Hormonal Control of Mammogenesis and Onset of
Lactation in C o w s - A Review ~
ABSTRACT
Estrogen stimulates development of
mammary ducts, and progesterone and
estrogen stimulate proliferation of secre-
tory tissues. In vivo, sequential addition
of insulin (step 1), glucocorticoid (step
2), and prolactin (step 3) leads to biosyn-
thesis of casein and lactose. In cows,
mammogenesis continues until termina-
tion of pregnancy and overlaps onset of
lactation. Progesterone probably inhibits
differentiation of secretory cells at step 2
or step 3. Sensitivity of individual cells to
progestational inhibition may decrease
variably which may be interdependent
upon relative increases in estrogen, pro-
lactin, corticoids, and growth hormone to
cause asynchronies among them at calv-
ing. Since prolactin in plasma is not
correlated with progesterone or the estro-
gens, factors other than feed-back effects
of ovarian steroids may be responsible for
its sustained increase periparturiently. Al-
so, elevated prolactin periparturiently
may be unrelated to subsequent rates of
lactation because its "basal" concentra-
tions may meet requirements when in-
hibiting effects of progesterone are re-
moved. This concept is attractive because
mammary ceils neither are synchronized
highly for biosynthesis nor secrete normal
milk for several days after calving. At the
latter time, concentrations in plasma are
low for progesterone and estrogen, similar
to 3 days before calving for glucocorti-
coids and prolactin, and increasing for
insulin. Evidence of lactation under un-
usual circumstances was discussed.
Received July 1, 1976.
~Journal Paper No. 6316, Purdue University Agri-
cultural Experiment Station. Text of invitational
paper presented during 71st Annual Meeting, Ameri-
can Dairy Scierice Association.
155
R. E. ERB
Department of Animal Sciences
Purdue University
West Lafayette, IN 47907
INTRODUCTION
Biology of lactation has been studied for
several centuries. The first documented reviews
were in 1765 and 1778 by VonHaller (23).
Subsequent reviews (Rothchild, 1901 to 1902;
Simon, 1968) included over 11,000 references
for 1700 to 1928, as cited by Cowie and Tindal
(23). Since 1928 an extraordinary number of
reviews and books have been published which
include discussions on endocrinology of lacta-
tion. Reviews cited in the text are (4, 19, 21,
23, 26, 27, 29, 47, 64, 65, 68, 80, 82, 101,
102, 103, 109, 110, 113).
The primary purpose of this paper is to
review hormonal control of mammogenesis
(growth and differentiation to stage prior to
active secretion) and onset of lactation (start of
active secretion) in dairy cows. Research on
other species (mainly goats, mice, rabbits, rats,
and sheep) will be cited as necessary to support
hypotheses regarding hormonal effects on lacta-
tion cycles.
HISTORICAL
Artificial development of udder function,
equivalent to that expected following preg-
nancy and parturition, has been attempted with
increasing frequency since 1897. It was demon-
strated initially that reintroduction of ovarian
tissue into ovariectomized animals permitted
continued growth of mammary glands (42, 61).
Hormonal stimulation was postulated in 1906
(63). Later (1909 to 1914), mammary growth
was associated with successive estrous periods
and with development of corpora lutea (eL)
following sterile copulation (23). This led to
conclusion that mammary growth was caused
by CL (corpus luteum) of pregnancy (3, 44).
The pituitary was implicated 14 yr later by
showing, in rabbits, that crude extracts of
pituitary initiated lactation in developed mam-
mary glands (97), and also caused mammary
proliferation in undeveloped glands (78). Then
in castrate male rabbits "estrus producing hor-
156 ERB
mone" (estrogen) in extracts of urine from
pregnant cows stimulated only mammary duct
development. Moreover, mixtures of estrogen
and progesterone stimulated mammary prolifer-
ation equivalent to late pregnancy in female
rabbits, but progesterone alone was without
effect (111, 112).
By 1931, it was understood in a general way
that both mammary proliferation and lactation
were stimulated by ovarian and pituitary hor-
mones (80, 109, 113). It was recognized that
maximal lactation response was associated with
a variety of hormonal changes characteristic of
pregnancy and parturition, which included a)
prolonged secretion of CL hormones, b) in-
creases in estrogen during late pregnancy, and
c) appropriate but undefined pituitary and
placental hormones.
Prior to 1928, substantial development of
mammary glands and variable amounts of secre-
tion were produced by implants of tissue (7) or
injection of extracts of various reproductive
tissues (7, 36, 38, 63). Steinach et al. (96)
apparently were first to report full milk secre-
tion in castrated animals (male and female
guinea pigs) after daily injections of purified
"estrus producing hormone" for 7 to 8 wk.
Evans (35) obtained milk from three virgin
goats, one dry goat, and a heifer by injecting
extracts from the anterior pituitary. However,
the technique was not successful when tried by
others (5, 11). Later, udders of virgin goats
were developed by inunction with estradiol
benzoate, and lactation was initiated by inject-
ing pituitary "lactogenic" hormone (25).
Artificial induction of lactation was at-
tempted with other synthetic estrogens (inunc-
tion of udder, implants, injections, and oral
administration) from about 1939 to 1946.
Results of these experiments have been sum-
marized by Meites (65). Diethylstilbestrol
(DES) was used most extensively, although
other synthetic estrogenic compounds were
investigated, including hexestrol and various
esters of hexestrol and DES. Lactation re-
sponses induced by treatments with synthetic
estrogens were variable and difficuh to repro-
duce. Finally, there was general agreement that
estrogen alone failed to develop normal mam-
mary secretory tissue of cows and goats and
that this defect could be corrected by extended
treatment (30 to 180 days) with estrogen and
progesterone (8, 71,100).
Journal of Dairy Science Vol. 60, No. 2
Continued efforts to initiate lactation by
treatment and nonpregnant heifers or dry cows
with various combinations of estrogen and
progesterone for 7 to 180 days has not resulted
in average milk production equal to amounts
expected after parturition (17, 34, 45, 49, 65,
72, 90, 91, 92, 110, 114, 115, 117, 118, 119).
Because of long treatment times, commercial
applications for hormonal induction of lacta-
tion were considered impractical prior to 1973.
However, a 7-day treatment, used subsequently
by Smith and Schanbacher (91), could have
practical application for salvage of cows non-
pregnant at end of normal lactations because
fertility seems normal after treatment. How-
ever, average milk yields after 7-day treatments
do not appear superior to those resulting from
long treatments. Successful induction rates of
about 70% and lactation yields 65 to 70% as
high as prior years among cows lactating after
treatment (34, 47, 90, 91), seem remarkable for
only 7 days of hormonal treatment (47).
However, the cows ordinarily do not initiate
lactation until 3 to 4 wk after first treatment,
and rate of increase in daily yields are subnor-
mal (33). Since dry periods of 6 to 8 wk are
recommended prior to calving and about 3 wk
are required after last milking before biochemi-
cal stasis can be observed" in the udder (85,
116), some cows may have only 3 to 5 wk to
regenerate secretory tissues before calving. The
preceding analogy suggests that full lactation in
induced cows should not be limited because of
short periods for treatment, and that it may be
possible to discover correct application of
hormones to induce regularly full lactation.
Q U A L I T A T I V E HORMONAL
REQUI REMENTS
Development and differentiation of parent
cells in mammary tissue from mice at mid-preg-
nancy, incubated as organ cultures, proceeded
in a time-dependent, hormone-dependent se-
quence (82, 101, 102 for review). The required
sequence was a) insulin which induced only one
division of parent cells, b) hydrocortisone for
differentiation of the daughter cells, c) prolac-
tin to initiate active synthesis and secretion.
Each sequence required about 2 days for
maximum response (102). If sequential devel-
opment was interrupted for as long as 4 days by
nonaddition of either hydrocortisone or prolac-
tin, daughter cells regressed to the cytological
 REVIEW: HORMONES FOR MAMMOGENESISAND LACTATION
appearance of parent cells. The same sequential
changes occurred when embryonic (12, 13) or
prepubertal (120) mammary tissues were incu-
bated under similar conditions. Cultured mam-
mary explants from nonlactating cows 40 days
prepartum required insulin for survival, hydro-
cortisone for differentiation, and prolactin for
biosynthesis (16). Requirements appeared simi-
lar when mammary explants were from heifers
pretreated with estrogen and progesterone al-
though addition of estrogen and progesterone
or calf plasma further improved indicators of
lactogenesis (76).
Based on in vitro results, parent cells should
advance to active secretion in vivo from a
variety of normal or abnormal conditions. Since
parent cells are at all stages after the embry-
onic mammary primary sprout stage, this con-
cept helps explain what appears to be diverse
observations regarding onset of lactation. Lacta-
tion may occur spontaneously in virgin goats
(37), in suckled virgin heifers (108), in heifers
milked as early as 120 days of first pregnancy
(5, 108) or following abortion (99), and follow-
ing hormone treatments prepartum (105), dur-
ing lactation (72), or during dry periods of
nonpregnant cows (17, 18, 23a, 34, 45, 65, 75,
90, 91).
The udders of heifers develop throughout
pregnancy. However, differentiation, synthesis
capability, and active synthesis, are relatively
incomplete in cattle until about 2 days before
parturition (4, 47, 59, 82) unless altered by
prepartum milking (1). Since mammary tissue is
exposed constantly to the metabolic hormones
and, at least to low concentrations of prolactin
in vivo, changing concentrations of progester-
one and estrogen in blood must be involved
until onset of lactation. This role includes a)
mammary duct development by estrogen, b)
lobule-alveolar development through synergistic
effects of estrogen and progesterone, and c)
nearly complete synchronization of secretory
cells ready for differentiation prior to secretion
(4, 82). It is unknown if roles of estrogen and
progesterone are primary or secondary, but
onset of lactation is associated with their rapid
changes in concentration periparturiently.
Progesterone appears the most probable in-
hibitor of onset of lactation (23, 46, 106).
Until the progesterone block is removed, pro-
lactin may be unable to initiate synthesis of
~-lactalbumin and lactose even though its con-
157
centrations in plasma and mammary secretions
may increase before progesterone decreases to
low concentrations. Whatever the mechanisms
in cows, cytologically (4, 7, 82) and otherwise
(26, 82), the effect is not simultaneous for all
cells perhaps due to differences in circulation to
their sites. Because estrogen continues to in-
crease until parturition, it may stimulate pitui-
tary release of prolactin (65, 66, 67, 83) and
enhance rate of milk secretion or perhaps
synergize mechanisms required for onset of
normal lactation, such as function of corticoids
(103), growth hormone (21), prolactin (21,
84), placental lactogen (9), and other factors in
blood plasma (76). However, estrogen decreases
to low concentrations within 72 h after calving
(19, 31). Therefore, lactation does not ap-
proach maximum rates until after plasma and
urinary estrogen are low. Moreover, basal con-
centrations of prolactin in blood are decreased
within 2 days after calving (55, 56) although
episodic release occurs thereafter from diverse
stimuli including milking (19, 62). Also oxy-
tocin release and regular removal of milk are
essential for increase to maximum rates and for
sustained lactation (82).
Q U A N T I T A T I V E C H A N G E S IN
HORMONES
Progesterone in blood decreases rapidly 2 to
3 days before calving (19, 30, 54, 95) whereas
prolactin (19, 30, 50, 55, 5 6 ) a n d growth
hormone (19, 55) increase 1 to 2 days before
calving and remain elevated for 1 to 2 more
days. Growth hormone decreases and insulin
increases during the 1st mo after calving (93).
Glucocorticoids increase significantly in blood
serum only during calving and decrease to
precalving concentrations within 12 h after
calving (19, 30, 53, 95). The parturient increase
in glucocorticoids may be due to stress of
parturition (20, 53), which suggests that the
increase per se may have little significance
regarding onset of lactation. Estrogens in blood
plasma (15, 28, 81) or serum (19, 95), urine
(54), and mammary secretion (15, 73) increase
rapidly during the last 1 wk of pregnancy.
Concentrations in blood are near maximum the
day before calving and in urine (31, 54) or milk
(31) during the 1st day after calving.
Data are more limited regarding periparturi-
ent changes in the 17/3 and 17& isomers of
Journal of Dairy Science Vol. 60, No. 2
158
estradiol. Although circulating estrone predomi-
nates, it seems clear that both isomers of
estradiol also increase during late gestation (15,
28, 81). Variations of up to 10-fold in amounts
of each estrogen in blood, urine, and mammary
secretions are not uncommon among cows on
the same day before calving. Biological signifi-
cance of such large variations is not understood,
especially as these may influence traits of
parturition, synchronies of biosynthesis and
secretion, and subsequent rates of milk yield.
Hormonal profiles at or near calving support
a hypothesis that progesterone inhibits either
the release or biological activity of pituitary
prolactin. In addition, either low ratios of
progesterone to estrogen or increased estrogen
alone triggers parturition and may enhance
release of prolactin from the pituitary (83, 88).
We have completed a study with 22 cows
and heifers. Blood and mammary secretions
were collected daily from 6.8 + .3 days before
calving to 2 days after calving (15). Concentra-
tions of immunoreactive prolactin, progester-
one, estrone, estradiol-17/3, and residual estradi-
ol (believed to be primarily 17-~) were mea-
sured to evaluate their relationships to onset of
normal lactation. Concentrations of lactose in
mammary secretions were measured to evaluate
synchrony of synthesis among secretory cells
(29, 82,102).
Average concentrations of the hormones in
plasma for periparturient intervals in Table 1
are typical of other data (19, 28, 95, 103).
Concentrations in mammary secretions (re-
ferred to as milk hereafter) mimicked changes
in plasma, but there were differences in ratios
among concentrations of hormones in milk
compared to plasma (31). Prolactin, progester-
one, and estradiol-17/3 were higher (P<.01) in
milk than in blood plasma whereas estrone was
lower (P<.01) in milk, and estradiol-17~ con-
centrations were not different (Table 1) (31).
Shifts in ratios of hormonal concentrations in
plasma and milk from 3 to 6 days prepartum to
day of calving (partum +- .5 day; Table 1)
suggest relative changes in hormonal transport.
Average ratios (milk:plasma) prepartum for
prolactin decreased progressively, whereas those
for progesterone and the estradiols increased.
Estrone ratios were unchanged. None of these
hormones was correlated significantly with per-
cent fat in the prepartum mammary secretions
(r = .15 to --.14).
Journal of Dairy Science Vol. 60, No. 2
ERB
Average changes prepartum in plasma for
progesterone and the estrogens are suggestive of
a temporal relationship with prolactin (Table
1). However, prolactin was not correlated with
progesterone or the estrogens in the same
samples of plasma, either 3 to 6 days before
calving or 2 days before to .5 day after calving.
Moreover, percent lactose in secretions was not
correlated with concentrations of the aforemen-
tioned hormones in plasma. This suggests that
concentrations in plasma of other hormones
[insulin (16, 76, 89), corticoids (16, 76, 89,
95), growth hormones (55), placental lactogen
(9), or other constituents in plasma (76)1 may
be related more directly to morphological
changes prior to onset of normal lactation.
Since differentiation, indicators of biosynthesis,
and rates of biosynthesis appear to occur
step-wise over several days in organ culture
(101, 102), correlations between prolactin and
lactose and other peripheral hormones on the
same day may not reveal the true significance
of the temporal changes.
Peripheral concentrations of hormones in
vivo may be inappropriate to define their
effects on mammary secretory cells or other
target activities for several reasons. Average
concentrations are predicted from samples usu-
ally collected in less than 1 rain at infrequent
intervals of hours to several days. Even if
"basal" concentrations are estimated reason-
ably well, these may be well above concentra-
tion gradients required to initiate or maintain a
specific biological action. Since biological ac-
tivity seems dependent on hormone-receptor
interaction at the cellular level (27), the degree
of receptor saturation should be of greatest
importance. Periparturiently, mammary tissue
may be capable of concentrating certain hor-
mones such as estradiol-17~ and progesterone
(Table 1) either dissolved in lipids or bound to
various high and low affinity binding sites.
For example, preparations from cow mam-
mary tissue contain a heat resistant component
which has low affinity hut large capacity for
binding cortisol (104). However, there is no
evidence that this binding "protein" is specific
for cortisol. The same preparation had high
affinity receptors which competed strongly for
progesterone and the corticoids (6, 104). Prepa-
rations from breast tumor tissue show evidence
of receptors specific for progesterone (52).
High affinity receptors specific for cortisol (41,
 T A B L E 1. Comparison o f h o r m o n e s in blood plasma and m a m m a r y secretions at intervals before, day of, and after parturition. Data s u m m a r i z e d from Chew et al.
(15).

Prepartum Postpartum
Comparison 3 to 6 days 1 to 2 days Partum -+ .5 days 1 to 2 days

Blood plasma (n) 59 41 42 44 :n

Prolactin (ng/ml) 28.8 ± 2.1 a 57.7 ± 7.0 73.5 ± 4.6 25.5 ± 2.5 o
Progesterone (ng/ml) 3.6 .2 2.8 .2 1.3 .1 .7 .02
Estradiol-17/3 (pg/ml) 276 23 423 41 388 40 74 14 o
Estradiol-17~ (pg/ml) 970 106 927 134 944 126 313 41 z
Estrone (pg/ml) 3146 307 3513 345 2095 347 373 89 r~

0
M a m m a r y secretions (n) 46 33 43 44
Prolactin (ng/ml) 148 13 201 26 212 18 83 8
Progesterone (ng/ml) 4.8 .5 5.1 •5 4.0 .4 1.4 .2
Estradiol-17~ (pg/ml) 509 64 509 39 609 74 124 14
Estradiol-17c~ (pg/ml) 630 90 620 86 1175 164 419 57
Estrone (pg/ml) 1719 192 1952 215 1620 210 288 67
Lactose (%) 2.6 .2 3.2 .2 3.2 .1 3.9 .1 Z
Fat (%) 3.3 .4 4.1 .6 5.3 .4 4.5 .4
r~
Total protein (%) 7.7 .4 6.9 .4 7.5 .4 5.6 .2

e- M a m m a r y secretions/plasma
(ratio)
Prolactin 5.1 3.5 2.9 3.2
Progesterone 1.3 1.8 3.1 2.0
Estradiol-17~3 1.8 1.2 1.6 1.7
Estradiol-17a .6 .7 1.2 1.3 0
Z
Estrone .5 .6 .6 .8

< aAverage ± standard error.

o

o
~o xO
160 ERB
87) have been found in udder tissue from
lactating cows (41). Receptors for progesterone
and corticoids may have special significance
because corticoids stimulate proliferation of
rough endoplasmic reticulum while progester-
one is inhibitory (82, 103 for review). There-
fore, if concentrations of progesterone de-
creased or corticoids increased, this should shift
ratios of "occupied" high affinity receptor
sites. Also, mammary tissue may metabolize
certain steroids (89), and metabolism may
occur at different rates for specific hormones.
If, for example, progesterone was metabolized
more efficiently than corticoids, then differ-
ences in rate of degradation, and possibly
disproportionate changes in blood flow among
local areas of the udder due to increasing
congestion, could account for variations among
alveoli in regard to whether or not their
secretory cells were cytologically mature or
juvenile prior to calving (82). A similar mecha-
nism may be operative, even when plasma
progesterone is relatively high, if basal concen-
trations of corticoids also are elevated. This
could explain variable rates of secretion among
cows milked prepartum. Finally, the above
hypothesis could explain why lactation was
initiated with a synthetic corticoid in preg-
nant heifers (105), or why rapid mammary
build-up and lactation seem to occur when
births are induced prematurely with dexa-
methasone (86). The latter synthetic gluco-
corticoid competes with cortisol in preparations
containing the mammary corticoid receptor
(41,104).
Interrelationships with other high affinity
receptors may be equally or more impor-
tant. These include receptors for insulin (77)
and prolactin (39) in plasma membrane of
secretory ceils, and for estrogens in cyto-
plasmic fractions and nuclear pellets (40).
Growth homaone and chorionic somato-
mammotropin appear to compete with pro-
lactin for the same receptor (39), and possi-
bly placental lactogen as well.
Cytosol receptors for estrogen appear specif-
ic and compete equally for estrone, estradiol-
17/3, and estriol (40). The significance of equal
competitive binding among estrogens is not
understood. Although estradiol-17~3 appears to
be the biologically active endogenous estrogen,
its concentrations are relatively low in bovine
plasma (15, 28, 31, 81) and mammary secre-
Journal of Dairy Science Vol. 60, No. 2
tions (15, 31) during late pregnancy as com-
pared to estrone and estradiol-t7a. Moreover,
temporal changes in concentrations of the
major estrogens appear proportional in mam-
mary secretions or plasma periparturiendy (Ta-
ble 1).
Selective uptake from plasma (14, 48) and
metabolism (89) of progesterone by mammary
tissue have been reported. Therefore, uptake,
storage, compartmentation within the ceils,
metabolism, and mechanism of action of estro-
gens in mammary tissue seem worthy of study.
Does the mammary gland differentially concen-
trate the estrogens? Differences in ratios of
their concentrations in mammary secretions
compared to plasma suggest differences in
mammary transport, especially between estrone
and the estradiols (Table 1). Also, to what
extent does mammary tissue convert estrone to
the estradiols? Conversion of estrone to estradi-
o1-17~3 may occur as indicated by a three-fold
average difference in their mammary secre-
tion/plasma ratios (Table 1). Changes in the
ratios for estradiol-17c~ from prepartum to
postpartum (Table 1), indicate either more
efficient removal of estradiol-17a or increased
formation from estrone and estradiol-17/L Such
conversions, if they occur at all in mammary
tissue, should occur throughout the gland,
including secretions stored in alveolar lumina.
This is postulated because enzymes appropriate
for such conversion apparently are in blood and
elsewhere (70 for review). Therefore, such
enzymes may be in mammary secretions. Con-
version of estrone to estradiol-17/3 near sites of
action may have a marked effect on their
relative concentration gradients in fluids in
contact with cell surface membranes. An alter-
native would be selective uptake, in or on the
plasma membrane, by mechanisms which favor
a specific estrogen. Since mammary cell recep-
tors for estrogen are in cytosol and nuclear
isolates, a two-step mechanism of action has
been postulated (40). Therefore, cellular entry
of at least the biologically active estrogen(s)
would be essential. Certain estrogens probably
enter mammary cells at all stages of function,
because of stimulatory effects on mammogene-
sis, and other effects which vary from stimula-
tion of lactation to udder regression as dosages
are increased (65, 72 for review). Moreover,
immunoreactive estrogens are in milk (31, 34,
72, 73).
 REVIEW: HORMONES FOR MAMMOGENESISAND LACTATION
EFFECT OF E X O G E N O U S H O R M O N E S
Mammogenesis and Induction of Lactation
Even though lactations induced exogenously
are nearly always inferior to those induced by
pregnancy, it seems worthwhile to review what
has been learned from experiments with domes-
tic ruminants. Milk production at subnormal
rates can be induced inconsistently in nonpreg-
nant, dry cows, goats (65 for review), and ewes
(79) by chronic treatment with various estro-
gens. Mammary morphology is generally abnor-
mal (65 for review). Brief pretreatment with
estrogen, prolonged treatment (30 to 180 day)
with a mixture of progesterone and estrogen
(ratios of at least 1000:1), and continuation of
estrogen after withdrawal of progesterone in-
duced lactation consistently (65, 115, 118).
Subsequent milk yields were equivalent in some
cows to yields expected after calving (65, 110,
115, 118).
Although no direct comparisons have been
made, it appears that mammogenesis and subse-
quent rates of lactation are more variable
following treatment for 7 days with progester-
one (P) and estradiol-17/3 (E2/3) at a ratio of
2.5:1 (.25 mg and .1 mg/kg/day) than for long
treatments. Limited evidence suggests that
synchrony among secretory cells at onset of
lactation after 7-day treatment is substantially
less than for mammary tissue at calving (18,
23a, 47, 75). Also, failures, or otherwise re-
duced lactation yields, occurred most frequent-
ly among cows in mid to late luteal phase when
7-day treatment was initiated (90). The forego-
ing observations suggest that mammary cells
may be refractory unless there is prior priming
with estrogen as would be the case just prior to
estrus. Amounts of estrogen required for prim-
ing may be low when progesterone is low, as in
ovariectomized cows, or high when progester-
one is high. Circulating estrogen is characteris-
tically low between days 10 and 17 of the
estrous cycle (32, 103). Average total estrogen
and progesterone in plasma at end of 7-day
treatment with E2~3 and P approximate concen-
trations observed during the last 3 to 7 days of
pregnancy (15, 31, 95, 103). However, varia-
tions were large among individuals not only
during treatment but for several weeks after
treatment (33, 34). Moreover, among cows
depletion of plasma hormones occurred at
161
different rates, and onset of lactation and
maximum rates of milk production were vari-
able (34, 58, 117).
Lactational Performance and
Hormone Concentrations
We recently compared concentrations of
progesterone, estrogen, and prolactin in blood
plasma with lactations traits induced in 29 cows
by treatment with E2/3 and P for 7 days (33).
Superior, median, and inferior lactations were
identified by ranking both weight-age adjusted
milk yields for 7 consecutive days at peak
lactation and days for milk yield to increase
from 5 to 10 kg/day. Plasma hormones were
measured on day 0 before first treatment (day
1), and on days 7, 14, 17, 21, 24, 28, and 35.
Cows having superior lactations a) had below
average progesterone and estrogen before treat-
ment (day 0), b) decreased rapidly in progester-
one and estrogen after treatment ended and
remained low thereafter, and c) had increased
prolactin in plasma from day 14 to 35. Al-
though prolactin also was elevated in the
median group, estrogen and progesterone were
higher for days 21 to 35 as compared with the
superior group. In comparison to superior and
median groups, the inferior group had above
average estrogen at end of treatment (day 7)
which decreased more rapidly between days 7
and 14. Also, plasma prolactin was chronically
lower from days 21 to 35. On day 0 plasma
concentrations of progesterone as well as pro-
lactin were significantly correlated (positive)
with their concentrations on certain subsequent
days (14 to 35 for progesterone and days 7, 21,
28, and 35 for prolactin). In contrast, day 0
plasma estrogen was correlated only with its
day-7 concentrations. Concentrations of plasma
estrogen as well as plasma progesterone consis-
tently were correlated (r = .33 to .92) among
subsequent days starting with day 17. Plasma
prolactin was correlated significantly with plas-
ma estrogen in the same sample only on day 21
(r = .52), and not correlated with progesterone
at any time. Failure to observe significant
correlations between plasma prolactin and ovar-
ian steroids, except with estrogen on day 21, is
consistent with similar comparisons in peripart-
urient cows (see preceding section). From our
studies of temporal changes in plasma hor-
mones prior to, during, and after onset of
Journal of Dairy Science Vol. 60, No. 2
162 ERa
lactation induced with E23 and P, several facets
are of interest: a) concentrations of progester-
one pretreatment were correlated positively
with concentrations thereafter (33); b) pro-
longed elevation of progesterone (above .5
ng/ml) after day 17 either inhibited lactation,
and/or delayed onset of lactation (33, 117);
and c) occurrence of significant differences in
hormonal profiles associated with inferior lacta-
tions as compared to superior lactations (33).
The major differences associated with inferior
lactations were high titers of estrogen in plasma
on last day of treatment (day 7) and failure to
maintain above average liters (290 to 900
pg/ml) through days 14 and 17 concurrent with
rapid decreases in progesterone (33, 117). Also,
the group with inferior lactations showed slow
decrease in total estrogen (33) or specifically
total estradio[ (58) and had chronically low
average prolactin (less than 24 ng/ml) after day
17 (33, 58).
Requirements for Prolactin
Consistent increases in plasma prolactin peri-
parturiently (15, 31, 55, 56, 103) or after
termination of treatment with E23 and P for 7
days (33, 34, 72, 117) suggest, but do not
prove, its requirement for onset of lactation in
cows. Depression of plasma prolactin with
ergocryptine appears to have little or no effect
on established lactation (57, 94) and appears to
inhibit onset of lactation and subsequent milk
yield only ~if treatment is started prior to
calving and is continued until after calving (57,
84). Cessation of treatment prior to calving had
less effect even though prolactin in plasma was
reduced temporarily to low concentrations
(84).
Synthetic thyrotropin releasing hormone
(TRH) causes immediate but short term in-
creases in thyroxine, prolactin and growth
hormone of plasma (20). However, response to
TRH as an additional treatment for hormonally
induced lactation in cows (74) and ewes (24,
and unpublished data) does not appear promis-
ing. Lactational responses were similar for
groups injected with saline or TRH 7 to 10 days
(cows; 74) or 10 to 13 days (ewes; unpub-
lished) after last treatment with E23 and P.
When TRH or saline was injected into estrogen-
ized ewes 14 to 17 days after last E23 and P,
TRH caused faster increase in percent lactose
Journal of Dairy Science Vol. 60, No. 2
(24). However, effects on milk yield were
inconclusive. Inability to demonstrate superior
initiation of hormonally induced lactations by
treatment with TRH may be due to several
factors. Endogenous prolactin (24, 33, 34, 88,
117) and growth hormone (24) were increased
before TRH was administered. Therefore, fur-
ther increases may have had no effect on the
average individual. Temporal increases in pro-
lactin and growth hormone could have been
ineffective because a majority of secretory cells
were not differentiated at time of treatment or
because concentrations were elevated only
briefly. Finally, timing of treatments with TRH
may have been incorrect.
Various attempts to evaluate the relative
importance of prolactin for initiation and main-
tenance of lactation in intact ruminants have
been inconclusive. However, its requirement for
initiation of lactation has not been questioned
(19, 65 for review). Milk yield of a goat
decreased rapidly after hypophysectomy (21,
22). Synthetic glucocorticoid was administered
after hypophysectomy. Additional treatment
with growth hormone and triiodothyronine
iT3) begun about 70 days after hypophysecto-
my resulted in about 30% restoration of milk
yield. When ovine prolactin also was included,
about 25 days later, lactation was restored
completely. After restoration, lactation was
maintained without prolactin. Then hormonal
requirements for maintenance of lactation in a
hypophysectomized goat were synthetic gluco-
corticoid, growth hormone, T3, and presum-
ably, endogenous insulin and glucagon. It is
intriguing that secretory structures apparently
remained intact for over 3 mo of minimal
secretory activity before restoration. It seems
unlikely that regression occurred and new
secretory structures regenerated in the absence
of estrogen and progesterone. Continued mam-
mary stimulation and removal of secretion may
explain partially the apparent maintenance of
the goat's secretory tissues (21, 82).
A CONCEPT OF HORMONAL
CONTROL-RUMINANTS
There has been general agreement for many
years that lactation cycles are controlled by
estrogen and progesterone (4, 98, 103, 109,
111). The changes in each are synchronized for
mammary development, completion of preg-
 REVIEW: HORMONES FOR MAMMOGENESISAND LACTATION
nancy, and lactation. In heifers, allometric
growth of the udder starts prior to puberty and
plateaus between 9 and 12 mo of age (98).
Estrogen alone stimulates duct proliferation,
but normal lobule-alveolar development re-
quires synergistic effects of estrogen and pro-
gesterone (4 for review). Moreover, since alveoli
are absent prior to first pregnancy (82 for
review), except under anomalous circum-
stances, it is assumed that concentrations of
estrogen and/or progesterone are either too low
or elevated too briefly during estrous cycles to
synergize lobule-alveolar development. Develop-
ment of mammary glands, capable of milk
production consistent with genotype of the
individual, requires sustained high concentra-
tions of estrogen and progesterone followed by
decreases in both periparturiently. Apparently,
maximum time required for regression and
regeneration of mammary secretory ceils does
not exceed 6 to 7 wk in dairy cows because
subsequent lactation yields are lowered only if
dry periods are less than 6 wk (60). Actual time
for complete regeneration during late pregnan-
cy of dairy cows may be 3 wk or less unless
regression and regeneration occur simultaneous-
ly.
Progesterone is the primary inhibitor of
parturition, and it seems appropriate to assign it
a similar role in onset of lactation. Based on
recent studies (1, 16, 76, 89, 101, 102), a
concept should account for sequential develop-
ment of secretory cells in vitro (step 1 - a t least
one division of parent cells requiring insulin,
step 2-organelle formation in daughter cells
requiring cortisol, and step 3-secretory capa-
bility requiring prolactin). Why should it not
postulate that progesterone inhibits step 2 via
the corticoid and progesterone receptors? Then
it is relatively easy to visualize asynchronies
among cells regarding their stage of differentia-
tion during late pregnancy (82). Incomplete
inhibition affecting rate of differentiation could
occur in certain treatments and certainly should
exist during terminal stages of pregnancy when
concentrations of progesterone are decreasing
at different rates among cows. Also, degree of
inhibition of step 2 by progesterone could be
dependent on concentrations of glucocorticoids
and need not have a direct effect on release or
action of prolactin. Even large amounts of
prolactin may be ineffective until step 2 is
completed which could explain absence of
163
negative correlations periparturiently between
progesterone and prolactin in blood plasma. It
seems necessary at least in cows to consider
that lactation by all cells is not normal until
their secretions are characteristic of normal
milk. The latter does not occur for several days
postpartum even when cows are milked pre-
partum (unpublished). Therefore, the prior
variable indicators of onset of lactation, such as
cytological and biochemical indicators, and
accumulation of secretions, only represent
stages associated with maturity of secretory
function.
Ratios of progesterone to estrogen in plasma
decrease rapidly for 1 to 3 days before parturi-
tion (103) (Table 1) and may be more impor-
tant in triggering traits of parturition and onset
of lactation than their absolute concentrations
at any specific time. Although concentrations
in plasma of pituitary prolactin, growth hor-
mone, corticoids, insulin, and oxytocin increase
periparturiently, such changes may be mediated
by anxiety and discomfort prior to initiation of
parturition and to stress during parturition (19,
53). Our failure to find significant correlations
periparturiently between concentrations of pi-
tuitary prolactin and ovarian steroids in plasma
and percent lactose in mammary secretions
during the same day suggests other mechanisms.
However, the hormonal comparisons with per-
cent lactose may not be realistic due to
asynchronies among secretory cells prior to the
end of parturition (82). In this case, any effects
on percent lactose in mammary secretions may
not occur for several days (101, 102).
Even though perip/arturient changes in other
circulating hormones may be inhibited partially
by progesterone or ;stimulated partially by one
or more of the estrogens, their average increase
may not be reflected by averaging gross mea-
surements of rates of synthesis and secretion. In
other words, the possibility remains that "ba-
sal" concentrations of other hormones meet
requirements for onset of lactation once the
postulated block of step 2 by progesterone is
removed. Under the latter concept the effect of
estrogen on mammary secretory ceils could be
simplified. Why should it not postulate positive
effects similar to its action on myometrial
tissues when not inhibited by progesterone?
Then direct effects of estrogen on mammary
tissues would be a) increased permeability of
cellular membranes, b) accumulation of fluid in
Journal of Dairy Science Vol. 60, No. 2
164
extracellular spaces (congestion and edema), c)
increased vascularity and, presumably, blood
flow rate, and d) stimulation of RNA-directed
synthesis of cellular proteins (107). Copious
synthesis and secretion of casein, ~-lactalbumin,
and lactose cannot be explained well if assigned
directly to effects of estrogen. This aspect may
be assigned more properly to catalyzing effects
of insulin, corticoids, prolactin, growth hor-
mone, and oxytocin, along with the mandatory
requirement that secretions be removed regular-
ly postpartum. In fact, the conceptual role
assigned to estrogen may not be necessary for
normal or near normal lactation once the
progesterone block of step 2 is removed or is
otherwise overcome by endogenous or exoge-
nous glucocorticoids via appropriate receptors.
At least two natural phenomena in cows can be
cited as evidence. Cows with prolonged gesta-
tion characteristically have elevated progester-
one and low estrogen (10, 51). Moreover, the
udder may remain completely collapsed until
the fetus is removed and then the udder fills
with secretions in a few days. Lactation is then
comparable to those initiated by normal calving
(10). Udders of heifers and cows with regressed
udders show no evidence of filling prior to
abortion if this occurs 2 to 3 mo before end of
normal gestation. Lactation can be established
by milking, but total lactation yields are de-
pressed and are proportional to stage of gesta-
tion when abortion occurs (99). Such cows
increase slowly to maximum daily rates (99), as
do those induced to lactate by 7-day treatment
with E2/3 and P (33, 91). Rate of excretion of
estrogen in urine of cows increased at time of
abortion induced by ovariectomy (69), and in
two cases of spontaneous abortion (unpub-
lished).
Several anomalous factors require rationali-
zation even though we cannot accept that
ensuing lactations are equivalent to that follow-
ing birth. These anomalies include spontaneous
lactation in virgin goats (37) and lactation after
udder stimulation (manual or suckled) of non-
pregnant (5, 108) or pregnant heifers (105), or
a nonpregnant, dry ewe (88). It seems necessary
to recognize the probable hormonal conse-
quences of mammary stimulation before onset
of lactation by calving. Presumably, udder
stimulation would increase vascularity as well as
cause additional release of oxytocin, prolactin,
and ACTH (19 for review). Under these condi-
Journal of Dairy Science Vol. 60, No. 2
ERB
tions secretory cell proliferation may occur.
Such cells should differentiate either because of
chronically low progesterone before puberty or
when a corpus luteum regressed. Substantial
yields of milk by heifers manually stimulated
during pregnancy or injected with synthetic
glucocorticoid (105), emphasizes potential for
stimulating secretory structures prior to first
calving. The effect of exogenous glucocorticoid
presumably would override inhibitory effects of
progesterone and probably also inhibit luteal
secretion rates as well since treated heifers
calved 25 days early. It is also possible that
manual stimulation and milking reduced luteal
function since untreated controls calved 12
days before expected due dates (105).
Prepartum milking also can be considered
anomalous. Low yields of secretion by some
individuals milked only during terminal stages
of pregnancy could be due to above-average
concentrations of estrogen and progesterone.
This rationale is based on observations that high
doses of estrogen (43) or estrogen and proges-
terone (72) inhibit established lactation where-
as small doses are stimulatory (65 for review).
Since mammary secretions prepartum con-
tain elevated concentrations of prolactin, pro-
gesterone, estrogens (Table 1), and probably
other hormones (corticoids, growth hormone,
etc.), their removal could alter the hormonal
environment of secretory cells. This may affect
only timing of events associated with onset of
lactation, but data available currently are inade-
quate to evaluate biological significance.
If roles assigned to progesterone and estro-
gen regarding onset of lactation should be
proven correct in a general way, then it is
doubtful that their variations periparturiendy
would be predictive of subsequent intensity of
lactation or clearly associated with periparturi-
ent variations in other hormones essential for
function of the mammary gland. A more
promising approach may be to understand the
mechanisms causing proliferation of alveoli and
continued replication of their secretory cells
during pregnancy. This includes more research
on mammary receptors and, especially, on
factors controlling entry of precursors and
steroids into mammary cells in vivo. It seems
essential to recognize that regulation of a cell's
activity is dependent upon characteristics of its
plasma membrane. Synergistic or inhibitory
effects of steroids on intracellular functions
 REVIEW: HORMONES FOR MAMMOGENES1S AND LACTATION
seem under control of receptors and other
mechanisms in plasma membranes which regu-
late uptake by cells. Therefore, rate and type of
intracellular activity in vivo should be depen-
dent upon transport mechanisms, as well as
upon temporal concentration gradients and
interactions of essential hormones in intercellu-
lar spaces and in or upon the plasma membrane.
Regardless of what the in vivo mechanisms of
c o n t r o l m a y be, greater mass o f s e c r e t o r y tissue
o u g h t to be associated w i t h i n c r e a s e d p o t e n t i a l
for milk yield. S u b n o r m a l l a c t a t i o n curves, a n d
yields a f t e r a b o r t i o n a n d t r e a t m e n t o f n o n p r e g -
n a n t , dry cows w i t h E2/3 a n d P to i n d u c e
l a c t a t i o n s u p p o r t t h e latter. Cows n o r m a l l y do
n o t a p p e a r to c o n t i n u e s e c r e t o r y cell prolifera-
t i o n a f t e r calving (4 for review). Moreover, it is
u n k n o w n if s e c r e t o r y cells are r e p l a c e d d u r i n g
l a c t a t i o n or if t h e y c o n t i n u e to increase for a
longer p e r i o d f o l l o w i n g a b o r t i o n , h o r m o n a l l y
i n d u c e d l a c t a t i o n , or o t h e r a n o m a l o u s c i r c u m -
stances t h a t f o l l o w calving. W h a t e v e r t h e case in
the n a t u r a l state, a t t e m p t s to s t i m u l a t e existing
l a c t a t i n g cells or to i n d u c e n e w s e c r e t o r y cells
d u r i n g l a c t a t i o n s h o u l d be c o n t i n u e d . Finally,
we s h o u l d r e m a i n o p t i m i s t i c t h a t practical
t e c h n i q u e s can be d e v e l o p e d to i n d u c e reliably
full l a c t a t i o n w i t h h o r m o n e s to salvage cows
t e m p o r a r i l y infertile.
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