PROGESTERONE IN BOVINE REPRODUCTION: A REVIEW 1

W . R. G O M E S AND R. E. ERB
Department of Animal Sciences, Purdue University, Lafayette, Indiana

ABSTRACT
Portions of an extensive literature were reviewed to evaluate the status of
our knowledge relative to progesterone in the bovine. Studies related to the
secretion and metabolism of progesterone have been hampered by the lack of
chemical methods sufficiently sensitive and specific to quantitate the hormone in
snmll samples. Certain bioassays are extremely sensitive but lack specificity and
have undefined limits of error. Spectrophotometric determination of progester-
one or progesterone derivatives appears specific but is sensitive to only 0.1-0.5
t~g. Fluorometric, gas chromatographic, and double-isotope derivative methods
promise sensitivity to 0.01 /zg with adequate specificity and precision.
Progesterone and 20 fl-hydroxy-A~-pregnene-3-one a p p e a r to be the prin-
cipal progestins in the corpus luteum (CL) of cycling and pregnant cows.
Progestins have also been reported in bovine ovaries, adrenals, and blood. The
CL appears to be the most important source of progesterone throughout bovine
pregnancy. The CL from a previous cycle contains measurable progesterone for
two or three days after estrus. Progestin in the CL and ovarian vein blood
declines rapidly during proestrus from maximum levels observed 14-16 days
post-estrus. I n the pregnant cow, CL progestin levels decline about 40% from
mid-cycle levels by 42-56 days of pregnancy, further decline slowly until 200
days, then rise again to the 28-day level; after 250 days, a decline to parturition
begins. Similar changes are seen in blood levels of the hormones during preg-
nancy. Progesterone appears important in pregnancy nmintenance, expression
of estrus, nornml cyclic function, and in hypophyseal-gonad interrelationships.

Thirty years have elapsed since the first iso-
lation of progester(me as a crystalline com-
pound. Since that time, most attempts to use
the hormone therapeutically to correct faulty
reproductio~ ia the bovine have generally
proven unsuccessful. Basic mechanisms in-
volved in the different phases of reproduction
are only generally understood, and many de-
tails are ahnost completely unknown. Specific
sites or qualitative and quantitative aspects of
progesterone action and the precise role of the
hormone in the hypothalamo-hypophyseal-ovar-
Jan axis (5, 60, 64, 127) remain obscure.
F o r many years progesterone (A4-pregnene-
3,20-dione) was thought to be the only naturally
occurring progestational agent. I n more recent
years, however, several other compounds have
been isolated. The most important of these,
quantitatively, appear to be 20 a-hydroxy-A~-
pregnene-3-one (20 a-ot), 20 fl-hydroxy-A4-
pregnene-3-oue (20 fl-ol) and 17 a-hydroxy-A~-
pregnene-3, 20-dione (17-hydroxyprogesterone).
I n 1936, the A.I~I.A. Council on Pharmacy and
Chemistry (3) adopted the term progestin to
indicate progestational agents without regard
to the state of chemical purity. Progestin will
~Journal Paper No. 2442, Purdue ~Jniversity
Agricultural Experiment Station.
314
be used in this p a p e r to refer to this group of
hormones in biological media or in pure fornL
The purpose of this paper is to review the
methodology in progestin research and the
levels and importance of progestin in the bo-
vine, using comparative data where they appear
applicable. No hormonal action is completely
independent of the effects of other hormones,
and progesterone is no exception; nonetheless,
the effects of progestin on reproduction will
be isolated here, insofar as is possible. F o r a
more complete presentation of hormonal inter-
relationships, the reader is referred elsewhere
(5, 10, 38, 40, 41, 114, 140, 157).
BIOLOGICAL A C T I O N S OF P R O G E S T E R O N E
The physiological and pharmacological ac-
tions of progestins have been recently listed
(98); therefore, no attempt will be made here
to present an all-inclusive discussion of the
properties. A general listing of these actions,
however, should be valuable for a better under-
standing of the complexity of the hormonal
aspects of reproduction.
F i r s t of all, progestins are essential for the
uterine development necessary for implantation~
blastocyst development, and maintenance of
the fetus and of uterine tone during pregnancy.
 PROGESTERONE IN BOVINE
Moreover, progestin may be important as a
conditioning agent for normal bovine estrus
(92) and may have an effect on tubal and
uterine transport of sperm and ova (98). These
hormones may inhibit fertilization at certain
dose levels in vivo (98) and in vitro (27). Pro-
found effects are also noted on vaginal epi-
thelium (108, 157), ovarian function (31, 49,
106, 142), gonadotrophin elaboration (45, 84),
and oxytocin (50) and relaxin (98, 157) ac-
tions. Pharmacological doses may antagonize
the actions of estrogens or corticoids (32, 98,
157).
Many of the above actions are mimicked to
one degree or another by progestins other than
progesterone, but no compound has yet been
found which duplicates exactly :all of the effects
of the parent hormone, progesterone. The bio-
logical significance of the 20-hydroxy com-
pounds, for example, is not understood; they
may well be naturally occurring metabolites of
progesterone. Both isomers have twice the ac-
tivity of progesterone in the Hooker-Forbes
intrauterine assay in mice (155). I n the Clau-
berg rabbit bioassay, 20 fl-ol has 10-20% of
the activity of progesterone (80, 155), and 20
a-ol is 30-50% as active (155). A f t e r studying
its effects on the endometrium, delay of men-
struation, production of withdrawal bleeding,
cessation of dysfunctional bleeding, cervical
mucus, vaginal smear, and several other factors
in the human, Lauritzen (80) reported that
20 fl-ol is a true progestational hormone with
nearly half the activity of progesterone.
Conversely, 20 fl-ol did not support deciduo-
mata formation in the ovariectomized r a t when
3-mg injections were used (0.5 mg progesterone
is effective) (147). I n addition, both 20-hy-
droxylated compounds failed to maintain preg-
nancy in the ovariectomized mouse, even with
large doses (146).
I t seems likely that the biological effectiveness
of progestins other than progesterone will re-
nlain obscure until considerably more is known
about the mechanisnl of action of these hor-
mones (25, 53, 79, 106, 141). I t is disappoint-
ing to note that few results have been forth-
coming in this area, although rapid advances
are being made toward the elucidation of the
p r i m a r y actions of other steroid classes (59,
74, 104, 141). I t is probable that the advent of
better methodology will facilitate such studies
with the progestins.
ASSAY METHODS FOR PROGESTERONE
Studies related to the secretion and metabo-
lism of progesterone have been hampered to
date by the lack of accurate chemical tech-
REPRODUCTION 315
niques sufficiently sensitive and specific to facil-
itate the quantitation of the hormone in small
plasma samples. Until quite recently it was
necessary to collect reproductive tissues at
surgery (55, 89) or slaughter (42, 158-161)
for the estimation of progesterone. Present
methods require the use of large volumes of
blood (118).
Certain biological assays are extremely sensi-
tive, but lack specificity and have undefined
limits of error (36, 98).
B I O A S S A ¥ OF P R O G E S T I l q S
Several excellent reviews of progestin bio-
assay methods are available (98, 108, 157). I n
general, the assays based on end points such as
complete progestational proliferation, uterine
granulosa/nlucosa areas, carbonic anhydrase ac-
tivity, deciduonlata formation, pregnancy main-
tenance, or parturition delay [for specific as-
says and references, see (98, 157)] lack sensi-
tivity and are often difficult and time-consum-
ing. The local progestational tests by McGinty
and Hooker and Forbes (see 108, 157) are the
most sensitive progesterone assays known, but
these tests utilize a subjective end point and
lack specificity. Furthermore, these methods
have consistently indicated progesterone levels
in biological fluids that cannot be verified by
chemical assay (36). I n fairness to the bio-
assay, it should be pointed out that animal re-
sponses are essential for the assessment of ac-
tivity of crude extracts and of individual
compounds and are valuable in the isolation
of unknown substances. The bioassay has
played a vital role in progestin research to date
and will be equally important in mechanism of
action studies and in the evaluation of the
newer synthetic progestins (45, 61, 62).
CHEMICAL ~IETI~ODS
Chemical methodology applicable to prog-
esterone research has been reviewed in recent
years (101, 114, 154). However, an abundance
of new methods has appeared which may be of
special significance, depending on the objectives
of various research programs dealing with large
animal production.
Extraction and purification. Extraction of
steroids from tissues and fluids has generally
been accomplished by the use of organic s01-
vents or mixtures of solvents (54, 154). Zander
(154) has shown that pretreatment of plasma
samples with sodium hydroxide as proposed by
Short (118) improves the precision of the final
progesterone quantitation. I n addition, NaOH
decreases the formation of solvent-plasma emul-
sions during ether extraction.
316 w.R. G O M E S AND R. E. E R B
One of the difficulties involved in solvent ex-
traction is the great quantity of lipid extracted.
Several methods have been utilized to remove
this f a t t y material, including centrifugation or
filtration in the cold (57, 134) or solvent par-
titioning (39, 118). A further suggestion has
been the use of an acetylation step following
extraction. I n this procedure (16), the crude
extract is subjected to acetylation following
preliminary chromatography. Since progester-
one cannot be acetylated, the other impurities
may be converted to derivatives which will not
remain with progesterone during subsequent
chromatography.
Zander (154) has reviewed the use of Girard's
Reagent T for the separation of ketones from
crude extracts, but points out that this proce-
dure has not been applied to routine micro-
analytical assays for progesterone.
To obtain a more nearly pure extract from
biological samples, Zaffaroni et al. (153) ap-
plied dialysis for the extraction of steroids
from blood, but recovery of added progesterone
was too low for this method to be valuable.
DeVenuto et al. (28) modified the dialysis pro-
cedure by extracting continuously in a Soxhlet
extractor. Referring to this procedure as per-
extraction, these workers rePorted 100% recov-
ery of progesterone-C ~ added to the blood.
Furthermore, no discernible lipid, protein, or
pigment was noted in the extract. The method
is limited by the relatively small volume of
fluid that can be extracted, but the lack of
lipids or chromagens in the extract might fur-
ther the use of perextraction as quantitative
• measurement of progestins becomes nmre sensi-
tive.
Separation of individual steroids. Counter-
current distribution has been used for the sepa-
ration and purification of steroids (83, 100,
101, 108, 114), but has been utilized in only
one routine method (83). Application of this
excellent method appears to have been limited
by the relatively long time required to separate
and p u r i f y a single sample.
Wilson et al. (149) devised a method using-
column chromatography for the separation of
steroids on synthetic aluminum silicate with
aqueous ethanol. Other workers have used simi-
lar systems with silica gel, eelite, aluminum
oxide (105, 130), or other supporting media in
the columns. Column chromatography is ad-
vantageous for the removal of large amounts
of impurities, but packing of columns and
analysis of individual fractions is very time-
consuming unless automation is applied (4,
48).
The widespread use of p a p e r chromatographic
techniques in the separation and identification
of progestins is evidence of the advantage of
this procedure (15, 16, 102). Since the early
use of these systems for the separation of
adrenal steroids (15, 16), p a p e r chromatography
systems have been devised and studied for the
separation of virtually all steroids. Extensive
reviews and books on the p a p e r chromatographic
separation of steroids are available (16, 102).
I n substance, steroid chromatographic systems
utilize biphase organic solvents for the separa-
tion of compounds on a p a p e r strip or sheet.
Resolution, sharp separation, and simplicity
are distinct advantages of this tool.
A new method of adsorption and partition
chromatography--an open column--has been
developed in recent years. Thin-layer chroma-
tography (TLC), though in its infancy in
steroid research, has already been heralded as
a micro-chemical technique that will supple-
ment or even replace p a p e r chromatographic
methods. The speed (20-40 min) compared to
that of p a p e r chromatography (4-24 hr), the
wide range of distinct resolution (5-500 ttg),
and the noticeably sharp separation of com-
pounds are distinct advantages of TLC (63).
Furthermore, the organic layer on a glass sup-
port allows the use of corrosive spray reagents
and can be prepared with luminous indicators
(115). The method has already been tested for
a great number of steroids in various solvent
systems and complete separation data are avail-
able for progestins (81).
Enzymatic methods have become more valu-
a b l e in recent years for the separation and
identification of steroids, althogh this has not
been their primary role in progestin research.
Henning and Zander (67) probably offered the
best example of this application of enzymatic
methods when they described the separation of
20 a-ol and 20 fl-ol by 20 fl-hydroxysteroid
dehydrogenase. The specific enzyme quanti-
tatively converts 20 fl-ol to progesterone but
leaves 20 a-ol unchanged. The separation can
then be accomplished by chromatography, and
the 20 fl-ol can be regenerated by the same
enzyme and different cofaetors (67).
Quantitation of progesterone. Zarrow et al.
(156) confirmed that the ±'-ene-3-one grouping
on the steroid nucleus must be present for
progestational activity. This same group may
be quantitated by its strong absorbance at 240
mg in alcoholic solutions (16, 34). Although
the A4-ene-3-one grouping is not specific for
progestins, ultraviolet absorption can be a re-
liable method for their detection and quantita-
tion if suitable separation and purification is ac-
 PROGESTERONE IN BOVINE REPRODUCTION 317

complished. Wiest (145) measured the absorp-
tion of progesterone at 240 mt~ by direct spec-
trophotometric scanning of the paper chro-
matogram.
Other spectrophotometric assays have been
reviewed by Zander (154) and are based on
the absorbance of ultraviolet light at wave
lengths other than 240 m~ by progesterone de-
rivatives. These include the progesterone bis-
thiosemiearbazone at 300 mtL, the progesterone
bisdinitrophenylhydrazone at 380 m~, the bis-
isonicotinic acid hydrazone at 380 mtL, and the
sulfuric acid-ethanol chromagen at 290 mtL.
As pointed out by Zander (154), these methods
have the advantage of shifting the quantitation
wave length away from that of interfering ma-
terials, but lack specificity and nmke it diffi-
cult to identify the isolated material as prog-
esterone.
The limit of sensitivity of the spectrophoto-
metric methods ranges from 0.5 to 1.0 t~g if
micro-cells are used in the spectrophotometer.
I f not, five times as much hormone is needed
(Table 1). Recently, Sommerville et al. (130)
reported a modification of the thiosemicarba-
zide reaction and reported sensitivity to 0.1 t~g
using nficro-cells. This is the most sensitive
spectrophotometric method reported for prog-
esterone to date, but the specificity problems
mentioned above still apply. The fact that
other (nonspectrophotometric) assays yield
lower results for progesterone levels in non-
pregnant women (130, 150, 152) suggests that
this method may measure thiosemicarbazone
derivatives of other compounds in addition to
progesterone, since the progesterone derivative
is not isolated prior to spectrophotometric
evaluation (130).
Fluorometric procedures have been used since
1948 for the quantitation of steroid estrogens
(71); however, progesterone exhibits less than
1% of the fluorescence shown by estradiol in
sulfuric acid (2, 143). For this reason, this
procedure was of little value for the determi-
nation of progesterone until it was shown that
pretreatment of the hormone with methanolic
potassium hydroxide would increase the peak
one hundred-fold (143). Using a micro-cell
adaptation of this method, Short and Levitt
(123) were able to detect 0.05 ~g of prog-
esterone standard. However, when samples as-
sayed by this method and the spectrophotometric
method of Short (118) were compared, the
fluorescence results tended to be higher at low
concentrations of progesterone and lower at
high concentrations. This prompted these work-
ers (123) to question the specificity of the
fluorescence reaction, especially because of high
and variable fluorescence of paper blanks.
Although the theory of gas chromatography
was recognized as early as 1941, it was not
until 1952 that this procedure was first suc-
cessfully used (72). By 1960, the technique
was only beginning to be applied to steroid
separation and qualitative identification (107).
As recently as 1962, the first quantitative as-
says of progesterone by gas chromatography
appeared. These were difficult, time-consum-
ing assays with less sensitivity than available
methods (51). I n 1964, the first highly sensi-
tive gas chromatographic assay for progester-
one was reported (152). The method is re-
ported sensitive to less than 0.02 tLg of prog-
esterone with excellent precision. Gas chroma-
tography also appears to offer specificity that
cannot be equalled by spectrophotometric or
fluorometric methods. The procedure can also
be adapted to isotope dilution methods for re-
covery estimates or quantitative measurements
(22, 75, 77).
The introduction of isotopic reagents for the
estimation of steroid hormones has overcome
the problem of inadequate sensitiviy of con-
ventional chemical procedures. Keston et al.
(76) first applied the isotope derivative prin-
ciple to the estimation of amino acids in 1946,

TABLE 1
Expected sensitivity of available methods for the quantitation of progesterone
Expected sensitivity (~g)
Standard Micro-
Quantit~tion method cells cells Reference
Spectrophotometry ~
In alcohol 2.5 0.5 13, 34, 82, 118, 154
In sulfuric acid: ethanol 2.5 0.5 34, 105, 154
Derivatives 2.5 0.1-0.5 34, 130, 154
Fluorometry 0.2 0.05 2, 123, 143
Gas chromatography 0.005-0.02 17, 75, 77, 152
Double-isotope derivative 0.005-0.02 111, 150, 151
For a more complete listing and comments on precision and recovery, see Reference 154.
318 w.R.
but it was not until recently that the principle
was used for steroid hormones (11, 14, 70).
The general method entails addition of an iso-
topically labeled steroid (e.g., C~-labeled) to
the unknown sample. The endogenous steroid
is extracted and pm'ified in a mixture with the
labeled compound, and both are converted to
a derivative, using a reagent labeled with a
second isotope (e.g., Hs). The amount of H ~
in the final isolated derivative quantitates the
amount of derivative formed and the C" meas-
ures procedural losses. This procedure appears
to be limited in sensitivity only by the specific
activities of the derivative-fornfing reagent and
the labeled steroid.
The double-isotope derivative principle was
first applied to the estimation of aldosterone
(11) and testosterone (70) by forming the ace-
tate of these hormones using acetic anhydride-
I t ~ and C~'-labeled steroids. This method could
not be applied per se to the estimation of prog-
esterone, since the hormone cannot be aeety-
luted; therefore, it was necessary to investigate
other derivatives. Riondel et al. (111) first
applied the double-isotope derivative principle
to progesterone estimation using progesterone-
H ~ with thiosemicarbazide-S~ as the derivative-
forming reagent. A later report by Woolever
and Goldfein (151) advocated the conversion
of progesterone to 20 fl-ol, using tritiated so-
dium borohydride and progesterone-C ~ as the
isotope sources. This method is reported sensi-
tive to 0.01 tLg of progesterone or less (150),
and appears highly specific and adequately
precise (151).
E V A L U A T I O N OF ]~IETtIODS AND P O S S I B I L I T I E S
FOR F U T U R E DEVELOPMENT
A major problem in progesterone research
has been to develop methods with greater sensi-
tivity without losing reliability. As shown in
Table 1, methods employing spectrophotometric
end points are sensitive only to 2.5 t~g of the
hormone unless micro-ceils are used. This adap-
tation increases sensitivity fivefold, but also
magnifies the problem of interference from im-
purities. Barring new developments in ultra-
violet instrumentation, or discovery of a new
color-forming reagent, it appears that the spec-
trophotometric methods have nearly achieved
their peak sensitivity. These methods, however,
should continue to be of great value where ex-
treme sensitivity is not the p r i m a r y criterion.
The fluorometric procedure used by Short
and Levitt (123) is quite sensitive (Table 1)
but requires adequate purification and extreme
care with reagents and p a p e r to insure the nec-
essary specificity. F u r t h e r advances might be
G O M E S AND R. E. E R B
made by the conversion of progesterone to a
more highly fluorescent compound such as
Finkelstein et al. (44) reported for estimation
of testosterone.
Judging from the very r a p i d advances in
gas chromatography, one must expect the ex-
cellent method of Yannone et al. (152) to be
superseded soon by even more sensitive (22),
equally specific methods. The developmental
possibilities for gas chromatography in quali-
tative and quantitative steroid assays are ex-
tensive.
Although the double-isotope derivative meth-
ods currently available for progesterone re-
search are extremely sensitive (Table 1), it is
not unreasonable to believe that sensitivity can
be further increased tenfold or more. Use of
borohydride of a higher specific activity (50
mC/mM was used in the original study--200
mC/mM and higher is now available) could, in
itself, increase sensitivity remarkably. Other
developments might include the chemical (103)
or enzymatic (67) conversion of progesterone
to a hydroxylated derivative (e.g., 20 fl-ol), fol-
lowed by aeetylation with labeled acetic anhy-
dride. The analysis could then be carried out
with higher recovery, using a conversion re-
agent avaiIa'ble with a very high specific activ-
ity. A less likely possibility is the conversion
of progesterone to 20 fl-ol by 20 fl-hydroxy-
steroid dehydrogenase (67), using tritium-la-
beled reduced nicotinamide adenine dinucleo-
tide ( N A D H ~) as a tritium donor. This con-
version should be quantitative, but one would
likely have to prepare N A D H ~ in the laboratory
(1) and the compound would probably fail to
meet the specific sctivity requirements.
PROGESTINS IN THE FtOVINE
SOURCES AND ENDOGENOUS COMPOUNDS
E d g a r (35), in 1953, appears to have been
the first to identify progesterone in the bovine
corpus luteum (CL). Following gonadotrophin
treatment of a calf, he isolated 20 tLg of prog-
esterone from 60 g of luteal tissue. A year
later, t I a y a n o et al. (66) demonstrated the
conversion of progesterone to 20 fl-ol by bovine
CL in vitro; and, in ]957, Zander et al. (155)
isolated 20 a-ol and 20/~-ol from human ovaries.
In 1958, Gorski et al. (57) reported the iso-
lation of progesterone and 20 fl-ol from ovaries
of nonpregnant cows. A third ultraviolet ab-
sorbing area, corresponding to 20 a-ol or A~-
androstene-3, 17-dione, was noted, but insuffi-
cient amounts were present for identification.
In a subsequent publication, Gorski et al. (58)
demonstrated the presence of progesterone and
20 fi-ol in corpora lutea, ovaries, and adrenals
 PROGESTERONE IN BOVINE REPRODUCTION
of a pregnant cow, but were unable to detect
either progestin in 1,400 g of placental tissue,
one liter of uterine venous blood, 305 ml of
uterine venous plasma, or blood or tissues of
the 258-day male fetus. Others have supported
the report of progesterone and 20 fl-ol in
ovarian and adrenal tissue in the bovine (9,
40, 41). Short (117) was unable to detect
progesterone in 1 kg of bovine placental tissue,
but Melampy et al. (93) reported high levels
of a compound thought to be progesterone in
bovine placenta. Later, Bowerman and Melampy
(13), using a different chemical method, could
detect progesterone in only one sample of eight
analyzed.
Progesterone and 20 fl-ol have been isolated
from mare placentae (116) and progesterone
and 20 a-ol from ewe placeutae (125). The 20
a-ol compound is very high in the rabbit (128),
rat (17, 145), and ewe (99, 125).
SYNTHESIS AND METABOLI~:~I OF PROGESTERONE
I N T E E BOVINE
Rapid developments in steroid biochemistry,
especially the use of radioactive tracers, have
helped to elucidate the biosynthetic and meta-
bolic pathways of progestins. Use of acetate-C 1'
in in vitro incubations with bovine CL has
shown that simple carbon compounds may be
precursors of the progestin nucleus (136). The
intermediate compounds probably include aeeto-
acetate, hydroxymethylglutarate, and squalene,
which is a precursor of the cholestane series of
compounds (96). Cholesterol is probably hy-
droxylated at the C-20 position and cleaved at
the 2]-carbon by a bovine side-chain desmolase
(137). The resulting pregnenolone is converted
by two enzymes to progesterone. Duncan et al.
(33) demonstrated in vitro synthesis of proges-
terone in swine CL and commented on sevel~l
factors affecting this synthesis.
Sweat et al. (136) have shown that a wide
spectrum of hormones is produced from proges-
terone by ovarian tissue in vitro. Using C~-
labeled compounds, these workers reported the
conversion of progesterone to both 20-hydroxy
derivatives, 17-hydroxyprogesterone, 6 fl-hy-
droxyprogesterone, and pregnanedione. I t seems
likely that more sensitive methods will demon-
strate this entire spectrum and probably still
more progestins in samples from endogenous
sources.
A f t e r progesterone enters the circulation, it
is subject to metabolism in the blood. Coyle
and Romanoff (24) have demonstrated the in
vitro conversion of progesterone-C ~ to 20 fl-ol
and three more polar compounds by bovine
blood. Miller et al. (97) estimated the half-life
319
of free progesterone to be between 18 and 59
rain, with an average for 11 cows of 33.8 rain.
These values must be considered maximal be-
cause progesterone-C 1~ was not isolated, but
rather total radioactivity per sample was meas-
ured and reported as progesterone. Short and
Rowell (126) estimated the half-life of proges-
terone in the blood of the ewe to be 7-8 min,
using values of isolated progesterone-4-C 14.
Similar studies in the human suggest a half-life
of 3-5 nfin (108).
Studies with progesterone-C 1' in women indi-
cate that hydroxylated, saturated metabolites
of progesterone are excreted mostly in the
urine, whereas the corresponding ketonic com-
pounds are excreted via the bile (20). I n both
cases, most of the hormone is conjugated as the
glucuronide. Mayer et al. (91) reported preg-
nanediol and two nonconjugated progesterone
metabolites in sow and gilt urine, and rabbit
urine has been shown to contain progesterone
metabolites (19). I n contrast to these animals,
progesterone metabolites have only occasionally
been reported in the urine of ruminants (10).
Will~ams (148) administered progesterone-4-C 1'
to dairy cows and recovered only 3% of the
radioactivity in urine, whereas nearly 50% was
found in the feces. Boscott (12), after in-
jecting 100 mg progesterone daily into goats,
found no progestin metabolites in the urine.
Other workers failed to find pregnanediol in
the urine of cows. The studies of Miller and
Turner (95, 96) indicate that progesterone is
converted to androgenic substances in the liver
and excreted via the bile into the feces. I n ad-
dition, progesterone metabolites may be ex-
creted in the milk (148) or as carbon dioxide
(54).
QUANTITATIVE ESTL~[ATES OF P R O G E S T I N S
Nonpregnant co,vs. One hesitates to sum-
marize reported quantitative levels of proges-
tins, because of the uncertainty of how effi-
ciently the hormones were extracted and
purified, especially in earlier studies. However,
enough evidence exists to warrant doing this to
show generalized trends.
As shown in Table 2, the progestin levels in
corpora lutea of cycling cows agree remarkably
well for three of the nmre complete studies (42,
55, 89). Data from other studies support these
three (47, 132, 133), but there are some con-
flicting data (23). In the majority of studies
using bovine corpora ]urea, progesterone con-
centration (tLg/g) showed an initial rise during
the early proliferative stages of the CL (Days
1-4 of the cycle), followed by a more or less
constant level (25-35 t~g/g) until Day 9 or 10.
 TABLE 2
P r o g e s t i n c o n t e n t of corpus l u t e u m a a n d o v a r i a n ve i n blood p l a s m a b of t he c y c l i n g cow ( 5 5 ) a n d ewe ( 3 7 )

C orpus l u t e u m Ovarian vein plasma

Edgar &
Mares et al. (89) Oomes ct al. (55) Gomes et al. (55) R o n a l d s o n (37)
Days
since No. NO. NO. NO.
estrus CL CL W t Prog. 20 fl-ol CL CL W t Prog. 20 fl-ol s~mples Prog. samples t'rog.
(g) (~g/g) (g) (Pg/g) (~g/ml) (~g/ml)
0 2 1.54 ¢ 6.8 d 2 d
1 3 1.22 ° 6.5 d 3 d 5
2 3 0.74 a d 3 0.9 6 d
3 6 0.54 24.7 1.6 3 0.79 19.1 0.1 3 0.2 4 0.5
4 3 1.98 34.7 d 3 0.9 5 0.4
5 5 1.88 24.7 1.6 3 2.24 38.1 d 3 0.9 4 0.6
6 3 3.63 32.8 d 3 1.4 8 0.9
7 4 4.06 25.8 1.1 3 3.62 35.3 0.4 3 1.3 8 1.9
8 3 6.20 22.5 d 3 2.1 12 1.6
9 4 3.70 35.0 0.6 3 6.38 24.8 0.6 3 2.5 17 1.5
10 15 1.6
11 4 5.54 33.8 1.4 1 4.29 66.4 4.2 1 4.3 9 1.4
12 11 2.0
13 4 4.20 41.9 4.1 6 1.5
14 4 6.70 73.2 7.0 4 3.2 6 2.3
15 4 5.27 44.7 6.0 3 5.83 54.9 6.1 3 6.2 14 1.8
16 2 4.32 32.2 3.0 2 2.0 5 2.0
17 4 3.53 7.0 2.1 14 d
18
19 6 3.05 14.2 4.9
20 2 1.86 10.0 d 2 0.6
21 1 2.92 d d 1 0.7

F o r a d d i t i o n a l d a t a a n d other species, see R e f e r e n c e s 13, 23, 33, 40, 41, 57, 58, 78, F i g u r e 1.
b F o r a d d i t i o n a l d a t a a n d other species, see T a b l e 3.
" Co rp ora l u t e a of p r e v i o u s cycle.
d NO p r o g e s t i n detected.
 P R O G E S T E R O N E IN B O V I N E R E P R O D U C T I O N
Thereafter, the concentration of the hormone
again increased to a peak on Days 14 and 15,
followed by a decline to the next estrus (Table
2). Detectable progesterone is present in the
regressing CL for two or three days after the
next estrus period. The levels of 20 fl-ol in the
CL contributed 5-20% of the total progestins,
apparently depending on the method of CL
collection. Since progesterone can be converted
to 20 fl-ol by the incubating CL, it seems likely
that collection of tissues at slaughter (42)
might be expected to show higher levels of 20
fl-ol than collection from the living animal
(55, 89). The data indicate that such is the
case. Progestin concentrations in the CL of
the bovine (Table 2) a p p e a r to be paralleled
by concentrations in the ovarian venous efflu-
ent (55). Table 2 shows also the concentration
of progesterone in the ovarian vein blood of
the cycling cow (55) and ewe (37).
Armstrong et al. (7) have shown that bovine
CL exhibits maximum in vitro progesterone
synthesis (270-300 tLg/g/2 hr) when the gland
is removed surgically 4-13 days post-estrus. A
gradual decline in synthetic capacity was noted
between Days 14 and 18 post-estrus (80 tLg/g/
2 hr). No detectable synthesis occurred with
19-day-old CL. Luteinizing hormone ( L H )
stimulated increased progesterone synthesis by
all CL collected prior to 19 days post-estrus,
but had no discernible effect on luteal tissue
obtained on Day 19 or later. Progesterone syn-
thesis in these latter CL's could be only par-
t i a l l y restored by addition of pregnenolone or
glucose-6-phosphate plus NADP* to the incu-
bation medium (7). Duncan et al. (33) re-
ported the in vitro synthesis of progesterone
by slices of porcine CL. As with the bovine
CL (7), glands collected 4-16 days post-estrus
were able to synthesize progesterone, but those
obtained on Day 18 contained no detectable
progesterone before or after incubation.
I t can be generalized from several studies
that growth in weight of the CL and proges-
terone content, synthesis, and secretion are
closely related to the degree of luteinization
as determined by histological studies (26, 89).
During the first five days of the cycle, the
granulosa cells of the CL are gradually in-
teinized (26), the CL grows in weight (55, 89),
and progesterone content and synthesis become
detectable (7, 55, 89). A f t e r Day 6, the theca
interna cells become luteinized (26), the CL
grows larger, and progesterone concentration
is higher (55, 89). Degeneration of the luteal
cells begins on Day 16 or 17 in the nonpregnant
bovine (26, 89), corresponding to a decrease
in weight and progesterone concentration (55,
321
89), rate of release (55), and a lowered ca-
pacity to synthesize (7). Similar changes in
R N A / D N A ratios and per cent Type I and
Type I I cells (46) were shown by Mares et al.
(89).
I n a study designed to compare luteal and
blood plasma progestins in the nonpregnant
bovine, Gomes et al. (55) showed a significant
correlation between CL progestins (t~g/g or
tLg/CL) and ovarian vein progestin concentra-
tion (#g/ml of plasma). However, they re-
ported that the peripheral blood progesterone
levels found in their study did not reflect the
stage of the estrous cycle, quantity of concen-
tration of progestin in the CL, or concentration
of progestin in ovarian vein blood plasma.
Ovarian vein progestin concentrations in the
cow and ewe show similar trends (Table 2)
and indicate that CL levels of the h o l ~ o n e may
adequately reflect secretion rates. The work of
Stormshak et al. (135) in the cycling ewe sup-
ports the hypothesis that luteal progestin con-
tent is indicative of the amount of hormone
released into the blood, but Clegg et al. (23),
in a study involving only seven cows, suggest
that CL levels of progesterone do not reflect
secretory rate.
I t is discouraging to note that Gomes et al.
(55) found no relationship between tissue or
ovarian vein plasma progesterone and periph-
eral blood plasma levels of the hormone. I t
should be pointed out, however, that micro-cell
attachments were not used for the spectro-
photometric quantitative step (55). Thus, read-
ings were often made near the limit of sensi-
tivity (Table 1), increasing the possibility of
error. ~¢[cCraeken (86) has shown that periph-
eral blood plasma progesterone levels begin to
decrease within 15 rain after CL removal and
fall to nondetectable levels within 24 hr, indi-
cating that a positive relationship may exist be-
tween ovarian and peripheral levels of the
hormone. This would suggest that a highly
sensitive assay allowing analysis of multiple
samples from one animal throughout the cycle
might yield results which would lead to a better
understanding of ovarian-peripheral proges-
terone relationships.
P r e g n a n t cows. Figure 1 was drawn in an
attempt to generalize available data on proges-
tin levels in corpora lutea and peripheral blood
plasma of pregnant cows. CL data are com-
bined from the studies of Zimbelman et al.
(158-161, control data), Stormshak and Erb
(134), Estergreen et al. (43), and Gomes et ah
(56). The data on peripheral plasma hormone
levels are taken from the reports of Short
322 w.R. G O M E S A N D R. E, E R B

00
.J
O.
2 D .J
,<
E
LIJ
-1-
(/) • CORPORA LUTEA ° i1
a. 4 0 A [] PERIPHERAL PLASMA b _ E
n-
O 1.5 "'
D.
o
b. .J
o 30 O
(.9 o
\
(.9 1.0
:k
20 !

Or) ul
Z
I--
o
Or) 0.5 ~
'" I0 I---
o
0t~
{1. o
0 I I I I I I I I I 0 o_
0 8 16 I00
2J4~'(~0 140 180 220 260
DAYS SINCE ESTRUS
Fro. 1. Progestin (progesterone and 20 fl-ol) in corpora ]utea and peripheral blood plasma
of pregnant cows. References 40, 41, 43, 56, 134, 158-161. h References 13, 56, 87, 117.

(119), Bowerman and Melampy (13), Gomes
et al. (56), and MeCracken (87).
CL levels of progesterone, both in total
(t~g/CL) and in concentration (tLg/g), are in
excellent agreement for several studies (56,
134, 158-161). Others (]3, 93) tend to report
lower quantitative values; therefore , these are
not shown on the curve in Figure 1. Of the
combined data in the CL curve in Figure 1,
the values of control cows reported by Zimbel-
man et al. (158-161) tend to be lower; but the
differences are not large when the other data
are restricted to cows under 10 yr of age. Since
earlier studies have shown very close parallel-
ism between total progesterone in the CL and
progesterone concentration (40, 41), only the
latter has been shown in Figure 1.
I n several studies, essentially no difference
was found in CL progestins 14-16 days follow-
ing estrus in pregnant or nonpregnant cows
(40, 47, 134). Pregnant cows show a slight
decline by Day 19, but the most pronounced
decline occurs after Day 42. A leveling off is
seen after Day 56 and a further decline at
about 120 days of pregnancy. A later rise
reaches a peak at about 250 days, followed by
a decline to the time of parturition (Figure
1). Similar changes are seen in peripheral
blood progesterone levels (Figure 1). Gomes
et al. (56) have shown that ovarian vein plasma
progestin concentration also declines after 250
days in the bovine. This concentration reaches
undetcctable levels after parturition (43).
Progestin was also undetectable in a CL ob-
tained eight days post-partum (134). Edgar
and Ronaldson (37) reported that ovarian vein
blood progesterone remained at about 1.5 t'g/
ml during Days 7-28 of pregnancy in the ewe,
then increased to over 2 tLg/ml on Day 35.
Thereafter, the concentration remained near 1.5
t*g/ml until Day 111, followed by a gradual
decline to nondetectable levels at 140 days.
These workers (37) suggested, from their data,
that uterine contents in the pregnant ewe are
not an important source of progesterone. How-
ever, other work has shown that the ewe does
not abort following ovariectomy after Day 60
of pregnancy (18), peripheral blood proges-
terone levels in the pregnant ewe do not fall
after ovariectomy, and the placenta of the ewe
has been found to be a source of progesterone
and 20 a-ol (125).
Other species. Although this paper is pri-
marily concerned with bovine progestins, and
it is often difficult to extrapolate between spe-
cies, a certain amount of reference to compara-
tive data may be helpful in elucidation of the
role(s) of progesterone in reproductive proc-
esses. For this reason, a summary of proges-
terone levels in the blood of a number of species
 PROGESTERONE IN BOVINE REPRODUCTION 323

is shown in Table 3. These d a t a have pre- early d i e s t r u s (40). M e C a n n (84) f o u n d t h a t

viously been s u m m a r i z e d in p a r t (35, 41, 54).
F o r tissue a n d follicular fluid levels of the
hormone, the r e a d e r is r e f e r r e d elsewhere (35,
40, 41, 113).
PROGESTIN AND NOR]'C[AL CYCLIC FUNCTION
O b s e r v a t i o n s f r o m several e x p e r i m e n t s indi-
cate t h a t p r o g e s t e r o n e is associated w i t h the
a v a i l a b i l i t y o f L H d u r i n g late p r o e s t r u s or
p r o g e s t e r o n e o r estradiol b e n z o a t e alone h a d
little effect on L I t release in the ovariectomized
rat, b u t p r o g e s t e r o n e i n j e c t i o n s into estrogen-
p r i m e d females reduced p l a s m a L H to non-
detectable levels. As reviewed earlier b y E r b
(40), r a t i o s of p r o g e s t e r o n e to estradiol of
12.5:1 to 7 5 : 1 a p p e a r to be o p t i m a l f o r the
i n d u c t i o n o f estrus in the ovarieetomized cow,
but ratios h i g h e r t h a n 7 5 : 1 will block this

TABLE 3
A summary: L e v e l s o f p r o g e s t e r o n e i n b l o o d o r p l a s m a as d e t e r n f i n e d b y c h e m i c a l a s s a y
Fraction of Progesterone
Species Reproductive status blood (~g/lO0 ml) Reference
Cow a Preovulatory-1 day Peripheral plasma 0.20 117
Cycle-12 days 0.19 117
-12 days 0.92 86
- 1 day Not detected 87
-13 days 1.06 87
- 1 day 0.71 55
-2-5 days 0.36 55
-6-8 days 0.46 55
-9-15 days 1.68 55
-19-20 days 0.09 55
P r e g n a n t - 9 0 days 1.18 87
-10-49 days Peripheral blood 0.9 93
-50-89 days 1.4 93
-90-]29 days 0.4 93
-130-169 days 3.1 93
-170-209 days 3.4 93
-210-249 days 4.0 93
-250-280 days 3.1 93
-111-117 days Ovarian plasma 264 54
-250-254 days 259 56
-281-282 days 140 56
Ewe a Cycle
Mid-htea] Ovarian plasma 95 124
Late luteal 104 124
P r e g n a n t - 6 days 125 85
-1-4 wk Ovarian blood 130 37
-5-6 wk 215 37
-7-16 wk 160 35,37
-17-18 wk 125 37
-19 wk 40 37
-20 wk Not detected 37
-15 days Peripheral plasma 0.43 99
-60 days Not detected 117
-96 days 0.68 117
-99 days 0.91 !17
Sow Cycle-Mid-hteal Peripheral plasma 0.75 117,120
P r e g n a n t ~ 0 days 1.70 120
-48 days 3.]2 120
-49 days 2.92 120
-53 days 2.04 120
-55 days 0.91 120
-99 days 1.04 120
-102 days 1.20 120
-112 days 0.34 120
-113 days 0.67 120
-114 days 0.49 ]20
Goat P r e g n a n t - 1 3 4 days Ovarian blood 230 109
-112 days Peripheral plasma 0.71 117
* For additional data, see Table 2 and Figure 1.
324 w.R. G O M E S AND R. E. E R B
condition (92). I n intact cows, 10 mg of
progesterone administered during estrus re-
duces the time to next ovulation by about 10 hr
(62). W o r k with the bovine and the rat indi-
cates that progesterone accomplishes this end
by enhancing the hypothalamo-hypophyseal re-
lease of ovulating hormone (likely L H ) .
Foote et al. (47) observed a high incidence
of cystic ovaries following supra-vaginal re-
moval of the CL on D a y 14 of the cycle. Sta-
ples et al. (132) produced cystic ovaries in
significant proportion of heifers when CL in-
hibition and precocious ovulation were induced
with oxytocin. Conversely, 50 mg of proges-
terone daily will inhibit ovulation and estrus
effectively, but large follicles still appear on
the ovary (144). This suggests that a minimal
level of progesterone is necessary to enhance
release of gonadotrophin, but a higher level
may be inhibitory.
The inhibition and synchronization of estrus
and ovulation in cattle by progesterone and
other progestins have been widely used for
both practical and experimental reasons. The
practica~ aspects have been adequately re-
viewed elsewhere (49, 61), but the value of
such compounds in the experimental nmnipu-
lation of cyclic functions is worthy of note here.
The injection of repositol progesterone (76
mg/cwt) into heifers on the day of heat or on
D a y 8 or 16 of the cycle significantly decreased
pituitary levels of follicle-stimulating hormone
( F S H ) and L H and reduced the diameter of
the CL. When progesterone (100 mg/ewt)
was injected on D a y 1 or Day 4 of the cycle,
CL weight, progesterone content, and func-
tional cells in the CL were reduced when in-
spected on Day 14; but estradiol had no a p p a r -
ent effect on the total amount of progesterone
in the CL (83). This a p p a r e n t luteolytic--or
anti-luteotrophic--effect of progesterone offers
a basis for the study of CL growth and main-
tenance. I f one were to determine which go-
nadotrophin(s) were decreased in bovine pi-
tuitary effluent (30) following progesterone
administration, it might be possible to deter-
mine t h e nature and normal level of the pi-
tuitary luteotrophin in the bovine and to add
to our meager knowledge of progesterone-(uter-
ine) -hypothalamo-hypophyseal-owrian relation-
ships. Such studies might also be helpful in
determining whether other progestins act in
the same manner as progesterone itself (106).
CORPUS L U T E U M MAINTENANCE
The most important factor in the onset of
pregnancy, other than the presence of a zygote,
is maintenance of the CL. The mechanism of
this maintenance or, conversely, the mechanism
of luteolysis in the nonpregnant bovine, is
poorly understood. Our knowledge of these
processes is most advanced for the rat and
mouse; but it appears that prolactin, the luteo-
trophic hormone ( L T H ) in these species, is
ineffective for CL maintenance in other species
(122). As mentioned earlier, Hansel and Wag-
ner (62) reported the anti-]uteotropic nature
of oxytocin in the bovine. Simmons and Hansel
(129), in an ingenious experiment using the
prevention of this effect as an end point, re-
ported that a luteotrophic factor is present in
crude anterior pituitary extracts of the bovine.
The oxytocin-induced effect was also overcome
by human chorionic gonadotrophin (HCG),
but was unaffected by bovine growth hormone,
ovine prolactin, or equine LH. I t is disappoint-
ing that bovine L H was not available for this
study (129), since H C G exhibits predominantly
L H action (157), and ovine L H increased the
in vitro synthesis of progesterone synthesis in
human CL, as does HCG, but ovine L H is
ineffective (110).
A n y theory of CL maintenance or luteolysis
must necessarily include a uterine effect, since
hysterectomy will result in CL maintenance
(5), oxytocin repression of the CL fails in the
hysterectomized cow (133), and insertion of
foreign bodies into the uterus can cause pre-
cocious estrus and ovulation (62). These re-
sults suggest that a certain normal uterine con-
dition is necessary during the early cycle for
continued secretion of L T H in the bovine.
Oxytocin, hysterectomy, or foreign objects dis-
r u p t this necessary condition. I f a zygote does
not enter the uterus, a substance from that
organ may induce luteolysis, either by inter-
rupting pituitary L T H release or by a direct
LTH-over-riding effect on the ovary (122).
PROGESTI~qS A:ND PREGNANCY
Essentiality of the CL. As reviewed earlier
(41), the CL appears essential throughout
pregnancy in the rabbit, rat, sow, goat, and
bitch, but for less than full term in primates,
mares, ewes, and guinea pigs. In all of the
species studied to date, however, progesterone
or its equivalent activity is required for the
normal span of pregnancy. In the cow, the
necessity of the CL during pregnancy remains
controversial. I t is generally concluded that
the CL is required for about 200 days in this
animal, but in one study removal of the CL
after 200 days did not result in abortion prior
to 250 days (88). I n another study, when
cows were ovariectomized after 200 days, eight
of nine aborted between 238 and 274 days of
 PROGESTERONE IN BOVINE REPRODUCTION
pregnancy (43). Furthermore, peripheral blood
progesterone concentrations dropped to unde-
tectable levels a f t e r ovariectomy (56), whereas
this operation does not lower the hormone lev-
els in peripheral plasma of the p r e g n a n t ewe.
As pointed out earlier in this paper, attempts
to isolate progesterone f r o m bovine placenta
have generally been unsuccessful (58, 117),
but occasional positive results have been noted
(13, 43, 93).
F r o m the above evidence, one might propose
the following hypothesis to explain the sources
of progesterone in p r e g n a n c y nmintenance in
the bovine :
The m a j o r source of progesterone throughout
pregnancy in the cow is the CL (58), but
ovaries and adrenals might contribute a sig-
nificant amount (58, 134) and body fat may
store the hormone to be used if needed (87).
The placenta in the cow produces a v e r y low
level of progesterone. I n most cases, this level
is insufficient to maintain p r e g n a n c y following
ovariectomy but m a y be adequate when only
the CL is removed. Because of individual vari-
ation in placental production of the hornione,
an occasional cow may fall to abort following
ovariectomy.
This postulated low level of progesterone
synthesis is not unique among animals. I f
fetuses are removed f r o m the rat in such a
way that the placentae remain intact, 12 pla-
centae will maintain one fetus in the ovarieeto-
mized rat (65). The general failure to find
progesterone in bovine placenta cannot be con-
sidered p r o o f of an absence of the hormone,
since the levels may be so low as to have
escaped detection by the methods used but high
enough to exert an i m p o r t a n t local uterine
effect (25, 50). Use of more sensitive assays
(Table 1) should enable one to better resolve
this question.
REFEREI~CES
(1) ABRAHAM, S. 1964. Biosynthesis of La-
beled Carbohydrates and Other Compounds
of Biochemical Interest. Atomlight, 36: 1.
(2) AB~A]~AM, R., AND STAUDINGER, II. 1964.
Spektorscopische Untersuchungen zur Fluo-
rometrischen Steroidanalyse. Z. Klin. Chem.,
2: 16.
(3) AMERICAN MEDICAL ASSOCIATION COUNCIL
ON PHARI~ACY AND CHEMISTRY. 1936.
J. Am. Med. Assoc., 106: 1808.
(4) ANDERSON, F. O., CRISP, L. R., RIGGLE,
G. C., VERUK, G. G., HEFTMANN, E., JOHN-
SON~ D. F., FRANCOIS~ D., AND PERRINE,
T.D. 1961. Steroid Analyzer. Anal. Chem.,
33 : 1606.
325
(5) ANDERSON, L. L., BOWERMAN, A. M., AND
MELAMPY, R. M. 1963. Neuro-Utero-Ovarian
Relationships. I n A. V. Nalbandov, Ed.
Advances in Neuroendocrinology. Univer-
sity of Illinois Press, Urbana.
(6) ANDERSON, L. L., NEAL, F. C., AND ME-
LAMPY, R. M. 1962. Hysterectomy and
Ovarian Function in Beef Heifers. Am. J.
Vet. Research, 23: 794.
(7) ARMSTRONG, D. T., BLACK, D. L., AND
CONE, C. E. 1964. Metabolic Studies with
Active and Involuting Bovine Corpora Lu-
tea. (Abstract.) Federation Proe., 23:
462.
(8) AXELROD, L. R., AND WERTHESSEN, N. T.
1961. Blood as a Site for the Conversion
of the Steroid Hormones. Endocrinology,
68:180.
(9) BALFOUR, W. E., COMLINE, R. S., AND
SHORT, R. V. 1959. Changes in the Secre-
tion of 20 a-Hydroxypregn-4-en-3-one by
the Adrenal Gland of Young Calves. Na-
ture, 183: 467.
(10) BENSON, G. K., AND COWIE, A. T. 1957.
Reviews of the Progress of Dairy Science.
Section A. Physiology of Dairy Cattle.
Hormones in Reproduction and Lactation.
J. Dairy l%search, 24: 252.
(11) BOJESON, E., AND DEGN, H. 1963. A
Double Isotope Derivative Method for the
Determination of A]dosterone in Peripheral
Plasma. Acta Endocrinol., 37: 541.
(12) BOSCOTT, R. J. 1952. The Metabolism of
Progesterone in the Goat. The Separation
of Urinary Cortical Steroids. Colloq. on
Endocrinol. CIBA Found., 2: 327.
(13) BOWERMAN, A. M., AND MELAMPY, R. M.
1962. Progesterone and A~-Pregnen-20 fl-ol-
3-one in Bovine Reproductive Organs and
Body Fluids. Proc. Soc. Exptl. Biol. Med.,
109 : 45.
(14) BURGER, H. G., KENT, J. R., AND KELLIE,
A . E . 1964. Determination of Testosterone
in Human Peripheral and Adrenal Venous
Plasma. J. Clin. Endoerinol. Metabolism,
24:432.
(15) BURTON, R. B., ZAFPARONI, A., AND KEUT-
MANN~ E. ]:I. 1951. Paper Chromatography
of Steroids. II. Cortieosteroids and Re-
lated Compounds. J. Biol. Chem., 188: 763.
(16) BusH, I. E. 1961. Chromatography of
Steroids. Pergamon Press, New York.
(17) CARLSON, I. It., BLAIR, A. J., AND MEYER,
R. K. 1964. Analysis of Rat Ovarian
Venous Effluents by Gas-Liquid Chromatog-
raphy. (Abstract.) Proc. AmL Meet. En-
docrine Soc.
(18) CASIDA, L. E., AND WARWICK, E. J. 1945.
The Necessity of the Corpus Luteum for
Maintenance of Pregnancy in the Ewe.
J. Animal Sci., 4: 34.
(19) CHAMBERLAIN, J., KNIGHTS, B. A., AND
THOMAS, G. H. 1963. Analysis of Steroid
326 w. 1~. GOMES AND I%. E. ERB
Metabolites by Gas Chromatography. J. :En-
docrinol., 26 : 367.
(20) CtIANG, :E., SLAUNWttITE, W. R., AND SAND°
FEral, A. A. 1960. Biliary and Urinary
Metabolites of 4-C~-Progesterone in Human
Subjects. J. Clin. :Endocrinol. Metabolism,
20 : 1568.
(21) CI~ANNING, C. P., AND VILLEE, C. A. 1964.
Some :Effects of Luteinizing ttormone on
Metabolism of the Corpus Luteum of the
Rat. (Abstract.) Proc. Ann. Meet. Endo-
crine Soc.
(22) CLARK, S. J., AND WOTIZ, H. tI. 1963.
Separation and Detection of Nanogram
Amounts of Steroids. Steroids, 2:535.
(23) CLEGG, M. T., SQUIRES, C., AND GANONG,
W . F . ]962. Progesterone Output from Cow
Ovaries. (Abstract.) Proc. Ann. Meet.
:Endocrine Soc.
(24) COYLE, M. G., AND R0~ANOPF, :E. B. 1964.
Transformation of 4-C~-Progesterone by
Bovine Blood. (Abstract.) Federation
Prec., 23: 462.
(25) CSAFO, A. 1961. The Role of Progesterone
in the Maintenance and Termination of
Pregnancy. I n A. C, Barnes, Ed. Progester-
one. Brook Lodge Press, Augusta, Michi-
gan.
(26) CUFFS, P. T., LABEN, R. C., AND MEAD,
S. W. 1959. Histology of Pituitary,
Adrenal, and Reproductive Organs in Nor-
real Cattle and Cattle wi~h Lowered Re-
productive Efficiency. Hilgardia, 29: 383.
(27) DANIEL, 5. C., JR., AND LEVY, J. D. 1964.
Action of Progesterone as a Cleavage In-
hibitor of Rabbit Ova in Vitro. J. Reprod.
Fertil., 7: 323.
(28) DEVENUTO,F., MULE, S., AND WESTPItAL, U.
1958. Perextraction, A Procedure to Ex-
tract C_~ Steroids from Tissues and Blood.
Arch. Biochem. Biophys., 73:451.
(29) DOI~INGUEZ, O. V., SEELY, ,]-. R., AND
GORSKI, J. 1963. Studies on the Ace~yla-
tion of Steroids Using l-Carbon-14-Acetic
Anhydride. Anal. Chem., 35: 1243.
(30) DONALDSON, L. E., AND HANSEL, W. 1964.
Collection of Blood from the Cavernous
Sinus in the Cow. J. Dairy Sei., 47: 98.
(31) DONKER, J. D., NICHOLS, J. R., GRAHAIV[,
:E. F., AND PE~ERSON, W. E. 1958. Con-
trolled Estrus in Cattle. ProF. I I I Sympos.
Reprod. and InfertiI. Pergamon Press, New
York.
(32) DORF!~[AN,R. I., I~INCL, F. A., AND RINGOLD,
H. J. 1961. Anti-Estrogen Assay of Neu-
tral Steroids Administered by Gavage. En-
docrinology, 68: 43.
(33) DUNCAN, G. W., BOWERMAN, A. N., ANDER-
SON, L. L., HEARN~ W. R., AND MELA1VIFY~
R. M. 1961. Factors Influencing in Vitro
Synthesis of Progesterone. :Endocrinology,
68 : 199.
(34) DUSZA, J. P., HELLEI~, M., AND BERNSTEIN,
S. 1963. U]travlolet Absorption. I n L. L.
Engel, : E d . Physical Properties of the
Steroid Hormones. MacMillan Co., New
York.
(35) :EDGAR,D. G. 1953. The Progestin Content
of Body Fluids. J. Endocrinol., 10:54.
(36) EDGAR, D. G., FLUX, D. S., AND RONALDSON,
J. W. 1959. Comparison of Chemical and
Biological :Estimations of Plasma Progestin.
J. Endocrinol., 19: 44.
(37) :EDGAR, D. G., AND RONALDSON, J. W. 1958.
Blood Levels of Progesterone in the :Ewe.
J. :Endoerinol., 16: 378.
(38) EMMENS, C. W. ]959. Role of Gonadal
Itormones in Reproductive Processes. In
H. H. Cole and P. T. Cupps, Eds. Repro-
duction in Domestic Animals. Vol. I. Aca-
demic Press, New York.
(39) ENGEL, L. L., A~]) CARTER, P. 1963. Parti-
tion Coefficients. I n L. L. :Engel, Ed.
Physical Properties of the Steroid Hor-
mones. MacMillan Co., New York.
(40) :EBB, R. E. 1961. Significance of Female
Sex Steroids in Bovine Reproduction. V.
Biennial Sympos. Animal Reprod., Knox-
vi]le, Tenn.
(41) EBB, R. :E., AND GOMES, W. R. 1962. Cyclic
Changes in Levels of Female Sex Steroids.
Sympos. No. Atlantic See., Am. Soc. Ani-
mal Sci., Princeton, N. J.
(42) EBB, R. E., AND STORMSttAK, F. 1961.
Progestins in Corpora Lutea, Ovaries, and
Adrenals A f t e r :Estrus and Breeding of
l~ormal and Abnormal Cows. J. Dairy Sci.,
44 : 888.
(43) :ESTERGREEN, V. L., JR., GORES, W. R.,
FROST~ 0. L., AND :EBB, R. :E. 1964. Un-
published data.
(44) FII~KELSTEIN, M., FORCIt~ELLI, :E., AND
DOlCF~AN, R. I. 1961. Estimation of Testos-
terone in Human Plasma. J. Clin. :Endo-
crinol. Metabolism, 21: 98.
(45) FroST, N. L., S ~ T ~ A N , F. W., RmOR,
E. M., AN]) CASIDA, L. E. ]963. Factors
Affecting Ovulation and Follicular Cyst
Formation in Sows and Gilts Fed 6-Methyl-
17-Acetoxyprogesterone. J. Animal Sci.,
22 : 66.
(46) FOLEY, R. C., AND GREENSTEIN, J. S. 1958.
Cytological Changes in the Bovine Corpus
Luteum During Early Pregnancy. Proc.
I I I Sympos. Reprod. ]nfertil. Pergamon
Press, New York.
(47) FOOTE, W. D., ZIMBEL~AN, R. G., LOY,
R. G., AND CASIDA, L. :E. 1959. Endocrine
Activity of Corpora Lutea from First-Serv-
ice and Repeat-Breeder Dairy I~eifers.
J. Dairy Sei., 42: ]944.
(48) FRANCOIS, D., JOHNSON, D. F., AND HEFT-
~[ANN, E. 1963. Programmed Gradient :Elu-
tlou Chromatography with the Steroid Ana-
lyzer. Anal. Chem., 35: 2019.
(49) FRIDI~IANDLER, L., AND PINCUS, G. 1964.
Pharmacology of Reproduction and Fer-
tility. Ann. Rev. Pharmacol., 4: 177.
 PROGESTERONE IN BOVINE REPRODUCTION
(50) FVCHS, A. R. 1964. Effect of Intra-Am-
nionic Administration of Progesterone on
Oxytocin-Induced Labour in ]~abbi%s. Aeta
Endocrinol., 46: 235.
(51) FUTTERWEIT, W., McNIvEN, N. I., AND
I)ORFMAN, R. I. 1963. Gas-Chromatographic
Identification of Progesterone in Human
Pregnancy Plasma. Biochim. et Biophys.
Aeta, 71: 474.
(52) GARDNER., M. L., FIRST, N. L., AND CASIDA,
L . E . 1963. Effect of Exogenous Estrogens
on Corpus Luteum Maintenance in Gilts.
J. Animal Sci., 22:132.
(53) GLASSER, S. R., AND SOUPART, P. 1964.
Actinomycin D Effects on Implantation and
Decidual Cell Response. (Abstract.) Proe.
Ann. Meet. Endocrine Soc.
(54) GOMES, W. 1~. 1962. Assay for Progestins
in Peripheral and Ovarian Venous Blood
from Cows. Thesis, Washington State Uni-
versity.
(55) GOMES, W. R., ESTER~REEN, V. L., JR.,
FRost, O. L., ANI) EzB, i~. E. 1963. Prog-
estin Levels in Jugular and Ovarian Venous
Blood, Corpora Lutea, and Ovaries of the
hIonpregnant Bovine. J. Dairy Sci., 46: 553.
(56) GOMES, W. R., FROST, O. L., AND ESTER-
0REEN, V. L., JR. 1962. Progestins in
Ovarian and Peripheral Blood of Cows Dur-
ing Late Pregnancy. J. Dairy Sci., 45: 670.
(57) GORSKI, J., DOMINGUEZ, O. V., SAi'~UELS,
L. T., AND ERB, R. E. 1958. Progestins of
the Bovine Ovary. Endocrinology, 62: 234.
(58) GORSKI, J., EEB, R. E., DICKS01q, W. M.,
AN]) BUTLER, tI. C. ]958. Sources of Prog-
estins in the P r e g n a n t Cow. J. Dairy Sei.,
41 : 1380.
(59) GORSKI, J., AND 1N"ICOLETTE, J. A. 1963.
Early Estrogen Effects on Newly Synthe-
sized RNA and Phospholipid in Subcellular
Fractions of Ra¢ Uteri. Arch. Biochem.
]3iophys., 103: 418.
(60) HANSEL, W. 1961. The Hypothalamus and
Pituitary Function in Mammals. Intern. J.
Fertil., 6: 241.
(61) HANSEL, W., VAN BLAKE, H., AND MAL-
YEN, P. 1964. Artificial Estrous Cycle Syn-
chronization in Cattle by Feeding Tech-
niques. Proc. Cornell Nutr. Conf. for Feed
Manufacturers.
(62) HANSEL, W., AND WAGNEI~ W. C. 1960.
Lutea] Inhibition in the Bovine as a Re-
sult of Oxytocin Injections, Uterine Dilata-
tion, and Intrauterine Infusions of Senfinal
and Preputial Fluids. J. Dairy Sci., 43:
796.
(63) I-IARA, S., TANAKA, ]:L, AND TAKEUCttI, M.
1964. Systematic Analysis of Thin-Layer
Chromatogram. Chem. Pharm. Bull., 12:
626.
(64) I'IARTMAN, C. G. 1960. Physiological Mech-
anisms Concerned with Conception--An In-
ventory of Unanswered Questions. Pers.
Biol. Med., 4: 77.
327
(65) HATERIUS, H. O. 1936. Reduction of Litter
Size and Maintenance of Pregnancy in the
Oophorectomized Rat: Evidence Concern-
ing the Endocrine Role of the Placenta.
Am. J. Physiol., 114: 399.
(66) HAYANO, M., LINDBERG, M. C., WIENER, M.,
ROSENKRANTZ~ i . , AND DORFMAN, R. I.
1954. Steroid Transformations by Corpus
Luteum Tissue. Endocrinology, 55: 326.
(67) HENNING, H. D., AND ZANDE4¢, J. 1962.
Verwendung von Hubeners 20 fl-Hydroxy-
steroid-Dehydrogenase bei mikrochemischer
Identifizierung und Trennung yon Steroiden.
Z. Physiol. Chem., 330: 31.
(68) I'IILLIARD, J., ARCHIBALD, D., AND SAWYER,
C. H. 1963. Gonadotropic Activation of
Preovulatory Synthesis and Release of Prog-
estin in the Rabbit. Endocrinology, 72: 59.
(69) It0LM, L. W., AND SHORT, R. V. 1962.
Progesterone in the Peripheral Blood of
Guernsey and Friesian Cows During Pro-
longed Gestation. J. Reprod. Fertil., 4: 137.
(70) HUDSOn, B., COG~LAN, J., DULM~NIS, A.,
WINTOUR, M., AND EKKEL, I. 1961. The
Estimation of Testosterone in Biological
Fluids. Australian J. Exptl. Biol. Med.
Sei., 41: 235.
(71) JAILER, J. W. 1948. A Fluorometric Method
for the Clinical Determination of Estrone
and Estradiol. J. Clin. Endoerino]. Metabo-
lism, 8: 564.
(72) JAMES, A. T., AND MARTIN, A. L. P. 1952.
Gas-Liquid Partition Chromatography: The
Separation and Micro-Estimation of Vola-
tile F a t t y Acids from Formic to Dodecanoic
Acid. Biochem. J., 50: 679.
(73) JOHNSOh~, K. R., AND ERR, R. E. 1962.
Maintenance of Pregnancy in Ovariecto-
mized Cattle with Progestin Compounds
and Their Effect on Progestin Levels in
the Corpus Luteum. J. Dairy Sci., 45: 633.
(74) KARLSON, P. ]963. New Concepts on the
Mode of Action of Hormones. Pers. Biol.
Med., 6: 203.
(75) KARMEN, A., MCCAFFREY, I., AND KLIMAN,
B. 1962. Derivative Ratio Analysis: A
New Method for Measurement of Ster()ids
and Other Compounds Using Radioassay
by Gas-Liquld Chromatography. A~ml. Bio-
chem., 6: 31.
(76) I~ESTON, A. S., UNDEFRIEND, S., AND CAN-
NON, R. K. 1946. Microanalysis of Mix-
tures (Amino Acids) in the Form of Iso-
topic Derivatives. J. Am. Chem. Soc., 68:
]390.
(77) KLII~AN, B., AND BRIEFER, C., JR. 1964.
Gas-Liquld Chromatography of Carbon-14
and Tritium Labeled Steroids. (Abstract.)
Froc. Ann. Meet. Endocrine Soc.
(78) ]~RISTOFFERSEN, J. 1960. Ges~cogens in
Corpus Luteum of Cattle. Acta Endocrinol.,
33 : 417.
(79) I~U~AR, D., AND BARNES, A. C. 1963.
Studies on the Mechanism of Action of
328 W. 1%. GOMES AND R. E. ERB
Progesterone on the Human Myometrium.
Bull. Johns Hopkins Hosp., 113: 330.
(80) LAURITZEN, C. 1963. Biologische Wirkung
des 20 fl-Hydroxy-Pregn-4-en-3-on. Acta
Endocrinol., 44: 225.
(81) LISEOA, B. P. 1963. Separation and Char-
aeterisation of A*-3-Ketosteroids of the
Pregnane Series by Means of Thin-Layer
Chromatography. Acta Endocrinol., 43:47.
(82) LOY, R. G., MOSIIAN, W. H., AND CASIDA,
L . E . ]957. A Rapid Method for Purifica-
tion and Quantitative Estimation of Prog-
esterone from Luteal Tissue. J. Biol. Chem.,
229 : 583.
(83) LoY, R. G., ZIMBELMAN, i~. G., AND CASIDA,
L. E. 1960. Effects of Injected Ovarian
Hormones on the Corpus Lutemn of the
Estrual Cycle in Cattle. J. Aninlal Sei.,
19 : 175.
(84) McCANN, S. M. 1962. Effect of Progester-
one on Plasma Luteinizing Hormone Ae-
%ivity. Am. J. Physiol., 202:601.
(85) MOCLURE, T. J., AND RONALDSON, J. W.
]962. Progesterone Levels in the Ovarian
Venous Plasma of Ewes with Abnormal
Ova. New Zealand J. Agr. Research, 5: 507.
(86) MCCRACKEN,J. A. 1963. Plasma Progester-
one Concentration A f t e r Removal of the
Corpus Luteum in the Cow. Nature, 198:
507.
(87) McCRACKEN, J. A. 1964. Progesterone in
the Body F a t of the Dairy Cow. J. Endo-
crinol., 28 : 339.
(88) McDoNALD, L. E., MCNUTT, S. H., AND
NICHOLS, R. E. 1953. On the Essentiality
of the Bovine Corpus L~teum of Preg-
nancy. Am. J. Vet. Research, 14: 539.
(89) MARES, S. E., ZIMBELMAN, R. G., AND
CASIDA, L. E. 1962. Variations in Prog-
esterone Content of the Bovine Corpus Lu-
teum of the Estrual Cycle. J. Aninlal Sci.,
21 : 266.
(90) MASON, N. R., AND SAVARD,K. 1964. Speci-
ficity of Gonadotropin Stimulation of Prog-
esterone Synthesis in Bovine Corpus Luteum
in Vitro. Endocrinology, 74: 664.
(91) MAYEI%~D. T., GLASGOW, B. R., AND GAW-
IENOWSKI, A. 1961. Metabolism of Prog-
esterone and Their Physiological Signifi-
cance in the ~/rine of Pregnant and Non-
Pregnant Sows and Gilts. J. Animal Sci.,
20 : 66.
(92) MELA/~(PY,R. M., EIKMERSON, M. A., RAKES,
J. M., HANKA, L. J., AND ENESS, P. G.
1957. The Effect of Progesterone on the
Estrous Response of Estrogen-Conditioned
Ovariectomized Cows. J. Animal Sci., 16:
967.
(93) MELAIV~PY,R. M., HEAKN, W. R., AND RAKES,
J. M. ]959. Progesterone Content of Bo-
vine Reproductive Organs and Blood Dur-
ing Pregnancy. J. Animal Sei., 18: 307.
(94) MIKHAIL, J., NOALL, M. W., AND ALLEN,
W. M. 1961. Progesterone Levels in the
Rabbit Ovarian Vein Blood Throughout
Pregnancy. Endocrinology, 69 : 504.
(95) MILLER~ W. R., AND TURNER, C. W. 1961.
Evidence for an in Vivo Conversion of C21
Steroids to C19 Androgens in the Bovine.
J. Dairy Sei., 44: 2278.
(96) MILLER, W. R., AND TURNER, C. W. 1963.
Biosynthesis of Steroids from Pregnenolone-
16-H ~ by Bovine Ovarian Tissue in Vitro.
Steroids, 2: 657.
(97) MILLER, W. R., WILLIAMS, R., PIPES, G. W.,
AND TURNER, C. W. 1963. Conjugation,
Distribution, and Biological Half-Life
(t 1~) of Radioactive Progesterone in
Plasma and Red Cells of Bovine Blood.
J. Dairy Sei., 46: 1402.
(98) MIYAKE, T. 1962. Progestational Sub-
stances. I n R. I. Dorfman, Ed. Methods in
Hormone Research. Vol. II. Academic
Press, New York.
(99) MOORE, N. W., ROWSON, L. E. A., AND
SHORW, R. V. 1960. Egg Transfer in Sheep.
Factors Affecting the Survival and Devel-
opment of Transferred Eggs. J. Reprod.
and Fertil., 1: 332.
(100) MORRIS, C. J. O. R., AND MORRIS, P. ]963.
Separation Methods in Biochemistry. In-
terscience, New York.
(101) NATELSON, S. 1962. Progress in Biochem-
ical Investigations. I. Steroids : 1961.
Microchem. J., 6: 381.
(102) NEHER, R. 1963. Chromatographic Mobil-
ities. I n L. L. Enge], Ed. Physical Prop-
erties of the Steroid Hormones. MacMillan
Co., New York.
(103) NEUSTAEDTER, P. J. 1963. Selective Oxida-
tions of Polyhydroxyl Steroids and Non-
Catalytic Selective Reductions of Poly-
carbonyl Steriods. I n C. Djerassi, Ed.
Steroid Reactions. Holden-Day, Inc., San
Francisco.
(104) NOTEBOOM, W. D., AND GORSKI, J. 1963.
An Early Effect of Estrogen on Protein
Synthesis. Prec. Natl. Acad. Sci., 50: 250.
(105) OERTEL~ G. W., WEISS, S. P., AND EIK-NES~
K . B . 1959. Determination of Progesterone
in Human Blood Plasma. J. Clin. Endo-
crinol. Metabolism, 19: 213.
(106) OVF~EE~K, G. A., AND DE VISSER, J. 1964.
Different Modes of Action of Two Anti-
ovulatory Compounds. Aeta Endocrinol.
Suppl., 90: 179.
(107) PATTI, A. A., AND STEIN, A. A. 1964.
Steroid Analysis by Gas Liquid Chromatog-
raphy. C. C Thomas, Springfield, Illinois.
(108) PEARL]~IAN, W. H. 1960. Progesterone. In
H. N. Antonaides, Ed. Hormones in Human
Plasma. Little, Brown and Co., New York.
(109) RAESlDE, J. I., AND TURNER, C. W. 1955.
Chemical Estimation of Proges*erone in the
Blood of Cattle, Sheep, and Goats. J. Dairy
Sci., 38: 1334.
(110) RICE, B. F., HAIV/MERSTEIN, J., AND SAVARD,
K. 1964. Steroid Hormone Formation in
 PROGESTERONE IN BOVINE REPRODUCTION
the Human Ovary. II. Action of Gonado-
tropins in Vitro in the Corpus Luteum.
J. Clin. Endocrinol. Metabolism, 24: 606.
(111) RIONDEL, A. M., TAIT, J. F., TAIT, S. A. S.,
LITTLE, B., AND GUT, M. 1962. The Deter-
ruination of Progesterone and Testosterone
in Peripheral Blood with S~-Thiosemicarba-
zide. (Abstract.) Proc. Ann. Meet. Endo-
crine Soc.
(112) ROMANOFP, E. B., DESHPANDE, N., AND
PINCUS, G. 1962. Rate of Ovarian Prog-
esterone Secretion in the Dog. Endocrinol-
ogy, 70: 532.
(113) ROWLANDS, I. W., AND SHORT, R. V. 1959.
The Progesterone Content of the Guinea
Fig Corpus Lutemn During the Reproduc-
rive Cycle and After IIysterectomy. J. En-
docrinol., 19: 18.
(114) SOHWEPPE, J. S. 1961. A ]Review of Steroid
Hormones. I. Basle Physiological Steroid
Concepts with Comments upon Measurement
and Fraetionation Techniques. Northwestern
Medical School, Evanston, Ill., Quart. Bull.,
35 : 248.
(115) SEASE, J. W. 1947. The Use of a Fluores-
cent Adsorbent for the Chromatography of
Colorless Compounds. J. Am. Chem. Soe.,
69 : 2242.
(116) SNORT, R. V. 1956. Progesterone in the
Placentae of Domestic Animals. Nature,
] 78 : 743.
SHORT, R. V. 1957. Progesterone and Re-
(117)
lated Steroids in the Blood of Domestic
Animals. Colloq. on Endocrinol., CIBA
Found., 11: 362.
(118) SNORT, R. V. 1958. Progesterone in Blood.
I. The Chemical Determination of Prog-
esterone in Peripheral Blood. J. Endo-
crinol., 16:415.
(119) SHOR% R. V. 1958. Progesterone in Blood.
II. Progesterone in the Peripheral Blood
of Pregnant Cows. J. Endocrinol., 16: 426.
(120) SHOR% R. V. 1960. Blood Progesterone
Levels in Relation to Parturition. J. Re-
prod. Fertil., 1: 61.
(121) SNORT, R. V. 1961. Progesterone. I n C. H.
Gray and A. L. Bacharach, Eds. IIormones
in Blood. Academic Press, New York.
(122) SNORT, R. V. 1964. Ovarian Steroid Syn-
thesis and Secretion in Vivo. Recent Prog.
HHormone Research, 20: 303.
(123) SNORT, R. V., AND LEVITT, I. 1962. The
Fluerometric Determination of Progester-
one in Human Plasma During Pregnancy
and the Menstrual Cycle. J. Endocrinol.,
25 : 239.
(124) SHORT, R. V., McDONALD, M. F., AND
ROWSON, L. E. A. 1963. Steroids in the
Ovarian Venous Blood of Ewes Before and
After Gonadotropic Stimulation. J. Endo-
crinel., 26 : 155.
(125) SNORT, R. V., AND MOORE, N. W. 1959.
Progesterone in Blood. V. Progesterone
and 20 a-iiydroxypregn-4-ene-3-one in the
329
Placenta and Blood of Ewes. J. Endo-
crinol., 19: 268.
(126) SttORT, R. V., AND ROWELL, J. G. 1962.
The HHalf-Life of Progesterone in the Per-
ipheral Blood of a Ewe at Two Stages of
Gestation. J. Endocrinol., 25: 369.
(127) SILBIGER, M., AND ROTHCHILD, I. 1963.
The Influence of the Uterus on the Corpus
Luteum-PJtuitary Relationship ill the Ra~.
Acta Endocrinol., 43: 521.
(128) SIIVIMER, H. H., HILLIARD, ,I., AND ARCHI-
BALg, D. 1963. Isolation and Identification
of Progesterone and 20 a-iiydroxypregn-4-
en-3-one in Ovarian Venous Blood of Rab-
bits. Endocrinology, 72: 67.
(129) SIM1VIONS, K. R., AND HANSEL, W. 1964.
Nature of the Luteotrophic Hormone in
the Bovine. J. Animal Sci,, 23:136.
(130) SO1VIMERVILLE, I. F., PICKETT, M. T., COL-
LINS, W. P., AND DENYER, D. C. 1963.
A Modified Method for the Quantitative
Determination of Progesterone in Human
Plasma. Acta Endocrinol., 43: 101.
(131) SPIES, H. G., ZIlYII~ERI~£AN, D. R., SELF,
If. L., AND CASIDA, L. E. 1960. Effect of
Exogenous Progesterone on the Corpora
Lurer of Hysterectomized Gilts. J. Animal
Sci., 19: 101.
(132) STAPLES, R. E., AND IIANSEL, "~V. 1961.
Luteal Function and Embryo Survival in
the Bovine. J. Dairy Sci., 44" 2040.
(133) STAPLES, R. E., McENTEE, K., AND RAN-
SEL, W. 1961. Luteal Function as Related
to Pituitary and Ovarian Cytology and
Embryo Development in the Bovine.
J. Dairy Sci., 44: 2049.
(134) STORIV[SHAK~ F., AND EI~B, R. E. 1961.
Progestins in Bovine Corpora Lutea, Ovar-
ies, and Adrenals During Pregnancy.
J. Dairy Sci., 44: 310.
(135) STOK~SIIAK, F., INSKEEP, E. K., LYRIC,
J. E., POPE, A. L., AN]) CASIDA, L. E. 1963.
Progesterone Levels in Corpora Lutea and
Ovarian Effluent Blood of the Ewe. J. Ani-
mal Sci., 22: 1021.
(136) SWEAT, M. L., BERLINEI~, D. L., BEYSON,
M. J., NABORS, C., JR., HASKELL, J., AND
I-IOLMSTRO~, E. G. 1960. The Synthesis
and Metabolism of Progesterone in the
Ituman and Bovine Ovary. Biochim. et
Biophys. Acta, 40: 289.
TAI~IAOKI, B., AND PINCUS, G. 1961. Bio-
(137)
genesis of Progesterone in Ovarian Tissues.
Endocrinology, 69: 527.
(138) TELEGDY, J. 1963. Die Veranderungen des
Progeteron-und A'-Pregnenol-(20 a)-on-(3)-
Gehalts in Ovargewebe von Ratten wahrend
des Ostruszyklus. Endokrinologie, 44: 29.
(139) TELEGDY, J. 1963. The Ovarian Secretion
of Progesterone and 20 a-Hydroxypregn-4-
en-3-one in Rats During the Estrous Cycle.
Steroids, 2. 119.
(140) TEPPEI~I~AI~, J. 1962. Metabolic and En-
330 W. IK. GOMES AND R. E. Et~B
docrine Physiology. Year Book Medical
Publishers, Chicago.
(141) TEPPERI~£AN~ J., AND TI~PPEI~I~AN~ I~. M.
1960. Some Effects of Hormones on Cells
and Cell Constituents. Pharmacol. Revs.,
12 : 301.
(14~) TO~'H, F. 1964. Effect of Synthetic Prog-
estogens on the Ovaries. Intern. J. l%rti].,
9 : 151.
(143) TOUCHSTONE, J. C., AND MUI~AWEC,T. 1960.
Enhancement of the Fluorescence of Prog-
esterone (and Other Steroids) in Sulfuric
Acid. Anal. Chem., 32: 822.
(144) TI~IMBEI~GEI~ G. W., AND HANSEL, W. 1955.
Conception Rate and Ovarian Function Fol-
lowing Estrus Control by Progesterone In-
jection in Dairy Cattle. J. Animal Sei.,
14: 244.
(145) WIEST, W. G. 1959. Progesterone and 4-
Pregnen-20 a-ol-3-one in the Tissues of
Pregnant Rats. Endocrinology, 65:825.
(146) WIEST, W. G., AND FORBES, T. R. 1964.
Failure of 20 a-Hydroxy-A~-Pregnen-3-one
and 20 fl-Hydroxy-A4-Pregnen-3-oneto Main-
rain Pregnancy in Ovariectomized Mice.
Endocrinology, 74: 149.
(147) WILCOX, R. B., AND WIEST, W. G. 1960.
Comparative Effectiveness of 4-Pregnen-20
a-ol-3-one in the Development of Deciduo-
mata. Endocrinology, 67: 281.
(14s) WILLIAMS, W. F. 1962. Excretion of
Progesterone and Its Metabolites in Milk,
Urine, and Feces. J. Dairy Sci., 45: 1541.
(149) WILSON, IX., BOI~RIS, ~. J., AND GARRISON,
M. M. 1958. Chromatographic Procedure
for the Determination of Urinary Corti-
costeroids and Clo Steroids. J. Clin. Endo-
crinol. Metabolism, 18: 643.
(150) WOOLEVER, C. A. 1963. Daily Plasma
Progesterone Levels During the Menstrual
Cycle. Am. Y. Obstet. Gyneco]., 85: 981.
(~51) WOOLE%rER, C. A., AND GOLDFEII~, A. 1963.
A Double-Isotope Derivative Method for
Plasma Progesterone Assay. Intern. J.
Appl. Radiation and Isotopes, 14: 163.
(152) YANNON:E, M. E., McCoMAS, D. B., AND
GOLDFEIN, A. 1964. The Assay of Plasma
Progesterone. J. Gas Chromatog., 2: 30.
(153) ZAFFARONI,A. 1953. Micromethods for the
Analysis of Adrenocortical Steroids. Recent
Prog. Hormone Research, 8: 51.
(154) gANDER, J. 1962. Progesterone. I n R. I.
Dorfman, Ed. Meihods in Hormone Re-
search. Vol. I. Academic Press, New York.
(155) ZANDER, J., FORBES, T. R., V0N MUNSTEI~-
I~ANN, A. M., AND NEHER, R. 1958. A~-3-
Ketopregnene-20 a-ol and n~-3-Xetopreg-
nene-20 fl-o], Two Naturally Occurring Me-
tabolites of Progesterone. Isolation, Identi-
fication, Biologic Activity and Concentra-
tion in Human Tissues. J. Clin. Endocrinol.
Metabolism, 18: 337.
(156) Z~mROW, M. X., NEHER, G. M., LASOWASEM,
E. A., AND SALHANICK, H. A. 1957. Bio-
logical Activity of Certain Progesterone-
Like Compounds as Determined by the
Hooker-Forbes Bioassay. J. C]in. Endo-
crinol. Metabolism, 17: 658.
(157)
ZAI~ROW, M. X., YOCHI~i, J. M., AND MC-
CARTHY, J. L. 1964. Experimental Endo-
crinology. Academic Press, New York.
(158) ZI~BEL~AN, R. G., LOY, ]:~. G., AND CASIDA~
L. E. 1959. Biochemical Studies of tile
Bovine Corpus Luteum of Early Pregnancy.
J. Animal Sci., 18: 1551.
(159) ZIIM[BELMAN, n. G., LOY, R. G., AND CASIDA,
L . E . 1961. Variation in Some Biochemical
and Histological Characteristics of Bovine
Corpora Lutea During Early Pregnancy.
J. Animal Sci., 20: 99.
(160) ZI~[REL1ViAN, R. G., LOY, R. G., AND CASIDA,
L . E . 1961. Effect of Exogenous Progester-
one on the Bovine Corpus Luteum of Early
Pregnancy. J. Animal Sci., 20:106.
(161) ZIMBELMAN, R. G., MCSHAN, W. H., TYLER,
W. J., AND CASIDA, L. E. 1961. Effect of
a Pituitary Extract on the Bovine Corpus
Luteum of Late Pregnancy. J. Animal Sei.,
20 : 246.