Frederic Dessauge

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.

Hot Topic: Prepubertal Ovariectomy Alters the Development of Myoepithelial Cells in the Bovine Mammary Gland

Prepubertal ovariectomy can dramatically inhibit mammary development, but the mechanism of inhibition is not well characterized. Holstein heifers were ovariectomized (OVX) or sham operated but left intact (INT) at d 40 and then sacrificed at d 55, 70, 85, 100, 130, or 160 to provide tissues for histologic analysis of cell proliferation. Our histologic analyses unexpectedly revealed a pronounced effect of ovariectomy on myoepithelial cell development. Myoepithelial cells were identified on the basis of location, morphology, and immunohistochemical staining for alpha-smooth muscle actin (SMA). Vascular smooth muscle staining served as an internal positive control for all immunohistochemical analyses. Mammary tissues from d 40 heifers had an abundance of SMA+ cells associated with the ductal parenchyma. In INT heifers, the frequency of SMA+ cells decreased as development progressed. Only a limited number of isolated SMA+ cells were observed in d 70 to d 160 INT heifers. In OVX heifers, SMA+ cells were abundant, had elongated morphology, and frequently stained more intensively than vascular smooth muscle cells. The intense SMA staining and altered morphology was most prominent in older heifers. Limited analysis of gene expression revealed that maspin, a protease inhibitor expressed by myoepithelial cells, was expressed in parenchyma from both INT and OVX heifers. Our hypothesis is that ovarian secretions stimulate epithelial proliferation, and block myoepithelial differentiation. Myoepithelial cells are known to limit parenchymal cell proliferation. Ovariectomy may thus remove an estrogenic growth stimulus and permit the emergence of inhibitory cell populations that further limit parenchymal expansion. Our observation has important implications for control mechanisms that regulate parenchymal development.

Staphylococcus aureus phenol-soluble modulins impair interleukin expression in bovine mammary epithelial cells

ABSTRACT The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis.

Suppression of ovarian secretions before puberty strongly affects mammogenesis in the goat.

The objective of this study was to provide insight into the biological mechanisms underlying mammary development and the role of the ovaries in prepubertal caprine mammogenesis using a serial ovariectomy approach. Young Alpine goats were ovariectomized (Ovx) or sham-operated (Int) at three periods before puberty (G1=1 month, G2=2 month and G3=3 months of age) and one after puberty (G7=7 months of age). The goats were slaughtered at 9 months of age and mammary glands were removed. Ovariectomy performed at 1, 2 and 3 months of age caused a 50% reduction in DNA concentration, in mammary tissue taken from the parenchyma-stroma border region. Morphological analysis of mammary tissue sections indicated that the parenchymal structures of Ovx goats were negatively affected by ovariectomy. Goats ovariectomized before 2 months of age (Ovx-1 and Ovx-2) showed a significant decrease in the percent of cells proliferating in mammary glands of 9-month old goats (proliferating cell nuclear antigen expression and antigen Ki67-positive cell number). Also, goats ovariectomized at 1 and 2 months of age had reduced matrix metalloprotease 2 activity at 9 months of age. E-cadherin was strongly decreased in goats ovariectomized before 2 months of age (80 and 85% in Ovx-1 and Ovx-2 goats, respectively). Quantitative PCR analysis of transcripts encoding for oestrogen (ERα) and progesterone receptors (PR) and immunodetection of ERα showed that ovariectomy at 1 and 2 months of age strongly inhibited the transcription of ERα and PR in the mammary gland. We conclude that ovariectomy before 3 months of age markedly impaired parenchymal development. These findings suggest that prepubertal mammogenesis in goats depends on the ovaries to initiate mammary epithelial cell proliferation and mammary gland remodelling.

Oestradiol enhances apoptosis in bovine mammary epithelial cells in vitro

Ovarian steroids, oestradiol and progesterone, are required for normal mammary growth at puberty and during pregnancy. They contribute to mammary parenchyma development by stimulating mammary epithelial cell (MEC) proliferation. However several studies demonstrate that oestradiol negatively affects milk production during the declining phase of lactation, but the oestradiol effect on MEC in lactating mammary gland remains unclear. The objective of this study was to investigate the differential effect of oestradiol on bovine MECs mimicking two physiological statuses: active and early apoptotic MECs. We demonstrated that oestradiol has a major effect on early apoptotic MECs and might accelerate MEC apoptosis by activation of caspases rather than by inducing apoptosis in active MECs. Early apoptotic MECs could be compared with senescent cells in the late-lactation mammary gland. These results suggest that the negative effect of oestradiol on milk production during the declining phase of lactation would be due to an enhancement of apoptotic processes in MECs.

Cabergoline inhibits prolactin secretion and accelerates involution in dairy cows after dry-off

Dairy cattle require a dry period between successive lactations to ensure optimal milk production. Because prolactin (PRL) is necessary for the initiation and maintenance of milk production, strategies that can inhibit PRL secretion might hasten the involution process. The objective of this study was to determine the effect of the PRL release inhibitor cabergoline on markers of mammary gland involution during the early dry period. To assess the effect of cabergoline treatment on mammary gland involution, 14 Holstein dairy cows in late lactation were treated with either a single i.m. administration of 5.6mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n=7) or placebo (n=7) at the time of dry-off. Blood samples and mammary secretion samples were collected 6d before dry-off and again 1, 2, 3, 4, 8, and 14d following the abrupt cessation of lactation. Blood samples were used to determine plasma PRL concentrations. Mammary secretion samples were used to determine somatic cell count, milk fat, lactose, true protein content, and concentrations of α-lactalbumin, lactoferrin, and citrate. Following the cessation of lactation, changes in mammary secretion composition indicated diminished milk synthesis, including reduced concentrations of α-lactalbumin, citrate, and lactose. In contrast, milk somatic cell count, percent total protein, percent fat content, and lactoferrin concentrations significantly increased as involution progressed. Cabergoline treatment decreased the plasma PRL concentrations during the first week of dry-off, compared with the control treatment. No significant differences in citrate, α-lactalbumin, or protein content were observed between treatment groups. The most dramatic changes in secretion composition as a consequence of cabergoline treatment occurred during the first week of the dry period, when lactose concentrations and the citrate:lactoferrin molar ratio were lower and lactoferrin concentrations higher than in the control cows. Cabergoline treatment also tended to increase fat content and somatic cell count more rapidly following dry-off compared with the control group. These changes in mammary secretion composition following the abrupt cessation of lactation indicate that cabergoline treatment facilitated dry-off and effectively accelerated mammary gland involution.

Bovine mammary gland development: new insights into the epithelial hierarchy

Milk production is highly dependent on the extensive development of the mammary epithelium, which occurs during puberty. It is therefore essential to distinguish the epithelial cells committed to development during this key stage from the related epithelial hierarchy. Using cell phenotyping and sorting, we highlighted three sub-populations that we assume to be progenitors. The CD49fhighCD24neg cells expressing KRT14, vimentin and PROCR corresponded to basal progenitors whereas the CD49flowCD24neg cells expressing luminal KRT, progesterone and prolactin receptors, were of luminal lineage. The CD49flowCD24pos cells had features of a dual lineage, with luminal and basal characteristics (CD10, ALDH1 and KRT7 expression) and were considered to be early common (bipotent) progenitors. The mammary stem cell (MaSC) fraction was recovered in a fourth sub-population of CD49fhighCD24pos cells that expressed CD10/KRT14 and KRT7. The differential ALDH1 activities observed within the MaSC fraction allowed to discriminate between two states: quiescent MaSCs and lineage-restricted “activated” MaSCs. The in-depth characterization of these epithelial sub-populations provides new insights into the epithelial cell hierarchy in the bovine mammary gland and suggests a common developmental hierarchy in mammals.

Molecular signature of the putative stem/progenitor cells committed to the development of the bovine mammary gland at puberty

Milk production is highly dependent on the extensive development of the mammary epithelium, which occurs during puberty. It is therefore essential to distinguish the epithelial cells committed to development from the related epithelial hierarchy. Using cell phenotyping and sorting, we highlighted four cell sub-populations within the bovine mammary gland at puberty. The CD49fhighCD24neg cells expressing CD10, KRT14, vimentin and PROCR corresponded to cells committed to the basal lineage. The CD49flow sub-population contained two cell subsets (CD49flowCD24neg and CD49flowCD24pos). Both subsets expressed hormone receptors including ER, PR and PRLR, as well as ALDH1 activity but only the CD49flowCD24pos subset expressed ELF5. These data indicated that the CD49flow sub-population is mainly composed of cells displaying a luminal phenotype and that this population comprises two luminal cell subsets, namely the CD24neg and CD24pos cells, likely committed to ductal and alveolar lineage, respectively. The putative mammary stem cell (MaSC) fraction was recovered in the CD49fhighCD24pos sub-population which were shown to form mammospheres in vitro. These cells differentially expressed CD10, KRT14 and KRT7, suggesting the existence of several putative MaSC sub-fractions. In-depth characterization of these epithelial sub-populations provides new insights into the bovine mammary epithelial cell lineage and suggests a common developmental lineage in mammals.

Technical note: Quantification of caseins from a crude extract of mammary epithelial cells

En masse secretion of milk proteins, notably the caseins in the form of casein micelles, is a unique feature of the milk-secreting mammary epithelial cell. Caseins are therefore specific markers of these cells and constitute an ideal tool to monitor their differentiation, as well as functional status, during the development of the gland. To use them as such, a reliable method for quantitative analysis of the caseins from mammary cells or tissue is needed. Here we show that the caseins are heat-stable, a feature that leads to their complete extraction from a complex cellular extract by boiling. This allows for high enrichment and direct analysis of the caseins, even when they are poorly expressed in the starting material.