1 Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot and 2The Institute for Human Reproduction, and the Reproductive Genetics Institute, Chicago, IL 60657, USA
3 To whom correspondence should be addressed. Email: m.eisenbach{at}weizmann.ac.il or iturkaspa{at}aol.com
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Abstract |
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Key words: fertilization site/oviduct/ovulation/sperm storage site/temperature gradient
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Introduction |
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Guidance in the form of sperm chemotaxis to substances secreted from the egg or its surrounding cells, a process demonstrated to occur in mammals (Eisenbach, 2004), is likely restricted to the immediate surroundings of the egg. The reason is peristaltic movements of the oviduct (Battalia and Yanagimachi, 1979
), which probably prevent the formation of a chemical gradient in the oviduct over a long distance from the egg (Eisenbach, 1999
). On the basis of findings that, at least in rabbits (David et al., 1972
) and pigs (Hunter and Nichol, 1986
), there is a temperature difference of 2°C and 0.7°C, respectively, within the oviduct, Bahat et al., (2003)
recently demonstrated that rabbit and human spermatozoa have the capacity to sense such a temperature difference and respond to it by thermotaxis, i.e. by swimming from the cooler to the warmer temperature.
These findings raise two major questions, which are highly relevant for evaluating the function of thermotaxis in reproduction: Is the temperature difference time-dependent and synchronized with ovulation? If so, how is the temperature difference establishedby temperature rise at the fertilization site or by temperature decrease at the storage site? Here we address these questions.
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Materials and methods |
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Pre-operative procedures
Primary analgesia of the animals was induced with 1 ml of Diazepam (5 mg/ml), given intravenously. Anesthesia was achieved by an intramuscularly injection of ketamine hydrochloride (35 mg/kg) and xylazine 2% (5 mg/kg). The animals were placed on a wrapped operating table and an abdominal laparotomy was performed by an experienced reproductive surgeon. The reproductive tract was exposed and the ovaries were examined carefully to determine whether ovulation occurred.
Temperature measurements
Temperatures within the lumen of the oviduct and within the rectum were measured by using two thermistor probes (Technomad Ltd., Israel). Each probe (stainless steel; 0.5 mm in diameter and 30 cm in length) was connected to a separate digital thermometer with a temperature accuracy of ±0.2°C. Before each set of surgeries, the thermometers were adjusted by putting both probes together in the same 37°C water bath. While performing the experiment, one probe, placed constantly within the animal rectum (23 cm deep), was used as a reference for the temperature loss due to anesthesia and the open abdomen. [The normal rectal temperature in healthy rabbits is 38.540.0°C (Harkness and Wagner, 1989).] The second probe served for alternating temperature measurements within the isthmus (near the uterotubal junction) and within the isthmic-ampullary junction. The probe sited at the isthmus was inserted via a section in the vagina wall, through the uterus, until its tip reached the beginning of the oviduct. The probe sited at the isthmic-ampullary junction was inserted via the fimbria to a depth of 34 cm. The temperatures in each region were recorded 24 times. The temperature measurements in each animal took no more than 15 min in total. At the end of the experiments the animals were sacrificed by an intravenous injection of sodium pentobarbital (133 mg/kg).
Statistics
The significance of the differences between different time points was determined by the ANOVA test or nonparametric (MannWhitney) test, as indicated, using InStat 3 software package (Graph Pad Software, San Diego, CA).
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Results |
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Discussion |
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We cannot deduce from our experiments the duration of the temperature difference. In the rabbit it lasts for at least 10 h (Figure 2), more than enough for attracting the capacitated spermatozoa by thermotaxis. This is because, in the rabbitan induced ovulator in which the timing of the capacitated state is synchronized with egg availability in the female genital tractspermatozoa are capacitated during a time window of 8 h, ranging from
12 h to
20 h post-mating (Giojalas et al., 2004
).
Our findings indicate that although the temperature difference between the storage and fertilization sites increases around the time of ovulation, a small temperature difference already exists prior to ovulation. The spermatozoa probably do not utilize this small temperature difference prior to ovulation for thermotaxis because, at this pre-ovulatory time, the spermatozoa are not yet capacitated (Giojalas et al., 2004) and, consequently, are not yet thermotactically responsive (Bahat et al., 2003
).
Although human spermatozoa are thermotactically responsive too (Bahat et al., 2003), it is not known whether or not an ovulation-dependent temperature gradient exists in humans. Assumingon the basis of the two mammals in which the temperature was measured and on the basis of the study of Cicinelli et al. (2004)
demonstrating temperature differences in the female genital tract of humans before and after ovulationthat it does, the time window in humans may be much longer. This is because in humans, where no linkage between ovulation and sperm capacitation exists, capacitated spermatozoa are available for extended time periods (Giojalas et al., 2004
).
An intriguing question is how the time-dependent temperature difference is established between two sites, 10 cm apart, within the same abdominal organ. On the basis of current knowledge, at least three mechanisms appear plausible.
One potential mechanism for the generation of a temperature difference within the oviduct is a biochemically generated endothermic process such as hydration. Such a mechanism was originally proposed by Luck et al. (2001) to explain the lower temperature in human (Grinsted et al., 1985
), pig (Hunter et al., 1997
), and rabbit (Grinsted et al., 1980
) follicles relative to the adjacent tissue. Luck et al. (2001
) proposed that the follicle growth involves continuous secretion of a large molecule(s) [e.g. mucopolysaccharide (Zachariae, 1959
)], whose hydration results in the endothermic cooling of the fluid. We propose that such a mechanism may also apply to the cooling of the storage site relative to the fertilization site. The basis for this possibility is the finding of Jansen and Bajpai (1982)
that in the rabbit, acid mucus glycoproteina highly hydrated macromoleculeis secreted predominantly from the isthmus, to a lesser extent from the isthmicampullary junction, and not at all from the ampulla, and the finding of Gandolfi (1995)
that this glycoprotein secretion is controlled by the level of circulating steroid hormones. Thus, it is possible that acid mucus glycoprotein or a similar hydratable molecule, the secretion of which is hormone-dependent and which is mainly confined to the sperm storage site in the isthmus, causes the oviductal fluid in this specific region to undergo hydration with a resultant temperature decrease.
Another potential mechanism is counter-current heat exchange, proposed by David et al. (1972) and Hunter and Nichol (1986) to explain their findings of temperature differences within the oviduct. The vessel that supplies blood to the storage site is different from the blood vessel to the fertilization site, and both of them are aligned close to the ovarian vein, flowing blood in the opposite direction (Leese, 1988
). According to this possibility, cold blood from the ovary cools the blood that enters the storage site by counter-current heat exchange, in a manner similar to the heat transfer known to occur between the testicular veins and artery (Glad Sorensen et al., 1991
).
The recent finding that, in women, the source of blood supply to the storage site appears to change at ovulation (Cicinelli et al., 2004) may provide another mechanism. Thus, prior to ovulation, the blood supply to the storage site appears to be mainly carried out by the ovarian artery. However, subsequent to ovulation, the supply is mainly by the uterine artery. It is, therefore, possible that the blood in this artery, thought to be cooled by counter-current heat exchange with blood flowing from the vagina (Cicinelli et al., 2004
), is responsible for the temperature drop found at ovulation. It could also account for our observation that the basal temperature at the storage site is lower than that at the fertilization site.
The temperature difference within the rabbit oviduct, demonstrated in this study to be generated at ovulation by lowering the temperature at the sperm storage site, may play an important role in mammalian reproduction. Sperm thermotaxis from the cooler storage site to the warmer fertilization site should be further explored as a natural mechanism for sperm selection in both in vivo and in vitro fertilization.
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Acknowledgements |
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References |
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Submitted on December 6, 2004; resubmitted on February 8, 2005; accepted on March 4, 2005.
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