Mammary development and function during lactation 16

In   the   sheep,   there   is   very   little   mammary   gland   growth   post-­‐partum.   Anderson,   (1975)   reported  that  in  sheep,  based  on  DNA  content,  98%  of  mammary  gland  growth  from  the  beginning   of  pregnancy  occurs  during  pregnancy  while  only  2%  occurs  in  early-­‐lactation.  

1.4.7.1 Lactogenesis  

Lactogenesis  is  the  term  given  to  the  process  of  functional  differentiation  of  MECs  in  order   to   produce   milk   during   lactation   (Akers   &   Capuco,   2002).   During   lactogenesis   biochemical   and   structural   changes   occur   in   MECs   to   prepare   them   for   the   onset   of   milk   synthesis   and   secretion.   Lactogenesis  is  broken  into  two  stages;  lactogenesis  stage  1  and  lactogenesis  stage  2.  Stage  1  occurs   in   late   pregnancy   prior   to   parturition,   and   is   characterised   by   an   increase   in   synthesis   of   milk   proteins   and   cytological   changes   (e.g.,   increase   of   cell   cytoplasm   in   relation   to   the   nucleus   and   increased  presence  of  milk  fat  globules  and  vacuoles,  Akers  &  Capuco,  2002).  Lactogenesis  stage  2,   which  initiates  secretion  of  milk,  is  triggered  by  hormones  at  parturition  and  by  external  stimuli  such   as  a  suckling  lamb  (Akers  &  Capuco,  2002;  Hovey  et  al.,  2002).  Lactogenesis  stage  2  is  characterised   by   structural   changes   including   increased   cell   size,   increased   size   and   development   of   organelles   such  as  the  mitochondria,  rough  endoplasmic  reticulum  (RER)  and  Golgi  apparatus,  and  polarisation   of   cells   (Akers   &   Capuco,   2002).   In   fully   differentiated   cells   the   baso-­‐lateral   region   is   the   site   of   uptake  of  metabolic  precursors  and  protein  and  lipid  synthesis.  The  basal  membrane  has  receptors   for   transporters   to   facilitate   the   intake   of   nutrients   into   cells   for   milk   biosynthesis.   The   apical   membrane  is  populated  with  Golgi  apparatus  and  is  the  site  at  which  post-­‐translation  modification   of   proteins,   protein   packaging   and   lactose   synthesis   occurs   (Stelwagen,   2011a   &   b).   Vesicles   containing  protein  and  lactose  bind  to  the  apical  membrane  and  are  subsequently  exocytosed  into   the  alveolar  lumen.  Lactogenesis  stage  2  also  promotes  increased  metabolic  activity,  closure  of  tight   junctions   and   increased   translation   of   milk   proteins   to   support   milk   production   (Nickerson   and   Akers,  1984).    

1.4.7.2 Milk  protein  synthesis  

Milk   protein   provides   an   essential   source   of   amino   acids,   antibodies   and   minerals   to   the   neonate   and   is   a   commercially   valuable   component   of   the   milk   (Ng-­‐Kwai-­‐Hang,   2011;   Stelwagen,   2011a).  Proteins  are  synthesised  in  MECs  from  amino  acids  which  can  be  transported  from  the  blood   or  synthesised  de  novo  (Bionaz  &  Loor,  2011).  The  casein  and  whey  proteins  make  up  the  majority  of   milk  proteins  In  addition  IgA,  lactoferrin,  transferrin  and  serum  albumin  are  also  found  in  milk  (Ng-­‐ Kwai-­‐Hang,  2011;  Stelwagen,  2011a).  Casein  proteins  are  phosphoproteins,  of  which  there  are  four   different   types:   αs1-­‐casein,   αs2-­‐casein,   β-­‐casein   and   κ-­‐casein.   Whey   proteins   synthesised   in   the  

sheep   mammary   gland   include   beta-­‐lactoglobulin   and   alpha-­‐lactalbumin   (Ng-­‐Kwai-­‐Hang,   2011;   Ramos  &  Juarez,  2011).  Alpha-­‐lactalbumin  is  important  for  lactose  synthesis,  as  part  of  the  lactose   synthase   enzyme   complex,   and   for   milk   secretion   (Brew,   2011).   The   biological   function   of   beta-­‐ lactoglobulin  is  unknown,  although  it  has  been  suggested  to  be  some  sort  of  transporter  or  merely  a   convenient   nutritional   protein   (Creamer   et   al.,   2011).   There   are   two   main   variants   of   beta-­‐ lactoglobulin  (A  and  B).  

Synthesis   of   milk   proteins   can   be   regulated   at   the   transcriptional   level   and   also   at   the   translational  and  post-­‐translational  levels  (Chevalier,  2011;  Stelwagen,  2011a).  Lactogenic  hormones   regulate   transcription   of   milk   proteins.   On   a   molecular   level,   this   may   be   mediated   by   changes   in   chromatin   conformation,   nuclear   receptors,   transcription   factors   or   transcriptional   repressors,   facilitating   or   inhibiting   the   transcription   of   genes   (Rijnkels  et   al.,   2010;   Singh  et   al.,   2010a).   Milk   protein  gene  expression  is  typically  used  as  a  marker  of  differentiated  MECs.  Expression  of  the  casein   genes  and  whey  protein  genes  increases  from  late  pregnancy,  dramatically  increasing  at  parturition   (Naylor  et   al.,   2005).   Prolactin   (Prl),   growth   hormone   (GH)   and   glucocorticoids   are   known   to   promote  expression  of  milk  protein  genes.  Both  Prl  and  GH  can  activate  the  Janus-­‐kinase  and  signal   transducer   and   activator   of   transcription   (JAK-­‐STAT)   signalling   cascade.   Mitogen-­‐activated   protein   kinase   (MAPK)   signalling   and   the   mammalian   target   of   rapomycin   (mTOR)   are   also   thought   to   be   involved  in  transcription  of  milk  protein  genes  (Naylor  et  al.,  2005;  Bionaz  &  Loor,  2011).  Following   translation,  milk  proteins  are  folded  and  post-­‐translational  modifications  occur  in  the  endoplasmic   reticulum,  after  which  they  are  packaged  for  transport  out  of  the  cell  in  the  Golgi  apparatus  (Mather,   2011;  Stelwagen,  2011a).    

1.4.7.3 Lactose  synthesis  

Lactose  is  the  main  carbohydrate  and  osmotic  component  of  milk.  Lactose  is  a  disaccharide   synthesised  from  glucose  and  galactose  (which  is  derived  from  glucose)  within  the  Golgi  apparatus   (Stelwagen,  2011b).  Glucose  is  the  sole  precursor  required  for  synthesis  of  lactose.  The  mammary   gland   is   unable   to   generate   glucose   due   to   the   lack   of   glucose-­‐6-­‐phosphatase;   therefore,   glucose   must   be   supplied   to   the   mammary   gland   from   the   circulation   (Finucane  et   al.,   2008;   Stelwagen,   2011b).   Synthesis   of   lactose   is   catalysed   by   the   lactose   synthase   enzyme   complex,   which   is   comprised   of   beta-­‐1,4-­‐galactosyl-­‐transferase   and   the   whey   protein   alpha-­‐lactalbumin   (Stelwagen,   2011b).  Synthesised  lactose  is  then  packaged  into  secretory  vesicles,  along  with  milk  proteins,  which   are  pinched  off  from  the  Golgi.  These  secretory  vesicles,  assisted  by  the  microtubule  network,  move   through  the  cytosol  to  the  apical  membrane  where  they  fuse  and  release  their  contents  into  the  milk   in  the  alveolar  lumen  (Mather,  2011).  The  osmotic  gradient  between  the  vesicles  and  cytosol,  due  

largely   to   the   lactose   content,   pulls   water   into   them   increasing   the   volume   of   milk   (Stelwagen,   2011b).  

1.4.7.4 Lipid  synthesis  

Milk  fat  provides  an  essential  source  of  energy  for  the  neonate.  The  sheep  has  an  average  of   15%   lipid   content   in   their   milk,   which   is   higher   than   the   cow   which   has   ~10%   (Ramos   &   Juarez,   2011).   Milk   fat   is   predominantly   triacylglycerols   (TAG),   but   also   includes   a   small   amount   of   diacylglycerides,   monoacylglycerides,   cholesterol,   phospholipids   (includes   sphingolipids)   and   free   fatty   acids   (Bauman  et   al.,   2011).   Synthesis   of   TAGs   occurs   in   the   smooth   endoplasmic   reticulum   (SER)   from   glycerol   and   fatty   acids.   In   ruminants,   milk   fat   TAGs   are   largely   from   short   to   medium   chain  (4  to  18  carbon  length)  fatty  acids.  These  short  to  medium  chain  fatty  acids  are  synthesised  de   novo  while  longer  chain  fatty  acids  are  supplied  from  circulation  (Bionaz  &  Loor,  2008;  Harvatine  et   al.,  2009;  Bauman  et  al.,  2011).  De  novo  synthesis  of  fatty  acids  in  the  mammary  gland  increases  at   parturition   and   increases   to   peak   milk   yield,   then   declines   as   milk   yield   declines.   Expression   of   lipogenic   genes   also   increases   at   parturition   under   the   regulation   of   endocrine   hormones,   in   particular  decreased  progesterone  and  increased  prolactin  (Naylor  et  al.,  2005;  Rudolph  et  al.,  2007;   Bionaz  &  Loor,  2008;  Finucane  et  al.,  2008;  Bionaz  et  al.,  2012).  In  non-­‐ruminant  species,  glucose  is   used   as   a   precursor   for   fatty   acid   synthesis.   In   ruminants,   however,  a   glucose   sparing   mechanism   (Randle’s   effect,   Randle,   1998)   enables   the   use   of   acetate,   and   beta-­‐hydroxybutyrate   (BHB)   to   a   small  extent,  as  a  precursor  for  fatty  acid  synthesis  (Bauman  et  al.,  2011).    

1.4.7.5 Galactopoiesis  

The  increase  in  milk  production  at  the  onset  of  lactation  is  largely  due  to  increased  numbers   of   secretory   cells   as   indicated   by   increased   mammary   DNA,   while   an   increase   in   the   metabolic   activity  of  these  cells  then  becomes  an  important  factor  for  milk  yield  until  peak  lactation  (Knight  &   Peaker,  1982;  Knight,  2000;  Akers  &  Capuco,  2002;  Capuco  &  Ellis,  2013).  The  balance  between  cell   proliferation  and  apoptosis,  while  at  low  levels  during  lactation,  is  an  important  factor  of  maintaining   the  secretory  cell  population.  As  lactation  progresses  this  balance  favours  apoptosis,  demonstrating   that  apoptotic  cell  death  may  be  responsible  for  the  decline  in  lactation  (Knight  &  Peaker,  1982).    

Galactopoiesis  refers  to  the  maintenance  of  established  lactation.  Increased  galactopoiesis   means   increased   lactation   persistency   which   is   of   benefit   in   animal   production   systems.   Lactation   persistency  depends  upon  maintenance  of  the  population  of  secretory  cells,  therefore,  factors  which   promote  cell  proliferation  and  survival  and  inhibit  apoptosis  may  improve  milk  production  in  animals   (Figure  1.3,  Capuco  &  Ellis,  2013).  Factors  such  as  hormones,  systemic  and  locally  produced  growth   factors  and  signalling  molecules,  changes  in  milking  frequency,  antioxidants  or  exposure  to  reactive  

oxygen  species/oxidative  stress,  disease  such  as  mastitis,  blood  flow  and  nutrient  partitioning  and   other  stressors  may  affect  lactation  persistency  (Capuco  &  Akers,  2011;  Collier  et  al.,  2011;  Wall  &   McFadden,  2012).    

Figure   1.3   Factors   contributing   to   changes   in   milk   yield   during   a   bovine   lactation.   Prior   to   peak   lactation,   milk   yield   increases   owing   to   increased   secretory   activity   per   cell.   After   peak   lactation,   milk   yield   declines   primarily   because   of   a   decline  in  epithelial  cell  number  owing  to  apoptotic  cell  death.  Factors  that  decrease  loss  of  cells  decrease  the  decline  in   milk   yield   (i.e.,   increase   persistency).   Factors   shown   above   the   lactation   curve   increase   milk   yield   and   persistency,   and   those  below  decrease  milk  yield  and  persistency  (Capuco  &  Ellis,  2013).