Accepted Manuscript

Physiology of Milk Secretion

Sandrine Truchet, PhD, scientist, Edith Honvo-Houéto, RT

PII: S1521-690X(17)30106-9
DOI: 10.1016/j.beem.2017.10.008
Reference: YBEEM 1172

To appear in: Best Practice & Research Clinical Endocrinology & Metabolism

Please cite this article as: Truchet S, Honvo-Houéto E, Physiology of Milk Secretion, Best Practice &
Research Clinical Endocrinology & Metabolism (2017), doi: 10.1016/j.beem.2017.10.008.

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 ACCEPTED MANUSCRIPT
Title: Physiology of Milk Secretion

Authors: Sandrine Truchet, PhD, scientista* and Edith Honvo-Houéto, RTb

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Affiliations:

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a
VIM, UR 892 INRA, Université Paris-Saclay, Jouy-en-Josas, France.
b
GABI, INRA/AgroParisTech/Université Paris-Saclay, Domaine de Vilvert, 78352

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Jouy-en-Josas, France.

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*Corresponding author. VIM, UR 892 INRA, Université Paris-Saclay, F-78352, Jouy-

en-Josas Cedex, France. Tel.: +33 (0)1 34 65 25 49. Fax: +33(0)1 34 65 28 73.
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E-mail adresses: [email protected] (S. Truchet), [email protected]

(E. Honvo-Houéto).
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Running head: Physiology of Milk Secretion

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Key words: mammary gland, breast, mammary epithelial cell, lactation, secretion,
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exocytosis, budding, milk, hormone, physiology, breastfeeding.

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Total words: 8730

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Abbreviations: ACTH, adrenocorticotropic hormone; APM, apical plasma

membrane; AQP, aquaporin; BPM, basolateral plasma membrane; BM, basement

membrane; BTN1, butyrophilin; ECM, extracellular matrix; EGF, epidermal growth

factor; ER, endoplasmic reticulum; EV, extracellular vesicles; FGF, fibroblast growth

factor; FIL, feedback inhibitor of lactation; FSH, follicle stimulating hormone; GC,

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glucocorticoid; GH, growth hormone; GnRH, gonadotropin-releasing hormone; 5-HT,

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5-hydroxytryptamine; Ig, immunoglobulin; IGF, insulin-like growth factor; IL,

interleukin; LD, lipid droplet; LH, luteinizing hormone; LTF, lactoferrin; MEC,

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mammary epithelial cell; MG, mammary gland; miRNA, micro-RNA; MFG, milk fat

globule; MMP, matrix metalloprotease; MVB, multivesicular body; OT, oxytocin; PG,

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prostaglandin; hPL, human placental lactogen; PLIN2, perilipin2/adipophilin; PRL,
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prolactin; SNAP, synaptosomal-associated protein; SNARE, soluble N-
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ethylmaleimide-sensitive factor attachment protein receptor; PUFA, polyunsaturated

fatty acid; SV, secretory vesicle; TEB, terminal end bud; TGF, transforming growth
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factor; TJ, tight junction; VAMP, vesicle-associated membrane protein; XOR,

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xanthine oxidase.
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Abstract

Milk is a unique and complete nutritive source for the mammal neonate, also

providing immune protection and developmental signals. Lactation is a complex

process, proper to the mother and child dyad, and including numerous variables

ranging from psychological aspects to the secretory functioning of the mammary

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epithelial cells, all contributing to a successful breastfeeding. This review gives an

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integrated overview of the physiology of lactation with a particular focus on cellular

and molecular mechanisms involved in milk product secretion and their regulations.

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Introduction
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Milk production and secretion is a complex physiological process resulting
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from both the previous development of the mammary gland (MG) and tight

regulations by systemic hormones and local factors. All these aspects ultimately
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contribute to the coordinated secretory functioning of the mammary epithelial cells

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(MECs) in order to provide milk of adequate composition and in sufficient quantity to

the newborn.
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A. Mammary gland development

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The mammary gland (MG) is a dynamic exocrine organ that can undergo

repeated cycles of growth, functional differentiation, and regression, closely

intertwined with the reproductive processes. Indeed, mammary development begins

during early fetal life, occurs only slightly during estrous cycles, while complete

mammogenesis only takes place during pregnancy to become fully functional after
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parturition to provide a nutritional support to the newborn. Once the child is weaned,

the mammary tissue declines during involution and can re-differentiate if a new

pregnancy starts (Fig. 1). All steps of the physiological development of the MG are

tightly spatio-temporally coordinated, both by systemic hormones and local factors.

From about 7 weeks of gestation, the human MG develops from a single

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ectodermal ridge localized along the anterior body wall which extends from the

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epidermis into the underlying mesenchyme. Concomitantly, a loose condensation of

mesenchyme extends sub-dermally to form the fat pad precursor. The ectoderm

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elongates to form a mammary sprout, invades the fat-pad precursor (10th-12th

weeks), branches (13th-20th weeks), and canalizes to form the primary mammary

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ductal system (32th weeks), which opens onto the area that gave rise to the nipple

(Fig. 1, birth) [1, 2].

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After birth, the MG remains as a rudimentary network of small branching ducts

ending in short ductules called terminal end buds (TEBs) lined by one to two layer of
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epithelial and one of myoepithelial cells. These structures regress at ~4 weeks

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postpartum along with a decrease in the secretion of prolactin (PRL) from the anterior

pituitary gland of the infant. Until puberty, the growth of the breast is isometric. Of
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note, diet and/or metabolic pathologies such as diabetes may impair mammary
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development and subsequent lactation performance [3].

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After a period of quiescence during childhood, the increase in ovarian estrogen

secretion at puberty (8-12 years of age) stimulates the allometric growth of both the

epithelial network and the adipose tissue within the MG (Fig. 1, puberty). Thus, while

the increase in breast size is merely due to the enhanced deposition of adipose

tissue, the mammary epithelium progressively elongates and further branches

resulting in an extensive ductal network. These maturational changes occur in the

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course of ovulatory cycles and are then regulated by both systemic and locally

released factors such as estrogen, progesterone, PRL, luteinizing hormone (LH),

follicle stimulating hormone (FSH), growth hormone (GH) and epidermal growth

factor (EGF) [4]. During the follicular phase of the menstrual cycle, the lobules are

small, with few alveoli, and there is low mitotic activity. During the luteal phase,

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ovarian progesterone stimulates lobulo-alveolar development, e.g. mitotic activity of

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the bilayered MECs, and opening of lumens in TEBs to form small alveoli [4]. TEBs

generate new branches, twigs, and small alveolar structures which cluster around a

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terminal duct, forming a lobule (~11 alveoli/duct, Fig. 2B). Lobule formation occurs

within 1-2 years after onset of the first menstrual period. Alveolar clusters grow and

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increase in complexity during each luteal phase and slightly regress with the onset of
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the menses and the loss of hormonal support, thus leading to a gradual accretion of
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the epithelial tissue with each successive cycle. In women, 3 types of lobules have

been identified based on the size of the composing alveolar buds and their
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differentiation state (see Fig. 1 in [1]). With increasing years, mitotic activity slightly
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decreases until ~35 years of age. Then, full differentiation of the MG is a gradual

process taking many years, which is achieve only if pregnancy supervenes.

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While in the non-pregnant adult woman connective and adipose tissues

predominate and epithelial tissue is sparse, the onset of pregnancy induces

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progressive changes in both cellular and functional organization in the MG. Early
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pregnancy is characterized by growth due to a marked proliferation of both ductal and

alveolar cells, concomitantly with the reduction of the fat pad. This leads to the

formation of an extensive branched ductal system with a high number of alveoli of

variable size and shape, gradually derived from TEBs (mammogenesis, Fig. 1,

gestation). The surrounding stromal and myoepithelial cells provide essential cues for
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MEC survival, proliferation and differentiation. In newly formed lobules, the alveolar

MECs not only increase in number due to active cell division but also increase in

size, mainly because of cytoplasm enlargement. These modifications are regulated

by numerous systemic hormones, including estrogen, progesterone, PRL, GH,

insulin, glucocorticoids (GCs) and parathyroid hormone-related protein, as well as

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local factors such as insulin-like growth factor-1 (IGF-1), EGF and fibroblast growth

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factor (FGF), which are likely produced by the stromal cells [2, 4]. Moreover, both

MECs and stromal cells produce various extracellular matrix (ECM) components (e.g.

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proteoglycans, hyaluronan, fibronectin, and laminin), which are important for MG

growth and function [5]. The definitive structure of the ductal tree is essentially settled

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by the end of the first half of pregnancy and further changes until parturition are
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chiefly continuation and accentuation of branching and alveoli formation. Hence,
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small amounts of secretion product (colostrum) can be observed in enlarged lumen of

alveoli and milk ducts signing the functional secretory differentiation of MECs
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(referred as lactogenesis I). In the last trimester, there is a reduced proliferation of

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new alveoli with a further increase in their size due to distension of their lumen

(terminal differentiation of MECs) by accumulation of colostrum. In addition to

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progesterone, PRL and/or human placental lactogen (hPL) appeared to be involved

in the final stages of secretory MECs growth and differentiation. Concomitantly with
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the increased metabolic activity of MECs, the mammary blood flow approximately
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doubles in volume during pregnancy and persists during lactation until weaning.

1. Secretory differentiation of MECs

Secretory differentiation of the alveolar MECs or lactogenesis starts around

mid-pregnancy and has been divided into two successive phases: initiation or
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lactogenesis I and activation or lactogenesis II. These critical stages rely on

variations of gene expression, structural and functional properties of alveolar cells, all

of which being hormonally regulated [6, 7].

During lactogenesis I, MECs differentiate morphologically and become

competent to produce and secrete some milk components referred as colostrum [8],

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due to the activation of the expression of some milk protein genes and biosynthetic

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enzymes, as well as the production of lactose and accumulation of lipid droplets

(LDs) [6]. However, production and secretion of milk components appear to be

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restricted to a limited number of alveolar MECs with incompletely developed

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secretory mechanisms. As colostrum is not removed by suckling, its components are
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reabsorbed into the blood through the paracellular pathway. At late pregnancy, milk

secretion is inhibited by high plasma concentrations of progesterone and estrogen

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until parturition.
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After parturition, the expulsion of the placenta results in a rapid withdrawal of

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progesterone [9], estrogen and hPL during the 4-6 days after birth, while PRL

concentrations remain high in the presence of insulin and cortisol, thus triggering
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lactogenesis II [6, 10].

Colostrum is produced during the first 4 days postpartum, followed by a 10-15

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days period of transitional milk secretion, before copious production of mature milk
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(after 15 days) [8]. Milk composition is dramatically altered: sodium and chloride

concentrations fall while those of lactose, immunoglobulins A (IgA), lactoferrin (LTF)

and other components of mature milk increase. These changes are completed by 72

hours postpartum and precede the increase of milk volume by ~24 hours, accordingly

to the terminal differentiation of alveolar MECs into lactocytes [11]. These changes

result from substantial variations of milk protein genes (e.g. α-lactalbumin) and
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biosynthetic enzymes (e.g. acetyl-CoA carboxylase and fatty acid synthetase)

expression [12], supported by alveolar MECs reorganization, including apico-basal

polarization of organelles, expansion of mitochondria and RER, maturation of the

Golgi apparatus, appearance of secretory vesicles (SVs) containing casein micelles

and of numerous microvilli at the APM, increase in the number of bigger LDs and

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closure of TJs that blocks the paracellular pathway, to adapt to their high secretory

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state [13]. Moreover, there is an increase of transport activities for all substrates for

milk production such as amino-acids, glucose and fatty acids, as well as ions. Indeed,

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with the closure of TJs, ions such sodium and chloride can no longer pass from the

interstitial space into the lumen of the alveolus and then must be secreted by the

cellular route.
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Milk volume produced rapidly increases in the first 24 hours postpartum,
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accordingly to the increase of both the frequency of breastfeeding and the volume

consumed by the newborn, and stabilizes after ~1 month (~750-800 ml/day) to

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remain fairly constant up to 6 months postpartum [11]

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Although not essential within the first hours after birth, milk removal by day 3 is
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critical for the establishment of a successful lactation. Both the time of the first

breastfeeding and the breastfeeding frequency on day 2 were positively correlated

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with milk volume on day 5 postpartum, suggesting that milk removal soon after birth
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increases the efficiency of milk secretion. Once lactation is established, the volume of

milk produced is merely determined by the baby’s appetite [14]. Indeed, the breast is

rarely completely drained during a suckling (on average 67% of the available milk is

consumed). Thus, in connection with the frequency and effectiveness of the

drainage-filling cycle of the alveoli [14, 15], there is a switch from endocrine to

autocrine control and milk removal becomes the primary regulatory mechanism for
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galactopoiesis (milk synthesis) and to adjust milk volume to the requirements of the

newborn. Milk can be stored for up to 48 h before the rate of milk synthesis and

secretion begins to decrease. However, incomplete/inefficient milk removal or milk

stasis induce multiple local effects on milk secretion: 1) an autocrine whey protein,

termed ‘feedback inhibitor of lactation’ (FIL) regulates milk secretion according to

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frequency or completeness of milk removal in each MG [16]; 2) other factors such as

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osmolarity and mechanical stress [17] influence milk synthesis; 3) expression of the

PRL receptors in MECs decreases, thereby uncoupling the stimulatory effects of PRL

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on milk synthesis; and 4) prolonged milk stasis triggers MECs apoptosis. Lactation is

prolonged as long as milk is regularly removed from the MG [14].

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2. Delayed and impaired lactogenesis
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Some particular conditions may delay lactogenesis, including placental

retention caesarean section, diabetes or stress during parturition. Obese women are
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more likely to experience delayed lactogenesis II, potentially due to hormonal

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influences on milk production, increased difficulty attaining a successful infant latch to

the breast, and/or socio-cultural factors [18].

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Early milk removal, correct attachment of the baby to the nipple, as well as the

frequency and the efficiency of suction are the main key conditions contributing to a
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successful breastfeeding. Therefore, irregular or incomplete removal of milk leading

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to breast engorgement may be due to a mother’s pathology such as an impaired milk

ejection, inverted nipples or mastitis, as well as poor attachment and/or positioning,

ineffective suckling, infrequent feeds of the infant. The best indicator of an adequate

milk supply is the infant weight gain during the early neonatal period.
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3. Effects of lactation on reproduction

Lactation has a marked effect on fertility in breastfeeding women. After

parturition, the systemic levels of LH and FSH, both controlled by the pulsatile

release of gonadotropin-releasing hormone (GnRH), are low due to the suppression

of the hypothalamic-pituitary axis by placental steroids. While fertility returns

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approximately 6-9 weeks postpartum in non-breastfeeding women [19], in

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breastfeeding women GnRH secretion is suppressed by various factors such as

maternal nutrition, PRL levels [20] and the suckling stimulus [21] and appears to be

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highly correlated with the breastfeeding pattern, e.g. the frequency and duration of

suckling. The inhibition of GnRH release results in the disturbance of pulsatile LH

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secretion, which in turn suppresses ovarian activity. In addition, the increased
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sensitivity of the hypothalamo-pituitary system to the negative feedback effects of
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ovarian oestrogen after parturition also contribute to the suppression of fertility during

lactation.
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4. Post-lactational changes and involution

At the end of lactation, when regular removal of milk ceases, the MG enters a
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tissue-remodeling process known as involution [22]. Early after weaning, the

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epithelial architecture is maintained by the recent exposure to elevated systemic

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hormones, e.g. PRL, GCs and IGF-1, which are also critical survival (anti-apoptotic)

factors for MECs. During this first phase of involution or “reversible phase”, the MG

can revert to a state of milk production if the suckling stimulus occurs again [23].

However, extended milk stasis in the ducts and alveolar lumen, concomitantly with

PRL and GC withdrawal due to the absence of suckling, leads the MG to enter an

“irreversible phase” (or phase 2 of involution) and to become unable to return to

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lactation without being re-stimulated by pregnancy levels of hormones [23]. Milk

stasis directly inhibits milk protein synthesis and secretion through both mechanical

stretch and local production of various pro-apoptotic factors such as serotonin (5-

hydroxytryptamine, 5-HT), LTF, Interleukin (IL)-6 family of cytokines, transforming

growth factor-β (TGFβ) and α-lactalbumin. These factors lead to the inhibition of milk

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production by inducing the desensitization of MECs to lactogenic hormones. The fine

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balance between survival factors (PRL, GC, IGF-1) and cell death factors (5-HT, ILs,

TGFβ, Vitamin-D receptor, IGF-binding protein-5) regulates the coordinated,

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multifocal and asynchronous processes resulting in a massive epithelial tissue

regression (~80%), mainly via apoptosis and autophagy of MECs . TJs gradually

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breakdown and the ECM is progressively remodeled by the action of both the matrix
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metalloproteases (MMPs) and the plasminogen system [24]. Loss of attachment-
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dependent survival through integrins signaling (e.g., anoikis) together with pro-

apoptotic signals leads to the elimination of MECs, collapse of acinar structures and
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narrowing of the tubules, while myoepithelial cells remain relatively well-organized

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during involution around residual ductal buds [25]. In addition to immune cells

present in the MG at all stages of development [26], surviving MECs play a major role
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in the clearance of residual milk and cell debris as they engulf casein micelles, MFGs

and apoptotic cells [27]. They also release anti-inflammatory cytokines which limit the
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action of the recruited leukocytes and neutrophils during early involution.

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Macrophages, local acute-phase response activation, and a late B-lymphocyte

response complete the clearance of cell debris. These events ultimately lead to rapid

regression of the epithelial tissue resulting in a rudimentary ductal tree

morphologically similar to a virgin MG with some persisting alveoli (Fig. 1, involution).

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Concomitantly, pre-adipocytes re-differentiate (adipogenesis) and colonize a major

part of the MG, while the vascular tissue is also remodeled [28].

5. Menopausal breast

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After menopause, accompanied by an almost complete cessation of ovarian

estrogen and progesterone production, the breast undergoes a slight regression.

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Nulliparous and parous breasts appear quite identical with only minimal quantitative

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differences in the proportion of lobule subtypes. However, nulliparous women exhibit

a higher incidence of breast cancer than parous women and differentiation is

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suggested to protect the MG against carcinogenesis [29].
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B. Functional anatomy of the lactating breast

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During lactation, the MG consists of a highly branched tubulo-alveolar

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glandular epithelium (or parenchyma) (Fig. 1, lactation), embedded in a stroma of

both connective- and white adipose-tissue, and supported by a loose framework of

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fibrous connective tissue referred as Cooper’s ligaments (Fig. 2A) [30].There is a

decrease in the amount of adipose tissue relative to glandular tissue (ratio ~1:2),
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which is not correlated to milk production or storage capacity, and the size and
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weight of the breast increases. Human breast consists of 15 to 20 lobes, the size of

which is highly variable, subdivided into lobules containing between 10 and 100

alveoli or alveoli (~0.12 mm in diameter), which are the basic secretory units

producing milk (Fig. 2B). Alveoli are clustered around ductules connected to the

interlobular duct of the lobules that coalesce to form larger ducts, which are drained

towards the nipple by a lactiferous duct (1.2 to 2.5 mm in diameter) that only dilate
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during milk ejection (no storage, only transport) [31]. The significant variation in

lobule size observed may reflect the difference in secretory activity from lobule to

lobule. Moreover, growth and differentiation of MECs can occur in the same lobule,

concomitantly with milk production. Each alveolus is surrounded by contractile

myoepithelial cells responsible for milk ejection [32] and an extensive capillary

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network [28]. In addition to their role in milk ejection, myoepithelial cells also regulate

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mammary development through secreting various growth factors [4], spatially restrict

MECs to form ducts during puberty, and act as tumor suppressors. Alveoli are

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embedded in a connective-tissue stroma containing adipocytes, fibroblasts and some

plasma cells, which produce the Igs found in milk, as well as non-cellular components

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such as collagen and proteins of the ECM (Fig. 2C),. Lymph is drained by two main
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pathways: the axillary nodes and the internal mammary nodes which mostly drain the
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medial and lateral portions and the deep portion of the breast, respectively. The

lymphatic network transports lipid-soluble nutrients (e.g., vitamin K and lipids) to the
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lactocytes, while the lymph nodes, which contain leukocytes (mainly lymphocytes and
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macrophages), provide an immune defense system to the MG in response to bacteria

or foreign material. The MG contain only few internal innervations. Nerve fibers
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associate with the major duct system and are rather sparse in the region of the

smaller ducts, areola, and nipple [33]. Sympathetic nerves are associated with the
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arteries but not the alveoli and there is no parasympathetic innervation of the MG.
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However, sensory nerves present in the nipple are critical for initiating the afferent

neural pathway of the milk ejection reflex. As there is no motor innervation of the

mammary epithelium nor the myoepithelial cells, milk production and ejection are

independent of the neural stimulation.

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In the alveoli, the secretory MECs form a sealed epithelial monolayer upon the

closure of their apical adherens- and tight-junctions (TJs), which segregate the lumen

from the interstitial space, thus preventing paracellular transport (Fig. 2D). TJs also

delimit the apical (APM) from the basolateral (BPM) plasma membranes of MECs,

thus contributing to the establishment and the maintenance of the functional

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asymmetry (polarity) of MECs required for the vectorial secretion of milk [34]. The

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basal side of alveolar MECs contacts myoepithelial cells and the basement

membrane (BM), a specialized ECM, which separates the epithelium from the stroma

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and the vascular system. The BM results from the secretion by both stromal cells and

MECs of specific ECM components further assembled in a 100 nm thick matrix at the

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basal surface of the mammary epithelium [5]. Integrins are hetero-dimeric ECM
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receptors localized on the BPM of MECs and mediate cell-matrix adhesion and
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regulate various aspects of MEC development and function through integration with

other signals [5]. Integrin/BM interaction leads the formation of focal adhesion centers
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integrating both the assembling of the cytoskeleton and cell survival signals. Integrins
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signaling is thus involved in the establishment of the apico-basal polarity (e.g. apical

side speciation) of MECs and lumen formation during pregnancy, enables PRL
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signaling through its effectors Jak2 and Stat5 to activate milk protein genes during

lactation, as well as remodeling of the mammary tissue during involution [35].

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Moreover, the composition and stiffness of the BM change during MG development

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according to the variations in the ratio of the mammary cell types, there is a critical

interdependency of tissue architecture and cell fate for the spatio-temporal regulation

of both mammary development and function [35]. Thus, by transducing both

biochemical (survival, differentiation and functional) and biophysical signals (changes

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in cell shape or membrane tension through cytoskeletal changes), integrins

determine the fate of MECs [35, 36].

The APM of MECs borders the lumen of the alveoli, where milk product are

released. As their principal function is to produce and secrete huge amounts of milk

to feed the newborn, the intracellular organization of MECs reflects their highly

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secretory state. Indeed, the cytoplasm of alveolar MECs is filled with an extensive

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rough ER network, enlarged Golgi apparatus, and contains numerous mitochondria

and SVs containing casein micelles. Lactose is synthesized in the Golgi, and is

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transported with casein micelles into the SVs towards the APM. Secretory MECs also

produce LDs emerging from the ER by accumulation of neutral lipids and which grow

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during their transport before being released as milk fat globules (MFGs) by budding
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(Fig. 2D) [37].
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C. MECs secretory routes

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After reaching the MG through the blood stream or the lymph system, nutrients
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and other components used to synthesize milk constituents diffuse in the interstitial

space and reach the BPM of MECs. Depending on their molecular nature, they enter
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MECs and are secreted in milk by several routes. Most of the transport pathways are
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tightly regulated and coordinated, so that sufficient milk of adequate composition is

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available for the newborn, even during inadequate food intake by mothers.

1. Paracellular pathway

Molecules can enter milk through paracellular or transcellular pathways (Fig.

2D), which are affected by the functional state of the MG and regulated by hormones,

growth factors and probably mechanical constrains. While the mammary epithelium is
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leaky before lactation, the direct bi-directional paracellular exchanges of molecules

between the interstitial space and the alveolar lumen (Fig. 2D, 1) is inhibited during

the first days of lactation after the closure of TJs triggered by the hormonal changes

[13]. Consequently, large trans-epithelial concentration gradients are established and

maintained for ions and macromolecules between blood and milk.

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2. Transcellular pathways

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After TJs closure, the composition of the milk reflects the highly coordinated

functioning of four main transcellular pathways in MECs, which operate to produce

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milk components from blood-borne and interstitial molecules [6, 38]. Many
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transporters (Fig. 2D, 2) are involved in the transfer of ions, glucose, amino acids and

water are present on both the BPM and the APM [39]. Transcytosis (Fig. 2D, 3)
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allows the transport numerous components originating from the bloodstream or the
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stroma, such as Igs, albumin, transferrin, insulin, PRL, estrogen, cytokines and
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lipoprotein lipase [40]. Endogenously produced constituents such as major milk

proteins, oligosaccharides, lactose, citrate, phosphate and calcium are secreted

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through the exocytic pathway (Fig. 2D, 4) [41], while lipids (mainly triglycerides) are

secreted by a specialized budding process (Fig. 2D, 5) [37].

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a. Membrane transporters

Water is drawn across the alveolar MECs in a transcellular manner, driven by

an osmotic gradient largely created by the lactose content of the milk. Water is

transported by small transmembrane proteins of the aquaporin (AQP) family. AQPs

are quite ubiquitous and, in addition to water, may also facilitate entry of gases such
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as CO2, NO and ammonia within cells. Various AQPs have been identified in the MG

of various species including human, and localized in MECs, endothelial and

myoepithelial cells. For example, AQP3 is localized in the BPM of alveolar MECs and

may participate in the regulation of milk isotonicity by diluting milk components. The

permeability of AQPs is strongly dependent on the molecular weight of the osmolytes

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they are exposed to. Moreover, the activity of AQPs could be up-regulated after their

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rapid membrane translocation in response to hormones [42].

Membrane transport pathway (Fig. 2D, 2) relies on the concerted activity at

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both the BPM and the APM, as well as in cellular membranes, of various transporter

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proteins, allowing transcellular transfer of ions, trace elements, glucose and amino
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acids from blood to milk. Ion transporters or channels for sodium, potassium and

chloride are found on the BPM and the APM of MECs, while calcium, phosphate,
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iodide and citrate transporters appear to be limited to the BPM [39, 43]. Sodium and

potassium are also actively transported by Na+/K+ ATPase pumps localized in the
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BPM but not APM of MECs. Active transport of calcium and trace elements including
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iron, zinc, copper, selenium, iodide, fluoride, and manganese have also been

described in MECs but the underlying mechanisms have not been fully characterized.
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Moreover, the activity of some of these transporters, such as Ctr1 and ATP7A for
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copper and Zip3 for zinc, has been shown to be up-regulated by PRL, which induces
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their targeting to the BPM. Adequate supply of trace elements from milk is crucial to

ensure neonate survival and both their uptake from blood and release in milk are

tightly regulated by MECs, so that trace element concentrations remain remarkably

stable, independently of the mother’s diet [44].

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As human milk contains up to 8 mM of calcium , large quantities of calcium are

transported from the blood, which contains ~3 mM of calcium, before being

concentrated in milk [11]. The presence of calcium channels has been described in

the BPM and some intracellular membranes of MECs [44, 45], while the intracellular

compartmentalization of calcium depends on cytoplasmic binding proteins. In milk,

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calcium is found associated with casein micelles (~20%), free ionized or non-ionized

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(~32%) or complexed to inorganic anions such as phosphate and citrate (~46%).

In addition, the expression and/or the activity of some ion transporters such as may

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be hormonally regulated. For example, potassium uptake [39] and chloride transport

may be up-regulated by PRL via phosphorylation of transporters, while the

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expression of the sodium/iodide symporter is regulated by PRL and OT [43].
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Plasma-derived glucose is a substrate for several key metabolic processes in
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MECs, including fatty acid and amino acids synthesis, triglyceride esterification, and

is the obligate precursor for lactose synthesis. Hence, several types of glucose
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transporters (predominantly GLUT1) are found at both the BPM and the APM of
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MECs, as well as on Golgi where lactose is synthesized from UDP-galactose and

glucose, and SVs membrane. Lactogenic hormones such as PRL control both the
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expression of glucose transporters and their activity through translocation from

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intracellular sites to the BPM [46].

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As amino acids are building blocks of proteins, large amounts of these

precursors are required to support milk protein synthesis in the lactating MEC. Both

sodium-dependent and -independent amino acid transporters are present at the BPM

of MECs, but their presence at the APM remains unclear, although milk contains

some amino acids. Amino acid transport has been shown to be modulated by PRL

and milk stasis [47].

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b. Transcytosis

After their receptor-mediated endocytosis at the basal side of MECs, some

interstitial molecules enter milk through the transcytic pathway (Fig. 2D, 3). After

endosomal maturation, these molecules are transported alone or in complex with

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their receptor to the APM of MECs, where they are secreted by exocytosis, while

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their receptor are degraded or recycled back to the BPM. Transcytosis has been

described for IgA, insulin, PRL, serum albumin, transferrin, IGF-1 and low-density

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lipoprotein. Of note, the fusion of some transcytic vesicles with SVs may occur in the

apical area of MECs before the exocytosis of their content [40].

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c. Protein secretion pathway
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Milk proteins are synthesized in a classical secretory pathway (Fig. 2D, 4),

beginning with the transcription of their genes into mRNA, then translated in proteins
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and folded in the rough ER. Major milk proteins, namely caseins (α-, β-, γ-, and κ-
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caseins) also undergo post-translational modifications [48], mostly in the Golgi,

associate with calcium and phosphate to form supramolecular structures called

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casein micelles (~140 nm in diameter), which are packed in SVs. SVs also contain
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water, LTF, oligosaccharides, and high concentration of lactose, phosphate, calcium

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and citrate. SVs are vectorially transported via microtubules and fuse with the APM,

then releasing their content into the lumen of the alveolus by exocytosis (Fig. 2D, 4).

Interestingly, this pathway may be regulated by lactogenic hormones at several

levels. Indeed, independently of activating casein gene expression, PRL exerts a

secretagogue effect on the last steps of apical transport and possibly the exocytosis

of caseins through the production of arachidonic acid [49]. On the other hand, after
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binding to its cognate receptor in MECs, OT has been shown increase the number of

SVs and to accelerate their transport towards the APM [50].

The molecular machinery responsible for membrane fusion has been

characterized in many cell types, particularly in neuronal cells, and more recently in

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MECs [51]. SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein

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Receptor) proteins mediate specific fusion of transport vesicles with target cellular

membranes. To do so, the vesicular SNARE (v-SNARE) binds to cognate SNAREs

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located on the target membrane (t-SNAREs), thus forming a tripartite SNARE

complex that promotes the fusion of the vesicle with the target membrane (Fig. 3A

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and B). The whole process of exocytosis is highly regulated by numerous proteins
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working in close association with the SNARE complex. In MECs, specific SNAREs
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have been observed associated with the APM, SVs and MFGs during lactation [51].

On the other hand, several studies have shown that SNARE proteins are the targets
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of arachidonic acid in different neuroendocrine cell types [52]. Thus, it is tempting to

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speculate that the SNARE proteins may be the target effectors of arachidonic acid

produced in response to PRL, then providing a link between signal transduction,

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secretagogue effect and exocytosis in MECs (Fig. 3C), [53]. Moreover, the

expression of some SNARE genes has been found to be regulated by PRL [54]. In
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MECs, the expression of some genes encoding SNAREs involved in casein

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exocytosis (e.g. SNAP23 and VAMP8) is strongly up-regulated during lactation [51]

and our unpublished results, potentially in response to lactogenic hormones [55].

d. Lipid secretion pathway

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MECs import, synthesize and store lipids as LDs which are mainly formed by

accumulation of triglycerides between the two leaflets of the ER and coated with

some specific proteins (Fig. 2D, 5) [37, 56]. Precursors of triglycerides include

acetate, ß-hydroxybutyrate, acetoacetate, fatty acids, glycerol, and

monoacylglycerides, which are taken up by MECs, as well as ketone bodies. Free

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cholesterol also associates with LDs [57]. LDs are thought to grow by fusing with

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each other during their apical transport, and are released by budding, enwrapped by

APM, as MFG [37]. Although proteins such as butyrophilin (BTN1), adipophilin

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(PLIN2) and xanthine oxidase (XOR) appear to play a critical role in this unique

secretory process, the molecular mechanisms of MFG release have not been fully

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deciphered [58]. Moreover, it occasionally results in the inclusion of a cytoplasmic
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crescent in the MFG, thus virtually enabling any cellular components to reach
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milk.The MFG is a major energy source for the newborn and also contains numerous

enzymes, immunomodulatory factors, such as lactadherin/MFG-E8 and BTN1. Lipid

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secretion is regulated by hormones such as PRL [59]and OT through mechanical

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deformation of MECs upon myoepithelial cells contraction [60].

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D. Secretory pathways coupling

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As the release of milk constituents involves at least two distinct mechanisms

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(e.g. exocytosis and budding, Fig. 2D, 4 and 5) synchronized at time of suckling, it is

likely that common activation switch and/or molecular effectors may exist to

coordinate their activities. On the other hand, because of their large size (~4 µm in

diameter) and their high number, the membrane surface needed to enwrap the MFGs

could exceed that of the APM of MESCs. Thus, at time of suckling, there is both

membrane supply and loss at the APM of MESCs, due to SV fusion and MFG
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budding, respectively. Various data also reinforce the possibility of coupling of these

two processes: 1) the association of SVs with the APM and the basal part of the

budding MFG (Fig. 3A) has been extensively described, 2) some SNARE proteins

are localized at the interface between SVs and the budding MFG [51] (Fig. 3B), and

3) the membrane supplied by the fusion of a high number of SVs with the APM is

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used to enwrap the MFG [57, 61]. As depicted in Fig. 3C, a possible scenario could

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be that, in response to PRL (secretagogue effect), local production of arachidonic

acid, potentially from neutral lipid core of the MFG, stimulate membrane fusion

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through interacting with SNARE proteins [40, 53]. Both heterotypic (SVs with APM)

and homotypic (SVs with SVs) fusion may then occur (Fig. 3C), leading to the

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coordinated release of milk products. Furthermore, this would also partly balance the
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membrane loss caused by MFGs release, concomitantly with the efficient resealing of
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the APM (Fig. 3D). Recently, the final expulsion of MFGs has been shown to occur

after OT-mediated contraction of the myoepithelial cells [60]. This observation is

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compatible with the above scenario and suggest that milk secretion processes are
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spatio-temporally coupled and regulated by both hormonal and mechanical factors.

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E. Hormonal regulation of milk secretion

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As MG growth and differentiation, lactation is regulated by hormones, but also

by interactions between the MG and the central nervous system. PRL signals through

the JAK2/STAT5 pathway to regulate the expression of target genes, and also

stimulates lipid synthesis and exocytosis. On the other hand, OT is rapidly released

in response to suckling and induces the contraction of myoepithelial cells surrounding

the alveoli, thus triggering milk ejection [6].

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1. Prolactin

PRL is a pleiotropic hormone produced by the anterior pituitary which is

involved in homeostasis, reproduction, and lactation. During the MG development

and differentiation, PRL exerts morphogenic effects, while during lactation this

hormone displays lactogenic effects by stimulating milk protein and lactose synthesis

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and secretion, as well as other metabolic processes in MECs. PRL is thus required to

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maintain milk yield but also for alveolar MECs survival and maintenance of tight

junctions (TJs) [6, 13]. During pregnancy, the serum PRL level slightly increases from

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~10 ng/mL in the non-pregnant women up to ~200 ng/mL at term [62]. In the course

of lactation, levels of circulating PRL gradually decrease to return to ~10 ng/mL after

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~6 months postpartum. PRL is episodically released in response to suckling to reach
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a peak in concentration in the blood 45 minutes after the beginning of breastfeeding,
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for up to 75 minutes in duration [63]. However, while the amount of PRL released is

related to the intensity of nipple stimulation, plasma PRL concentration does not
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appear to be directly correlated with the volume of milk produced. Interestingly, in

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serum and milk, several molecular forms of PRL are found, which arise from PRL

processing such as cleavage [64]. Whether this molecular heterogeneity can account
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for the various effects of PRL remains unclear. For example, while binding of the 23-

kDa PRL to its cognate receptor on the BPM of MECs stimulates milk protein genes
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transcription, the internalization of the PRL/PRL receptor complex enable the

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transcytosis of PRL to the lumen, which is required for milk protein secretion [40].

2. Oxytocin

As soon as it begins, suckling is detected by mechanoreceptors of sensory nerve

terminals in the areolus of the nipple which send afferent cholinergic impulses to the
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paraventricular nuclei and supraoptic nuclei in the hypothalamus, that in turn

stimulate the pulsatile release of OT, a nonapeptide hormone, from the posterior

pituitary [65]. Once in the bloodstream, OT reaches the MG where it interacts with

specific G-protein-coupled receptors localized on myoepithelial cells, and induces

their asynchronous contraction. As OT receptors are also present in MECs [66], this

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hormone may also exert direct effects on the secretory activity of MECs (Fig. 3D) [50,

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60]. Milk is then expelled out of the alveoli into the ducts and lactiferous sinuses.

Contraction of the myoepithelial cells also shortens and widens the ducts, thus

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increasing the intraductal pressure and consequently the milk flow rate, ultimately

leading to milk ejection from the nipple. Thus, OT mediates the milk ejection reflex (or

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let-down reflex), which is essential for the efficient removal of milk from the breast. As
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OT is released in a pulsatile manner, there are several ejections of milk during a
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feeding [31]. The number of ejections is significantly correlated to the volume of milk

consumed but not to the duration of the feeding [31]. Suckling also causes an
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inhibition of the release of LH-releasing hormone by the hypothalamus that results in

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the inhibition of ovulation and a natural form of birth control.

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There is also a significant psychological component in the let-down reflex, as

OT release also occurs in response to such stimuli as the sight or sound of the baby
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[67]. In addition to mediate the milk ejection reflex, OT also has significant roles on
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the central nervous system for the psychological integration of the interactions

between the mothers and the suckling neonate, and in maternal behavior.

Furthermore, physical and psychological stress or pain of the mother has been

shown to decrease milk output through the inhibition of OT release [68]. However,

responses to stress seem to be reduced, e.g. plasma levels of adrenocorticotropic

hormone (ACTH), cortisol, and epinephrine are significantly decreased in lactating

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women stressed with graded treadmill exercise as compared to those found in non-

lactating women [69]. OT release is likely to be involved as its pulsatile release in

response to suckling is accompanied by a decrease in plasma ACTH and plasma

cortisol levels in lactating women.

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F. Milk composition

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The composition of milk varies between and within species and is specifically

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and ideally adapted to the needs of the neonate mammals to properly develop.

Indeed, milk composition varies according to gestation, time postpartum and even

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during suckling.
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During pregnancy, pre-colostrum contains high concentrations of protective

Igs, lysozyme, and LTF, sodium, chloride, and low concentrations of casein, lactose,
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potassium, citrate, calcium, and phosphate. Colostrum persists for 4 or 5 days after

parturition, followed by transitional milk for a further 5 days until mature milk is
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produced [11].
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Mature milk is a complex emulsion of fat and aqueous fluid containing proteins
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(~3.5%), sugars (~7%), lipids (~4%), minerals (~0.5%) and water, constituting a

unique complete nutritive source for the newborn.

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Milk protein fraction includes four major proteins [70], e.g. α-lactalbumin and
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LTF (an iron-binding immunomodulatory protein with antibacterial properties), which

are the most nutritionally important, caseins, and Igs (IgA for up to 10% of human

milk protein, IgM and IgG). Igs provide passive immunity to the newborn and also

serve as part of the immune system of the MG [71].

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The aqueous fraction of milk (or whey) contains serum albumin, some

hormones (e.g. PRL and insulin, leptin and adiponectin), growth factors (EGF, IGF-1,

Ghrelin, and TGF), cytokines, lysozyme (a heme peroxidase with antibacterial and

anti-oxidant properties), more than 30 enzymes (including lactoperoxidase which

oxidizes bacterial components; proteases, protease activators, nucleases,

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glycosidases, amino-acid oxidases), vitamins, non-protein nitrogen, nucleotides, as

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well as minerals (sodium, potassium, chloride, citrate, calcium, magnesium, free

phosphate, trace elements), and water. Growth factors such as EGF may regulate

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the intestinal growth, while hormones may modulate metabolism and body

composition of the newborn [72]. Factors with antimicrobial activities play important

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roles in protecting both the gastrointestinal tract of the newborn and the mother’s
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breast [73]. Of note, the sodium concentration in breast milk within the first 3 days
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postpartum may be predictive of lactation success, particularly in some mothers at

high risk for insufficient milk supply [74]. Indeed, high concentrations of sodium in
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milk are found in some clinical situations such as mastitis, inhibition of PRL secretion,
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and premature birth [75].

Milk also contains various types of carbohydrates, mainly lactose, a

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disaccharide unique to milk, glucose, galactose, and oligosaccharides, which display

substantial protective effect against a variety of pathogens [76].

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In human, the fat accounts (MFGs) for ~4% of milk volume and contributes for
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up to 50% of the energy content. MFGs mainly contain triglycerides as well as a

variety fatty acids, cholesterol, phospholipids, and steroid hormones (GCs,

progesterone and estrogen [77]. Fat is the most variable fraction as its fatty acid

composition varies with the maternal diet, and even during suckling. Bioactive lipids

such as prostaglandins (PGs, including PGE2, PGD2, PGF2, PGI2), and thromboxane
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A2 are, which are synthesized from arachidonic acid by cyclooxygenases are also

present in milk and may exert protective effects.

Extracellular vesicles (EVs) such as exosomes (40-100 nm diameter) have

been identified in milk. Exosomes are vesicles formed in the multivesicular bodies

(MVBs) derived from the endocytic pathway. During MVBs biogenesis, cargos, such

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as proteins, lipids, non-coding RNAs including micro-RNAs (miRNAs), and mRNAs

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are sorted into internal vesicles (e.g. exosomes), which are released into milk after

exocytosis of the MVBs. In addition to play a role in nutrition, exosomes may

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participate to cell-to-cell communication, regulate developmental and immune

processes, intestinal microflora, as well as cellular metabolism and gene expression

after ingestion by the newborn [78].

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Human milk is particularly rich in microRNAs, which are potentially involved in
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infant protection and development. MicroRNAs are small non-coding RNA molecules

that regulate gene expression at the post-transcriptional level, modulating important

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cell functions such as cell cycle, metabolism, proliferation, differentiation, apoptosis,

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and immune response [78].

Some cells such as MECs, macrophages, neutrophils, lymphocytes and stem

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cells are also found in milk [79, 80].

Human milk has also been identified as the first probiotic food as it contains a
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large microbial community including more than 200 phylotypes. Although not clearly
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established, these bacteria may be present on the mother’s skin or may come from

the maternal intestine after reaching the MG via lymph and/or blood circulation [81].

In addition to enrich the intestinal flora of the newborn, milk bacteria could influence

the long-term microbiota composition and activity, thus playing a key role to prevent

various diseases such as allergies, disorders, and metabolic syndrome [82, 83].
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Therefore, breast milk not only functions as a nutritive source but also delivers both

developmental and immune modulatory factors to the newborn.

Although the gross composition of mature human milk appears fairly constant

with only slight changes for major components with stage of lactation, there are

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declines in the total fat content of the milk between 1 and 2 months, in the

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concentration of protein between 1 and 6 months [84], and in the concentration of

calcium between 4 and 6 months. In addition, subtle variations occur in some

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constituents, such as fatty acids, vitamins, selenium and iodide, according to the

maternal diet [3]. Indeed, although the total fat content of breast milk appears

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unaffected by diet, the proportions of some fatty acids, e.g. omega-3 and omega-6
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polyunsaturated fatty acids (PUFAs) vary substantially with the mother’s diet [77].
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These variations may have important consequences due to the positive correlation

between the quantity of omega-3 PUFAs in the mother’s diet and the infant brain
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development [85]. The fat content of milk is also known to increase with the duration
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of breastfeeding in proportion to the emptying of the alveoli [86]. Thus, even if the

storage capacity influences fat concentration in milk, it does not affect the total
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amount of fat consumed by the child [87]. While the concentration of lactose shows

no significant change with stage of lactation, variations in milk glycans, e.g. complex
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oligosaccharides free or covalently bound (glycolipids, glycoproteins, glycopeptides,

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and glycosaminoglycans), have also been observed both between lactating women

and during the course of lactation, according to the newborn’s needs. These complex

glycostructures are important dietary factors during early life as they regultate

multiple functions [88]. However, the growth rate of breast-fed babies is related to the
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total amount of milk they consume, rather than the concentration of fat, protein, or

lactose [89].

Maternal nutrition affects both the quantity and quality of milk, which vary

among countries, and lactation requires adjustments of maternal metabolism to adapt

to the energetic demands of breastfeeding [90]. As the milk production is almost

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entirely regulated by the infant demand, the maternal metabolism can be increased

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up to 20% of the metabolic output of the mother. This can be achieved by an

increased food intake or increased weight loss to compensate for the metabolic

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needs to produce milk.

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G. Breastfeeding patterns

Milk production works according to the ‘use it or lose it’ principle and current
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recommendations are to feed babies on demand [91]. Indeed, babies feed according
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to their appetite and the mother’s milk production is regulated to match the baby’s
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needs. Although PRL stimulates the synthesis of milk proteins, it does not control the

amount of milk produced once lactation is established. In fact, the quantity of milk
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produced is correlated to the draining efficiency of the suckling and is accordingly up-

regulated if the breast is well-drained [87]. Moreover, the efficient draining of the
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breast appears to be more important than the frequency of feeding to stimulate milk
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production. According to its appetite, a baby drains the breast one or more times per

day but on average takes only 67% of the available milk [15]. Therefore, the feeding

frequency appears significantly increased for mothers with low storage capacities.

Milk contents in proteins and lactose also seem to have more influence on the

frequency of feeding, which is independent of the volume of milk consumed, than the

quantity of lipids or the calorie value of the meal [92]. Furthermore, the fat content of
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milk is related to the degree of “fullness” of the breast: the more the breast is filled

with milk, the more the fat content of milk is low, while conversely, the more the

breast is drained, the more the fat content of milk is high.

H. Storage capacity

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During exclusive breastfeeding, the lactating breast has a limited capacity

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(from 80 to 600 ml) to store milk, which varies to adapt to the child’s needs [15].

Storage capacity also varies from one breast to the other, independently of the ability

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to produce enough milk, but potentially affecting the feeding frequency. This may be

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related to the frequency and the efficiency of milk removal and to the local negative
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feedback regulation of milk secretion occurring when alveoli and ducts are filled with

milk. As supplementary feeds are introduced, the milk storage capacity decreases
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along with the reduction of milk production [87].

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I. Extended lactation
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The World Health Organization recommends exclusive breastfeeding for the

first 6 months of life, and partial breastfeeding into the second year [91]. When
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lactation is extended beyond 6 months, a significant decrease of the mammary tissue

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occurs gradually accompanied with a slight decline of the volume of milk produced
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and changes of its composition [93]. Breast returns to its preconception size after ~15

months of lactation.

J. Breastfeeding and associated outcomes

Human milk is an optimal food for newborns as it contains both nutrients and

bioactive compounds which contribute to both the short and long-term health benefits
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that have been reported to be directly correlated with the duration of breastfeeding.

Breast-fed infants experience fewer and shorter infections, exhibit different growth

patterns, have different gut microflora, show better cognitive development and even

face differences in the risk of chronic diseases, such as obesity, type 1 and type 2

diabetes and cardiovascular disease. Breastfeeding also appears to be protective

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against sudden infant death syndrome, the risk of diarrhea, respiratory infections, and

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malocclusion, but does not seem to provide a protection towards either eczema or

food allergy [94, 95]. Breastfeeding outcomes are also related to mother genotype,

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phenotype, diet, disease, and lifestyle [90].

Human milk is also recommended to feed preterm infants as it significantly

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reduces complications associated with prematurity such as necrotizing enterocolitis,
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retinopathy of prematurity, broncho-pulmonary dysplasia and late-onset sepsis and
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promotes brain development and neurocognitive outcome [96].

Extended breastfeeding has also beneficial effects for the mother as it leads to
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birth spacing due to longer periods of amenorrhoea, reduces risk of developing a

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longer-term diabetes type 2, overweight/obesity, and leukemia [97]. However,

breastfeeding seems not to have a protective effect towards hypertension and/or

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hypercholesterolemia [97]. Extended breastfeeding also reduces the incidence of

ovarian and breast cancer. Numerous studies suggest that high parity is associated
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with a decreased risk of developing breast cancer but that lactation itself, even
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extended, contributes no extra protection. The incidence of breast cancer appears to

be reduced among pre- and post-menopausal breastfeeders, but a direct relationship

between the duration of lactation and the reduction in the risk is found only for

women with premenopausal cancer. Nonetheless, the mechanisms by which

lactation could protect against breast cancer are not clearly identified although they
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probably involve the hormonal changes associated with breastfeeding and their

effects on both the breast and the inhibition of ovulation [29].

Conclusion

Numerous aspects of the lactation process still remain to explore and to

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understand. The emergence of more efficient approaches to decipher mammary

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development and secretory functioning and milk composition. Moreover, the

integration of multi-scaled data from clinical trials to cellular biology should highlight

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new aspects of breastfeeding and help to improve both mother and child’s health, as

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well as infantile formulae. AN
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Footnotes

The authors declare that there are no conflicts of interest.

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Practice points

• Breast development and function under physiological and pathological

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conditions.

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• Milk composition and influence of maternal and environmental factors.

• Consider important aspects to help mothers who want to breastfeed their

newborn.
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Research agenda

•
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Meta-analysis of clinical data.

• Integration of multi-scaled data, from populations to cell biology.

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• Elucidate what and how milk can transfer information to the child and study of
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trans-generational effects.

• Milk composition and microbiota outcomes in neonate’s development, and in

short- and long-term pathologies.

• Intra-vital imaging to decipher molecular mechanisms of breast development

and milk secretion.

• Pre-term alimentation and improvement of infantile formulae.

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Legends to Figures

Fig. 1. Mammary gland development. The postnatal development of female

mammary tissue occurs in several steps regulated by hormones. At birth, the

mammary epithelium consists of limited ducts. At puberty, high levels of circulating

hormones stimulate both the proliferation of the MECs and the enlargement of the

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surrounding fat pad. At the onset of pregnancy, epithelial ducts elongate, branch and

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alveoli develop. During lactation, the mammary epithelium reach its maximal

development containing numerous alveoli, which produce huge amounts of milk.

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Upon weaning, milk production ceases, the mammary alveoli regress (involution) and

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the mammary epithelium returns to a non-pregnant state.
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Fig. 2. Anatomy and functional organization of the lactating mammary gland. A)
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During lactation, the mammary epithelium is organized in lobes containing numerous

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lobules and connected to lactiferous ducts, which drain milk towards the nipple. B)
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Each lobe contain numerous lobules formed by several alveoli, which are the milk

secreting units. C) The alveolus is defined by a monolayer of polarized alveolar

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mammary epithelial cells (MEC) arranged around a lumen, where milk is secreted.

The alveolus is connected to the lactiferous duct formed by a bilayer of MECs.

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Alveolar MECs contact the BM, a specialized ECM and are surrounded by contractile
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myoepithelial cells. Mammary alveoli are embedded in a stroma containing collagen,

endothelial cells, adipocytes and fibroblasts. D) Milk products are secreted in the

lumen of the alveolus by various pathways in polarized MECs. Transport of plasma

components and sometimes leukocytes through the paracellular pathway (1) occur

only during pregnancy, early lactation before the closure of TJs, and involution or

during inflammation. Membrane transporters (2) allow the direct movement of ions,
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water and glucose across the BPM and the APM of the MEC. Some plasma proteins

such as Igs and PRL reach the lumen after crossing the MEC by vesicular

transcytosis (3). Milk proteins, lactose, calcium and other components of the aqueous

phase of milk are transported in secretory vesicles (SVs) and released after

exocytosis (4). LDs are formed in the endoplasmic reticulum (ER) and grow during

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their transport to the apex where they are released as milk fat globule (MFG) by

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budding, being enwrapped by the apical plasma membrane of the MEC (5). BV,

blood vessel; M, mitochondrion; Myo, myoepithelial cell.

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Fig. 3. Potential mechanism for the coupling of exocytosis and budding. A) In the
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lactating MEC, secretory vesicles (SV) containing casein micelles (black dots) are

observed just beneath the apical plasma membrane (APM) and around the
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cytoplasmic part of the budding milk fat globule (MFG). SNARE proteins are
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associated both with the APM (STX and SNAP23) and the SVs (VAMP). B) The
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SNARE proteins associate to form a tripartite complex called SNARE complex, which

promote both the docking of the SV to the APM and the fusion of the SV with the
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APM (exocytosis). SNARE complex may also form between SVs. C) During suckling,

PRL (PRL) stimulate a phospholipase A2 (PLA2) to locally produce arachidonic acid

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(AA), potentially from neutral lipids contained in the MFG, which in turn may promote
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membrane fusion through binding to the SNARE complexes. This signaling pathway

may lead to both heterotypic fusion between SVs and the APM (1), and homotypic

fusion between SVs (2). D) Finally the MFG is released enwrapped by a membrane

bilayer, while the APM is resealed. The contraction of the myoepithelial cells in

response to oxytocin (OT) leads to the deformation of the MECs and the pressure

exerted on the APM may promote the final release and the ejection of milk products
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towards the nipple. This scenario is advantageous for MECs as it limits membrane

loss and cell death, while promoting the spatio-temporally coordinated secretion of

milk products. SNAP23, synaptosomal-associated protein 23; STX, syntaxin; VAMP,

vesicle-associated membrane protein.

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