Reprod Dom Anim 41, 522–526 (2006); doi: 10.1111/j.1439-0531.2006.00707.x
ISSN 0936-6768
The Role of Oestradiol in the Uterine Peristalsis in the Perfused Swine Uterus
T Maltaris, R Dittrich, W Widjaja, C Sindhuwinata, I Hoffmann, MW Beckmann and A Mueller
Department of Obstetrics and Gynaecology, University-Hospital Erlangen, Erlangen, Germany
Contents
This study was designed to examine the effects of oestradiol
(E2) on sperm transport in the swine uterus. The bicornuate
swine uterus is optimal for the study of the uterine transport
and peristalsis because the influence of various factors can
be examined on each uterine horn independently. Forty
swine uteri (with or without ovarectomy) were perfused for
a period of up to 7 h. Two different E2 concentrations (3 or
30 pg/ml) in the perfusion medium were administered for
30 min unilaterally. Through an intracervical catheter 1 ml
of a high concentrated dextran blue solution was administered directly in the upper part of the cervix. After bilateral
perfusion of the swine uterus with a bolus of 0.3 IU
oxytocin the distribution of coloured particles was assessed
macroscopically before and after incision of the uterine
horns. Coloration was evaluated by two observers blinded to
the site-specific administration of E2. In the 10 ovarectomized uteri with the 3 pg/ml E2 concentration a unilateral
distribution towards the side of oestradiol administration
was observed in six uteri, in four it was a bilateral
distribution. In the 10 non-ovarectomized uteri with the
3 pg/ml E2 concentration a uni- and ipsilateral coloration
was observed in five uteri, in five it was a bilateral
distribution. In the 20 uteri with 30 pg/ml E2, a unilateral
coloration of the uterus horns was observed in all uteri.
Oestradiol is one of the main factors, which influences the
direction of the sperm transport in a dose-dependent
manner, in the perfused swine uterus.
Introduction
In mammals oestradiol is the dominating hormone
during the periovulatory phase when uterine contractions rise in frequency and amplitudes. In humans the
oestradiol peak during the menstrual cycle occurs
when an effective transport mechanism towards the
fundus uteri and the Fallopian tube is needed for
sperm transport (Lyons et al. 1991; Fukuda and
Fukuda 1994). In the sow uterine contractility is
maximized at the time around oestrus and can be
increased after oestrogen administration (Langendijk
et al. 2002a).
Perfusion models of various organs have been of great
interest, particularly in the field of transplantation
medicine and in studies of the physiology and metabolism of tissues. We have previously demonstrated that
the perfusion model used here is capable of keeping the
swine uterus in a functional condition for up to at least
7 h and that it is appropriate for the study of physiological questions (Dittrich et al. 2003; Maltaris et al.
T. Maltaris and R. Dittrich contributed equally to the study.
2005a,b; Müller et al. 2006). The experimental system
detects electrical and mechanical activities in the whole
organ, as it maintains the architecture and intercellular
relations of the uterus (Bulletti et al. 1988, 1993; Richter
et al. 2000, 2003, 2004, 2006).
The swine uterus is a bicorneate organ and therefore
ideally appropriate for examining the influence of
various factors on the uterine transport and peristalsis
because each uterine horn can be perfused independently. The swine produces many offspring in contrast to
the humans and has no single dominant follicle.
Therefore, under normal physiological conditions, there
is no difference in oestradiol concentration in the uterine
horns. The uterine lumen is a relatively hostile environment for the semen and uterine contractions are
probably important for the transportation of sperm
cells to the uterotubal junction as soon as possible after
insemination (Langendijk et al. 2002b). There is few and
sometimes contradictory literature available concerning
the role of uterine contractility on the transport of
sperm cells and fertilization (Baker et al. 1968; Rodriguez-Martinez et al. 1987a,b,c; Claus 1990; Langendijk
et al. 2002b).
The present study examined the direct and isolated
effect of two oestradiol concentrations regarding sperm
transport, with or without ovarectomy in the perfused
swine uterus. The aim of this study was to examine
whether the increased oestradiol concentration in one
uterine horn could stimulate the uterine contractility
and thus direct the sperm transport towards this specific
horn.
Methods
Swine uterus
Swine (Sus scrofa domestica) are widely used in research.
The female reproductive system in the swine has a
bicornuate uterus with tortuous Fallopian tubes. The
Fallopian tubes in an adult female have the same
diameter as those in the human, but they are much
longer. Forty swine uteri were obtained from a local
slaughterhouse. They were selected on the basis of their
size, overall condition and the condition of the uterine
arterial stumps. The mean weight of the swine uteri was
149.8 g (105.6–230.2 g). They all came from healthy
animals aged 5 months to 1.5 years. On the basis of
previous observations involving perfusion experiments
(Dittrich et al. 2003), it was decided that the ideal size of
uteri for the experiments would be approximately 90–
200 g. The swine uteri were very easily separated from
the rest of the body in approximately 2 min shortly after
the animal had been killed by electric shock (1.5 A,
400 V, 4 s).
Ó 2006 The Authors. Journal compilation Ó 2006 Blackwell Verlag
Perfused Swine Uterus as a Model for Sperm Transport Experiments
Perfusion system
After cannulation of both uterine arteries with 16- to 24gauge needles (Abbocath-T; Abbott, Sligo, Ireland)
depending on the size of the uterus, the organ was
placed in a controlled-temperature perfusion chamber
(Karl Lettenbauer, Erlangen, Germany) filled with the
perfusion medium (Fig. 1a). The uterus was then
connected bilaterally to two reservoirs containing the
perfusion buffer (Krebs–Ringer bicarbonate-glucose
buffer; Sigma, Deisenhofen, Germany).
The perfusion medium was oxygenated with carbogen
gas (a mixture of 95% oxygen and 5% carbon dioxide)
and then pumped into the uterine arterial catheters with
two roller pumps. The flow rates of the perfusion
medium and oxygenation were constantly monitored
and kept at 15 ml/min and 0.05 bars, respectively, with
an ideal pressure rate of 100 mmHg being maintained
throughout the duration of the experiments.
17-b-oestradiol (3 or 30 pg/ml b-oestradiol, E4389;
Sigma-Aldrich, Deisenhofen, Germany) was added in
the perfusion medium and administered always only in
one uterine horn. It is known (Almond and Dial 1990)
that in the sow the maximum E2 serum concentration is
at the periovulatory period (approx. 30 pg/ml), whereas
the minimum E2 concentration is present at the
diestrous (approx. 10 pg/ml).
523
was ‘bilateral’. The uterus was then incised and photographed (Fig. 1b, c).
Results
In all 40 uteri there was a significant blue coloration of
at least one of the uterine horns. In none of the 40 uteri
could a contralateral blue coloration be detected to the
oestradiol-treated uterine horn.
Group A (ovarectomized swine uteri)
In the 3 pg/ml group six uteri showed a significant
ipsilateral coloration and four uteri showed a bilateral
coloration (Table 1). In the 30 pg/ml oestradiol administration group all 10 uteri showed a significant ipsilateral blue coloration.
Group B (intact swine uteri)
In the 3 pg/ml group five uteri showed a significant
ipsilateral blue coloration and five uteri showed bilateral
Vitality parameters
Samples of the perfusate were taken at 1-h intervals after
collecting the medium for measurements of pH, PO2,
PCO2, HCO3, lactate and oxygen saturation (for details,
see Dittrich et al. 2003).
Study design
Twenty swine uteri were perfused after ovarectomy with
two oestradiol concentrations (30 or 3 pg/ml) in order to
simulate the hormonal environment of the sow in the
oestrus and diestrus. In order to examine the effect of
oestradiol on the sperm transport in the intact swine
female genital tract, the experiment was also performed
on twenty non-ovarectomized uteri.
Evaluation of blue dextran distribution
Thirty minutes after the continuous oestradiol administration was started, blue dextran solution (20 mg/ml
Blue Dextran, D5751; Sigma-Aldrich) was administered.
Blue dextran cannot be absorbed by the tissue and was
used to simulate the semen fluid. Oxytocin (Syntocinon;
Novartis Germany Ltd, Nuremberg, Germany) was
used as a bolus through the uterine arterial catheters to
induce contractions of the uterus at a dose of 0.3 IU.
Uterine contractions occurred normally immediately
after oxytocin administration.
Five minutes after blue dextran administration, two
observers, blinded to the side in which oestradiol was
administered, examined the uterus independently. Only
if both examiners stated that the same uterus horn was
blue coloured was the test ‘unilateral’, if one horn was
not significantly more blue coloured, then the decision
Fig. 1. (a) Perfused swine uterus in the organ bath. (b, c) Two swine
uteri with unilateral blue coloration after incision
Ó 2006 The Authors. Journal compilation Ó 2006 Blackwell Verlag
524
T Maltaris, R Dittrich, W Widjaja, C Sindhuwinata, I Hoffmann, MW Beckmann and A Mueller
Table 1. Ipsilaterally blue coloured swine uteri after a 17-b-oestradiol
perfusion
Ovarectomized swine uteri (n ¼ 20)
Intact swine uteri (n ¼ 20)
3 pg/ml
17-b-oestradiol
perfusion
30 pg/ml
17-b-oestradiol
perfusion
6
5
10
10
blue coloration. In the 30 pg/ml oestradiol administration group all 10 uteri showed a significant ipsilateral
blue coloration.
Discussion
In humans adequate uterine contractility is involved in
transport of the semen and gamete and in successful
embryo implantation while inadequate uterine contractility may lead to ectopic pregnancies, miscarriages,
retrograde bleeding and endometriosis (Leyendecker
et al. 2004; Bulletti and de Ziegler 2005). Intact uterotubal transport function and directed sperm transport
due to uterine contractility are therefore of critical
importance in processes of human reproduction (Kissler
et al. 2004, 2005). In fertile women the directed unilateral transport of immotile sperm-like material through
the female genital tract to the side bearing the dominant
follicle can be assessed using hysterosalpingoscintigraphy (Kunz et al. 1996, Kissler et al. 2004).
In swine, sperm cells have to be transported from the
cervical end to the tubal end of the uterine horns after
mating or artificial insemination. The length of the
uterine horns is about 0.8 m during oestrus. Sperm
transport is thought to be a combination of both passive
and active transport (active sperm movement). The
active sperm transport is thought to be responsible for
the migration of sperm cells from the proximal uterus
into the uterotubal junction and the oviduct. There a
sperm reservoir is built, until about the time of
ovulation, which acts as a barrier to sperm cells, because
of its morphology, the consistency of the luminal mucus,
oviductal motility, sperm-epithelial adhesion and ciliary
movements of oviductal cells (Rodriguez-Martinez
2000). Passive transport is more important in the initial
phase of transport from the side of the deposition to the
proximal uterus and the uterotubal junction (Scott
2000). The passive transport is probably driven by the
flow of intrauterine fluid-containing sperm cells, due to
gravitational force, and uterine contractions (Baker
et al. 1968; Scott 2000). This phase is also called rapid
phase and lasts only some minutes (Rodriguez-Martinez
et al. 2005). The transport of the spermatozoa to the site
of fertilization constitutes the third phase and is
regulated by the disappearance of the intraluminal
mucus in the sperm reservoir, the increase in the flow
of fluid by increasing ciliary activity towards the
ampullary-isthmic junction, and/or the conspicuous
motility of the myosalpinx (Rodriguez-Martinez et al.
1982; Rodriguez-Martinez 2000).
The bicornuate swine uterus is optimal for the study
of the uterine transport and peristalsis because the
influence of various factors can be examined on each
uterine horn independently.
Swine have been increasingly used as biomedical
research models during the last 20 years, as they are
recognized as a suitable animal model for human disease
on the basis of comparable anatomy and physiology.
The use of uteri from freshly killed animals from the
slaughterhouse had the logistical advantage that obtaining a large number of uteri every day only took a matter
of minutes, and in addition, approval from an ethics
committee or animal experimentation board was not
needed, as no animals were killed for experimental
purposes. Moreover, the uteri were derived from healthy
young animals in their reproductive years.
Normally, the uterus would rapidly degenerate without constant perfusion. A complex perfusion system was
therefore established that provided the uteri with a
steady flow rate of circulation, a constant temperature
and continuous oxygenation of the perfusion medium,
simulating physiological conditions in every possible
way. In contrast to previous swine uterus perfusion
experiments the intrauterine pressure changes were not
registered in order to avoid manipulation of the cervix
by the intrauterine catheter which might be expected to
affect the uterine activity or the migration of blue
dextran solution. In half of the animals, ovariectomy
was performed to avoid an influence in the hormone
levels of the own ovaries. In the other half of the animals
the ovaries were not removed, in order to examine the
effect of uterine peristalsis in an intact swine uterus.
This study demonstrates that uterine contractility is
mainly responsible for sperm transport from the cervical
to the tubal end. Furthermore, we were able to
demonstrate that oestradiol is a major factor determining sperm transport. As there were no other hormones,
serum factors or seminal plasma factors in the perfusion
system, it can be assumed that oestradiol was the only
variable factor that influenced the transport of dextran
blue solution towards the one uterine horn that received
oestradiol treatment.
Our experiments showed that within 5 min the whole
uterine horn was coloured with blue dextran solution.
Other researchers have already found out that uterine
contractility can promote the distribution of spermcontaining fluid throughout the whole genital tract, even
enabling unilaterally, deep intrauterine deposited semen
to be distributed over both horns (Rath 2000). The
question remains as to how oestradiol which fluctuates
characteristically during the menstrual cycle is involved
in influencing uterine peristalsis. Richter et al. (2003)
reported that oxytocin receptor expression is upregulated by oestrogens not only in pregnant but also in nonpregnant human uteri (Richter et al. 2003, 2004).
Franczak et al. (2002) also stated that estrogens are
evident stimulators of uterine oxytocin receptors synthesis in gilts. Also an increase in intrauterine pressure in
response to oxytocin was most evident in sows in estrous
(Zerobin and Spörri 1972; Langendijk et al. 2002a) and
parturition (Zerobin and Spörri 1972).
Therefore, it can be logically suggested that oestrogens may support uterine contractility and act synergistic with oxytocin in the regulation of uterine peristalsis
and transport mechanism towards the tubal ends.
To our knowledge this is the first study of unilateral
administration of drugs by perfusion in an extracorpo-
Ó 2006 The Authors. Journal compilation Ó 2006 Blackwell Verlag
Perfused Swine Uterus as a Model for Sperm Transport Experiments
really perfused uterus. These experiments show that the
perfused swine uterus is an appropriate system for
examining the physiology of the genital tract and offers
the possibility of examining the action of specific
hormones or pharmaceuticals directly on one or both
uterine horns. In these experiments the cervical-tubal
sperm transport promoting effect of oestradiol was
proved.
Acknowledgements
This work was supported by the University of Erlangen-Nuremberg
ELAN funds.
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Submitted: 18.12.2005
Author’s address (for correspondence): Dr Ralf Dittrich, Department
of Obstetrics and Gynecology, University of Erlangen-Nuremberg,
Universitaetsstrasse 21–23, D-91054 Erlangen, Germany. E-mail:
[email protected]
Ó 2006 The Authors. Journal compilation Ó 2006 Blackwell Verlag