STUDIES ON THE MOVEMENT OF GLUCOSE, PYRUVATE AND LACTATE INTO THE AMPULLA AND ISTHMUS OF THE RABBIT OVIDUCT

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1 Quarterly Journal of Experimental Physiology (1983) 68, Printed in Great Britain STUDIES ON THE MOVEMENT OF GLUCOSE, PYRUVATE AND LACTATE INTO THE AMPULLA AND ISTHMUS OF THE RABBIT OVIDUCT H. J. LEESE Department of Biology, University of York, York YOJ SDD (RECEIVED FOR PUBLICATION 19 MARCH 1982) SUMMARY The appearance of glucose, pyruvate and lactate in fluid recirculated through the lumen of the ampulla and isthmus of the rabbit oviduct has been followed for 2 h in vivo. Each nutrient 8 times its rate in the lumen of the appeared in the lumen of the ampulla at approximately 1 isthmus. The circumference of the mucosa lining the two regions was measured together with the distribution of ciliated and secretory cells. The surface area of the mucosa lining the ampulla was 343 mm2, and that lining the isthmus, 191 mm2. It is tentatively suggested that the capacity of the ampulla and isthmus to transport small molecules is a function of their respective mucosal surface areas. INTRODUCTION The mammalian oviduct may be divided into four regions: the preampulla, which includes the fimbriae and infundibulum, the ampulla, the isthmus, and the junctura (Nilsson & Reinius, 1969; Reinius, 1970). In all species studied, the preampulla and the ampulla are thin-walled muscular tubes with a pronounced lumen lined by a highly convoluted epithelium. By contrast, the isthmus has a much thicker wall, and the lumen is narrow, with fewer mucosal folds (El-Banna & Hafez, 1970). In each region the epithelium consists of two main types of cell: ciliated and non-ciliated, or secretory. The distribution of the two cell types has been reported to change along the length of the oviduct, ciliated cells predominating at the preampulla end, secretory in the isthmus. (Nilsson & Reinius, 1969; Hafez, 1972; Kanagawa, Hafez, Pitchford, Baechlar & Barnhart, 1972). The ciliated cells are thought to play a role in gamete transport, the secretory cells in the formation of oviduct fluid. David, Brackett, Garcia & Mastroianni (1969) ligated regions of the rabbit oviduct, and found significant differences in the composition of the fluid accumulated after three days by the ampulla, the ampullary-isthmic region and the isthmus. Leese & Jeffries (1977) examined the appearance of endogenous glucose in fluid recirculated through the lumen of the rabbit oviduct for 4 h in vivo, and obtained evidence that the glucose moved by facilitated diffusion. Similar experiments were carried out on pyruvate, lactate and amino acid transport (Leese & Aldridge, 1979; Leese, Aldridge & Jeffries, 1979). This approach has now been used to compare the transport of glucose pyruvate and lactate into the lumen of the rabbit ampulla and isthmus. The circumference of the mucosa lining the two regions has also been measured at various points together with the distribution of ciliated and secretory cells.

2 90 H. J. LEESE Preampulla Ampulla aj l) \ Isthmus \ Uterus ( ) A Fig. 1. Diagrammatic representation of the position of the cannulae perfusing the lumen of (A) the ampulla and (B) the isthmus. The arrows show the direction of flow of the perfusion medium. B METHODS Experimental procedure New Zealand white rabbits (3-4 kg) were used in all the experiments. They were anaesthetized with 35 mg pentobarbitone sodium (Sagatol, May & Baker Ltd, Dagenham per kg body wt. and kept on a warm tray to assist in maintaining body temperature. The procedure to perfuse the lumen of the ampulla and isthmus was similar to that described for the whole oviduct by Leese & Jeffries (1977) and Leese & Aldridge (1979). In order to cannulate the ampulla, a small incision was made at the junction of the preampulla and the ampulla, i.e. at the top of the pronounced bend in the oviduct that occurs at this point. A glass cannula (i.d. 0-6 mm, o.d. 1 2 mm) was inserted into the lumen of the ampulla and gently pushed down in the direction of the isthmus. The point at which the oviduct became visibly constricted, and resistance to further movement of the cannula was felt, was taken as the ampullary-isthmic junction. A second incision in the oviduct wall was made at this point, and the cannula was pulled through this incision and tied in place. A second glass cannula (i.d. 1 0 mm, o.d. 2 0 mm) was then tied into the first incision, at the junction between the preampulla and the ampulla. In this manner, fluid could be recirculated through the ampulla via cannulae at each of its ends. To cannulate the isthmus, an incision was made in the uterus below the utero-tubal junction, and a glass cannula (i.d. 0 6 mm, o.d. 1 2 mm) was pushed through and tied into the isthmus approximately 1-25 cm above the utero-tubal junction. A second glass cannula (i.d. 0-6 mm, o.d. 1 2 mm) was introduced into the lumen of the ampulla, as described above, and tied in place at the

3 TRANSPORT IN THE RABBIT AMPULLA AND ISTHMUS E 0 0 O 1 2 Time (h) Fig. 2. The time course of appearance of glucose in fluid circulating through the lumen of the rabbit ampulla (0) and isthmus (A) in vivo. Values are means +S.E.M. of fifteen oviducts. ampullary-isthmic junction. The cannulations were carried out with the minimum of disturbance to the vascular supply to the oviduct. The positions of the cannulae are shown in Fig. 1. It should be emphasized that the preampulla of the oviduct was not perfused. It proved impossible to perfuse the ampulla and isthmus from the same oviduct. In most of the experiments, therefore, the ampulla on one side of the rabbit was perfused and the isthmus on the other. The perfusion circuit has been described previously (Leese & Jeffries, 1977). The perfusion medium was 2 ml 0-9% (w/v) NaCl at 37 C which was recirculated through the lumen at a rate of 50,ul/min. The medium entered the ampulla and isthmus segments via the cannulae in the ampullary-isthmic junction and lower part of the isthmus respectively. Analytical measurements Serial samples of medium were deproteinized with 0-6 M-HCIO4 and the supernatants neutralized with 30% (w/v) K2CO3. The pyruvate and lactate content of the extracts were measured by a modification (Leese & Aldridge, 1979) of the automated method of Leese & Bronk (1972). Glucose was also analysed by an automated method (GOD-Perid: Boehringer Corporation (London) Ltd, Lewes, East Sussex. Histological measurements Approximately 2 mm thick segments of tissue were cut from the same six regions of four oviducts, each from a separate rabbit. Expressed as fractions of the total length of the ampulla and isthmus, and beginning at the top of the ampulla, the segments were located at points 0, 0-15, 0-30, 0-47, 0-65 and 0-84 of the way along the oviduct. They were placed in a combined formaldehyde-glutaraldehyde fixative at 4 C, dehydrated in acetone and embedded in Epon. 1 #m thick transverse sections were used for the counts of ciliary and secretory cells, and 3,m sections for the measurement of the internal circumference. The sections were stained with Toluidine Blue and photographed under a Zeiss photomicroscope. The secretory and ciliated cells in a given section could easily be distinguished and their numbers counted. The circumference of the lumen was determined with a map measurer with appropriate correction for the magnification of the section. In all the experiments the data from the left and right ampulla and isthmus were pooled, since no consistent differences were ever detected between them. Values are given as means + S.E.M.

4 92 H. J. LEESE Fig. 3. I Time (h) The time course of appearance of pyruvate in fluid circulating through the lumen of the rabbit ampulla (@) and isthmus (A) in vivo. Values are means +S.E.M. of nine oviducts. 2-0 r E c.) cx.- C) IV c.) ci J2 I ' I I! 0 I Time (h) Fig. 4. The time course of appearance of lactate in fluid circulating through the lumen of the rabbit ampulla (0) and isthmus (A) in vivo. Values are means + S.E.M. of nine oviducts. RESULTS Biochemical measurements The appearance of glucose, pyruvate and lactate in fluid recirculated for 2 h through the lumen of the ampulla and the isthmus is shown in Figs. 2, 3 and 4. The rate of appearance of the compounds was linear. Although the absolute values differed considerably, the ratios of the rates of appearance of glucose, pyruvate and lactate in the ampulla compared with the isthmus were very similar, at 1 84, 1 83 and 1 88 respectively.

5 TRANSPORT IN THE RABBIT AMPULLA AND ISTHMUS E C E Fractional length Fig. 5. The circumference of the rabbit oviduct lumen (i.e. the mucosal surface) at six points, from the top of the ampulla (zero on the abscissa) to within 1-25 cm of the bottom of the isthmus. The dotted line shows the position of the ampullary-isthmic junction. Values are means + S.E.M. of at least four oviducts. Anatomical measurements The total length of the oviduct (i.e. ampulla plus isthmus but excluding the preampulla) was mm (n = 1 1). Of the total, an average of 32% was ampulla ( mm) and 68% was isthmus ( mm). In cross-section, the ampulla appeared thin-walled with highly convoluted mucosal folds whereas the wall of the isthmus was much thicker and the mucosal folding far less pronounced. A diagrammatic illustration of the gradation of these differences along the length of the rabbit oviduct has been given by Hafez (1973). The circumference of the oviduct lumen (i.e. the surface of the mucosal lining) at the six points from the top of the ampulla to within 12 5 mm of the bottom of the isthmus is shown in Fig. 5. The dotted line shows the position of the ampullary-isthmic junction. The circumference at the top of the ampulla was mm and then declined as the ampullary-isthmic junction approached. The lowest value ( mm) was found in the upper part of the isthmus. The circumference then increased very slightly towards the utero-tubal junction. On the assumption that the lines joining the six points taken for the anatomical measurements approximated the circumference at an infinite number of points along the oviduct, the total surface area of the mucosa could be calculated from the areas

6 94 H. J. LEESE I Fractional length Fig. 6. The percentage of ciliated (@) and secretory (A) cells at six points along the length of the rabbit oviduct, from the top of the ampulla (zero on the abscissa) to within 1 25 cm of the bottom of the isthmus. The dotted line shows the position of the ampullary-isthmic junction. Values are means + S.E.M. of four oviducts. under the lines in Fig. 5. The values of the ampulla and the isthmus were 343 and 191 mm2 respectively and their ratio was The relative proportions of ciliated and secretory cells at the same six points along the oviduct are shown in Fig. 6. Ciliated cells predominated in the ampulla, their proportion decreasing in the direction of the isthmus, where secretory cells became the more abundant. DISCUSSION An attempt has been made to correlate the structures of the ampulla and the isthmus of the rabbit oviduct with their capacity to transport small molecules. Transport capacity was measured by the appearance of glucose, pyruvate and lactate in saline medium recirculated through the lumen of the two regions. Each nutrient appeared in the lumen of the ampulla at approximately 1 8 times its rate in the lumen of the isthmus. David et al. (1969) reported that the concentration of lactic acid in the rabbit isthmus after three days ligation was 64-78,tg/ml and in the ampulla, 34-36,tg/ml. Taking into account the volume of fluid accumulated in the two regions (032 and 1 15 ml respectively), the total lactate appearance

7 TRANSPORT IN THE RABBIT AMPULLA AND ISTHMUS in the experiments of David et al. was 39-5,ug/3 d in the ampulla and 20-7,g/3 d in the isthmus; the ratio of total lactate appearance in the ampulla to that in the isthmus (1 92) is therefore very close to that reported for the acute experiments in the present paper, in which fluid transport was not measured. Further indirect support for the present results comes from the work of Misra, Sahib, Gupta & Karkum (1977) who found that 1 more protein was released by the rabbit ampulla than the isthmus when incubated in 8 times vitro. The distribution of ciliated and secretory cells in the two regions (Fig. 6) was virtually identical to that reported by Hafez (1972) for the oviduct of the rabbit and the macaque, and in agreement with the reports of Fredricsson (1959), Kanagawa etal. (1972) and Nilsson & Reinius (1969). The pattern of mucosal surface was very similar to that reported for the area of the lumen, mucosa and submucosa of the rabbit and bovine oviducts, by El-Banna & Hafez (1970). The question then arises as to which structural parameter(s), if any, correlates best with the movement of small molecules. Since the ratio of mucosal surface 95 in the ampulla to that the isthmus was similar to the ratio of transport capacity in the two regions, the answer is the total surface area of the mucosa bordering the lumen. This conclusion must, however, be tentative. For example, different forms of ciliated and secretory cells and possibly other cell types could be present in the ampulla and the isthmus. Ciliated cells may have extensive surface, and some secretory cells may have pedicles and microvilli which would provide additional surface area. The present work should therefore be considered first approach to the problem. However, if the capacity of the ampulla be interesting and the isthmus is function to examine only of their respective mucosal surface areas, it would the cells involved, but the junctions between them. I thank Mr N. Astley excellent technical assistance, and The Wellcome Trust grant. for a research REFERENCES DAVID, A., BRACKETT, B. G., GARCIA, C.-R. MASTROIANNI, L. JR. (1969). Composition of rabbit ligated segments of the Fallopian Tube. Journal of Reproduction and Fertility 19, EL-BANNA, HAFEZ, E. S. E. (1970). Profile analysis of oviductal wall in rabbits and cattle. Anatomical Record 166, FREDRICSSON, (1959). Proliferation of rabbit oviduct epithelium after estogenic stimulation, with relationship between ciliated and secretory cells. Acta morphologica neerlandoscandinavica 2, HAFEz, E. (1972). Scanning electron microscopy of female reproductive tract. Journal of Reproductive Medicine , HAFEZ, E. S. E. (1973). Endocrine control of the structure and function of the mammalian oviduct. of Physiology, ed. GREEP, R. 0. ASTWOOD, E. B., p Chicago: Univ. Chicago Press. KANAGAWA, H., HAFEZ, E. S. E., PITCHFORD, W. C., BAECHLAR, C. A. & BARNHART, M. I. (1972). patterns reproductive tracts of the rabbit observed by scanning electron microscopy. Anatomical Record 174, LEESE, H. J. & ALDRIDGE, 5. (1979). The nrovement of pyruvate, lactate and lactate dehydrogenase rabbit oviductal fluid. Journal of Reproduction and Fertility 56, LEESE, H. J., ALDRIDGE, S. & JEFFRIES, K. 5. (1979). The movement of amino acids into rabbit oviductal fluid. Journal of Reproduction and Fertility 56, LEESE, H. J. & BRONK, J. R. (1972). Automated fluorometric analysis of micromolar quantities of ATP, glucose and lactic acid. Analytical Biochemistry 45, LEESE, H. J. & JEFFRIES, K. 5. (1977). Evidence for the facilitated diffusion of glucose into rabbit oviductal fluid. Journal of Reproduction and Fertility

8 96 H. J. LEESE MISRA, D., SAHIB, M. K., GUPTA, D. N. & KARKUN, J. N. (1977). Quantitative and qualitative aspects of proteins contributed by different parts of rabbit Fallopian Tube and the uterus in vitro. Indian Journal of Experimental Biology 15, NILSSON, 0. & REINIUS, S. (1969). Light and electron microscopic structure of the oviduct. In The Mammalian Oviduct. ed. HAFEZ, E. S. E. & BLANDAU, R. J., pp Chicago: Univ. Chicago Press. REINIUS, S. (1970). Morphology of oviduct, gametes and zygotes as a basis of oviductal function in the mouse. I. Secretory activity of oviductal epithelium. Fertility 15,