J. Phyaiol. (1975), 248, pp. 307-315 With 1 plate and 2 text -ft gurea Printed in Great Britain

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THE BIQELECTRIC PARAMETERS OF THE VAGINA DURING THE OESTROUS CYCLE OF THE RAT

By D. FRANCES EDWARDS ANDRB. J. LEVIN From the Department of Physiology, University of Sheffield, Sheffield S10 2TN

(Received 20 August 1974) SUMMARY

1. A new in vitro preparation of the rat vagina was devised to allow the measurement of the electrical potential difference (p.d.), short-circuit current (s-c.c.) and tissue resistance. 2. In vivo and in vitro, the magnitude of the transvaginal p .d. (adventitia positive to lumen) was greatest at oestrus and smallest at dioestru s and metoestrus. The p.d. at pro-oestrus was not significantly different

from the latter values. 3. The s-c.c. was significantly greater at oestrus than at metoestrus, dioestrus and pro-oestrus. 4. The vaginal resistance was maximal at pro-oestrus and was significantly greater than that at metoestrus, dioestrus and oestrus. The value at oestrus was significantly greater than that at metoestrus and dioestrus. These changes in resistance correlate well with known changes in vaginal morphology during the oestrous cycle. 5. It is proposed that the cellular site for the generation of vaginal bioelectric activity is the basal cell layer of the vagina and that the variations of the transvaginal p.d. measured in vivo are caused by the changes in vaginal current and tissue resistance. INTRODUCTION

Rogers (1935-6, 1938) was the first to observe that the magnitude of the electrical potential difference (p.d.) between the vaginal lumen and the pubic symphysis of rats (the abdomiino-vaginal potential) varied with the phase of the oestrous cycle. The highest value was at oestrus with the lowest values at metoestrus and dioestrus. Removal of the ovaries caused the periodicity to disappear and to be replaced by a day-to-day patternless rise and fall in potential. Since the morphology of the vaginal epithelium is controlled by the ovarian hormones the inference drawn from these

D. FRANCES EDWARDS AND R. J. LEVIN studies is that tie cyclical variation in p.d. during the oestrous cycle is also the result of the action of ovarian hormones on the vaginal epithelium. Because the abdomino-vaginal potential measured by Rogers includes potentials generated by non-vaginal tissues, unequivocal statements about the exact nature of the vaginal bioelectric activity were not possible until the development by Levin & Camfield (1967) of preparations of the vagina that allowed direct measurement of the transvaginal potential (the p.d. between the adventitial surface of the vagina and the vaginal lumen). The in vitro preparation developed by Levin & Camfield precluded the measurement of the short-circuit current and resistance of the vagina. Consequently a new preparation has been devised which permits these parameters to be measured. The results reported in this paper were obtained with this preparation. A preliminary account of the work has been published (Edwards & Levin, 1970).

'308

METHOD

Experimental animals. Virgin female albino rats bred in the Sheffield Field Laboratories from a specific pathogen free strain and weighing 230-260 g were used. They were maintained on a commercial diet (diet 86, Oxoid London) and water, both available ad lib. The temperature of the animal house was 170 C and the lights were on from 6 a.m. to 6 p.m. each day. Vaginal smears. Vaginal smears were obtained from each rat by using a platinum loop which had been dipped in 0 9 % NaCl. The loop was flame sterilized between successive smears to remove cell debris from the previous smear. Smears were fixed in a solution consisting of equal volumes of ether and 95 % (v/v) ethyl alcohol. They were air dried and stained by the method given by Hartman (1944). The stage of the oestrous cycle (Dioestrus, Pro-oestrus, Oestrus and Metoestrus) was assessed by the criteria of Long & Evans (1922). The smears were stored and re-examined several weeks later; if necessary they were reclassified (Mandl, 1951). The critical period of the hypothalamo-pituitary axis has not been determined for this colony but rats were not anaesthetized after 12.00 hr unless they were to be used for an experiment. Recording the transvaginal p.d. in vivo. The rats were anaesthetized i.P. with pentobarbitone sodium (Nembutal, Abbott Laboratories) and the adventitial surface of the vagina was exposed by a mid line abdominal incision taken through the pubic bone at the symphysis. Once severed, the halves of the bone were pushed apart. A glass cannula was inserted into the vagina and filled with bicarbonate saline (Krebs & Henseleit, 1932) previously warmed to 38° C and gassed with 95% 02/5 % COS (v/v). One end of an agar-salt bridge (3 % agar in molar KCl) was placed inside the cannula and the other end was connected to a calomel half cell. The circuit was completed by placing a cotton wick electrode (soaked in 0-9 % NaCl) on the adventitial surface of the vagina. The free end of this electrode was placed in a beaker containing 0-9 % NaCl. An agar-salt bridge connected the beaker of saline to a calomel half cell. The two half cells were arranged back-to-back across the input terminals of a Vibron electrometer (Model 33B-2, Electronic Instruments Ltd, Richmond) the output of which was displayed on a recording potentiometer (EUW20A, Heath, Benton Harbor, Michigan or Model 93503, Beckman Instruments Ltd, Fife). The transvaginal p.d. was recorded for 1 min.

309 VAGINAL BIOELECTRIG PARAMETERS30

Preparation of the vagina for the recording in vitro of the tran~svaginat p.d., shortcircuit current and resistance. To prepare the vagina for the in vitro measurements the organ was removed by making a transverse cut just below the cervix uteri and two longitudinal cuts on either side of the vagina. A final transverse cut at the level of the vulva freed the vagina. It was placed on filter paper moistened with 0-9 % NaCi and a longitudinal cut along its length allowed the tissue to be mounted as a flat sheet between two Perspex chambers mounted in a Perspex sleeve. The chambers each had a well of 10 ml. capacity and communicated by opposing holes (area 0-6 cm2) drilled near the bottom of the well. The vagina was held in position by a series of small pins surrounding the aperture on one of the chambers. Small holes in the Perspex surrounding the other aperture matched the position of these pins. The adventitial surface of the vagina was placed against the pins. To make the apparatus water-tight the surfaces of the two Perspex chambers surrounding each aperture were coated with Vaseline. Once the vagina had been clamped in position approximately 10 ml. bicarbonate saline (prewarmed to 380 C) were placed on each side of the vagina. The chambers were mounted in a Perspex sleeve and were kept tightly opposed to one another by means of a screw at one end of the sleeve. The whole apparatus was partially immersed in a Shandon Bath (Model Circotherm II, Shandon Scientific Co. Ltd, London) where the temperature of the water was maintained at 40' C. Perspex is a poor conductor of heat, but the temperature of the Krebs buffer was back to 38' C well within 1 hr. Immediately following the setting up of the vagina the buffer on each side of the tissue was gassed with 95 % 02/5 % C02 which was moisturized by passing the gas through a Dreshel bottle, filled with distilled water, which was immersed in the water-bath. When the short-circuit current (s-c.c.) and resistance of the tissue were measured the motor of the Shandon bath was switched off to prevent interference with the measurement. The time taken to set up the vagina from the moment of its removal to the commencement of gassing was never greater than 3 min. The transvaginal p.d. was measured by calomel half cells which were arranged back-to-back across the input terminals of the Vibron electrometer and connected to the preparation by two agar-salt bridges. The bridges were placed as close to the tissue as possible. The short-circuit current and the resistance of the vagina were obtained by passing a current from an external battery circuit through the tissue via agar-salt bridges and calomel half cells. The current was measured by a microammeter (Scalamp, Pye & Co., Cambridge). The 'true' s-c.c. of the vagina was obtained in vitro by the technique used by Asano (1964) for intestinal tissue. Before mounting the vagina in vitro a current-voltage plot was obtained for bicarbonate saline alone with approximately 20 mlA. buffer in the Perspex chambers. Another current-voltage plot was obtained in a similar manner with the vagina mounted between the two chambers. The point of intersection of the two current-voltage plots gave the s-c.c. in microamps (flA). The slope of each line gave the resistance of the buffer and that of the buffer and vagina. The resistance of the tissue alone was obtained by subtracting the former resistance from the latter. The s-c.c. (#aA) and resistance (0) reported are those for 0-6 cm2 tissue. They have not been converted to 1 cm2 since the proportion of edge damage produced by clamping tissues in vitro varies with the aperture size (Dobson & Kidder, 1968). Both the p.d. in vivo and in vitro were corrected for any imbalance between the calomel half cells and the bridges. All the in vitro results quoted are those obtained 1 hr after incubation. After 1 hr in the majority of vaginal preparations, the various electrical parameters had stabilized and remained stable for the next 2 hr. In some preparations stable measurements could be obtained for as long as 7 hr of incubation. 14

PH Y

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310

D. FRANCES EDWARDS AND R. J. LEVIN

Statistical methods. The data were treated by an analysis of variance and the differences between groups located by the least significant range test (LSR) given by Sokal & Rohlf (1969). LSR

=

Q

1CI 41s1within 2n +n (^/ ni n

Q is obtained from tables (Rohlf & Sokal, 1969), and MS,,,t,. is taken from the analysis of variance table while nj and n2 are the number of observations in each of the two groups compared. The results are taken as significant when the computed difference between two means exceeds the actual difference of two means. The test was applied at P = 0-05. The results in the text are given as the mean + S.E. of mean. RESULTS

The data for the vaginal potential measured in vivo and the vaginal potential, short-circuit current and resistance measured in vitro are shown in Text-figs. 1 and 2 respectively. The results were treated by an analysis of variance and the differences were then located by the Least Significant Range test (Table 1). TABLE 1. Least significant range test. The test compares the computed difference between two means with the actual difference between two particular means (Sokal & Rohlf, 1969). The abbreviations are as follows: D, dioestrus; P, prooestrus; 0, oestrus; M, metoestrus; PD, transvaginal potential; s-c.c., vaginal short-circuit current. The results that were significantly different at P 6 0-05 are marked with an asterisk (*)

Cycle stages compared O and M o and D o and P P and M P and D D andM

PD in vivo 7.66* 6.0* 5.28* 7-96

5-9 6-4

PD

in vitro 4.56* 3-7* 3.45* 5-0 4-6 4-6

s-c.c. 3.62* 2.7* 4.0* 3-65 4-8 4-0

Resistance 411* 305* 342* 546* 542* 451

Transvaginal potential in vivo and in vitro The polarity of the p.d. both in vivo and in vitro was adventitia positive to the lumen of the vagina. The highest p.d. recorded in vivo at oestrus (38-2 + 1-8 mV) was significantly greater than the values at dioestrus (9-9 + 0-9 mV) and metoestrus (9-3 + 2-1 mV). It was also significantly greater than the value at pro-oestrus (15-3 + 2-2 mV). The p.d. at pro-oestrus was not significantly greater than the metoestrus and dioestrus values. The transvaginal p.d.s measured in vitro, although always smaller than those observed in vivo, show an identical pattern. The highest p.d. at oestrus (13-8 ± 1-2 mV) was significantly greater than the p.d. at metoestrus (3-6 + 0-4 mV), dioestrus (3-9 + 0-3 mV), and pro-oestrus (5-7 + 1-0 mV) but

311 VAGINAL BIOELECTRIC PARAMETERS the pro-oestrus p.d. was not significantly greater than the metoestrus and dioestrus values. The percentage decrease in the transvaginal p.d. caused by mounting the vagina in vitro at the different stages of the cycle was found to be 53% at dioestrus, 61*3% at pro-oestrus, 65% at oestrus and 52-5% at

metoestrus. 40

The transvaginal potential

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M Text-fig. 1. The transvaginal p.d. measured in vivo at different stages of the rat oestrous cycle. The results are given as the mean + S.E. of mean. The number in the bases of the bars represent the number of animals used. The abbreviations are: D, dioestrus; P, pro-oestrus; 0, oestrus; M, metoestrus.

The vaginal short-circuit current The highest s-c.c. was measured at oestrus (10.2 + 1.0 /%A). This was significantly greater than the s-c.c. at dioestrus (5-1 + 0.5 ,uA), metoestrus (4.4 + 0*5 ,uA) and pro-oestrus (3.0 + 0 7 ,uA). The current at pro-oestrus was not significantly smaller than the currents measured at dioestrus and metoestrus, although in some preparations the short-circuit current at prooestrus could be as low as 0*2 ,uA. The vaginal resistance The resistance of the vagina was highest at pro-oestrus (2366 + 205 Q) and was significantly greater than the resistance at dioestrus (841 + 80 Q), I4-2

312

D. FRANCES EDWARDS AND R. J. LEVIN

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1 (13) 1 | (18) _I (36) 1 1| (13)|2. D 0 P M Text-fig. 2. The transvaginal p.d., s-c.c. and vaginal resistance measured in vitro at different stages of the rat oestrous cycle. The results are given as the mean + S.E. of mean. The number in the bases of the bars represent the number of animals used. The abbreviations are as in the legend to Text-fig. 1.

l

313 VAGINAL BIOELECTRIC PARAMETERS metoestrus (827 + 70 Q), and oestrus (1490 + 96 fl). The resistance at oestrus was also significantly greater than that at dioestrus and metoestrus. DISCUSSION

The transvaginal p.d. measured in vivo at all four stages of the oestrous cycle correlate well with the previous pattern obtained by Levin & Camfield (1967). In both studies the highest value was obtained at oestrus and the lowest values at metoestrus and dioestrus. Similarly the values for the transvaginal p.d. measured in vitro in the present study correlate remarkably well with those of Levin & Camfield despite the fact that in their preparation the vagina was everted and tied into a sac while in our study the vagina was mounted as a flat sheet. The s-c.c. measured in vitro clearly varied with the phases of the oestrous cycle, being maximal at oestrus and minimal at pro-oestrus. The resistance of the vagina also changed over the oestrous cycle, being highest at prooestrus (where it may reach 4000 Q) and least at metoestrus and dioestrus. The oestrous value was less than that at pro-oestrus but was still significantly greater than those measured at metoestrus and dioestrus. Interpretation of these electrical data from in vitro measurements suggest that the changing values of the transvaginal p.d. observed in vivo are probably the result of variations in current and resistance of the vaginal epithelium. The known changes in vaginal morphology (Long & Evans, 1922) and in ultrastructure (Stegner & Iwata, 1967; Parakkal, 1974) correlate well with the observed changes in vaginal resistance. The low resistances of the vagina at metoestrus and dioestrus match the thin (two to three cells) epithelium of these phases. At pro-oestrus, the epithelium becomes ten to twenty cells thick and possesses an outer layer of nucleated cells. This layer, together with the thickness of the epithelium and the compactness of the cell layers offers a satisfactory morphological explanation for the very high resistance of the organ measured at pro-oestrus. At oestrus, the epithelium is still ten to twenty cells thick but the layer of nucleated cells has been shed and the outer layers of cells are now fully keratinized. The spaces between the cells appear greater at oestrus than at the other stages (Stegner & Iwata, 1967). The loss of the outer layer of nucleated cells, the loose keratinized layers and the expanded intercellular spaces account for the reduced resistance of the oestrous vagina compared to the pro-oestrous state. R. J. Levin (unpublished observations) has noted that in dioestrus, metoestrus and oestrus the vital dye nigrosin will penetrate and stain the vaginal epithelium after the tissue has been treated with the surface-active agent nonylphenoxypolyethoxyethanol (Edwards, Kugler & Levin, 1970). The dye does not stain the epithelium at

D. FRANCES EDWARDS AND R. J. LEVIN prooestrus, indicating that solutes of the size of nigrosin do not readily penetrate. In the experiments where the transvaginal p.d. is measured both in vivo and in vitro the values for the latter are nearly always much lower than the former. Two experimental factors can cause the decrease in vitro. The first is that clamping the tissue between chambers will cause edge damage (Dobson & Kidder, 1968) which will shunt out part of the potential. The second is that the vagina may suffer from partial anoxia when it is mounted in vitro despite gassing with 95 % oxygen in both adventitial and mucosal fluids. If the vagina was partially anoxic in vitro, one would expect to find that the p.d. (and s-c.c.) would be most affected in those phases of the oestrous cycle when the vaginal epithelium is thickest, i.e. at pro-oestrus and oestrus. However, the percentage decrease of the transvaginal p.d. caused by mounting the organ in vitro was nearly as great at dioestrus and metoestrus (53 %) as observed at oestrus (65%) and pro-oestrus (61 %). Comparing the similar decrease in the transvaginal p.d. at pro-oestrus and oestrus with the disparity in their short-circuit currents strongly suggests that while some of the decrease in p.d. in vitro may be attributed to partial anoxia most of the decline is due to edge damage. It is germane at this juncture to examine the cellular site for the generation of the vaginal bioelectric activity. At the present moment the available evidence suggests that it is located in the basal/parabasal layer of cells. The reasons for such a supposition are as follows. At dioestrus and metoestrus the vaginal epithelium is but a few cells thick yet the tissue generates a measurable current and p.d. The cells in this basal layer contain numerous mitochondria which could provide the energy necessary for the 02-dependent ion transporting processes. Levin & Camfield (1967) showed that hypoxia depressed the transvaginal p.d. in vitro. Cells not in the basal or parabasal layer contain few, if any, mitochondria. A final feature of the cells in the basal layer is that they are polarized anatomically; one part of their membrane is embedded in the connective tissue close to the basement membrane (P1. 1). Such an arrangement is ideal for the generation of electrical p.d. across the cell by a pump/leak mechanism. The ion pump would presumably be at the basal (embedded) membrane while the 'leaky membrane allowing ion entry would be the more luminally orientated membrane. The s-c.c. that we have measured across the vagina must be created by the active movement of ions across the tissue. We assume that the magnitude of this current depends upon the number of cells and/or pumps undertaking the active ion movement and the availability of ions for the pumps. It is not possible at present to distinguish the mechanism for the increase in s-c.c. at oestrus. However, oestrogens must have some action 314

The Journal of Physiology, Vol. 248, No. 2 ::; .m.m-

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315 VAGINAL B,0ELECTRIQ PARAMETERS on the transport of ions at this stage of the cycle since the s-c.c. is greater than that measured at dioestrus and metoestrus despite the fact that the resistance at oestrus is nearly twice as great. One of us (D.F.E.) was supported by a grant from the British Empire Cancer Campaign. Acknowledgement is given to Richard Gawin for excellent technical assistance. The electron microscope study was undertaken by J. H. Kugler. REFERENCES AsANo, T. (1964). Metabolic disturbances and short-circuit current across the intestinal wall of the rat. Am. J. Physiol. 207, 415-422. DOBSON, J. G. & KIDDER, G. W. (1968). Edge damage effect in in vitro frog skin preparations. Am. J. Physiol. 214, 719-724. EDWARDS, F., KUGLER, J. H. & LEVIN, R. J. (1970). Effects of a surface active contraceptive agent (nonylphenoxypolyethoxyethanol) on the vagina; a new functional approach to assessing the actions on the vagina of spermicides. J. Physiol. 208, 35-36P. EDWARDS, F. & LEvIN, R. J. (1970). Mechanisms involved in the electrogenic action of oestrogens on the vaginal epithelium. J. Physiol. 207, 22-23P. HARTMAN, C. G. (1944). Some new observations on the vaginal smear of the rat. Yale J. Biol. Med. 17, 99-112. KREBS, H. A. & HENSELEIT, K. (1932). Untersuchungen fiber die Harnstoffbildung in Tierkorper. Hoppe-Seyler's Z. physiol. Chem. 210, 33-66. LEVIN, R. J. & CAMFIELD, J. (1967). The isolated everted vagina - a preparation for studying vaginal bioelectric phenomena in vitro. Life Sci. Oxford 6, 1871-1881. LONG, J. A. & EvANs, H. M. (1922). The oestrous cycle in the rat and its associated phenomena. Mem. Univ. Calif. 6, 1-148. MANDL, A. M. (1 951). The phases of the oestrous cycle in the adult white rat. J. exp. Biol. 28, 576-584. PARAxKAL, P. F. (1974). Cyclical changes in the vaginal epithelium of the rat seen by scanning electron microscopy. Anat. Rec. 178, 529-538. ROGERS, P. V. (1935-6). Changes in electrical potential during normal and experimentally induced oestrus in rats. Anat. Rec. 64 (suppl.) 40, abstr. 88. ROGERS, P. V. (1938). Changes in electrical potential in normal, castrate and theelintreated rats. Am. J. Physiol. 121, 565-573. ROHLF, F. J. & SoKAL, R. R. (1969). Statistical Tables, p. 200. San Francisco: W. H. Freeman and Co. SOKAL, R. R. & ROHLF, F. J. (1969). Biometry. The principles and practice of statistics in biological research, p. 244. San Francisco: W. H. Freeman and Co. STEGNER, H.-E. & IWATA, M. (1967). Elektronenmikroskopische Untersuchungen am Scheidenepithel der Ratte Walhrend des Oestrischen Zyklus. Z. Zellforsch. mikroslk. Anat. 76, 491-508. EXPLANATION OF PLATE

A basal cell of rat vagina. The nucleus, the collection of mitochondria, the granular cytoplasm and the basement membrane just beneath the basal (embedded) membrane of the cell are shown. Intercellular spaces between basal cells shown to the right and left of the central cell. The connective tissue into which the cell's processes penetrates contains collagen (osmium tetroxide fixation embedded in Araldite (Ciba-Geigy), section cut at approximately 500 A and examined in Philips EM 200). Magnification x 20,000.

The bioelectric parameters of the vagina during the oestrous cycle of the rat.

1. A new in vitro preparation of the rat vagina was devised to allow the measurement of the electrical potential difference (p.d.), short-circuit curr...
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