R. Michael Akers

Mammary Involution in Dairy Animals

Lifetime milk production is maximized when dairycows are pregnant during approximately 70% of eachlactation. Between lactations, a nonlactating period isnecessary for optimal milk production in the succeeding lactation. With cessation of milking, alveolarstructure is largely maintained and little or no loss ofcells occurs. However, increased apoptosis and cellproliferation, relative to that in lactating glands during the same stage of gestation, suggestthat a nonlactating period serves to promote cellturnover prior to the next lactation. Even in theabsence of pregnancy, mammary involution in dairyanimals occurs at a slower rate than in rodents;alveolar structure is maintained for several weeks andlactation can be reinitiated after four weeks or more ofinvolution. Although apoptosis appears to be initiated within a similar time frame to that in rodents,the maximum proportion of apoptotic epithelial cellsappears to be lower than in rodents, and apoptosis maybe accompanied by an initial increase in cell proliferation. The ability to manipulateapoptosis and cell proliferation during the nonlactatingperiod and during lactation is expected to provideenormous benefits to the dairy industry.

Radioimmunoassay for measurement of bovine α-lactalbumin in serum, milk and tissue culture media

A sensitive, specific radioimmunoassay for bovine alpha-lactalbumin (alpha-la) suitable for measurement of serum, tissue culture media and milk was developed. alpha-La was not detected in serum from prepubertal male or female cattle, but was detected as early as 60 d of gestation in nulliparous Holstein heifers, the level being greatly increased during the last one third of gestation. Serum from cross bred beef heifers contained less alpha-la and it was not detected until late gestation. Concentrations of alpha-la in serum samples from pregnant multiparous Holstein cows decreased at drying-off and subsequently increased just before parturition. Secretion of alpha-la by mammary tissue explants from steroid-primed prepubertal Holstein heifers was induced by the addition of bovine prolactin, ovine prolactin or human growth hormone to tissue culture media.

Mastitis and its Impact on Structure and Function in the Ruminant Mammary Gland

It is a given in biology that structure and function go hand-in-hand. At the level of the mammary alveoli, copious milk production depends on the proliferation of mammary epithelial cells and the biochemical and structural differentiation of these cells after parturition. For example, data from quantitative structural studies demonstrate that differences in milk production between beef and dairy cows correspond with a relative failure of alveolar cell differentiation in cattle not specifically selected for milk yield. It is likely, but not proven, that production differences within or between dairy breeds are also determined by differences in the capacity of alveolar cells to differentiate or to maintain an adequate state of differentiation. These observations strongly support the belief that insults from mastitis that lead to losses in mammary function are directly related to disruption of alveolar cell integrity, sloughing of cells, induced apoptosis, and increased appearance of poorly-differentiated cells. Ironically, reduced milk production in cases of subclinical mastitis, is also associated with increases in milk somatic cell count. Thus the elevated neutrophil migration evoked to fight inflammation can inadvertently rendered alveolar epithelial cells non-secretory. A challenge to future researchers will be to devise mastitis treatments and therapies that prevent and/or repair damage to alveolar structure and maximize subsequent secretory cell differentiation.

Local IGF-I Axis in Peripubertal Ruminant Mammary Development

The regulation of mammary growth and development in heifers is accomplished by complexinteractions of hormones, growth factors, and extracellular matrix molecules. Many of thesegrowth stimulators are believed to be locally produced in the mammary gland and to beaffected by developmental and nutritional status. Although estrogen and growth hormone areconsidered critical to pubertal mammogenesis, results summarized in this review suggest thatIGF-I6 and IGF binding proteins are especially important locally-produced growth regulatorsin peripubertal ruminants. This assertion is supported by studies of ovariectomized heifers, inwhich increased stromal IGFBP-3 and reduced IGF-I correspond with a failure of udderdevelopment. Similarly, reduced mammary development with overfeeding coincides withreduced mitogenic activity of mammary tissue extracts and altered concentrations of IGF-Iand IGFBPs. In vitro studies convincingly demonstrate that much of the mitogenic activity ofmammary extracts or serum can be attributed to IGF-I and that alterations in IGFBP-3 modulateits effectiveness. Thus by analogy to second messenger mechanisms of action for proteinhormones, local mammary-derived growth factors likely explain many of the effects attributedto the classic mammogenic hormones.

Expression of ovine insulin-like growth factor-1 (IGF-1) stimulates alveolar bud development in mammary glands of transgenic mice

To determine whether murine mammary growth is modulated by local insulin-like growth factor-1 (IGF-1) production, expression of recombinant IGF-1 was directed to the mammary glands of transgenic mice using an ovine prepro IGF-1 cDNA under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter. Bioactivity of recombinant IGF-1 in transgenic mouse milk extracts was demonstrated by a concentration-dependent increase in [3H]thymidine incorporation in clonal bovine mammary epithelial cells (MAC-T) compared with control mouse milk extracts; moreover, addition of excess recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3) abolished the increase in [3H]thymidine incorporation attributed to recombinant IGF-1 in transgenic mouse milk. Recombinant IGF-1 was produced in mammary tissue of virgin and pregnant transgenic mice, and secreted into milk of lactating mice. However, recombinant IGF-1 was not detected in serum from transgenic mice; and ligand blot analysis of serum insulin-like growth factor binding proteins (IGFBPs) indicated no differences owing to transgene presence. In peripubertal virgin mice at 49 d of age, the frequency of appearance of mammary alveolar buds was significantly higher in MMTV-IGF-1 than in CD-1 mice, and was unaffected by ovariectomy or estradiol treatment. In conclusion, mammary synthesis of recombinant IGF-1 enhances the rate of development of alveolar buds in mammary glands of virgin transgenic mice.

IGF-I-Induced IGFBP-3 potentiates the mitogenic actions of IGF-I in mammary epithelial MD-IGF-I cells

Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29-32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditions by MAC-T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The origin and evolution of lactation

The presence of mammary glands is the defining morphological feature of mammals. The recent assembly of the bovine genome and a report in Genome Biology that links the milk and lactation data of bovine and other mammalian genomes will help biologists investigate this economically and medically important feature.

Insulin-like growth factor (IGF) binding protein-3 regulation of IGF-I is altered in an acidic extracellular environment

While extracellular acidification within solid tumors is well‐documented, how reduced pH impacts regulation of insulin‐like growth factor‐I (IGF‐I) has not been studied extensively. Because IGF‐I receptor binding is affected by IGF binding proteins (IGFBPs), we examined how pH impacted IGFBP‐3 regulation of IGF‐I. IGF‐I binding in the absence of IGFBP‐3 was diminished at reduced pH. Addition of IGFBP‐3 reduced IGF‐I cell binding at pH 7.4 but increased surface association at pH 5.8. This increase in IGF‐I binding at pH 5.8 corresponded with an increase in IGFBP‐3 cell association. This, however, was not due to an increase in affinity of IGFBP‐3 for heparin at reduced pH although both heparinase III treatment and heparin addition reduced IGFBP‐3 enhancement of IGF‐I binding. An increase in IGF‐I binding to IGFBP‐3, though, was seen at reduced pH using a cell‐free assay. We hypothesize that the enhanced binding of IGF‐I at pH 5.8 is facilitated by increased association of IGFBP‐3 at this pH and that the resulting cell associated IGF‐I is IGFBP‐3 and not IGF‐IR bound. Increased internalization and nuclear association of IGF‐I at pH 5.8 in the presence of IGFBP‐3 was evident, yet cell proliferation was reduced by IGFBP‐3 at both pH 5.8 and 7.4 indicating that IGFBP‐3‐cell associated IGF‐I does not signal the cell to proliferate and that the resulting transfer of bound IGF‐I from IGF‐IR to IGFBP‐3 results in diminished proliferation. Solution binding of IGF‐I by IGFBP‐3 is one means by which IGF‐I‐induced proliferation is inhibited. Our work suggests that an alternative pathway exists by which IGF‐I and IGFBP‐3 both associate with the cell surface and that this association inhibits IGF‐I‐induced proliferation.

Chronic pulsatile shear stress alters insulin-like growth factor-I (IGF-I) binding protein release in vitro.

AbstractInsulin-like growth factor-I (IGF-I) is a potent smooth muscle cell mitogen indicated to have a role in vascular disease. IGF-I stimulates proliferation via receptor activation but its activity is mediated by IGF binding proteins (IGFBPs). Since hemodynamics have been linked to vascular proliferative disorders, we studied how pulsatile low (5±2 dynes/cm2) and high (23±8 dynes/cm2) shear stresses impacted IGFBP metabolism in bovine aortic endothelial cells using the Cellmax capillary system. We modeled the pulsatile flow in our system using the Womersley model for flow inside a rigid tube and harmonic analysis revealed that the flow was sinusoidal with a frequency of ∼0.3 Hz for both shear stress treatments. Laminar flow was confirmed and the phase lag between the pressure and the flow found to be insignificant. Thus, our study provides a necessary characterization of this in vitro system as well as an investigation into how shear impacts the IGF axis. We found a significant difference in IGFBP distribution between treatments and, given that IGFBPs regulate IGF-I activity and that IGF-I-independent activities have been suggested for IGFBP-3, suggest that shear stress may indirectly regulate IGF-I activity, and, by extension, the effect of IGF-I on vascular pathologies.

Hormonal and extracellular matrix regulation of plasminogen activator in a bovine mammary epithelial cell line

The effects of lactogenic hormones on urokinase plasminogen activator (u-PA) produced by bovine mammary epithelial cells (MAC-T) were examined. High levels of u-PA activity were detected in growth arrested cells cultured on plastic. This suggests that high levels of PA activity alone are not sufficient to induce proliferation of bovine mammary epithelial cells. Cells were cultured on various extracellular matrices: plastic, fibronectin, collagen, matrigel, and laminin. Basal levels of u-PA activity in media from MAC-T cells cultured on matrigel were 1.6-, 2-, 2- and 3.5-fold higher than that of cells cultured on plastic, fibronectin, collagen, and laminin, respectively. Insulin increased (P<0.01) mammary epithelial u-PA activity on a per cell basis, an effect observed irrespectively of the type of extracellular matrix onto which cells were cultured. Not unexpectedly, insulin-like growth factor (IGF)-I, Des(1–3) IGF-I and IGF-II increased mammary epithelial u-PA activity on a per cell basis and u-PA mRNA levels, thus, mimicking the effect of insulin. Dexamethasone suppressed (P<0.01) u-PA activity but was unable to suppress the insulin-induced increase in u-PA activity of cells cultured on various extracellular matrices. These data indicate that u-PA activity is modulated by both lactogenic hormones and the type of extracellular matrix.