BIOLOGY
OF REPRODUCTION
Coital
Stimuli
32, 925-933
Controlling
(1985)
Luteinizing in the
R. S. CARROLL,2
Hormone
Female
M. S. ERSKINE, and
Department Massachusetts
P. C. DOHERTY, M.
and Ovulation
L. A. LUNDELL,
J. BAUM
of Nutrition Institute
Cambridge,
Secretion
Ferret’
and Food Science of Technology
Massachusetts
02139
ABSTRACT A series of izing hormone ment investigated
experiments focused on the masculine coital behaviors controlling pituitary lutein(LH) secretion and reflex ovulation in the estrous female ferret. An initial experiwhich coital stimuli from the male are required to induce ovulation. It was found luteum formation, which served as an index of ovulation, occurred in estrous female
that corpus ferrets only if the male achieved a penile intromission. Neck gripping, mounting, and pelvic thrusting behavior without intromission by the male failed to induce ovulation. A second experiment investigated the timing and magnitude of the coitus-induced LH surge associated with ovulation. Blood was obtained via jugular catheters from estrous females in various mating situations. Plasma LH concentrations were measured by a heterologous radioimmunoassay that was validated for use in the ferret. A significant surge in plasma LH occurred only when an intromission was achieved by the stud male. Plasma LH was significantly elevated 2.0 h after the introduction of the male, peak values were reached 6.0 h later, and this elevation lasted on average 5.7 hours (5/5 females). No LH rise occurred in 2/2 female ferrets in which only neck gripping, mounting, and pelvic thrusting, but no intromission, were allowed to occur. The ferret mating pattern and the resultant LH response differ from those seen in three other induced ovulators (cat, vole, and rabbit) in which the male’s intromission latency and duration are much shorter than in the ferret, and in which a distinctive peak in plasma LH often occurs within 1 h after mating.
INTRODUCTION Much
variation
species precedes hormone
in
peak
and
most
of
ferret) mission have
it
(Dufy-Barbe
among
these
after species
has been is necessary very
various
mating. (vole,
presumed to induce
few
studies
components
plasma LH surge vole (Charlton
Accepted Received This
ovulating
Although
rabbit, that penile ovulation,
on the
actual
LH
and
after
values
duration
introthere
(2
effects copula-
now.
secretion in the coitus-
than
Goodman
Neill,
and
and
in
LH the
rise rabbit
of
neural cycle
gonadotropins the
control
is subject
photoperiod.
1904). the
Estrus
While the to
begins
the tonic
to
syn-
occurs
with
in
and may not occur the
male
female on the dorsal surface of her the male maintains a neck grip, he female
display
which is
Mating
of the
and
lengthening photoperiod, up to 5 mo if copulation does
thrusting,
925
(5-6
to
begins
vole cat
by
h)
The
in the
the until
mounts
sion
shorter
reached
later.
in
gripping
neck.
was
h
the
chrony
vice grants HD-13634 and RCDA MH-00392 to M.J.B., MH-15761 training grant support to R.S.C., and National Service Award HD-06333 to P.C.D. R. S. Carroll, Boston University, Dept. of Biology, 2 Cummington St., Boston, MA 02215.
levels
4
and
secretion
Ser-
these
h). The LU response to coitus ferret has not been characterized
(Marshall, Health
coitus,
In the ferret the seasonal reproductive
response last for Public
1973;
approximately
of the
h)
(10-12 female
have been carried out et al., 1975), rabbit
10, 1984. 23, 1984. supported by
mm
30-60
peak
December
August work was
et al.,
1976), and cat (Wildt et al., 1980; Johnson and Gay, 1981). In general, a 20-40-fold increase in plasma LH concentrations occurred within
in
cat,
of masculine
tory behavior on coitus-induced females. Studies characterizing induced for the
reflex
the pattern of mating behavior that an ovulatory surge in luteinizing (LH) and in the timing of this LH ovulation
been
of the
exists
achieved.
and
after
intermittent
terminate In
one
several periods
when study
minutes of
pelvic
an intromis(Baum
and
CARROLL
926
Schretlen, from
1975) 2.5
to
addresses two coital stimuli induce
mm in
intromissions duration.
related questions. from the male
ovulation
Second, surge male’s
single
151.3
in the
is there
estrous
and
varied This
First, which are needed to
a coitus-induced
ferret?
plasma
LH
in females and, if so, which aspects of the sexual behavior cause this surge? MATERIALS
AND
50
EDTA
paper
female
ET AL. antiserum
1:500
(1:32,000
normal
in
rabbit
PBS/0.05
serum;
M
Antibodies
Incorporated, Davis, CA). Tubes were incubated 5#{176}C for 24 h and then 25 M1 of ‘25I-Iabeled LH luted
1:50
in PBS/1%
hours
later
the precipitating
BSA)
were
added.
antibody,
at (di-
Twenty-four
50
M’
of sheep
antirabbit gainmaglobulin (Antibodies Incorporated; diluted 1:40 in PBS/1% BSA), was added. After an additional 24 h at 5#{176}C,the tubes were centrifuged (30 mm at 4#{176}C,1006 X g), the unbound label in the supernatant was decanted, and the activity
in the
METHODS
M1 of with
pellet
was counted on a Micromedic gamma A standard curve ranging from 0.02 to 10 ng LH (NIH-LH-S20) was used (Fig. 1). Increasing volumes of serum from ovariectomized ferrets and dilutions of homogenized ferret pituitaries yielded competition curves parallel to the ovine LH standard (Fig. 1). The pituitaries were first weighed and then homogenized in an equivalent volume of normal saline. Serial dilutions were taken from this stock. It counter.
Animals Adult female Fitch ferrets (Mustela furo) in postpartum estrus were purchased from Marshall Research Animals (North Rose, NV). The ferrets were housed individually in rabbit cages with water available ad libitum. Purina Cat Chow was provided in bowls each morning. The lights in the ferret colony were off between 2200 and 0600 h.
was also found that a single intrajugular g of gonadotropin-releasing hormone man
Materials
Catheters for blood collection were comprised of an extrajugular portion (i.d. 0.040 mm X o.d. 0.070 mm, polyvinyl; approximately 8” in length) and an intrajugular portion (i.d. 0.030 mm X o.d. 0.048 mm, polyethylene PE-60; approximately 6 cm in length). The catheters were constructed by using a hemostat to enlarge the end of the polyvinyl tube, into which the small polyethylene tube was then inserted. 0-rings, used to secure the catheter (cut from #5 French intragastric feeding tube; Argyle), were placed 5 mm apart at the junction of the two tubes. All junctions were sealed with dichloromethane. Catheters were left to air dry for 24 h and then were gas sterilized. Surgical
Procedures
Animals anesthesia were used
were anesthetized using pentobarbital (45 mg/kg), and sterile surgical procedures at all times. Females were ovariectomized
via a single midline incision. Females were given jugular catheters using a procedure similar to that described by Florczyk and Schurig (1981). A small skin incision was made on the top of the head, between the ears, and the catheter was threaded subcutaneously to emerge at this site. The catheter was maintained patent by flushing it with 2 ml of heparinized saline (100 U/mI) every other day.
Luteinizing
dins,
1983).
A
Palo
Alto,
CA)
of 4 Beck3-fold
increase in plasma LH, which peaked in about 15 min and returned to baseline after an additional 45 min (Fig. 2). The intraassay coefficient of variation was 8.8%. This value was based on 40 samples run in duplicate on two assays with LH values ranging from 0.5 to 4.5 ng/ml. All samples collected in this study were run in one of two LH assays, each of which included samples from females in the intromitted group and the nonintromitted group. The peak LH values for both groups were comparable in the two different assays. Concentrations of LH were calculated using the four-parameter logistic method (Rodbard and Huts, 1974). The levels of LH measured by RIA in the ferret were very low compared to those reported in other species. Therefore, increasing volumes of ferret plasma from mated estrous females (n=3) were run on a mouse Leydig cell bioassay (courtesy of Dr. Tony Plant) in order to determine whether the LH being measured using the ovine:antiovine RIA was physiologically active. Plasma samples containing basal levels of LH failed to stimulate testosterone secretion in the Leydig cell bioassay. However, increasing volumes of postmating samples, which contained peak levels of LH (approximately 4 ng/ml by RIA), caused dosedependent increases in testosterone secretion that were parallel to those caused by the ovine LH standard. Histology
Hormone
A heterologous measure ferret LH and terHaar, 1977;
Instruments,
injection (GnRH; caused a
radioimmunoassay
was
used
to
(Niswender et al., 1969; Donovan Ryan et al., 1983; Sisk and Desjarrabbit antiserum (GDN 15) raised
against ovine LH was used at a final concentration of 1:32,000. Highly purified ovine LH (LER-1056-C3) was radiolabeled with ‘“1 (New England Nuclear, Boston, MA) using a solid-phase oxidizing agent, 1, 3,4,6-tetrachloro-3o,6a-diphenylglycoluril (lodogen, Pierce Chemical Co., Rockford, IL; Franker and Speck, 1978; Salacinski et al., 1981). Each assay tube contained 85 MI of 1% bovine serum albumin (BSA) in 0.05 M phosphate-buffered saline (PBS), 40 M’ of plasma or standard (NIH-LH-S20) in PBS/1% BSA,
Ovaries
mersed
were
in Bouin’s
removed
from
their
capsules,
im-
for 24 h and embedded
in
sections were cut, mounted gel-coated and stained with hematoxylineosin prior to being examined for the presence corpora lutea. The total number of corpora lutea two ovaries is presented in the Results.
on
paraffin.
General
solution
Serial 30-Mm glass slides,
of per
Procedures
I. Fourteen estrous females were put cage with a sexually experienced male. For of females (n=6), the male was allowed to neck grip, mount, and pelvic thrust. However, he was removed from the cage before penile intromission Experiment
into one
a test group
LUTEINIZING
HORMONE
p/Serum
SECRETION
(u)12.5
98
IN THE
FERRET
25
50
100
I
I
927 200
400 I
S
90
Ovariectomized
Ferret
Serum
50
10
I,
I,
ng NZH-LHS-2O(q Pituitary
Dilution FIG.
Fcc/or 1.
nate (m=-1.98),
0.05
0.01
10
iO
-
1O
standard
(m-2.03),
1O
102
(A)
Luteinizing and
hormone ovariectomized
inhibition ferret
curves for ovine serum (m=-2.47).
45
MINUTES 2. Effect
5
L_
30
FIG. ferrets.
0.5
0.1
of
intrajugular
injection
of
LU
60
AFTER 4 Mg of
GnRH
pituitary
homoge-
ovariectomized
female
ferret
(m=slope)
75
GnRH on
plasma
INJECT/ON LH
levels
in two
CARROLL
928 occurred. copulatory
In
the
group (n=8), all the male were allowed to occur, including an intromission. In some cases, the duration of the intromission received by various females was varied by removing the male from the test cage after different intervals. The durations of thrusting and intromission behavior were recorded during each test. One week after mating both ovaries were removed from all females and processed histologically. Experiment II. Seven estrous females were removed from their home cages and an extension (2.5 ft) was attached to their intrajugular catheters to allow repeated blood sampling during testing. Each female was put into the test cage alone for 45 mm, and second
behaviors
baseline
blood samples (0.7 ml) were taken every 15 A stud male was then put into the cage for two of three test situations and blood sampling continued every 15 min for 1 h. During this time an event recorder was used to record male and female sexual
mm.
behaviors
(duration
of
exposure
to
the
male;
was
in the test
allowed;
cage with
or
3) the
no male
female
present.
At
was the
RESULTS
placed
end
of
1
h (except if an intromission was still in progress), the male was removed from the test cage. Intromission was prevented in certain tests by using males that were known to have long intromission latencies. For the subsequent 3 h samples were taken every half hour, and then for the next 12 h samples were taken every hour. After the samples were centrifuged and the plasma taken, the red blood cells were resuspended in heparinized normal saline and were returned to the female through the jugular catheter. One week after mating both ovaries were removed from all animals and were processed histologically.
TABLE
1. Relationship
between
Animal number Ovulating
females
females
times
are given
Ovulation,
as evidenced
corpora lutea when the male 1).
An
intromission
sufficient behaviors were
by
the
in either ovary, was allowed to as
short
as
1 mm
The
other
not
by
themselves
Only
large
to
follicles
induce found
that received of luteinization
ever seen in these females. lation between intromission
There was duration
of
corpora
lutea
formed
no was
no correand the (r=-O.46;
n.s.).
of intromission
and corpus luteum formation in estrous
intromissiona
lutea/2
29.4 10.3 49.0 25.0 94.0 27.2
7.9 1.7 4.3 6.0 4.8 5.9
44.3 24.0
8.5 2.2
15.0 1.0 39.7 15.0 86.8 6.8 2.5 18.5
11 9 6 10 8 8 10 9
1A 2A
5.0 21.0
1.3
-
0
1.6
-
0
3A
7.0 20.6 25.0 25.0
0.3 2.4 0.4 0.2
-
0
in minutes.
to were
in the ovaries of females intromission. No evidence
number
was male
intromission
sufficient
antral
of
occurred (Table
induce ovulation. that occurred prior
to
ovulation.
presence
only intromit
thrustinga
1 2 3 4
identity
I
Duration
4A 5A 6A aAIl
occurrence
Experiment
Time with malea
5 6 7 8 Nonovulating
the
Luteinizing hormone values for the five animals that received an intromission were analyzed by a one-way analysis of variance (ANOVA) for repeated measures. For each animal we computed mean LH values for pretest samples (n4), preintromission samples (n=1-2), and samples (n=3-4) taken concurrently with intromission. These values were compared with the LH levels measured in individual samples taken 2.0-16 h after the introduction of the male. Post-hoc comparisons with preintromission values were made with Dunnett’s test (Bruning and Kintz, 1977). For all females the area under the LH peak was calculated from the 60-mm time point until the point at which the mean LH value was no longer significantly higher than the mean preintromission value (this occurred 12 h after introduction of the male).
neck
gripping, mounting, pelvic thrusting, intromission; frequency of female bites; and duration of female receptive postures). Three different test situations were used: 1) the male was allowed to neck grip, mount, pelvic thrust, and intromit; 2) the male was allowed to neck grip, mount, and pelvic thrust, but no intromission
ET AL.
of
Duration
of
Number
-
0
-
0
-
0
ferrets.
of corpora ovarie
LUTEINIZING
Experiment
HORMONE
SECRETION
II
measured
A surge in plasma LH was only observed in estrous females if the male achieved an intro-
the
mission
used.
(compare
Figs.
3 and
4).
Fig.
3 shows
the LH peaks (height and duration) for ual females that received an intromission; occurrence of different male copulatory iors
is also
indicated.
The
mean
individthe behav-
(± SEM)
pretest
LH value was 0.66 ± 0.04 ng/ml; the mission value was 0.74 ± 0.08 ng/ml; LH
concentration
rently
with
Plasma
LH
cantly
the
of testing
were
(i.e.,
8 h after
introduction
LH subsequently after introduction ± 0.21 ng/ml) from
the can
was
cause
levels.
Fig.
sampling
4 with
plasma
induce
LH.
Table
the number female, and
2 shows
formed n.s.,
in
about tions
LH
surge
frequent
lutea under
blood
no
relative
effect
on
As and
intromission On
ferret
increases
ferret LH
does that
exhibit results
in
occurs achieved
only by
behaviors
that
were
themselves
by
significant
rise
elevation
in
ferret
and
other
estrous and
mounts,
sion
suggest
the
a postcoital
the
surge
ovulation,
female
in plasma
and
that
this
after an a male.
intromission has been The masculine coital
occurred
prior
not
in
plasma
serum
LH
minimal
sufficient LH.
The
in one
to absence
unmated
LH
fluctuations
ferrets that pelvic thrusting
received without
that
the
significant
cause a of an estrous in
neck
two grips,
intromis-
rise
in
LH
time
to
in
number
in
corpora
in
lasting
the
Wildt
for
lutea
cat
more
h (Fig.
duration, LH
al.,
some aL,
mm of
was
(1981) cats
increased to were
and
plasma
magnitude those
ovulators.
in
of ferrets
previousIn
rabbits
Kanematsu
et
queens
(Concannon
et al.,
1980;
Johnson 1982;
reported following
at
an
even
a single
in 5 mm,
peaked in to
and
a peak
Gay,
mating:
et
within by
0.5-2
4 h. Johnson faster
al.,
Schille
20-40-fold
reached elevated
baseline allowed
in
1973;
copulation, still
rise
the
was
3).
from
reflex
In
mm
as 2.3
measured
different
other
male an
1 h or more, occur.
a clear-cut 6.0
plasma
et
for
as short
1981; Banks and Stabenfeldt, al., 1983), LH was elevated
LH
in single
the duration of duration of the
would
induce
et as in
returned queens
Johnson a
of
hand,
LH
were
found
Gay
were
concentrations
following
other
latency,
postcoitally
10 h, and
LH
ovulated.
et al., 1980;
ovulation
lasted
The
estrous
to an intromission
the
plasma
have
LH
intromission
sufficient
under the LH n.s., respec-
the
in plasma an
LH that
that
copula-
of
coital stimulation from the copulations), which induced that
1980;
show
multiple
in
to
increased
the
prolonged (multiple assured
DISCUSSION
rise
plasma
or
formed.
1974), results
a
an LH
In cats, as with the ferrets study, only those females that
1981),
(Dufy-Barbe
present
that
100%
copulation. Neither the area nor the LH peak was related to the
ly
The
1
intromission
infer
whereas
ovulation
ferrets
elevation
tively).
the
ferret lutea
induce ovulation. In the cat a induced ovulation in only
in the cat (Wildt
between number of
correlation either the
corpora
We therefore
confirmed
Gay,
durations
in each female’s
in both
every
to 94 mm. Corpora ferrets that showed
all
a definite
subsequently
behaviors,
formed each
or the area and r=-0.031,
or LH
time
from
though
cases,
present
female
plasma
of
a single
(Wildt et al., 1980). exhibited
LH
surge.
50%
that
of the
1
an
was sufficient
had
even
not
procedures
in
Ovaries
intromission
induced
of the
had
the
LH
ng/ml)
that them-
II.
varied from found in
were
Plasma
copulatory
There was no duration and
corpora lutea peak (r=-0.36,
peak
in
that
mating
after
male).
an
present
masculine of corpora the area
different
LH peak. intromission
shows male
wk
± 0.80
increase
also no
an
of
100%
I and
rise is needed to single copulation
over
in Fig. 4, the behaviors intromission were by to
ovulation
receiving
collection cats
was
and
an intromission,
=5.21,
value.
a significant
induce
intromission stimulus,
from
with
plasma
10 h later (i.e., 12 h male) to a level (1.55 not significantly differ-
preintromission
insufficient
even
of
that
differ
duration
noted
and
Ferrets copulation,
this
blood
the
signifi-
introduction
of the
declined of the
be seen prior to
occurred
selves
first
after
± 0.23 ng/ml), found 6 h later (3.8
values
As
was
h
of
929
following of
lutea
(F19,76
(2.35
male
ent
value 2.0
ferrets
result
artifact
to
FERRET
ng/ml.
elevation
(P
