Lactogenesis

“Lactogenesis” was initially defined as the action of lacto­genic hormones on mammary gland and histological alterations in the tissue. Later, lactogenesis was used to describe the onset of copious milk at the time of parturition and the appearance of organelles related to milk synthesis and secretion.

25.3.1 Enzymatic and Cytological Differentiation of Alveolar Cells Before the Onset of Lactation

The stage-I of lactogenesis is characterized by enzymatic and cytological differentiation of alveolar cells divided into four phases namely proliferative phase (early pregnancy), secre­tory differentiation phase (mid-pregnancy), secretory activa­tion (parturition), and lactation.

The proliferative phase initiates immediately after concep­tion and is characterized by extensive proliferation of mam­mary epithelial cells as indicated by increased DNA content. Around 25% of proliferative cells are evident till day 5 of pregnancy. Alveolar buds were developed from proliferating epithelial cells that progressively become milk-secreting lobules. The proliferation decreases during mid-pregnancy and a network of capillaries encompasses each alveolus.

The secretory differentiation starts around second half of pregnancy and is characterized by several biochemical changes required for initiation of milk synthesis such as the increased activity of lipid synthetic enzymes along with the expression of adipophilin protein and incorporation of ¹⁴C-acetate in the mammary glands of pregnant rabbits and rats.

The phase of secretory activation is characterized by the onset of copious milk secretion and coincides with the drop of plasma progesterone level and higher prolactin level after parturition. A higher level of prolactin initiates transcription of milk protein genes. The cytological alterations of mam­mary epithelium during this phase are characterized by an increased number of Golgi and endoplasmic reticulum which acts as a machinery for the synthesis of various milk components. The probable markers of secretory activation phase are lactose, protein, citrate, and sodium.

The last phase of lactogenesis is characterized by continu­ous production of milk. It is again subdivided into two sub-phases namely colostral phase (milk contains a large amount of immunoglobulins and immune defense proteins) and mature secretion phase (production of a large volume of milk to support newborn). Rapid proliferation of mammary epithelium together with the development of active enzy­matic machinery for the synthesis of milk constituents are the characteristic features of this phase. Activation of enzy­matic machinery required for lactose, fat, and protein synthe­sis during the initiation of lactation has been well documented in several species like pig, cow, rat, mouse, guinea pig, and rabbit and showed huge inter-species varia­tion in the enzymatic development of mammary gland. In rats and guinea pigs, the activities of these enzymes were increased during late pregnancy and early lactation and achieved maximal activity around the second or third day of lactation. Whereas, in cows, no significant increase in enzyme activities occurred during late pregnancy and early lactation. The probable explanation for this was the fact that the growth of mammary gland in cows required more time compared to rats and guinea pigs and during this proliferative phase only a small proportion of newly formed secretory cells are available in mammary tissue.

25.3.2 Milk Synthesis and Secretion

25.3.2.1 Metabolic Adaptations During Early LactationZNutrient Partitioning

During the initiation of lactation, the majority of the cows are in a state of negative energy balance (NEB) which indicates the energy required for maintenance and milk production are greater than the energy intake. NEB is not only due to limited feed intake but also due to high metabolic priority for milk production. In the dairy cow, energy requirements for lacta­tion can reach 80% of net energy from intake and lactose production can utilize 85% of circulating glucose. The energy requirement for milk production is obtained by mobilization of body fat and muscle proteins. Moreover, there is excessive partitioning of nutrients towards mammary gland is the fun­damental consequence of NEB which is most severe during the first week of postpartum. The partitioning of nutrients is regulated by homeostatic and homeorhetic mechanisms and under genetic control. The metabolic adaptations during peripartum period were aimed to increase endogenous glu­cose production together with the delivery of glucose and nonesterified fatty acids (NEFA) to the mammary gland for milk production which is facilitated by endocrine factors such as decreased insulin, leptin insulin like growth factors, and thyroid hormones together with the elevated level of GH, cortisol catecholamines, and glucagon. The major metabolic adaptions to cope with NEB are summarized in Table 25.10.

25.3.2.2 Biosynthesis of Lactose

Lactose is the major milk sugar in most species. However, sialyllactose is the predominant sugar present in rat and mouse milk. Lactose is a disaccharide composed of D-glucose and D-galactose, joined through β-1,4-glycosidic linkage whereas sialyllactose is an oligosaccharide. Lactose is the main contributor to milk osmolality and it is responsible for drawing water into the milk. It can also be noted that lactose is one of the least variable component of milk.

25.3.2.2.1 Precursors of Lactose

Blood glucose is the precursor of milk lactose and two molecules of glucose are required for the synthesis of one molecule of lactose. The predominant sources of glucose are dietary or endogenous production. In ruminants, the dietary carbohydrates are degraded in the rumen by microbial fer­mentation to volatile fatty acids and only 10% of the glucose requirement is filled up by dietary glucose and the animal has to produce the remaining 90% of required glucose through endogenous production by gluconeogenesis and glycogenol­ysis in liver. Gluconeogenesis and glycogenolysis contribute around 85% of glucose requirement in the mammary gland. Propionate is the main precursor for neoglucogenesis (45-60% of endogenous glucose is synthesized from propio­nate) together with lactate and amino acids, particularly ala­nine. Glycerol, produced from lipolysis of adipose tissue,

Table 25.10 The major metabolic adaptions during the state of NEB

also aids a little contribution to neoglucogenesis, particularly around parturition.

25.3.2.2.2 Glucose Sparing Action in the Mammary Gland of Cattle

In ruminants, blood glucose level is lower compared to non-ruminants (40-80 vs 80-120 mg/dL). During the initia­tion of lactation, the majority of glucose is channelized for milk lactose production and little glucose is available for maintenance energy requirement. So, under this high energy-demanding stage, the ruminants must have a well- developed glucose-sparing mechanism in mammary gland to ensure that glucose carbon is not used in synthetic and oxi­dative reactions whereas other substrates like acetate can effectively be used. One such example is seen in fatty acid synthesis in the mammary gland where the exclusion of glucose is linked with the absence of citrate cleavage enzymes essential for the generation of cytoplasmic acetyl CoA from glucose.

25.3.2.2.3 Utilization of Glucose in the Mammary Gland

The majority of glucose (60-70%) in the mammary gland is utilized for milk lactose synthesis and the remaining (20-30%) goes through the pentose phosphate shunt to gen­erate NADPH (used as reducing equivalents in milk fatty acid synthesis). A small amount of glucose is also utilized for the generation of ATP, ribose sugar (DNA and RNA), and syn­thesis of glycerol (used for milk triglyceride synthesis).

25.3.2.2.4 Glucose Uptake in the Mammary Gland

Glucose uptake in the mammary epithelial cells is mediated by two processes namely passive facilitative diffusion process and sodium-dependent glucose transport. The concentration gradient established across plasma membrane favors this dif­fusion process. Two distinct classes of glucose transporters are involved in the glucose uptake process namely, facilitative glucose transporters (GLUT) and sodium-linked glucose cotransporters (SGLT), for facilitative diffusion and sodium­dependent glucose transport, respectively.

The expression of these glucose transporters is lactation stage-specific, lower expression was reported during virgin state which was reported to be increased around 40-folds during mid-lactation and declined sharply around involution.

25.3.2.2.5 Biochemical Pathway of Lactose Synthesis

Glucose is phosphorylated at its sixth position to form glu- cose-6-phosphate with the help of hexokinase. Phosphoglu­comutase then transfers phosphate group from position 6 to 1 to form glucose-1-phosphate which combines with uridine triphosphate to form uridine diphosphate glucose and liberates pyrophosphate catalyzed by the enzyme UDP-glucose pyrophosphorylase. UDP-glucose is converted to UDP-galactose by UDP-galactose 4-epimerase. Finally, UDP-galactose combines with glucose to form lactose with the help of lactose synthetase enzyme.

The enzymatic machinery required for lactose synthesis particularly UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase increase markedly after the onset of mammary gland in rat and guineapig but not in cattle suggested that the enzymes already present in the mammary gland of cattle during pregnancy.

The enzyme lactose synthetase is a complex of two proteins that combines reversibly, in 1:1 stoichiometry. The protein A of this enzyme complex is galactosyltransferase which transfers galactose from UDP-galactose to terminal non-reducing N-acetylglucosamine residues of glycoproteins as follows:

UDP — galactose + N — acetyl — D — glucosamine → N

— acetyllactosamine + UDP

The protein B of lactose synthetase complex, i.e., α-lactalbumin inhibits this reaction and allows UDP- galactose to combine with glucose to form milk lactose.

UDP — D — galactose + D — glucose → Lactose + UDP

25.3.2.2.6 Secretion of Milk Lactose

After synthesis, lactose is transported to Golgi complex and encapsulated in Golgi membrane to form secretory vesicles. These vesicles move towards apical surface and fused with the apical membrane and discharge their contents by exocytosis.

25.3.2.3 Biosynthesis of Milk Fat

The predominant constituents of milk fat in bovines are triglycerides (98%), diacylglycerol (1%), cholesterol (16 C) are obtained from circulating lipids. The short- (4-8 C) and medium-chain fatty acids (10-14 C) arise from de novo synthesis. Fatty acids with 16 C are produced from both sources.

25.3.2.3.2 Sources of Circulating Fatty Acids

Mammary gland obtains circulating fatty acids from circulating lipoproteins and nonesterified fatty acids (NEFA). There are two important sources of circulating fatty acids, viz., absorbed lipids from the digestive tract and the lipids mobilized from fat reserve of the body. In ruminants, the predominant source of circulating fatty acids are dietary though a small proportion (the Mammary Gland

The generation of fatty acids by de novo synthesis varies between species, in elephant de novo synthesis is the princi­pal source of fatty acids synthesis and in the seal, and it is derived from circulation. About half of the fatty acids in the milk of ruminants are derived from de novo synthesis from acetate. However, butyrate contributes the first four carbons of fatty acids originating from de novo synthesis. Reducing equivalent like NADPH is also required for fatty acid synthe­sis which is derived either from pentose phosphate cycle and the isocitrate cycle (ruminants) or from pentose phosphate cycle and the malate transhydrogenation cycle (non-ruminants). The other required cofactors are Mn²+, Bio­tin, and HCO^3—. The de novo fatty acid synthesis occurs in the cytosol and the basic building block is acetyl CoA. The reaction is catalyzed by fatty acid synthase (FASN) and acetyl CoA carboxylase (ACC). Fatty acid synthase is a multi-enzyme complex consisting of acyl carrier protein (ACP) and 6 different enzymes namely b-ketoacyl synthase (KS), acetyl/malonyl-CoA transferase (MAT), b-hydroxyacyl dehydratase (DH), enoyl reductase (ER), b-ketoacyl reductase (KR), and thioesterase I (TE I). The biochemical pathways of fatty acid synthesis are depicted below.

The enzyme thioesterase is the key regulator of chain length of fatty acids. It has two subtypes. TE I is a part of FASN whereas TE II is a tissue-specific enzyme observed only in nonruminants that is independent of FASN. Both TE I and II can interact with each other and FASN to produce all kinds of fatty acids (C8, C10, C12).

Glycerol is synthesized from glucose or circulating glycerol. The primary source of glycerol is glycerol-3-P derived either from glycolytic pathway or lipolysis of triglycerides.

25.3.2.3.4 Esterification of Fatty Acids

Synthesis of triglycerides through the esterification of glyc- erol-3-phosphate and fatty acids is evident in most cell types including mammary gland. C₁₂-C₁₆ fatty acids are concentrated on C-2 atom of glycerol whereas short-chain fatty acids (C₄-C₅) are concentrated on C-1 and C-3 atom of glycerol. ATP, Mg²⁺, and CoA are the cofactors required for etherification process. The proposed model of esterification of fatty acids is given in Fig. 25.2.

Fig. 25.2 Esterification of fatty acids

Christiesomes are the cell fragments that contain endo­plasmic reticulum, mitochondria, and lipid droplets, identified in goat milk. These are involved in triglyceride synthesis.

25.3.2.3.5 Secretion of Milk Lipids

Milk lipids are secreted as droplets of various sizes. These lipid droplets are mainly composed of triglycerides (95%) with lesser amounts of sterols, partial glycerides, phospholipids, glycolipids, and hydrocarbons. The droplets are covered by a membrane composed of polar lipids and proteins known as milk fat-globule membrane (MFGM). The membrane originates either from apical plasma membrane (primary membrane) or from endoplasmic reticulum and other intracellular compartments. Based on the size there are two types of milk droplets namely cytoplasmic lipid droplets (CLDs) (1-5 μm) and microlipid droplets (MLDs) (0.5 μm or less). There are two proposed routes of milk lipid secretions.

Apical vesicle route: Lipid droplets originate as MLDs in the rough endoplasmic reticulum and transit towards apical membrane alone or in combinations to form CLDs (MLDs are fused during their transit towards apical membrane).

Secretory vesicle route: In this mechanism, CLDs are surrounded by secretory vesicles which in turn fuse together to form intracytoplasmic vacuoles and transported towards apical membrane. In both cases, the contents of apical and secretory vesicles are released by exocytosis.

A combination of both apical and secretory vesicle routes may also help to release milk fat droplets.

Lipid droplet proteins called as adipophilin is involved in the production of CLDs during secretory differentiation of mammary alveolar epithelium and play a pivotal role in both formation and secretion of milk lipids.

25.3.2.4 Milk Protein Synthesis

25.3.2.4.1 Precursors of Milk Proteins

The predominant precursors of milk proteins are the free amino acids of the plasma free amino acids. It has been estimated that 70% of amino acids perfusing mammary gland are derived from blood. Some nonessential amino acids were synthesized in the mammary gland after utilizing the nitrogen of certain amino acids, particularly arginine and ornithine. Other minor sources of amino acids are the gluta­thione of erythrocytes and plasma oligopeptides.

Amino acid uptake by the mammary gland: The substrate, amino acids from blood, is transported through the basolateral membrane to mammary secretory cell. Differ­ent groups of amino acids require different transporting system. Based on the substrate specificity and transport mechanism, amino acid transporters have been classified into

Na⁺-dependent transporters utilize the electrochemical gradients of Na⁺ and other ions to actively transport amino acids. These transporters are also energy­dependent and facilitate unidirectional amino transport. Based on the amino acid specificity Na⁺-dependent transporters may be of two types (Table 25.11).

Na⁺-independent transporters are non-energy dependent and facilitate bidirectional transport of amino acids. The details of Na⁺-independent transporters have been presented in Table 25.12.

Genes for milk protein synthesis: Majority of the milk proteins are synthesized from transcription of tissue­specific genes under the influence of hormones.

Table 25.11 Na⁺-dependent transporters in the mammary gland in different species

Table 25.12 Na⁺-independent transporters in the mammary gland in different species

The localization of casein gene locus has been identified in chromosome 6 in cattle, sheep, and goat, chromosome 5 in mice, and chromosome 4 in human. β-lactoglobulin- encoding gene locus has been identified in chromosome 3 in sheep and chromosome 11 in cow and goat. The α-lactalbumin-encoding gene has been linked to chromo­some 12 in man, chromosome 3 in sheep, chromosomes 5 in bovine and goat, and chromosome 5 in pig.

25.3.2.4.2 Mechanism of Milk Protein Synthesis

Hormone-induced transcription factors initiate the expression of aforesaid genes and the protein biosynthesis is initiated by following steps.

Transcription: In this step, messenger RNAs are formed in the nucleus which carries the codes of specific proteins. Non-coding sequences (introns) are removed from coding sequences (exons) by mRNA splicing. The formed mRNAs are localized in ribosomes in the rough endoplas­mic reticulum.

Amino acid activation: Amino acids in the cytoplasm are activated by reaction with ATP and attachment to transfer RNA (tRNA). The tRNAs are specific for each amino acid.

Translation: The mRNA contains codes for amino acids (codon). The anti-codons in the tRNA recognize codon to form appropriate amino acid-tRNA complex. This complex moves and appropriate amino acid-tRNA com­plex is added to form peptide chain.

25.3.2.4.3 Intracellular Transport and Processing of Milk Proteins

Transport in the endoplasmic reticulum: Proteins are synthesized in the ribosomes of rough endoplasmic retic­ulum as a long polypeptide chain. A short peptide of 16-30 amino acids present at the N-terminus of newly synthesized proteins is released by the action of a protease. This peptide acts as an open reading frame encoding the rest of the protein called signal sequence. These signal sequences are recognized by receptors on the ER and the proteins are translocated in the endoplasmic reticulum. After translocation in the endoplasmic reticulum, cysteine-rich peptide residues undergo disulfide bond for­mation that allows the folding of nascent polypeptides in the lumen of the endoplasmic reticulum. The oxidizing environment inside the lumen of the endoplasmic reticu­lum favors this bond formation and protein disulfide isom­erase (PDI) catalyzes this reaction. Appropriate folding of these proteins is an essential prerequisite for the transport of these proteins into Golgi complex. The other co-translation modifications are N-glycosylation of α-lactalbumin and oligomerization of caseins.

Transport in the Golgi and trans Golgi network: The protein cargo emerging from the endoplasmic reticulum is transported to tubule vesicular network between endoplas­mic reticulum and Golgi complex known as ER-Golgi intermediate compartment (ERGIC). These ERGIC are responsible for the protein trafficking between endoplas­mic reticulum and Golgi. Inside the Golgi complex and trans Golgi network lots of posttranslational modifications of the proteins occur (Table 25.13).

Table 25.13 Posttranslational modifications of major milk proteins in Golgi and trans Golgi network

25.3.2.4.4 Transport in SecretoryVesicles and Secretion

After phosphorylation of caseins in the Golgi apparatus, calcium ions are attached with casein ester phosphate groups in the terminal Golgi to form calcium caseinate phosphate and micelles formation occurs. Two types of secretory vesicles are formed. One type of vesicle is composed of chains of small spherical particles and filamentous structures. The other honeycomb-type vesicle contains densely packed casein micelles. Two types of secretory pathways of proteins are proposed.

Exocytosis: The major milk proteins (Caseins, α-lactalbumin, and β-lactoglobulin) are secreted by exocytosis. The secretory vesicles are channelized to the apical pole guided by microtubules. At the apical pole, secretory vesicles are fused with the apical membrane and emptied their contents in alveolar lumen.

Transcytosis: Immunoglobulins, transferrin, and serum albu­min are secreted by transcytotic pathways. It involves a series of complex sorting events. Transcytosis begins with the uptake of material by the basal membrane of mam­mary epithelial cells and enters into basolateral early endosome (BEE) compartment. The substances within BEE undergo a rapid recycling pathway to a common endosome recycling (CER) compartment. Within the CER there may be further sorting and the vesicles are channelized either apical or back to basolateral membranes for secretion.

25.3.2.5 Fluid and Electrolyte Transport Across the Mammary Gland

Transport of water and small solutes across the epithelial cells is mediated by a family of 28 kDa membrane proteins with high permeability for water called aquaporins (AQP). AQP are activated by arginine-vasopressin via V2-R receptors and translocated to the cellular membrane for water transport. Various studies have confirmed the presence of different AQP in mammary gland of several species as summarized in Table 25.14.

The concentration of different ions between extracellular fluid, mammary epithelial cells, and milk has been depicted in Table 25.15.

Table 25.14 Aquaporins in mammary gland of different species

Table 25.15 The concentrations of different ions between extracellular fluid, mammary epithelial cells, and in guinea pig

Source: Linzell and Peaker (1971)

The electrochemical gradients of these ions keep the inside of the epithelial cell electrically negative compared to milk (-41 vs 3 mV). In the basolateral membrane, Na⁺ and K⁺ gradients are maintained by Na⁺-K⁺-ATPse pump and in the apical membrane Na⁺ and K⁺ are passively distributed.

Na⁺-K⁺-Cl^- cotransport system have also been identified in the apical surface of lactating rat mammary epithelium which confirmed that the uptake of K⁺ by the mammary tissue is dependent on both sodium and chloride.

Na⁺/H⁺ exchange and Na⁺/HCO₃^- cotransport are involved in the regulation of mammary cell pH.

Rat mammary tissue expresses a Na⁺-dependent phos­phate transport system which helps to absorb free inorganic phosphate from blood and utilizes this inorganic phosphate for casein micelle formation.

Mammary secretory cells are having less quantities of calcium (of the endocrine milieu allows the differentiation of mammary secretory epithelium. The synthesis of lactose and fatty acids starts as early as 30 and 15 days prepartum, respectively. Prolactin and glucocorticoids induce the onset of “copious mileticulum.” The appearance of IgG receptors (especially IgG1) in mammary secretory epithelia allows the uptake of IgG from blood immediately after calving. Synthesis of all these constituents cause increased mammary gland volume and colostrum secretion commences. The amount of colostrum secreted by an individual Holstein cow varies from 23.1 ± 2.5 to 36.4 ± 2.1 L (pooled first four milking). Composition of colostrum: The components of bovine colos­trum have been summarized in Table 25.16.

Functions of colostrum: Colostrum is an excellent energy­rich nutritional supplement. The predominant function of the colostrum is to transfer passive immunity in form of antibodies. IgG-1 is the principal immunoglobulin type in colostrum followed by IgM, IgA, and IgG-2. Feeding of colostrum significantly reduces calf mortality. The cellular components (polymorphonuclear leukocytes and macrophages) present in the colostrum produce lysozyme, complement components, and interferon which protects the newborn against enterocolitis. Lactoferrin, an iron- binding protein present in the colostrum is also having antibacterial and antiviral properties. Intake of colostrum affects metabolism, endocrine systems, and the nutritional state of the calves including the development and function of the gastrointestinal tract. The growth facts of the colos­trum favor cell growth and tissue repair. Colostrum is also having a mild laxative effect which allows the passage of the first stool (meconium) along with the excretion of bilirubin.

Table 25.16 Composition of bovine colostrum

Source: Mehta (2015)

25.4.2.1 MilkFat

Fat is one of the important milk constituents ranging from below 3.0% to more than 6.0%. The economic value of milk is determined by its fat content. Fat serves as the source of energy (9 kcal/g) and carrier of fat-soluble vitamins. The desirable flavor of milk products is largely depending on milk fat. The fat in milk is present as milk fat globule (MFG) with a diameter of 0.1 to more than 22 μm. The surface of this spherical globule known as milk fat-globule membrane (MFGM) protects the nonpolar core of fat globule and stabilizes the MFG in an aqueous environment. Fat has a lower density compared to surrounding aqueous phase and hence can be separated by centrifugation.

The milk fat comprises of triacylglycerols (95%), diacylglycerol (2%), cholesterol (bgcolor=white>0.32 18,283 162 0 5 5.2 α-Lactalbumin 0.12 4176 123 0 8 4.3 Serum albumin 0.04 66,267 582 0 35 4.8