Domestic Animal Endocrinology 23 (2002) 329–337

Between prepartum luteolysis and onset of expulsion

M.A.M. Taverne a,∗ , V.N.A. Breeveld-Dwarkasing a ,
F.M.F. van Dissel-Emiliani b , M.M. Bevers a , R. de Jong a ,
G.C. van der Weijden a
a
Department of Farm Animal Health, Faculty of Veterinary Medicine,
Utrecht University, Utrecht, The Netherlands
b
Department of Biochemistry, Cell Biology and Histology, Faculty of Veterinary Medicine,
Utrecht University, Utrecht, The Netherlands

Abstract
In the cow the foetal endocrine signals that initiate the calving process result in prepartum luteolysis.
Withdrawal of progesterone (P4) action is a prerequisite for a normal calving. The rather abrupt declining
influence of P4 is followed by a cascade of physiological processes in the myometrium and cervix. This
contribution will focus on some of these events. Like in many other species, the myometrium in cows
is not completely inactivated during pregnancy. So-called contractures have been registered during the
final weeks of gestation and their EMG-characteristics in cows show a low frequency (on average: 13.6
per day) and long duration (on average 12.1 min). They are not evenly spread over the day because they
occur less frequently when the cows are disturbed for feeding or cleaning their stables. Contractures af-
fect several foetal functions. In the cow these contractures disappear during a period of about 8–9 h when
maternal plasma P4 levels are rapidly declining before calving. There is experimental evidence that this
temporary inhibition is associated with prepartal luteal regression. The cause of this inhibition is still un-
known. Because nitrous oxide inhibits smooth muscle cells and evidence in laboratory animals indicates
that expression of the inducible form of nitrous oxide (iNOS) is downregulated in myometrium, but
upregulated in the cervix around the onset of parturition, we started to investigate the role of this enzyme
in bovine tissues around calving. By means of a RT-PCR technique, we obtained a first indication that
iNOS is hardly expressed in the myometrium during calving, while expression was clearly detected at
day 4 after calving. Analysis of prepartum en periparturient biopsies from myometrium and cervix with
quantitative PCR is still underway. In six pregnant cows, provided with uterine EMG-electrodes and
with ultrasonic crystals implanted on the caudal cervical rim to measure cervical dilatation, calving was
induced with an injection of prostaglandin (PG) F2␣. While maternal plasma P4 levels had significantly

∗
Corresponding author. Tel.: +31-302521887; fax: +31-302531183.
E-mail address: mam.taverne@vet.uu.nl (M.A.M. Taverne).

0739-7240/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 7 3 9 - 7 2 4 0 ( 0 2 ) 0 0 1 6 8 - 6
330 M.A.M. Taverne et al. / Domestic Animal Endocrinology 23 (2002) 329–337

declined within 8 h after PG treatment, the myometrium escaped from temporary inhibition with the
development of a parturient contractility pattern on average at 13.5 h after injection. However, it was
only at 28 h after PG treatment that the first sustained increase of the opening of the vaginal ostium of
the cervix was measured.
© 2002 Elsevier Science Inc. All rights reserved.

1. Introduction

Parturition in the cow is triggered by a steep rise in the fetal plasma level of cortisol [5]. This
induces hormonal changes in the pregnant uterus, such as an enhanced synthesis of oestrogens
and prostaglandin (PG), and these lead to functional regression of the corpus luteum and
a rapidly declining maternal plasma levels of progesterone [5,8]. Prepartal withdrawal of
progesterone (P4) appears to be essential for a normal calving process. At term pregnancy,
luteolytic doses of (analogues of) PG F2␣ will induce parturition in cows [11,17] and treatment
with the progesterone-receptor antagonist RU486 also induces calving [19]. Furthermore [14],
Janszen et al., demonstrated that pregnancies were maintained if pregnant cows were given
two norgestomet containing ear-implants before a luteolytic dose PG was injected on day 264.
It was only between 36 and 47 h after removal of these implants (on day 270) that the expulsive
stage of calving started. It seemed that norgestomet kept the cervix closed and/or suppressed
myometrial activity in these animals during the period (days 264–270) that a functional CL
was absent. Alternatively, progesterone of placental origin [24,26], as reflected by the low but
measurable plasma P4 levels after luteal regression, might have maintained the pregnancies in
this experiment. However, if that would have been the case, it is more difficult to understand that
a rather good synchronisation of the calvings was achieved upon withdrawal of the norgestomet
implants. Other experimental data also indicates that progesterone withdrawal is a prerequisite
for a normal calving process: when cows, in which the foetuses had been provided with vascular
catheters, were treated with exogenous progestagens from day 240 of gestation onwards, foetal
infusions of dexamethasone failed to induce premature calvings, in spite of an increase of
PG F levels in utero–ovarian plasma [7]. Yet, the cervix of these cows remained firm and
closed under progestagen treatment. The calves were expelled within 36 h after termination
of the progestagen treatment, but four of the six were born dead, most likely because of the
occurrence of ineffective uterine contractions during the period that cows were still under
progestagen treatment. This observation suggests that when two opposite forces (parturition
inducing signals, represented by PGs and the pregnancy maintaining signal, represented by
progesterone) are present at the same time, the see-saw [6] is bent, but it does not go out of
balance like in a normal parturition.
Yet, while prepartum P4-withdrawal appears an essential maternal event for the final expul-
sion of the foetus, relative little information is available as to its temporal relationship with
functional changes in the myometrium and cervix of the cow. This contribution will therefore
concentrate on (recent) experiments in which induction of parturition at term pregnancy (with
either PGs or corticosteroids) was used as a model to study some aspects of the activation of
uterine contractility and cervical dilatation.
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2. Experimental approaches

For the studies described below, pregnant cows (mainly pluriparous Dutch Friesians) with
known breeding dates were used. During a laparotomy under general anaesthesia, two to four
pairs of silver electrodes were sutured on the pregnant uterine horn [29] and a catheter was
installed in the maternal aorta through the circumflex artery. In some of the cows, a foetal
vascular catheter and an amniotic fluid catheter also was installed (for details see [31,15]). In
six of these cows, two ultrasound crystals (a transmitter and a receiver) were sutured opposite
to each other on the vaginal ostium of the cervix on the day on which induction of calving with
PG took place (technique described by [4]). This allowed continuous measuring of the transit
time of ultrasound between the two crystals, so that cervical dilatation could be investigated
in these cows in conjunction with myometrial EMG-activity and changes in the levels of P4
and PGFM in maternal plasma.
Instrumented cows were housed in an individual pen that did not allow them to turn around.
They received standard antibiotic treatment for 5 days after surgery and data collection usually
started between 5 and 10 days after surgery. Cows were fed twice daily and had free access to
water. They were observed by a closed television system and recording and storage of signals
took place in an adjacent room, leaving the animals undisturbed except for times that feeding,
bloodsampling or inspection of cables, catheters or equipment took place.
Blood sampling took place beside the cow’s pen through extended catheters. Samples were
immediately placed on ice and after centrifugation at 4◦ C plasma was stored at −20◦ C. Con-
centration of plasma progesterone and 15-keto-13.14-dihydro-PG F (PGFM) were measured
using RIAs validated for the cow.

3. Uterine EMG-activity during pregnancy

Throughout gestation the myometrium is not completely quiescent. Based on in vivo record-
ings of intrauterine pressure changes and/or electromyographic activity, a specific pattern of
so-called contractures has been found in different species, such as sheep [10,22,34], goat [30],
pig [28] and rhesus monkey [33]. Contractures represent longlasting (at least several minutes),
low frequent (less than 3 per hour) epochs of electrical and mechanical activity, characterised
by a relative small rise of intrauterine pressure and with EMG-activity persisting also during
the declining phase of the pressure cycle (Fig. 1). Myometrial contractures may affect several
foetal physiological functions, such as foetal blood PO2 , fetal behavioural states, breathing
activity and plasma ACTH levels (review by [22]). Early studies in our lab [29] had already
demonstrated that contractures also occur in pregnant cows. A more detailed and accurate
characterisation of contractures can presently be given for the cow, based on 46 complete 24-h
recordings, which were made in 15 chronically instrumented cows during the last 4 weeks of
their pregnancies (Table 1).
Analysis of variance showed that neither the day of gestation, nor the type of uterine in-
strumentation (with or without opening of the uterus for a fetal surgery) did affect these
characteristics. The rather low frequency of contractures implies that prolonged recording
sessions have to be made in cows. This is highlighted by the observation that occurrence
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Fig. 1. Example of a myometrial contracture, recorded in a cow on day 278 of gestation. The EMG-signal is from
an electrode implanted on the pregnant uterine horn, while the intrauterine pressure was measured by an amniotic
fluid catheter attached to a hindleg of the foetal calf.

of contractures was not equally distributed over the 24 h of the day (Fig. 2). A significantly
lower frequency was found between 05:00 h and 06:00 a.m. (P < 0.01), between 04:00 h and
05:00 h. (P < 0.05) and between 08:00 h and 9:00 p.m. (P < 0.01). Because feeding and
cleaning of the stable is routinely scheduled in our animal hospital during these hours, one
might assume that these activities disturbed the animals in such a way that contractures were
less likely to be initiated spontaneously. Experimental studies in pregnant sheep, in which both
the intact pregnant uterus and autotransplanted grafts of myometrium were provided with elec-
trodes, have demonstrated that contractures represent intrinsic myogenic activity. However, it
is also known that local and systemic hormones, such as oxytocin, PG and catecholamines,
may affect the occurrence of contractures [20,23,32]. In the rhesus monkey the presence of a
24-h rhythm in myometrial activity during the final stages of gestation has been attributed to
changes in circulating oxytocin levels and myometrial responsiveness to oxytocin [12,13]. In
late pregnant cows, physiological doses of exogenous oxytocin induce contractures [32], but
oxytocin is hardly detectable in maternal plasma before calving [25] and there are no data in
cows indicating that oxytocin secretion changes during the day.

Table 1
Characteristics (means±SEM) of myometrial contractures in pregnant cows, based on 46 complete 24-h recordings
in 15 cows between days 250 and 275 of gestation
Frequency of contractures 13.6 ± 0.9 per day
Duration of contractures 12.1 ± 0.3 min
Duration of intervals between contractures 76.5 ± 3.1 min
 M.A.M. Taverne et al. / Domestic Animal Endocrinology 23 (2002) 329–337 333

Fig. 2. Distribution of the mean ± SEM frequency of myometrial contractures for each of the 24 h of the day in 46
complete 24-h recordings. Frequencies labelled with an asterisk are significantly different from the overall mean
frequency.

4. Myometrial activity and cervical dilatation between luteolysis and

onset of expulsion

Another, yet unexplained, feature of contractures in pregnant cows is that they rather sud-
denly disappear shortly before calving, at the time that luteolysis occurs [15]. Using induction
of calving by means of a single flumethason injection on day 270 as a model, an almost com-
plete inhibition of uterine EMG-activity occurred during a period of about 8–9 h, starting at
about 16 h after flumethason injection.
During this period maternal plasma P4 concentrations had decreased already significantly,
while PGFM levels had increased. A similar, temporary disappearance of contractures was ob-
served during PGF2␣-induced luteolysis in cows, in which pregnancy was meanwhile main-
tained by progestagen containing implants [16]. Escape from temporary inhibition begins
between 12 and 18 h before the onset of second stage labour. During the subsequent hours, a
parturient pattern of myometrial activity gradually develops [15,18]. This pattern is charac-
terised by reappearance of contractures and the appearance of contractions. The latter ones
occur at increasing frequency, are short lasting (less than 90 s) and are well-propagated over
the pregnant uterine horn [29].
Janszen et al. [16] postulated that the prepartum period of uterine quiescence forms a
time window, allowing several functional changes to occur in both myometrium and cervix,
that are essential for the initiation of labour-like uterine contractions and the start of cer-
vical dilatation. In other species, increased synthesis of proteins, such as those involved in
the formation of gap-junctions or ion-channels, occurs during the immediate prepartum pe-
riod (review by [21]). More recently we found increasing maternal arterial plasma levels of
334 M.A.M. Taverne et al. / Domestic Animal Endocrinology 23 (2002) 329–337

interleukin-1 [18] and an increased in vivo sensitivity to physiological doses of oxytocin [32]
during and shortly after the hours that contractures had temporarily ceased to occur after
induction of calving with PG. Based mainly on experiments with laboratory animals, the in-
ducible isoform of the enzyme nitrous oxide synthase (iNOS) appears to play a role in the
change from myometrial inhibition to activation at early stages of the parturition process.
Together with progesterone, NO inhibits smooth muscle activity through a cGMP pathway
during pregnancy. Expression of iNOS is downregulated in the myometrium, both at spon-
taneous parturition at term and during progesterone-receptor blocked preterm labor in rats,
while expression in the cervix is upregulated at these occasions (reviews by [9,27]). Such
a reversion of expression at two different sites of the same pregnant uterus would explain
one of the enigmas of periparturient physiology, namely that the myometrium becomes acti-
vated while smooth muscle cells within the cervix are supposed to be relaxated. Although
iNOS activity could be present not only in smooth muscle cells, but also in endothelial
cells, stromal cells and in invading inflammatory cells within the genital tract, the iNOS
hypothesis stimulated us to investigate the expression of this enzyme also in tissues of the
periparturient cow.
As a first step, we recently isolated mRNA (RNeasy Kit, Qiagen) from myometrial sam-
ples taken from a parturient cow, undergoing to a caesarean section because of an oversized
calf. Another sample was obtained by means of a laparoscopy from a cow at 4 days after a
PG-induced, normal calving. Bovine granulosa cells were used as positive control. After a
reverse transcriptase step, cDNA of iNOS and ␤-actin (as a housekeeping gene) were submit-
ted to PCR, using primers based on the published sequence of human iNOS and used before
in experiments with bovine macrophages by Adler [1]. Gels of the PCR-products obtained
are shown in Fig. 3. These preliminary data indicated a low level of iNOS expression during

Fig. 3. The mRNA of iNOS and ␤-actin in myometrial samples from a cow during parturition and from another
cow at 4 days after calving. Note that while ␤-actin shows a similar pattern of expression in these two samples,
iNOS expression is very weak in the sample taken during calving, but is markedly expressed at 4 days after calving.
Bovine granulosa cells were used as a positive control.
 M.A.M. Taverne et al. / Domestic Animal Endocrinology 23 (2002) 329–337 335

Fig. 4. Dilatation (in centimeter) of the caudal cervix (recorded as mean values during each hour) in six cows, in
which calving had been induced by injection of a PGF2␣ analogue at time 0 (on day 274). The dashed line is from
a cow with the calf in posterior presentation [2].

calving, while pronounced expression was observed at day 4 after calving, i.e., at a stage that
myometrial contractility has almost disappeared again. In biopsies, repeatedly collected from
the myometrium and the caudal cervix of the same cows around calving, we are presently
investigating if a transition from high to low iNOS expression (myometrium) or vice versa
(cervix) can be found around the onset of parturition.
Repeated biopsing of the cervix will also allow to analyse changes in water content and
collagen biochemistry, in conjunction with hormonal changes, myometrial activation and the
actual final dilatation of the caudal cervix. Recently [3], we measured onset and rate of dilata-
tion around PG-induced calvings. Preliminary data are presented in Fig. 4. Mean peripheral
plasma progesterone concentrations had reached values ≤1 ng/mL within 8 h after PG-injection
in these cows. It is obvious from these data that dilatation of the caudal cervical canal starts at
some 28 h after PG injection and that dilatation is well synchronised between the six animals.
The mean time of onset of the parturient pattern of myometrial EMG-activity was already at
13.5 h after induction. This means that on average about 14 h elapse between escape from my-
ometrial inhibition and the first sustained increase of the caudal cervical opening. In fact such
a temporal relation between hormonal changes, myometrial activation and cervical dilatation
has not been investigated before in any other parturient mammal. This nicely programmed
sequence of events around PG-induced calvings makes the cow a very attractive model for
future basic and clinical studies [2].
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