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Mammary Gland Involution

Mammary Gland

Cows walking to the barn.

The lactation function of the mammary gland is maintained by a delicate balance of systemic blood-borne factors and local mammary-derived factors, many of which are directly affected by the process of milk removal. Systemic factors include galactopoietic hormones such as growth hormone and suckling-induced prolactin secretion which generally stimulate milk secretion, but also include factors arising from competing physiological states such as pregnancy which may inhibit lactation function. Additionally, local control of milk secretion is directly linked to physical removal of milk. The impact of these factors on mammary function in dairy cattle is evident from the known effects of frequency of milk removal on milk yield, the effects of galactopoietic hormones on milk secretion, and the effects of milk stasis-induced mammary involution on mammary function. For a review of galactopoietic factors see the sections in the Lactation Resouorces on Hormones and on Milk Removal.

Maintenance of Lactation Function

The role of galactopoietic hormones such as prolactin in maintenance of lactation is well established (reviewed by Tucker 1994), although specific cellular mechanisms of action continue to be investigated. Prolactin is considered the major galactopoietic hormone in nonruminants. Prolactin is released at the time of milk removal in ruminants and nonruminants, and it remains a key systemic modulator of milk secretion during lactation. Conversely, growth hormone is generally considered to be the predominant galactopoietic hormone in ruminants (Bauman 1992; Tucker 1994). Inhibition of prolactin secretion or administration of prolactin to lactating cows has little effect on milk yields (Karg and Schams 1974; Plaut et al. 1987). However, these apparently clear-cut roles of prolactin vs. growth hormone in maintenance of lactation in nonruminants vs. ruminants are probably an oversimplification (Wilde and Hurley 1996). For example, in lactating sheep both prolactin and growth hormone seem to be important for galactopoiesis (Hooley et al. 1978; Tucker 1994). Even in the rat, recent studies have demonstrated an important role for growth hormone, independent of the role of prolactin (Flint et al. 1992; Flint and Gardner 1994).

Regardless of the hormones involved, all attempts to evaluate milk secretion must account for continued removal of milk. This is a reminder of the critical role of local mammary factors in maintenance of milk secretion. One such factor that plays a major role in regulating milk secretion in many species is a feedback inhibitor of lactation (FIL) found in milk (Wilde et al. 1995). FIL is thought to be produced by the mammary cells as they synthesize and secrete milk. Accumulation of FIL in the milk-producing alveoli results in feedback inhibition of milk synthesis and secretion. Frequent removal of milk from the gland minimizes local inhibitory effects of FIL and increases milk secretion (Wilde et al. 1987; Wilde and Knight 1989; Wilde and Peaker 1990).

Lactation in Decline

In spite of continued milk removal (with associated removal of FIL and stimulation of a post milking prolactin surge), milk yield in dairy cattle declines as lactation progresses. This decline occurs even with routine administration of growth hormone (bovine somatotropin). Lactation persistency is of particular concern in the production of milk. Mammary tissue function declines after peak lactation and this is at least in part due to a decrease in mammary cell number (Knight and Peaker 1984; Wilde and Knight 1989). The cell loss during the declining phase of lactation in the goat and cow apparently is the result of programmed cell death, also called apoptosis (Quarrie et al., 1994; Wilde et al., 1997). The mechanisms that control lactation decline remain important areas of investigation. Mammary involution is a greatly enhanced extension of these processes leading to a complete cessation of lactation function.

Concurrent pregnancy also influences persistency of milk yield in the declining phase of lactation. Inhibitory effects of pregnancy on lactating cows do not become apparent until about mid pregnancy (Wilcox et al. 1959; Bachman et al. 1988). An inhibitory effect of pregnancy on lactation has been noted in a number of other species, as well (Wilde and Knight 1989; Tucker 1994). The mechanism of this effect is not fully understood. However, the timing of inhibition of milk yield in cattle coincides approximately with the period of increasing placentally-derived plasma estrogen (Robertson and King 1979). Estrogen may have an effect on the transition of mammary function from a lactating state to an involuting state (Athie et al. 1996; Bachman 1982).

Bovine Mammary Gland Involution

Cessation of milk removal leads to rapid changes in the mammary tissue and initiation of the process of mammary involution (Hurley 1989). Changes in composition of mammary secretions during the early phases of involution indicate rapid changes in the normal mechanisms involved in milk synthesis and secretion (see discussion below; also Hurley and Rejman 1986; Hurley 1987; Hurley et al. 1987; Rejman et al. 1989; Hurley 1989). These changes in mammary gland secretion composition include a rapid decline in lactose concentration in the mammary secretions, indicating that lactose synthesis, and the associated water transport mechanism, decline soon after cessation of milk removal. However, total protein concentrations increase in early involution, partially because of water resorption from the secretion and partly due to increased concentrations of lactoferrin, serum albumin and immunoglobulins. Lactoferrin is a major protein found in mammary secretions during involution (Rejman et al. 1989). Its synthesis is increased during involution in contrast to milk-specific proteins such as casein whose synthesis is decreased (Hurley and Rejman 1993; Hurley et al. 1994a). Lactoferrin has a number of potential functions in the mammary gland, particularly as a nonspecific disease resistance factor (reviewed by Sanchez et al. 1992).

Involution-associated ultrastructural changes in bovine mammary cells begin within 48 hours after cessation of milk removal (Holst et al. 1987; Hurley 1989). The most apparent change is the formation of large stasis vacuoles in the epithelial cells (Holst et al. 1987), formed largely as a result of intracellular accumulation of milk fat droplets and secretory vesicles (Hurley 1989). These vacuoles persist to at least 14 days of involution and are usually gone by day 28 (Holst et al. 1987). Alveolar lumenal area declines during this period, while interalveolar stromal area increases. A substantial reduction in fluid volume in the gland occurs between day 3 and 7 of involution (Hurley 1989), probably accounting for the reduction in lumenal volume. By day 28 the collapsed alveolar structures remaining are considerably smaller than during lactation, with a very small lumen. General alveolar structure is maintained thoughout involution in the cow.

Histological and ultrastructural work on the bovine mammary gland during involution (Holst et al. 1987; Hurley 1989) provides no evidence for the extensive tissue degeneration observed in other species, such as rodents and others (Helminen and Ericsson 1968a,b; Helminen and Ericsson 1971). Limited autophagocytic processes occur only transiently during the initial two days after cessation of milking. Formation of autophagocytic structures in rodent mammary tissue is characteristic of involution (Helminen and Ericsson 1968a,b; Helminen and Ericsson 1971). A detachment of epithelial cells from the basement membrane and their loss from the tissue has been reported in rodents and other species (Wellings and DeOme 1963; Verley and Hollman 1967; Helminen and Ericsson 1968b; Richards and Benson 1971). This leaves characteristic bare spaces on the basement membrane and myoepithelial cells are thought to fill the space. No such situations are observed in the involuting bovine gland (Holst 1987; Holst et al. 1987). More recently, the involution process in the mouse has been characterized by examining the role of apoptosis.

Apoptosis and Mammary Gland Involution

Mammary involution in the mouse is characterized by a rapid loss of tissue function and degeneration of the alveolar structure and massive loss of epithelial cells. This cell loss is due to programmed cell death or apoptosis (Strange et al. 1992, Walker et al. 1989). Apoptosis is a both a natural and systematic method of cell suicide which takes place during normal morphogenesis, tissue remodelling and in response to infection or irreparable cell damage (Wyllie et al. 1984, Schwartzman & Cidlowski, 1993). There are two distinct types of cell death, apoptosis and necrosis, which may be distinguished by morphological, biochemical and molecular changes in dying cells. The process of apoptosis was originally distinguished from necrosis on the basis of its ultrastructure (Kerr 1971, Kerr et al. 1972). Apoptosis may be identified by a characteristic pattern of morphological changes: nuclear and cytoplasmic condensation, nuclear fragmentation and formation of apoptotic bodies (Walker et al. 1989, Strange et al. 1992). These changes are associated with cleavage of chromatin into discrete sized oligonucleosome fragments by a calcium dependent endonuclease (Arends et al. 1990), resulting in the appearance of oligonucleosomal DNA laddering in ethidium bromide stained gels (Wyllie et al. 1980).

A morphology consistent with apoptotic cell death can be observed in the murine mammary gland within two days of milk stasis. The nucleus and cytoplasm condense, the chromatin becomes fragmented and marginated, and apoptotic bodies are formed (Walker et al., 1989; Strange et al., 1992). This cell loss results in extensive disintegration of alveolar structure during the early period of involution in the mouse. DNA laddering characteristic of apoptosis also has been detected in goat mammary tissue during early and late lactation (Quarrie et al., 1994) and during late lactation in the cow (Wilde et al., 1997). This would suggest that removal of secretory epithelial cells by apoptosis is a normal physiological event in the ruminant mammary gland, even during lactation. In addition, milk stasis has been demonstrated to stimulate DNA laddering in both goat and cow mammary tissue (Quarrie et al., 1994; Wilde et al., 1997). These observations suggest that mammary epithelial cells are indeed lost during involution in the bovine mammary gland. However, this process of cell loss does not seem to be as dramatic as that observed in the mouse. In spite of the loss of cells, bovine mammary alveoli retain general structural integrity throughout involution (Holst et al. 1987). While the role of cell loss in the mouse mammary gland during involution is clear, the impact of mammary apoptosis in the bovine is not fully characterized.

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