRL) is essential for maintaining lactation, and yet the short-term suppression of PRL during established lactation by bromocriptine has produced inconsistent effects on milk yield in cows and goats. To assess the effect of the long-term inhibition of PRL release in lactating dairy cows, 5 Holstein cows in early lactation received daily intramuscular injections of 1mg of the PRL-release inhibitor quinagolide for 9 wk. Four control cows received the vehicle (water) only. During the last week of the treatments, one udder half was milked once a day (1×) and the other twice a day (2×). Blood samples were harvested at milking in wk -1, 1, 4, and 8. The daily injections of quinagolide reduced milking-induced PRL release but not the basal PRL concentration. Quinagolide induced a faster decline in milk production, which was about 5.3 kg/d lower in the quinagolide-treated cows during the last 4 wk of treatment. During wk 9, the inhibition of milk production by quinagolide was maintained in the udder half that was milked 2× but not in the half milked 1×. Milk production was significantly correlated with the quantity of PRL released at milking. Quinagolide did not affect the release of oxytocin at milking. Serum concentration of insulin-like growth factor-1 was not affected by treatment or correlated with milk production. Serum concentrations of leptin and the calciotropic hormone stanniocalcin were not affected by the treatment. In conclusion, the chronic administration of the PRL-release inhibitor quinagolide decreases milk production in dairy cows. The effect is likely the result of the reduced release of milking-induced PRL and is modulated at the level of the gland by milking frequency.

Oxytocin stimulates secretory processes in lactating rabbit mammary epithelial cells

Oxytocin plays a major role in lactation mainly by its action on milk ejection via the contraction of myoepithelial cells. The effect of oxytocin on milk production and the presence of oxytocin receptors on different epithelial cells suggest that this hormone may play a role in mammary epithelial cells. To determine precisely the various roles of oxytocin, we studied localization of oxytocin receptors in lactating rabbit and rat mammary tissue and the influence of oxytocin on secretory processes in lactating rabbit mammary epithelial cells. Immunolocalization of oxytocin receptors on mammary epithelial cells by immunofluorescence and in mammary tissue by immunogold in addition to in situ hybridization showed that lactating rat and rabbit mammary epithelial cells expressed oxytocin receptors. Moreover, oxytocin bound specifically to epithelial cells. To determine whether oxytocin had an effect on lactating rabbit mammary epithelial cells, isolated mammary fragments were incubated in the presence or absence of 10−6 i.u. ml−1 of oxytocin. After 1 min of incubation with oxytocin, the morphology of epithelial cells and the localization of caseins and proteins associated with the secretory traffic suggested a striking acceleration of the transport leading to exocytosis, whereas the contraction of myoepithelial cells was only detectable after 7 min. Addition of 10−8 g ml−1 of atosiban before the addition of oxytocin prevented the oxytocin effect on secretory processes and on myoepithelial cell contraction. Addition of 10−6 i.u. ml−1 of vasopressin to the incubation medium did not mimic the stimulating effect of oxytocin on secretory traffic. These results show that lactating rabbit and rat mammary epithelial cells express oxytocin receptors and that oxytocin binds to these receptors. They strongly suggest that oxytocin has a dual effect on lactating mammary tissue: an acceleration of the intracellular transfer of caseins in mammary epithelial cells followed by the contraction of myoepithelial cells.

Mammary cell activity and turnover in dairy cows treated with the prolactin-release inhibitor quinagolide and milked once daily

To assess the regulation of mammary cell activity, survival, and proliferation by prolactin (PRL), 5 Holstein cows in early lactation received daily i.m. injections of 1mg of quinagolide, a suppressor of PRL release, for 9 wk, whereas 4 control cows received the vehicle (water) only. During the last week of treatment, one udder half was milked once a day (1×) and the other twice a day (2×). Mammary biopsies were harvested 1 wk before and 4 and 8 wk after the start of quinagolide treatment. The quinagolide injections reduced milk yield and resulted in lower levels of κ-casein and α-lactalbumin mRNA in the mammary biopsies at wk 4 compared with the control cows. In the mammary tissue of the quinagolide-treated cows at wk 8 of treatment, cell proliferation (as determined by proliferating cell nuclear antigen labeling) was lower and apoptosis (as determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay) was higher than in the mammary tissue of the control cows. During differential milking, mammary epithelial cells (MEC) were extracted from the milk by centrifugation and purified by immunocytochemical binding to allow variations in the levels of mammary transcripts to be observed. After 9 wk of treatment, levels of α-lactalbumin and κ-casein mRNA were lower in the MEC isolated from milk of the quinagolide-treated cows. This effect was associated with lower PRL receptor mRNA levels and a tendency toward lower viability in the milk-isolated MEC from the 2×-milked glands. The decrease from 2× milking to 1× milking also downregulated α-lactalbumin and κ-casein transcripts in the milk-isolated MEC. Viability was higher for the MEC collected from the 1×-milked udder halves compared with the 2×-milked halves. In conclusion, the reduction in milk yield after chronic administration of the PRL-release inhibitor quinagolide is associated with a reduction in mammary cell activity, survival, and proliferation in lactating dairy cows. Reduced milking frequency was also associated with a decrease in MEC activity.

New developments on the galactopoietic role of prolactin in dairy ruminants.

In most mammals, prolactin (PRL) is essential for maintaining lactation and its suppression strongly inhibits lactation. However, the involvement of PRL in the control of ruminant lactation is less clear because inconsistent effects on milk yield have been observed with short-term suppression of PRL by bromocriptine. By contrast, in vitro studies have provided evidence that PRL helps to maintain the differentiation state and act as a survival factor for mammary epithelial cells. Therefore, a series of experiments were conducted to assess the galactopoietic role of PRL. In a first experiment, daily injections of the PRL inhibitor quinagolide reduced milking-induced PRL release and induced a faster decline in milk production. Milk production was correlated with PRL released at milking. Quinagolide reduced mammary cell activity, survival, and proliferation. During the last week of treatments, differential milking (1× vs 2×) was applied. The inhibition of milk production by quinagolide was maintained in the udder half that was milked 2× but not in the udder half milked 1×, suggesting that the response to PRL is modulated at the gland level. In a second experiment, cows were injected with quinagolide, quinagolide + injection of bovine PRL at milking time, or water. As in the first experiment, quinagolide reduced milk, protein, and lactose yields. Although PRL injections at milking time were not sufficient to restore milk yield, they tended to increase milk protein and lactose yields and increased the viability of milk-purified mammary epithelial cells. Recently, we investigated the use of quinagolide at drying off. Treating late-lactation cows with quinagolide decreased milk production within the first day of treatment and induced faster increases in somatic cells and bovine serum albumin content in mammary secretions after drying off, which indicates an acceleration of mammary gland involution. In conclusion, these data, combined with data from other studies, provide a good body of evidence indicating that PRL is galactopoietic in dairy cows. However, the response to PRL appears to be modulated at the mammary gland level.

Effects of nutrient restriction on mammary cell turnover and mammary gland remodeling in lactating dairy cows

The aim of this study was to investigate the effects of a severe nutrient restriction on mammary tissue morphology and remodeling, mammary epithelial cell (MEC) turnover and activity, and hormonal status in lactating dairy cows. We used 16 Holstein × Normande crossbred dairy cows, divided into 2 groups submitted to different feeding levels (basal and restricted) from 2 wk before calving to wk 11 postpartum. Restricted-diet cows had lower 11-wk average daily milk yield from calving to slaughter than did basal-diet cows (20.5 vs. 33.5 kg/d). Feed restriction decreased milk fat, protein, and lactose yields. Restriction also led to lower plasma insulin-like growth factor 1 and higher growth hormone concentrations. Restricted-diet cows had lighter mammary glands than did basal-diet cows. The total amount of DNA in the mammary gland and the size of the mammary acini were smaller in the restricted-diet group. Feed restriction had no significant effect on MEC proliferation at the time of slaughter but led to a higher level of apoptosis in the mammary gland. Gelatin zymography highlighted remodeling of the mammary extracellular matrix in restricted-diet cows. Udders from restricted-diet cows showed lower transcript expression of α-lactalbumin and kappa-casein. In conclusion, nutrient restriction resulted in lower milk yield in lactating dairy cows, partly due to modulation of MEC activity and a lower number of mammary cells. An association was found between feed restriction-induced changes in the growth hormone-insulin-like growth factor-1 axis and mammary epithelial cell dynamics.

Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling

Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

New insights into the importance of prolactin in dairy ruminants1

In most mammals, prolactin (PRL) is essential for maintaining lactation, and the suppression of PRL inhibits lactation. However, the involvement of PRL in the control of ruminant lactation is less clear, because inconsistent effects on milk yield have been observed with the short-term suppression of PRL by bromocriptine. Therefore, several experiments have been conducted to assess the galactopoietic role of PRL. In an initial experiment, cows in early lactation received daily injections of the dopamine agonist quinagolide for 9 wk. Quinagolide reduced milking-induced PRL release and caused a faster decline in milk production. Quinagolide also reduced mammary epithelial cell activity, survival, and proliferation. In goats, cabergoline, another dopamine agonist, caused a 28% decrease in milk yield the day after injection. In another experiment, cows were injected for 5d with quinagolide, with quinagolide plus bovine PRL injected at milking time, or with vehicles only. Again, quinagolide reduced milk, protein, and lactose yields. Although PRL injections were not sufficient to restore milk yield, they tended to increase milk protein and lactose yields and increased the viability of mammary epithelial cells purified from milk. Recently, our team stimulated PRL secretion with daily injections of the dopamine antagonist domperidone for 5 wk. Milk production increased gradually and was greater in domperidone-treated cows during the last 4 wk of the treatment period. In most experiments where PRL secretion was manipulated, feed intake paralleled the changes of PRL concentration, supporting the idea that PRL increases feed intake to provide the nutrients necessary to support lactation in dairy ruminants. In late-lactation cows, quinagolide and cabergoline decreased milk production within the first day of treatment and induced more rapid changes in several markers of mammary gland involution after drying-off. In addition, quinagolide improved the resistance to intramammary infection, suggesting that PRL inhibition could be an alternative strategy for facilitating drying-off. Prolactin appears to directly affect mammary gland functions, but mammary gland responsiveness to PRL appears to be modulated by local and systemic factors. Therefore, the modulation of the number and isoforms of the PRL receptors as well as the expression of intracellular modulators of cell signaling in the mammary gland require further investigation. In conclusion, these data, combined with those from other studies, provide a good body of evidence that PRL is galactopoietic in dairy ruminants.

In vivo inhibition followed by exogenous supplementation demonstrates galactopoietic effects of prolactin on mammary tissue and milk production in dairy cows

It has been previously shown that the long-term inhibition of milking-induced prolactin (PRL) release by quinagolide (QN), a dopamine agonist, reduces milk yield in dairy cows. To further demonstrate that PRL is galactopoietic in cows, we performed a short-term experiment that used PRL injections to restore the release of PRL at milking in QN-treated cows. Nine Holstein cows were assigned to treatments during three 5-d periods in a 3×3 Latin square design: 1) QN: twice-daily i.m. injections of 1mg of QN; 2) QN-PRL: twice-daily i.m. injections of 1mg of QN and twice-daily (at milking time) i.v. injections of PRL (2µg/kg body weight); and 3) control: twice-daily injections of the vehicles. Mammary epithelial cells (MEC) were purified from milk so that their viability could be assessed, and mammary biopsies were harvested for immunohistological analyses of cell proliferation using PCNA and STAT5 staining. In both milk-purified MEC and mammary tissue, the mRNA levels of milk proteins and BAX were determined using real-time reverse-transcription PCR. Daily QN injections reduced milking-induced PRL release. The area under the PRL curve was similar in the control and PRL injection treatments, but the shape was different. The QN treatment decreased milk, lactose, protein, and casein production. Injections of PRL did not restore milk yield but tended to increase milk protein yield. In mammary tissue, the percentage of STAT5-positive cells was reduced during QN but not during QN-PRL in comparison with the control treatment. The percentage of PCNA-positive cells was greater during QN-PRL injections than during the control or QN treatment and tended to be lower during QN than during the control treatment. In milk-purified MEC, κ-casein and α-lactalbumin mRNA levels were lower during QN than during the control treatment, but during QN-PRL, they were not different from the control treatment. In mammary tissue, the BAX mRNA level was lower during QN-PRL than during QN. The number of MEC exfoliated into milk was increased by QN injections but tended to be decreased by PRL injections. Injections of PRL also increased the viability of MEC harvested from milk. Although PRL injections at milking could not reverse the effect of QN treatment on milk production, their effects on cell survival and exfoliation and on gene expression suggest that the effect of QN treatment on the mammary gland is due to QNs inhibition of PRL secretion.

Regulation of cell number in the mammary gland by controlling the exfoliation process in milk in ruminants

Milk yield is partly influenced by the number of mammary epithelial cells (MEC) in the mammary gland. It is well known that variations in MEC number are due to cell proliferation and apoptosis. The exfoliation of MEC from the mammary epithelium into milk is another process that might influence MEC number in the mammary tissue. The rate of MEC exfoliation can be assessed by measuring the milk MEC content through light microscopy, flow cytometry analysis, or an immuno-magnetic method for MEC purification. Various experimental models have been used to affect milk yield and study the rate of MEC exfoliation. Reducing milking frequency from twice to once daily did not seem to have any effect on MEC loss in goat and cow milk after 7 d, but increased MEC loss per day in goats when applied for a longer period. An increase in MEC exfoliation was also observed during short days as compared with long days, or in response to an endotoxin-induced mastitis in cows. Other animal models were designed to investigate the endocrine control of the exfoliation process and its link with milk production. Suppression of ovarian steroids by ovariectomy resulted in a greater persistency of lactation and a decrease in MEC exfoliation. Administering prolactin inhibitors during lactation or at dry-off enhanced MEC exfoliation, whereas exogenous prolactin during lactation tended to prevent the negative effect of prolactin inhibitors. These findings suggest that prolactin could regulate MEC exfoliation. In most of these studies, variations of MEC exfoliation were associated with variations in milk yield and changes in mammary epithelium integrity. Exfoliation of MEC could thus influence milk yield by regulating MEC number in mammary tissue.

Mammary epithelial cells isolated from milk are a valuable, non-invasive source of mammary transcripts

Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC from milk using immunomagnetic separation may be a useful non-invasive method to investigate transcriptional regulations in ruminants’ udder. This review aims to describe the process of isolating MEC from milk, to provide an overview on the studies that use this method to analyze gene expression by qRT PCR and to evaluate the validity of this method by analyzing and comparing the results between studies. In several goat and cow studies, consistent reductions in alpha-lactalbumin mRNA levels during once-daily milking (ODM) and in SLC2A1 mRNA level during feed restriction are observed. The effect of ODM on alpha-lactalbumin mRNA level was similarly observed in milk isolated MEC and mammary biopsy. Moreover, we and others showed decreasing alpha-lactalbumin and increasing BAX mRNA levels with advanced stages of lactation in dairy cows and buffalo. The relevance of using the milk-isolated MEC method to analyze mammary gene expression is proven, as the transcript variations were also consistent with milk yield and composition variations under the effect of different factors such as prolactin inhibition or photoperiod. However, the RNA from milk-isolated MEC is particularly sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies where gene expression was compared using qRT-PCR or RNA Sequencing analyses. As a conclusion, when the RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA to study various factors that impact milk yield and composition (ODM, feeding level, endocrine status, photoperiod modulation, and stage of lactation).