Hum. Reprod. Advance Access originally published online on January 26, 2006
Human Reproduction 2006 21(3):624-631; doi:10.1093/humrep/dei394
An intravital microscopy method permitting continuous long-term observations of ovulation in vivo in the rabbit
1 Department of Obstetrics and Gynecology, Sahlgrenska Academy, Göteborg University, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden, 2 Department of Obstetrics and Gynecology, Kanazawa University Medical School, Kanazawa, Japan and 3 Department of Zoology, Göteborg University, Göteborg Sweden
4 To whom correspondence should be addressed. E-mail: pernilla.dahm-kahler{at}vgregion.se
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BACKGROUND: A method for intravital microscopy of the rabbit ovary was developed to enable observations of real-time changes during ovulation in vivo. The aim was to correlate these events to biochemical events at specific stages of ovulation. METHODS: Virgin, female rabbits were primed with equine chorionic gonadotrophin (CG) (30–100 IU) then HCG (100 IU) 2 days later to induce ovulation. During anaesthesia, the right ovary was surgically exteriorized and submerged in an organ chamber with a microscopy lens positioned close to the ovary. Continuous video recordings were performed. RESULTS: Initial equine CG priming experiments revealed the highest ovulation rate, without premature luteinization, after 30 IU equine CG. This priming protocol subsequently demonstrated follicular ruptures 11.5–14 h after HCG. Numbers of ovulations from the exteriorized and contralateral non-exteriorized ovary were similar. The sequence of typical features of ovulation was: shutdown of microcirculation in the follicular apex, formation of petechiae in the follicular wall and a cone-shaped structure over the future rupture site, marked bleeding in connection with follicular rupture and a fairly steady extrusion velocity of granulosa cells and the oocyte. CONCLUSION: This method captured a sequence of structural changes during ovulation. It could be combined with blood and follicular fluid sampling for biochemical analysis and could be used in studies on biochemical reactions in relation to specific changes in the follicular structure during ovulation.
Key words: follicle/intravital microscopy/ovary/ovulation/rabbit
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Introduction |
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The biochemical features of ovulation have been studied extensively over recent decades and modern molecular biology techniques, such as DNA array analysis applied to the ovary (Leo et al., 2001
), have identified several new potential intraovarian mediators of ovulation. Knowledge about the biochemical cascade of ovulation has thus developed considerably (Brännström and Janson, 1991; Richards et al., 2002) but less attention has been given to structural and haemodynamic alterations during the ovulatory process.
Pioneering experiments with observations in vivo in the rabbit were able to capture the gross events of the ovulatory process (Walton and Hammond, 1928; Hill et al., 1935; Markee and Hinsey, 1936). Later on, histological techniques demonstrated several typical changes in the morphology of the follicle during the interval from the LH surge until follicular rupture (Bjersing and Cajander, 1974b–d). Moreover, specific features of the changes in the vascular system were captured by corrosion cast techniques (Kanzaki et al., 1982; Okuda et al., 1983a, b).
To obtain further insight into the dynamics of ovulation, intravital microscopy techniques were developed for studies in vitro in the perfused rabbit (Löfman et al., 1982) and rat (Löfman et al., 1989) ovary. These studies could be performed since the established methodology, which was originally designed for studies of biochemical changes during in vitro perfusion, was modified for cinematography and video recordings. However, it should be emphasized that these intravital microscopy observations were obtained in vitro, in the absence of several suggested endogenous ovulatory factors, such as a nerve supply (Owman et al., 1975), a number of blood plasma proteins, such as kinins (Brännström and Hellberg, 1989), and leucocytes (Hellberg et al., 1991). Thus, the results from these experiments may not have mimicked the in vivo situation precisely.
Further refinements of methodology, permitting longitudinal in vivo observations, may help to characterize the ovulation process at a more physiological level. To enable observations of ovulation in vivo, techniques were developed for the rat ovary, using cinematographic recordings on 16 mm film (Blandau, 1955) and modern high-resolution video recording techniques (Löfman et al., 2002). The in vivo observations in the rat (Löfman et al., 2002) were difficult to interpret since the ovary-enclosing bursa, which is a natural component of the ovarian pedicle of the rat, had to be mechanically opened and retracted over the ovarian surface before recording. This procedure may involve a risk of injury to the follicle surface and may also change the important physiological interplay between the follicle and the components of the bursa (Shinohara et al., 1986; Krishna and Jaiswal, 1994). Furthermore, the small size and restricted transparency of the preovulatory follicle of the rat made it very difficult to capture the changes in the exterior follicle wall, even at high magnification (Löfman et al., 2002).
The two species of experimental animal in which the greatest knowledge of biochemical changes of ovulation has accumulated are the rat and the rabbit (Brännström and Janson, 1991; Brännström et al., 1997). These two species have traditionally been used for in vitro studies of ovulation, involving morphological observations with the light microscope (Bjersing and Cajander, 1974b) and electron microscope (Bjersing and Cajander, 1974a) and biochemical analysis of levels of potential ovulatory mediators (Tanaka et al., 1991; Zanagnolo et al., 1996a). Furthermore, a large number of in vitro studies on isolated follicles (Brännström et al., 1993) and perfused ovaries have been conducted (Wallach et al., 1984; Mikuni et al., 1998b, c; Komar et al., 2001) in these two species. These facts prompted us to develop an intravital microscopy model in the rabbit to extend our previous in vivo studies on the rat (Löfman et al., 2002) and in vitro studies on the perfused rabbit (Löfman et al., 1982) and rat (Löfman et al., 1989) ovary.
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Animals
The experiments were approved by the animal ethics committee of Sahlgrenska Academy at Göteborg University. Virgin, female New Zealand white rabbits (age around 5 months) weighing approximately 4 kg were used. The animals (n = 16) were kept under controlled conditions (12 h daylight and 12 h darkness) in the animal quarter for at least 1 week before experiments and had free access to water and pelleted food. The rabbits were primed with equine chorionic gonadotrophin (CG) (Sigma-Aldrich, St Louis, MO, USA) at doses of 30, 50 or 100IU given s.c., and 100 IU HCG (Profasi®; Serono, Solna, Sweden) was injected i.v. around 48 h (mean 48 h ± 3.4 [SEM]) later to induce ovulation.
Anaesthesia
Two Teflon catheters (23 gauge; BOC Ohmeda, Helsingborg, Sweden) were inserted and secured in large veins of each ear to obtain full i.v. access throughout the experiments. A mixture of dormicum 0.3 mg/kg (Dormitor® 1 mg/ml; Orion Pharma, Espoo, Finland) and ketamine 10 mg/kg (Ketalar® 50 mg/ml; Pfizer, Täby, Sweden) diluted in NaCl (0.154 mol/l) to a total volume of 10 ml was used for induction of anaesthesia. The initial induction dose of the combined solution was 2 ml i.v. and for maintenance 1–2 ml was given at intervals of approximately 10 min until gas anaesthesia was secured. A tracheotomy was performed and a 3.0 tracheal tube (Mallinckrodt Medical, Athlone, Ireland) was inserted and fixed. Gas anaesthesia was then started with 2% isoflurane (Abbott Scandinavia, Solna, Sweden) with air (600 ml/min) and O2 (200 ml/min) using a ventilator (B. Braun Melsungen, Melsungen, Germany) with settings of tidal volume of 45 ml and 36 inhalations/min. The rabbits were given continuous parenteral fluid infusion with Ringer acetate (Baxter Medical, Kista, Sweden) of 25–33 ml/h throughout the experiments. At the end of the experiments the animals were given euthanasia with pentobarbital (100 mg/ml, Apoteket, Sweden) i.v. (15 ml) and intracardially (5 ml). The procedure was finalized by surgical opening of the heart. The ovaries were removed from the rabbits and fixed in formalin, embedded in paraffin, sectioned and stained with haematoxylin and eosin for subsequent estimation of numbers of ovulations and corpora lutea.
Surgery
A polyethylene catheter (outer diameter 0.96 mm, inner diameter 0.6 mm; Intermedic VWR, Göteborg, Sweden) was inserted into the femoral artery and flushed with heparin solution (100 IU/ml). This catheter was used to control blood pressure and pulse rate. Thereafter a flank incision of length 30–40 mm was made on the right side. The muscle layer was gently dissected and the peritoneum opened. The ovary was then identified and gently exteriorized (Figure 1a). Care was taken to avoid any bleeding during the dissection of the skin, muscle layer and peritoneum since bleeding would later disturb microscopic vision. Haemostasis during dissection was performed by bipolar diathermia (Coa-Comp Bikoagulator; Instrumenta, Billdal, Sweden). Two holding sutures (3.0 Vicryl) were placed just lateral to each ovarian pole to enable positioning of the ovary within the organ chamber.
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Technical equipment
A system was constructed to enable continuous microscopic observations of the exteriorized ovary under conditions that would control and monitor the physiological state of the animal. A V-shaped aluminium, thermostat-regulated animal bed was used to hold the rabbit during the entire experiment. Rectal temperature was monitored continuously and the heating of the bed was adjusted to maintain the body temperature at 38.5°C. Breathing was controlled by a mechanical ventilator (see above). Blood pressure and pulse rate were monitored by a pressure transducer coupled to an RPS 7D Grass polygraph (Grass Instrument Co., Quincy, MA, USA). The right exteriorized ovary was slipped through a slit in the bottom of the V-shaped animal bed and submerged into the specially designed organ chamber (Figures 1b and 2). The chamber was made of anodized aluminium with side walls of glass (thickness 1 mm), and contained a basal section allowing water circulation for heating (Figure 3). The organ chamber was filled with Ringer acetate (60 ml), which circulated and was kept at 38.5°C by heating from the bottom part of the chamber. Furthermore, the circulating Ringer acetate was preheated to 44°C before entering the organ bath to remove dissolved gas and to avoid the interposition of tiny air bubbles between the lens and the observed ovary.
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A schematic drawing of the experimental design is given in Figure 3. The microscope equipment was composed of parts assembled on an anodized brass tube (outer diameter 30 mm) to enable placement of a video lens (magnification 4x; Sony, Tokyo, Japan). The front of the lens was placed at a distance approximately 26 mm from the ovarian surface. The lens was connected to a video camera (3 CCD Colour camera; Panasonic, Osaka, Japan) via the brass tube. A diaphragm from a film camera lens was installed in the brass tube in the space just over the microlens to enable light adjustment. Two light sources with flexible light guide cables and focusing lenses (Zeiss) illuminated the tissue from the side and from behind. The level of exposure was controlled with an oscilloscope (Hamlet Video International, Chesham, UK) and the light was adjusted via the diaphragm of the microscope or a diaphragm on the light source. All video sequences were simultaneously recorded on a computer and the software Motion Studio (www.loudmotionstudio.com) was used to analyse of the recordings.
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Modifications
In the initial experiments it was obvious that leakage of blood from small vessels of the peritoneum and muscle layer interfered with the view of the ovary. To avoid this, bipolar diathermia was introduced as a means of obtaining complete haemostasis at the cut surfaces. During the course of the experiments it was also noted that the majority of the preovulatory follicles that would rupture were situated with their apical areas towards the intra-abdominal dorsal side. Thus, the animals would then be positioned exclusively in a direction so that the dorsal side of the ovary was facing the microscopy lens. Moreover, placement of two holding sutures at each ovarian pole were introduced to enable finer adjustments of the ovarian position within the organ chamber. To prevent the ovary from being in an unfavourable position regarding the distance from the microscopic lens, an extra wall of an acrylic sheet product (Plexiglas; 3 mm) was introduced into the organ chamber to keep the ovary at a set position, with only 1.5 mm distance between the dorsal ovarian surface and the front glass wall, where the microscopy lens was positioned.
Systemic parameters
During the prolonged anaesthesia, blood pressure was kept fairly constant (mean 64.1 mmHg ± 1.9 [SEM]) with stable pulse rate (mean 232 ± 4/min). The body temperature of the rabbits was kept stable and within the physiological range (mean 38.4 ± 0.1°C) and the fluid in the organ chamber had a temperature similar to that within the body (mean 38.4 ± 0.1°C).
Ovulation induction
Both ovaries (n = 32) from all rabbits (n = 16) were examined by histology and the numbers of ovulations (rupture points/newly formed corpora lutea) were counted in the groups with different doses of equine CG priming and alternate times from HCG injection (100 IU) to examination.
In the first set of experiments, with the priming dose of 50 IU equine CG followed 48 h later by HCG, the ovaries (n = 6) were obtained 27–31 h after HCG and both rupture points (mean 7 ± 0.6 per ovary) and clearly visible corpora lutea (mean 2 ± 1.3 per ovary) were present. Priming with 100 IU equine CG (n = 2) was also tested, but examination showed premature luteinization. To find out whether the visible corpora lutea represented ovulations that occurred before the expected ovulation time in relation to HCG, experiments using the same dose of equine CG (50 IU), but with the animals killed at an earlier (16–18 h after HCG) stage (within 3–5 h after ovulation) were conducted. This would show whether any corpora lutea had been formed during equine CG priming (before HCG). Thus, at this time point all ovulations induced by HCG would be visible as ruptured follicles. In the ovaries (n = 12), both ruptured follicles (mean 3 ± 1.2 per ovary) and well-developed corpora lutea (mean 5 ± 0.9 per ovary) were seen. This clearly demonstrates premature luteinization with this priming protocol using 50 IU equine CG.
In subsequent experiments using 30 IU equine CG followed by HCG (100 IU) examinations (n = 12 ovaries) were made at a similar periovulatory stage (16–18 h after HCG), and rupture points (mean 2.5 ± 0.8 per ovary) were found, but with the absence of corpora lutea. This priming protocol was used for detailed studies on ovulation (see below).
Taking all experiments together, no differences were seen between the numbers of ovulations on the exteriorized ovary (n = 16 ovaries; mean 3.6 ± 0.8 per ovary) and the contralateral non-exteriorized ovary (n = 16 ovaries; mean 4.3 ± 1.0 per ovary).
Ovulations
Several typical features of the ovulatory process were noted during video recordings for up to 18 h after 30 IU equine CG and 100 IU HCG (n = 6). During the period from around 1 to 3 h prior to follicle rupture, a typical change in the shape of the follicle was noted. The entire ovarian surface over the ovulatory follicle was gradually becoming more protruding, thereby making the follicle more obvious as a follicle undergoing ovulatory changes (Figure 4a, b). Furthermore, the large blood vessels (presumably venules) in the follicle wall decreased in diameter by approximately 50% of their original diameter. Some of the blood vessels over the apex of the follicle also became invisible, indicating a complete shut-down of blood flow (Figure 4a, b).
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The ovulatory process then proceeded with the formation of a transparent zone that was formed over around one-third of the exterior follicular wall (Figure 4b). Just basal to this area of increased transparency, several areas of petechiae (range 5–9 petechiae/follicle) were formed (Figure 4c) well before (time range 7–45 min) the actual rupture. The next common feature was the formation of a stigma cone at the coming rupture site (Figure 4c). The period from the appearance of this stigma until actual rupture was around 2 min (range 50–250 s).
Clear follicular fluid then suddenly emerged from the follicle (Figure 5a). The follicle rupture would shortly afterwards change from leakage of follicular fluid to extrusion of granulosa cells (Figure 5b), generally followed by large amounts of blood (Figure 5c–f) and a larger quantity of granulosa cells, including the oocyte–cumulus complex. The bleeding stopped (Figure 5f) within minutes (range 115–455 s). The granulosa cells with the oocyte stayed trapped within a sticky basket-like structure of follicular fluid, attached to the surface of the ovary. No contraction of the follicular wall was seen.
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Discussion |
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In this study a technique allowing observation of the entire ovulatory process in vivo in the rabbit was developed. The main reason to set up this in vivo microscopy technique was to achieve a method that can be used to observe the dynamics of the prolonged ovulatory process in the rabbit and to combine this knowledge with available data on the biochemical changes of ovulation.
The process of ovulation is the central event of the ovarian cycle. This process encompasses several hours of finely tuned and time-regulated structural and functional changes within the preovulatory follicle, and it ends in follicular rupture with extrusion of an oocyte. The fact that the ovulatory process continues over several hours and that it involves dynamic physiological events, such as alterations in blood flow (Zackrisson et al., 2000) and intrafollicular pressure (Matousek et al., 2001), that are orchestrated by a number of biochemical pathways (Brännström and Janson, 1991; Richards et al., 2002) warrants the development of techniques that also allow this process to be studied from an overall perspective and with the possibility of experimental manipulation. These manipulations involve, for instance, systemic or local administration of perturbants to affect the ovary.
Very few reports exist that describe the visual capture of the entire ovulatory process under in vitro or in vivo conditions. Typical features of rabbit ovulations that have been captured by cinematographic video recordings during in vitro perfusion conditions are the formation of an ovulatory cone and a spurt of granulosa cells through the ruptured follicle wall (Löfman et al., 1982). In our follow-up in vitro perfusion study in the rat (Löfman et al., 1989), it was noted that, at least in vitro, there are distinct differences between ovulation in the rabbit and the rat. The preovulatory follicle of the rat appeared to degrade more extensively at the apex and for a longer period before rupture in comparison with that of the rabbit (Löfman et al., 1989). Moreover, loss of single granulosa cells through this partly degraded apex of the rat follicle was seen for several hours before the actual rupture occurred. Observations of ovulatory events in vitro might not fully mimic the physiological situation since the in vitro perfusion system lacks blood components and the ovary has no nerve supply.
The results of the present study represent the outcome of the latest methodology achieved in our attempts to develop a method to capture ovulations in vivo in the rabbit. Initially, we developed an organ chamber that was constructed to be placed in an intra-abdominal position within the rabbit and could be used in making video recordings through a fibre-optic instrument of small diameter inserted through a laparoscopic port (our unpublished observation). Several technical problems, such as difficulty in the optimal positioning of the ovary and bleeding from nearby tissues, were encountered and made it difficult to capture the ovulatory process with a reasonable quality of recording. Thus, we set out to develop an intravital microscopy technique with the ovarian pedicle partly exteriorized with similarities to the in vivo technique developed previously for studies of cycle-related events of the oviduct and ovary of the rat (Löfman et al., 2002).
In the present study we tested several equine CG doses to achieve a high ovulation rate. Previous experience is that equine CG priming is also a means of increasing the ovulatory response in the rabbit (Zanagnolo et al., 1996b) by LH-and FSH-like action (Christakos and Bahl, 1979). The ovulation rates in both the exteriorized and the exteriorized ovary seen in the resent study are within the same range as those occurring after natural mating (Salhab et al., 2001, 2003). The reason for the premature development of corpora lutea after higher equine CG doses, as demonstrated in the present study, is likely to be that the LH-like activity (Christakos and Bahl, 1979) at this higher dose of equine CG reaches levels that will initiate ovulation and luteinization.
In earlier studies on the isolated perfused rabbit ovary, LH-induced progesterone production was enzymatically blocked without changing the ovulation rate (Holmes et al., 1985; Yoshimura et al., 1987). However, it was found in the rat that progesterone had an important mediatory role in LH-induced ovulation (Brännström and Janson, 1989) and studies with knockout mice have since verified the importance of progesterone receptor activation for ovulation (Chen et al., 1995; Lydon et al., 1995). Thus, species differences seem to exist regarding the role of progesterone in ovulation. In the present methodological work we did not measure progesterone, but in future applications of this in vivo model such measurements, with blockers of progesterone synthesis and progesterone receptors, light might be shed on the controversial issue of the role of progesterone in ovulation in the rabbit.
In our previous study of ovulation in vivo in the rat (Zackrisson et al., 2000) we observed distinct changes in follicular blood flow and also found that bleeding occurred at some ruptures. There are no published reports on the appearance of ovulation in the rabbit in vivo except for three historical notes (Walton and Hammond, 1928; Hill et al., 1935; Markee and Hinsey, 1936) with drawings and descriptions of the rabbit follicle in the periovulatory phases. Old techniques, such as the use of ether as an anaesthetic, observation through a binocular dissecting microscope and a complete open abdomen with the intestines wrapped in moist cloths without monitoring the physiological state of the animal (Walton and Hammond, 1928), or only light microscopic histology (Markee and Hinsey, 1936), were used. Only one of these notes (Hill et al., 1935) truly captured follicle rupture in the rabbit, but only the gross morphology of the rupture was described. In the present study we used both modern video recording techniques and computer software that allowed analysis of the observations. It is also important to point out that the animals in the present study were monitored closely to ascertain that blood pressure and temperature were kept stable. The recordings showed a normal temperature and a pulse rate, which was in the normal range of 230–260 beats/min (Hiruta et al., 2005). The blood pressure was somewhat lower than the normal range of mean arterial pressure of 80–95 mmHg (Hiruta et al., 2005); we think this is due to the systemic effects of isoflurane anaesthesia, which is well known to occur during prolonged anaesthesia in the human. This minor decrease in blood pressure should not be of any physiological significance in regard to ovulation.
The findings of the present study show for the first time that distinct vascular changes occur at ovulation in vivo in the rabbit. General phenomena of ovulation were the early appearance of a decrease in the diameter of blood vessels, followed by the formation of a translucent avascular area on the top part of the follicle and then a distinct shut-down of the blood flow in the large vessels that crossed the follicle from the base to the top. The shut-down of blood flow to the apex and the creation of an avascular zone correspond to the results of Doppler flow measurements in the human follicle, where a gradual decrease in blood flow in the apex and increased blood flow in the basal area of the follicle were indicated (Brännström et al., 1998). Further indication of the formation of an avascular area in the apex area comes from corrosion cast studies in the rabbit (Kanzaki et al., 1981). By the use of an injection–corrosion technique coupled with scanning electron microscopy, it was revealed that, in the midovulatory phase, the blood vessels of the enlarged ovulatory follicles were prominent and the vascular network of the apical region protruded above the surface of the ovary. However, at a later stage (2–3 h before rupture) the apical region of these follicular casts was defective, with an oval-shaped area at the apex, indicating the presence of microthrombi or arteriovenous shunts (Kanzaki et al., 1982). Moreover, the absence of blood flow in the apex was noted in the rat ovary prior to ovulation (Löfman et al., 2002).
It has been speculated that the avascular zone at the top of the follicle may facilitate a site-specific necrosis to enable rupture of the follicle. Another possibility is that the shut-down of the blood flow of the top of the follicle would prevent major blood loss during the ovulatory event. The cause of this vascular change in the apical region of the follicle prior to actual rupture is not yet known, but it may be related to the formation of thrombi, the redirection of blood flow, or the vasoconstriction or regeneration of blood vessels. Vasoactive substances could account for the shut-down or redirection of blood flow from the apex to the base of the follicle (Brännström et al., 1998). The vascular system of the follicle has a basket-like structure in the theca region formed by major vessels from the base and sides of the follicle. The action of vasoconstrictive agents on small arterioles that feed the vessels to the apex would promptly decrease the blood flow to that region. Angiotensin II and endothelins are potent vasoconstrictors that have been identified in the ovary. Angiotensin II exerts its action through binding to a group of receptors, which are subclassified into type 1 (AT1) and type 2 (AT2) receptors. The concentration of angiotensin II in human follicular fluid increases after treatment with HCG or LH (Lightman et al., 1987) and this result was confirmed by an in vitro study in the rabbit (Yoshimura et al., 1994). It was previously reported that the blockade of the AT2 receptor in the rat ovary did not inhibit ovulation, whereas a non-selective angiotensin II receptor antagonist reduced the ovulation rate in the in vitro perfused ovary (Mikuni et al., 1998a). It is also known that angiotensin II (Mitsube et al., 2003) and endothelins (Levy et al., 2003) decrease ovarian blood flow.
A novel finding in the present study is the appearance of petechiae (purplish reds spot caused by haemorrhage) in the follicle wall. The petechiae were formed basal to the avascular translucent area. The petechiae may be the in vivo correlate of the leakage of resin that was seen at various parts of the capillary plexus, especially those in the part near the avascular apex (Kanzaki et al., 1982). It may well be that the minor leakage from the vessels that gives rise to petechiae in the follicle may not progress to intrafollicular bleeding, since the positive intrafollicular pressure of the rabbit ovary (Espey and Lipner, 1963) would prevent dissection of the bleeding towards the centre of the follicle. When the major rupture occurs, the follicle is quickly emptied of fluid and there is an immediate drop in intrafollicular pressure, allowing considerable bleeding into the follicle. This is in line with the observation in this study of fairly marked bleeding on follicular rupture. The findings correlate with the clinical situation in the human, where there is ovulatory bleeding that may give the abdominal pain that is often experienced by women at the time of ovulation. This follicular rupture bleeding ends within minutes. The relatively short duration of the bleeding may be the result of clot formation. An early event of ovulation is a generalized increase in plasmin activator (PA) activity (Beers, 1975; Beers et al., 1975), which would result in the activation of proteases, such as matrix metalloproteinases, but also in fibrinolytic activity in regard to blood coagulation. In the follicle, the cytokine interleukin-1 (IL-1) is an important intraovarian mediator, having an effect on PA and several other ovulatory mediator systems (Brännström, 2004). Interestingly, IL-1 has no effect on the PA system during early events of ovulation, but decreases PA activity at the time of follicular rupture (Bonello et al., 1995). This may be the biochemical explanation for the clotting of blood in the top of the follicle and the sudden cessation of bleeding.
The first prominent sign of follicle rupture observed in the present study was the formation of a cone; this was immediately followed by leakage of clear follicular fluid. The structural correlate of this cone formation may be a bulging of the surface epithelium or stromal cell layer, which may be pushed up from the underlying follicular antrum by positive intrafollicular pressure. The rupture would take place as a consequence of increased pressure inside the cone by the smaller radius compared with the follicle with a much larger radius.
More knowledge about ovulation in vivo is required to answer some of today’s questions concerning the process leading up to and including follicular rupture. The method using intravital microscopy developed in the present study may be used in further studies to enhance knowledge about the entire ovulation process in vivo, by, for example, combined measurement of blood flow and intrafollicular pressure or the sampling of blood and follicular fluid with the administration of known perturbants, locally or systematically.
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This research was supported by grants from the Swedish Research Council (11607 to M.B.), the Medical Faculty at the Sahlgrenska Academy, Hjalmar Svensson’s Research Foundation and Göteborg Medical Society.
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Submitted on May 13, 2005; resubmitted on October 14, 2005; accepted on October 26, 2005.
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