1 Department of Clinical StudiesReproduction, Royal Veterinary and Agricultural University, Copenhagen, 2 Department of Physiology and Pharmacology University of Southern Denmark, Odense and 3 Department of Molecular Animal Breeding and Genetics, Ludwig Maximilian University, Munich, Germany
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Abstract |
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Key words: counter-current heat exchange/endothermic reactions/follicular fluid/infra-red endoscopy/ovary/pig
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Introduction |
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Turning to the female, there has clearly been no pressing requirement for the ovary to assume an extra-abdominal location, although a minor migration from its initial embryonic site close to the kidneys and adrenal glands is usually demonstrable (Patten, 1948; Byskov and Høyer, 1988
). However, if one or more stages of gametogenesis are vulnerable to deep body temperature in the male, with an accompanying increase in the risk of germ cell mutation (Ehrenberg et al., 1957
; Cowles, 1965
), then questions arise concerning species with intra-abdominal testes (Millar and Glover, 1973
; Harrison, 1975
; Glover et al., 1990
) and, in the present context, concerning ovarian physiology. In brief, even though situated deep within the abdomen, do ovaries or particular compartments thereof always function at deep abdominal temperature? Infra-red scanning in both rabbits and man revealed the existence of gradients in ovarian tissue temperatures. Rabbit pre-ovulatory follicles were 1.4 ± 0.2°C cooler than adjoining stroma as measured by the infra-red approach or using micro-electrodes introduced during mid-ventral laparotomy (Grinsted et al., 1980
). Human pre-ovulatory follicles could be as much as 2.3°C cooler than ovarian stroma (Grinsted et al., 1985
).
In domestic farm animals, ovarian temperatures were also examined with thermistor probes sited in the follicular antrum or adjacent stroma, an invasive approach open to criticism but which nonetheless suggested that pig Graafian follicles of 810 mm (pre-ovulatory) diameter could be 1.0°C cooler than the stroma (Hunter et al., 1995
). In a subsequent approach using infra-red thermo-imaging of pig ovarian tissues, gradients were again found between pre-ovulatory follicular temperatures and those of ovarian stroma, the mean follicular temperature being 1.7 ± 0.4°C cooler (Hunter et al., 1997
). Comparable studies in cattle revealed a folliclestroma difference of 1.5°C (Greve et al., 1996
). This locally reduced temperature was considered to be significant during resumption of meiosis in pre-ovulatory oocytes, and was suggested to be of immediate relevance to procedures of in-vitro oocyte maturation and in-vitro fertilization (IVF) (Shi et al., 1998
). Inappropriate temperatures might lead to subtle derangements in oocyte maturation processes, not least in the spectrum of nuclear and cytoplasmic proteins expressed during preimplantation development. Inappropriate culture temperatures could, therefore, offer one explanation for the seemingly low overall success of in-vitro maturation and IVF procedures in generating viable fetuses after transplantation of embryos to suitable recipients. Accordingly, it appeared important to continue with measurements of ovarian temperature close to the time of ovulation, with a specific focus on mature Graafian follicles.
In the experiments described below, we have developed and extended previous studies based on infra-red sensing of ovarian temperatures in domestic pigs. The body of evidence derived from these studies strongly supports the existence of temperature gradients in reproductive tissues deep within the abdomen of mature animals.
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Materials and methods |
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Pre-operative procedures, anaesthesia and surgery
All animals were operated on during pro-oestrus or oestrus in a pre-heated and sealed theatre (2830°C). In addition to background heating, the theatre and contents had been warmed for at least the previous 18 h by a space heater. This was switched off only at the commencement of the following procedures. Theatre temperatures had fallen to 2628°C by the onset of surgery.
Animals were starved overnight, injected intravenously with Mebumal (pentobarbital sodium; 50 mg ml1) to induce anaesthesia and raised hydraulically onto an insulated operating table. Using a Rowson laryngoscope (Penlon Ltd, Andover, Hants, UK) and cuffed McGill tube (Warne Surgical Products, Andover, Hants, UK), intubation was achieved with the animal on its side after which it was positioned on its back under semi-closed circuit administration of isofluran (Forene; Abbott Labs Ltd, Abbott Scandinavia, Kista, Sweden) and oxygen (1.01.5 l/min). Once full surgical relaxation was achieved, anaesthesia was routinely maintained at 2.02.5% isofluran in oxygen for the duration of the experiment (~1.0 h).
The abdominal field of operation was cleaned, shaved, sterilized and fully-draped. Strict aseptic procedures then followed throughout. A mid-ventral laparotomy was performed with temperature recordings (see below) on the body wall and sub-peritoneal fat. Using an incision no longer than 12 cm, the peritoneum was opened, the reproductive tract located swiftly, and an ovary brought into the incision site with minimum traction. Ovaries were visualized primarily by reflecting the body wall and peritoneum laterally rather than by lifting individual gonads to the level of the body surface. After almost instant infra-red sensing of temperatures with the fimbria in situ, this covering was displaced and the ovarian surface thermo-imaged whilst most of the reproductive tissues remained within the abdomen. The first ovarypicked at randomwas then replaced and the same procedures followed for the contralateral ovary. A fine silk marker thread placed in the mesovarium was used to distinguish the first ovary. The incision was closed temporarily using haemostats applied to the skin and the reproductive organs left for a period of 5min for temperature equilibration before further recording. For the purposes of this report, a `sensing' was an individual temperature measurement; an `experiment' involved one or more sensings during the same surgical intervention.
Extensive measurements were made of the rate of ovarian cooling upon surgical exposure. Such measurements followed the initial sensing of ovarian temperatures in a total of 17 animals, and the period of equilibration. Cooling was observed over a period of ~20 s or, in a further group of six animals, during precisely 30 s with measurements at 10 s intervals. In a final group of three animals, the rate of cooling was assessed during a period of 60s (Table I). In these experiments, the ovary under observation rested within the incision site with the minimum of traction applied to its ligaments.
In two further sets of observations involving a total of 16 animals, the influence on ovarian tissue temperatures of arresting blood flow was examined by placing an atraumatic tubular rubber ligature around the ovarian pedicle after the initial readings (Figure 1). The ovary was then equilibrated in a closed abdomen for 5 min before infra-red sensing. It was treated similarly after removal of the rubber ligature. In each animal, only the ovary with the larger number of prominent follicles was examined in this manner.
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In a further three experiments, the liberated ovary was suspended on a braided silk thread, plunged into liquid nitrogen at 196°C to deep freeze, thawed in warm phosphate-buffered saline (37°C), and re-equilibrated in a closed abdomen for at least 5 min before withdrawal into the incision site for sensing.
Post-operative procedures
After completion of all infra-red recording, and close inspection and photography of individual ovaries, the abdominal incision was closed in three layers. The peritoneum was joined with a continuous suture whereas interrupted sutures were placed in the body wall and skin. Animals remained in the post-operative pens of the Clinical Department. All recovered without incident. None was operated on a second time.
Infra-red temperature sensing
Temperatures were recorded using an infra-red camera for thermo-imaging. The camera was mounted on a fully adjustable tripod and positioned ~50 cm directly above the abdomen. Prior focusing enabled almost instantaneous (<2 s) recording of temperatures upon exposure and orientation of ovarian tissues.
The infra-red camera (THV550, without filter) was manufactured by Agema Thermovision (Stockholm, Sweden) and made available, together with associated computing equipment, by Praecisions Teknik in Copenhagen (DK-2610 Rødovre, Denmark). The computerized recording system enabled post-operative retrieval of individual pictures and analysis of all experimental data. The temperature scale was precise to within ±0.1°C, and the camera and recording system were periodically verified for such precision by Agema in Sweden. The temperature scale could be visualized in colour on a TV monitor screen during the surgical procedures, and the results could subsequently be recalled in colour for analysis of tissue differences (Figure 2). Precise pin-pointing of Graafian follicles or neighbouring stroma was thereby possible on screen, with both tissues visible in the same picture. All infra-red measurements and subsequent downloading of information were made by the same individual (I.B.B.). At least three follicular and three stromal observations were made per ovary for calculation of mean temperature differences.
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Jugular and deep rectal temperatures
Body temperatures were recorded during surgery by means of two Cu/CuNi thermo-probes (Ellab, DK-2610 Rødovre, Denmark). One (50 cm long, 0.8 mm diameter) was inserted its full length into the jugular vein through a short intravenous catheter (Venflon, 17 gauge, 45 mm) in a prominent ear vein; the other, a standard rectal probe, was positioned at least 1015 cm into the rectum. The probes were connected to a four channel data acquisition and analysis system (Ellab, Rødovre, Denmark; CMC 96 + IBM Think Pad). Temperatures were measured, collected and stored every 2 s with an accuracy of within ±0.1°C.
Insertion into the jugular vein via an ear vein had to be abandoned in four cases because of failure to pass the intravenous probe around the contour of the vessel at the base of the ear. Successful recordings were obtained from 14 animals.
Experimental design
The distribution of animals used for the various individual measurements is summarized in Table I. A sequence of measurements was usually made in each animal, so addition of the numbers given in Table I
does not give the total number of animals in the study.
Statistical analysis
Results were analysed by means of Microsoft Excel version 8.0 for Apple computers. The progamme was used to generate standard deviations, standard error of the mean, t-tests and in the plotting of graphs and tables.
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Results |
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In three animals in which recent ovulations rather than mature follicles were observed, within an estimated 46 h of ovulation, the collapsed haemorrhagic follicles were also cooler than adjoining stromal tissues. As determined in both ovaries, recent ovulation sites were 1.5 ± 0.2°C (range 0.92.2°C) cooler than ovarian stroma.
The influence of sensing the ovaries enveloped in the fimbriated extremity of the Fallopian tube in five animals compared with actual ovarian surface temperatures upon displacing the fimbria is shown in Figure 4. Although follicles were always cooler than stroma (38.5 ± 0.1°C versus 39.4 ± 0.1°C with the fimbria in situ), the apparent stromal temperature drop upon displacing the fimbria from the first exposed ovary (designated ovary 1) was 0.1°C greater than the follicular drop (data not shown). However, this difference was not apparent when the results from both ovaries were summarized.
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Further sets of cooling rate measurements in a new group of five animals were made at precisely 10 s intervals for a period of 30 s. These results are shown in Figure 5. The initial rate of cooling of mature follicles slightly exceeded that of stroma although the overall trend in cooling was essentially parallel.
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Influence of ovarian ligation
In the study involving placement of tubular rubber ligatures around the ovarian pedicle to arrest the blood supply (Figure 1), initial sensing of the ovaries recorded a mean temperature difference between follicles and stroma of 1.32 ± 0.1°C (n = 10). After precisely 5 min of ligation, during which the follicles had turned a deep blue in colour, the temperature difference was reduced to 1.27 ± 0.2°C. However, when blood flow resumed for a further 5 min during abdominal incubation which restored the normal healthy appearance of the follicles the differential was measured as 1.30 ± 0.1°C (Figure 8
). Accordingly, ligation for 5 min had a rather minor influence on the ovarian tissue temperature differentials.
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In an extension of this approach, tissues were not killed. Rather, the detached ovary was left in the incision site and temperature-sensed for a period of 30 s (two animals) or 60 s (one animal). After such recording, it was suspended in the abdomen and the incision closed for 5 min. Then, once again, the ovary was withdrawn and temperatures recorded for 30 or 60 s. In 18 sets of observations (composed of 11 sets of observations in two animals during 30 s and seven sets of observations in one animal during 60 s), the ovarian temperature differentials were maintained or increased between mature follicles and stroma, although of course the tissues were cooling (Figure 10). There was a close parallel between these experimental observations on ovarian cooling rates in living tissues after ovariectomy and those in ligated ovaries as reported above.
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In a further series of preliminary measurements (December 1998) to test a lens coating (anti-fogging) fluid (Synoptik) on the endoscope, thereby avoiding any problem of condensation, six sets of measurements were made on the two ovaries in one animal (no. 1558) with excellent pre-ovulatory follicles via a mid-ventral incision before exposing the ovary for conventional infra-red sensing. A 5 min period of temperature equilibration with the abdomen closed followed each pair of readings. The individual results are shown in Table II. The mean folliclestroma difference in this animal was 0.7 ± 0.1°C compared with a mean difference by direct scanning of the ovary in the incision site of 1.6 ± 0.1°C. This scanning was made after completion of the endoscopy readings and after a further 5 min of equilibration.
Due to scheduling problems of bringing the operating team from Odense, Munich and Edinburgh together in Copenhagen in January 1999, and because the six animals reserved for the experiment had not remained on precisely 21 day oestrous cycles from their last recorded heats in December 1998, a final set of preliminary endoscopy experiments had to be performed on Graafian follicles not exceeding 7 mm in diameter or on very recently ovulated follicles. The pre-heated infra-red endoscope was introduced through a gas-tight incision and, with a minimum of insufflation (air, not CO2), the ovaries were located with a quartz-fibre endoscope but not touched with the endoscopic forceps (Figure 2). Equilibration of the instruments occurred during the initial location and orientation of the gonad whilst the animal was angled head-down on an operating table tilted at ~24° below the horizontal. The mean temperature differential recorded between the 57 mm diameter follicles or recent ovulations and adjoining stroma in the six animals was 0.6 ± 0.1°C (Table II
). A larger differential would have been anticipated with Graafian follicles of a pre-ovulatory 910 mm diameter.
Urinary bladder temperatures
The moist surface of the distended urinary bladder was fully visible and sensed immediately upon opening the peritoneum during laparotomy in three animals. In the first animal, the bladder temperature was recorded as 39.1°C compared with a mean for the stroma of 39.2°C and for the follicles of 38.0° and 38.1°C on the two ovaries. In a second animal, the bladder temperature was recorded as 39.4°C, closely similar to the ovarian stroma (39.2°C), whereas the follicular mean was 38.0°C. In a third animal, the bladder temperature was again recorded as 39.4°C, the mean stromal temperature also 39.4°C, whereas the mean follicular temperature was 38.0°C. Hence, the surface temperatures of an exposed bladder did not match those of exposed follicles.
Jugular vein and deep rectal temperatures
Although no difference was recorded in three of 14 animals studied, 11 animals had a higher deep rectal than jugular vein temperature (0.251.0°C higher, usually 0.5°C) when these sites were measured simultaneously. The jugular temperature tended to remain reasonably constant whereas the rectal temperature either increased or decreased during surgery, suggestive of a thermo-regulatory mechanism. These findings are the subject of a separate report (Einer-Jensen et al., 1999). Ovarian follicular and stromal temperatures were invariably cooler than deep rectal temperatures at the start of recording (Figure 11
).
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Discussion |
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Turning to the actual experiments, and in a context of possible artefactual cooling of ovarian tissues exposed in the operating theatre, the fact that temperature gradients existed when the ovaries were tightly enveloped by the undisturbed fimbriated extremities of the Fallopian tube comes as valuable supportive evidence. In this condition, loss of heat by evaporation from moist protruding follicles would be minimized by the intimate embrace of these thin but well-vascularized membranes. In other words, the fimbriae should act to retard or prevent surface cooling. The fact that folliclestroma differences could be sensed at all with the fimbria in situ must be because the underlying ovarian structures were in immediate contact with portions of the membrane and were thereby able to transmit locally the influence of their own temperatures. This is even more impressive when the prominent capillary bed of such engorged peri-ovulatory fimbriae is taken into account.
Measurements of cooling rates for exposed ovaries are also instructive. If 710 mm diameter Graafian follicles protruding from the ovarian surface require ~20 s to cool by 1.5°C (Figure 5), then it is highly improbable that an almost instantaneous recording of a mean 1.3°C difference between pre-ovulatory follicles and neighbouring stroma could be accounted for simply by artefact. Although follicular tissue cooled initially more rapidly than stroma, perhaps in part because of the topography of follicles compared with the more compact adjoining tissues, Figures 5 and 6
nonetheless reveal a significant temperature differential between the respective tissues during the first few seconds of recording. Furthermore, the subsequent rate of cooling of these tissues between 10 and 60 s is almost parallel, indicating maintenance of a temperature differential between large follicles and stroma. If the reasonable assumption is made that mild traction on the ligaments and mesenteries to exteriorise an ovary did not perturb follicular blood supply to a markedly greater extent than the stromal supply, then the conclusion must be that reactions are occurring in large Graafian follicles that actively generate such temperature differentials by removing heat. Establishing the precise nature of such putative endothermic processes is an important research objective.
Further lines of evidence for the existence of intra-ovarian temperature gradients come from the ligation experiments and those involving ovariectomy. Arresting blood flow for a period of 5 min while keeping the ovaries in a closed abdomen did not alter significantly the temperature differential between Graafian follicles and stroma. Indeed, the mean differential after 5 min ligation was almost identical with that found after removal of the ligature and a further 5 min of abdominal equilibration. The favoured interpretation here is that arrest of blood flow during a period of 5 min was insufficient to damage irreversibly or arrest the putative endothermic mechanisms that generate such gradients in the physiological situation; hence the consistent histograms in Figure 8. This interpretation for living tissues receives support from the ovariectomy data in which the differentials between large follicles and stroma were essentially maintained during a 60 s period of cooling of live tissues (Figure 10
). By contrast, when ovarian tissues were killed upon plunging into liquid nitrogen and then thawed and sensed by the infra-red camera, the follicular-stromal temperature gradients were completely removed; these results thus confirm previous ones (Hunter et al., 1997
). This evidence, based on prompt examination of ovariectomized tissues that had previously been sensed in situ, is also supportive of endothermic reactions in living follicles, for which there is now physico-chemical evidence. An abstract reporting preliminary studies (Luck and Griffiths, 1998
) recorded a decrease in temperature in bovine follicular fluid subjected to saline dilution within an appropriate incubator; the decrease in temperature (0.140.2°C) was sustained for 713 min. It was concluded that such follicular fluid derived from undated slaughterhouse ovaries showed a net uptake of heat energy when diluted with saline, seemingly indicative of an endothermic reaction resulting from hydration of a large molecular weight component.
A current interpretation is that diverse components within mature Graafian follicles contribute to the intra-ovarian temperature gradients rather than the activities of a `single' molecule. This hypothesis may be incorrect but it would seem that the mature follicle is in such a dynamic condition actively growing, remodelling, synthesizing and secreting, and doubtless influenced in a key way by its antral oocyte that there is scope for more than one source of endothermic reaction. The present studies offer no guidance as to the precise nature of such reactions nor do current thoughts extend beyond our previous suggestions (Hunter et al., 1997). These included the synthesis and secretion of steroid hormones, prostaglandins and diverse peptides (including growth factors) and proteins (including heat shock proteins); extensive remodelling of follicular tissues including angiogenic proliferation of the thecal capillary bed; increased coagulability of the follicular fluid; and expansion and mucification of the cumulus oophorus. The last point remains of specific interest in relation to the hydration of proteoglycans the principal intercellular cement substance during pre-ovulatory loosening of follicular tissues and, latterly, during preparation for release of a secondary oocyte through a degraded follicular wall aperture. Putative endothermic reactions in Graafian follicles may still be active shortly after ovulation since very recently ovulated follicles also showed reduced temperatures compared with stroma in this study, in contrast to previous findings (Hunter et al., 1997
).
If the assumption is made that endothermic reactions are indeed involved in the establishment of intra-ovarian temperature gradients, as previously considered (Grinsted et al., 1980), then a means must also be put forward for their maintenance deep within the abdomen. Two physiological systems may need to be functional to avoid local cooling of follicles being overridden by an influence of the systemic circulation. First, a counter-current exchange of heat might be required in the ovarian pedicle so that returning venous blood could cool the incoming arterial blood. Second, there would certainly need to be a counter-current exchange of heat in the blood supply to individual Graafian follicles (Figure 12
). A counter-current exchange of heat in the ovarian plexus can be supported by the highly coiled and intimate arrangement of the ovarian artery on the ovarian vein. This shows close parallels with the male gonadal blood supply in which a counter-current heat exchange has been clearly demonstrated in species with scrotal testes (Setchell, 1978
; Waites and Setchell, 1990
; Glad Sørensen et al., 1991
). A separate line of argument would be that the ovarian plexus facilitates a counter-current exchange of hormones between vein and artery, especially of relatively small molecules such as prostaglandins and steroids, as well as inert gases (McCracken, 1971
; McCracken et al., 1971
; Einer-Jensen, 1988
, 1992
), so there would seem to be no problem in principle for other forms of transfer across the walls of the ovarian artery and vein. As for the blood supply to individual follicles, although experimental demonstrations of hormone or gaseous transfer have yet to be made at such a level, follicular drawings (Andersen, 1926
) in pigs and more recent scanning electron micrographs of ovarian vascular corrosion casts (Macchiarelli et al., 1997
) in rabbits demonstrate the intimate network of blood vessels and lymphatics and thus the potential for an exchange of heat and thereby the local maintenance of temperature gradients.
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In conclusion, what is clear from the body of evidence accumulated in these studies is that mature Graafian follicles are cooler than adjoining ovarian stroma, and that both compartments are cooler than jugular vein and deep rectal temperatures. What is not clear is the true extent of the temperature difference between follicles and stroma: is it the overall mean of 1.3°C revealed at laparotomy in a pre-heated operating theatre or the initial endoscopic mean of 1.1°C made following mid-ventral studies, or the final mean value of 0.6°C obtained after endoscopy without prior intervention (Table III)? The last value may be closer to the physiological differential, although it should be emphasized that this value comes from observations on small follicles (<7 mm diameter) representing only days 1718, not day 21 of a normal oestrous cycle. A realistic suggestion might be that the value for pre-ovulatory porcine follicles of 910 mm diameter lies somewhere between 0.6 and 1.1°C. Even so, the caveat must be added that all of the methods employed in these studies were unphysiological. All were invasive to a greater or lesser degree, and all involved anaesthesia with the animal inverted on the operating table an approach that could have influenced both ovarian bloodflow and the partition of such bloodflow. Although we plan to continue with the endoscopic approach in future studies using a modified instrument, the most satisfactory means of overcoming the above limitations would be some form of non-invasive whole body scanning of a fully conscious animal. Whilst this remains an important objective, current techniques do not offer the necessary precision and sensitivity for monitoring the temperature of individual Graafian follicles.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 21, 1999; accepted on October 12, 1999.