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Cell Biology International (2003) 27, 355–359 (Printed in Great Britain)
A microprobe study of element distribution in vaginal epithelial cells of the rat
Hubert R Catchpolea, Milton B Engelab* and Steven L Jordanc
aDepartment of Oral Biology, College of Dentistry, University of Illinois at Chicago, 801 S. Paulina, m/c 690, Chicago, IL 60612, USA
bDepartment of Orthodontics, College of Dentistry, University of Illinois at Chicago, 801 S. Paulina, m/c 690, Chicago, IL 60612, USA
cDepartment of Mathematics, Statistics, and Computer Science, 851 S. Morgan, m/c 249, Chicago, IL 60680, USA


Abstract

Microprobe analysis of vaginal epithelial cells shed during the estrous cycle of the rat was done to determine cellular elements present in successive stages: pro-estrus, estrus, and post-estrus. Smears of vaginal contents were placed on carbon planchettes, fixed by freeze-drying, and examined in a scanning microscope with an energy dispersive spectrometer. Concentrations of Na, Mg, P, S, Cl, K, and Ca were calculated (mmol/kg dry weight) and analyzed statistically. For phosphorus a significant fall at estrus correlates with a loss of nuclear and cytoplasmic nucleic acids and nucleoproteins. An increase in sulfur at estrus is consistent with an accumulation of keratins over pro-estrus and a greater increase over the post-estrus epithelial cells. The epithelial cells of pro-estrus are highest in Mg and Ca. By post-estrus, the cells have recovered their Mg, not Ca. Potassium concentrations exhibited no significant change between the successive stages.


Keywords: Rat vaginal epithelial cells, Estrous cycle, Microprobe analysis, Element distribution, Keratinization.

*Corresponding author. Tel.: +1-312-996-7551; fax: +1-312-996-6044.


1 Introduction

During the estrous cycle of the rat, which recurs over a 4–5-day period, the surface cells undergo dehiscence. The shed cells show characteristic changes in cytoplasm and nuclear morphology associated with cornification. These stages were delineated and described in the classical study of Long and Evans (1922).

Following a short period of inactivity (anestrus), rounded nucleated epithelial cells are shed from the stratified squamous epithelium in large numbers (stage 1, pro-estrus), followed within a day by progressively keratinized cells, presented singly or in sheets of now large angular polyhedral cells, either lacking or with a shrunken nucleus (stage 2, coinciding with estrus or sexual receptivity). This stage is followed by an influx of leucocytes, disappearance of keratinized cells, presence of ‘debris’ and mucus, and reappearance of nucleated epithelial cells (stage 3, post-estrus), a stage passing over into anestrus. The morphological features of these cyclical changes as seen by scanning electron microscopy (SEM) have been described by Centola (1978) in shed cells and by Parakkal (1974) and Sato et al. (1997) in the vaginal epithelium. The last named emphasized apoptotic aspects of cell death during the estrous cycle.

Morphological changes in the cellular content of vaginal smears have been studied for three-quarters of a century; elemental changes have not hitherto been studied. To further characterize the epithelial cells present at these stages we have fixed the cells by freezing and drying in order to avoid element loss and structural relocation. This was followed by microprobe analysis as previously described for erythrocytes (Catchpole and Engel, 1996) to determine simultaneously the distribution of some significant chemical elements which are components of intracellular macromolecules and electrolytes.

2 Materials and methods

Cells were recovered by smearing the vaginal mucosa of adult female rats, a model used as indicated above to delineate progressive stages of the rodent estrous cycle. Recovered cells were placed on a glass slide and stained in a drop of 0.5% aqueous toluidine blue. The cells were examined microscopically to determine the stage of the cycle, and classified as pro-estrus, estrus, or post-estrus, following criteria established by Long and Evans. Smears at stage 2 are highly definitive. Our aim was to obtain smears at stages 1 and 3 that were as characteristic as possible. Young adult Sprague–Dawley rats weighing 175–250g were used. Vaginal smears were taken from a total of six animals at daily intervals, over a period of 1 year at an arbitrary chosen point of vaginal smearing done at daily intervals. Quantitative results are presented for three animals.

Definitive smears were taken after the test smears, spread on carbon planchettes, rapidly frozen in isopentane chilled in liquid nitrogen at about −150°C and dried in vacuo over P2O5at −32°C. The freeze-dried sample was exposed to anhydrous vapor of paraformaldehyde for 1h at room temperature in order to stabilize the cells against any destructive effects of carbon coating and of the electron beam. The specimens were then coated with a carbon film in a vacuum evaporator to facilitate conduction. The cells were examined in a JEOL scanning (SEM) microscope, which was equipped with an energy dispersive X-ray detector. The detector was coupled with an analyzer and computer to record the element distribution and element counts. The SEM was operated at an accelerating voltage of 15kV and the gun current was maintained at 80–100μA. The operating conditions were standardized using a copper grid and adjusting the condenser lens to yield 2000 counts per 50s. We employed the method of bulk analysis according to theoretical considerations elucidated by Russ (1977).

Analysis of the cells was done at a magnification of 900–1000× using a limited raster of 50–100μm2, essentially covering cellular cytoplasm and nucleus. The depth resolution was calculated to be approximately 5μm. Under these conditions, which are representative of the cell contents as a whole, we sampled a volume of cytoplasm which included both its organelles and nuclear structures. As an entity, the contribution of cell membrane to element counts would be expected to be small.

Eight to ten SEM analyses were made on each preparation. For each stage of the cycle, 15 values were determined which included the elements Na, Mg, P, S, Cl, K and Ca (in ascending atomic weight). The Tracor bulk program, SQ, subtracts the background, deconvolutes and strips the peaks, and identifies the elements. The counts were converted to millimoles per kilogram dry weight using gelatin-element standards prepared according to the method of Roomans (1979).

We would have preferred to express the values also in terms of the water content of the shed cells. The presence of exudate and secretions to different degrees in smears renders cell weighings unreliable. Limited inferences regarding cell water content in individual epithelial cell layers were drawn from the literature on skin (Von Zglinicki et al., 1993; Warner et al., 1988).

3 Results

Typical cells from the three stages of the rat estrous cycle are shown (Fig. 1a–f) together with their corresponding X-ray spectra of element distribution. Values for elements measured are given as millimoles per kilogram dry weight of epithelial cells (Table 1). For each element, comparison of concentrations was made for stage 1 vs. stage 2, for stage 2 vs. stage 3, and for stage 1 vs. stage 3.


Fig. 1

(a–f) Cells from vaginal smears with accompanying spectra of element distribution. Scale bars=10μm. (a, b) Pro-estrus; (c, d) estrus; (e, f) post-estrus; l, lymphocyte.


Table 1. Element distribution in rat vaginal cells (mean±std error) (mmol/kg dry weight)

Image

The most striking finding is the low phosphorous value for cornified epithelial cells at estrus as compared to pro- and post-estrus cells. Highest sulfur levels were observed in estrus cells with lower values in pro-estrus cells and the lowest concentration in post-estrus cells. The concentrations of the cations Mg, K, and Ca were lowest in the estrus cells. The elevated values for Na and Cl in stages 2 and 3 are due to confounding by the secretion overlay of the cycle itself, and will be considered in the discussion. The levels reported for pro-estrus cells appear to be more representative of the intracellular concentrations of Na and Cl.

A statistical analysis is presented in Table 2. Element levels were tested for equality of means between stages (two-sample t-test with unequal variances) and for equality of variance (two-sample F-test). Analysis of variance (ANOVA) was performed. The more conservative two-sided tests were used. Differences in means show significant changes (bold type) in progressing from pro-estrus to estrus in ion and element levels based on dry weight of cell contents in Mg, P, and Ca. The ratios of variance differ significantly between these stages for Mg and P. A one-way ANOVA was performed for each element, grouping the values according to stages, yielding significant p-values for Mg, P, and S, again in terms of dry weight; differences for K were not significant.

Table 2. Statistical significance (p-value) between stages

Image

Significance beyond 0.01 is shown in bold type.

4. Discussion

Major morphological changes occur in the epithelial cells shed into the vagina during the estrous cycle of the rat. These cells progress from the nucleated cells of pro-estrus to the fully keratinized stage of late estrus with dissolution of the cell nucleus prior to complete cell loss. At the same time, progressive changes in composition occur in cell macromolecules (Barbee, 1962 and Bern et al., 1957).

The synthesis and accumulation of keratins in the cells of the stratum corneum described by Gimenez-Conti et al., 1994 and Horvat et al., 1992 is consistent with the increase in sulfur in stage 2 which we have reported. The marked drop in phosphorous follows the nuclear dissolution ( Bern et al., 1957 and Sato et al., 1997). The phosphorus level is regained by cells of stage 3. While the values for these two major elements apply to a cornifying mucosa, some interesting comparisons canbe made with skin epithelium. In earlier studies on skin keratinization ( Engström and Lundström, 1947 and Mercer, 1961) an increase in sulfur concentration in the stratum corneum was reported. More recent microprobe investigations on human skin ( Von Zglinicki et al., 1993 and Warner et al., 1988) and guinea pig skin ( Wei et al., 1982) confirm an increase of sulfur in passing from the inner epidermal layer through the stratum corneum. These authors also confirm the sharp drop in phosphorus level in the cornified surface. The element profiles described by Warner et al. are especiallystriking.

Barrington (1968) found that keratinized cells of the palatal epithelium had a higher dry cell mass than lower layers and the microprobe data of Von Zglinicki et al. (1993) further indicates that keratinizing cells are losing water. These authors found that the stratum corneum of human skin contains one-third less water than more proximal epithelial cell layers (54 vs. 78%). If this value were applicable to rat vaginal epithelial cells, stages 1 and 2, and the results of Table 1 were expressed in terms of cell water, then the sulfur value at estrus would be even higher, and cations Mg, K, and Ca would be increased over pre- and post-estrus levels. The phosphorus level would remain significantly low.

We need to comment on the effect of vaginal tract secretion, especially in stages 2 and 3. This confounds measurements of Na and Cl by forming an overlay to the cells during processing. Studies in cell-free areas of the preparations show their composition to be largely Na and Cl, with other ions negligible. Consistently lower values at stage 1 may more reasonably reflect the true intracellular Na and Cl values at this stage of proestrus.

While the methodology and materials of Cameron et al. (1980) come closest to ours as a hormonal study of ions and elements of rat vaginal epithelial cells, their results apply specifically to the cytoplasm of the vaginal basal cell layer of previously castrated female rats, treated once with estrogen and examined at 2, 17, and 24 h. In terms of ions and elements there was a fall at 2 h in Na, P, and Cl and subsequent increases by 24 h, most pronounced for P. There were highly significant increases in K and Mg by 24 h. Estrogen clearly affects major cell ions and elements. The behavior, especially that of K in relation to Na, was discussed in relation to prevalent theories of ionic distribution in cells. These authors also considered the possibility that ionic changes may reflect, secondarily, changes in the growth state of vaginal epithelial cells in response, in this case, to hormones directing the cycle. We support and generalize this latter concept as applicable to all cell ions and elements included in the present study. Differences in a range of elements and ions in all of the quoted experiments would find their basis in measurable physiochemical properties of the cells in question (Engel and Catchpole, 1989 and Catchpole and Engel, 1996). Among them we have considered composition and fixed charge of cell macromolecules, the content and state of the cell water, and intrinsic properties of the individual elements.

Acknowledgements

The microprobe analysis was done at the Electron Microscope Facility of the Research Resource Center, University of Illinois at Chicago. We thank Kristina Jarosius and Linda Juarez of the Facility for their technical assistance.

References

Barbee FE. Histochemical demonstration of some protein substances in mucosal epithelia of rat and man. MS thesis. Chicago, IL: University of Illinois; 1962.

Barrington EP. Dry residue density of cheek and palate mucosa of the rat. PhD thesis. Chicago, IL: University of Illinois; 1968.

Bern, H.A., Alpert, M. and Blair, S.M., 1957. Cytochemical studies of keratin formation and of epithelial metaplasia in the rodent vagina and prostate. J Histochem 5, pp. 105–119.

Cameron, I.L., Pool, T.B. and Smith, N.K.R., 1980. Intracellular concentration of potassium and other elements in vaginal epithelial cells stimulated by estradiol administration. J Cell Physiol 104, pp. 121–125. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1)

Catchpole, H.R. and Engel, M.B., 1996. Microprobe analysis of element distribution in rabbit and dog erythrocytes as examples of ‘high’ and ‘low’ potassium cells. Scanning Microsc 10 3, pp. 745–752. View Record in Scopus | Cited By in Scopus (3)

Centola, G.M., 1978. Surface features of exfoliated vaginal epithelial cells during the oestrous cycle of the rat examined by scanning electron microscopy. J Anat 127 3, pp. 553–561. View Record in Scopus | Cited By in Scopus (5)

Engel, M.B. and Catchpole, H.R., 1989. Microprobe analysis of element distribution in bovine extracellular matrices and muscle. Scanning Microsc 3, pp. 387–394.

Engström, A.J. and Lundström, B., 1947. Histochemical analysis by X-rays of long wave lengths. Experientia 3, pp. 191–193. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1)

Gimenez-Conti, I.B., Lynch, M., Roop, D., Bhowmik, S., Majeski, P. and Conti, C.J., 1994. Expression of keratins in mouse vaginal epithelium. Differentiation 56, pp. 143–151. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (10)

Horvat, B., Vrcic, H. and Amjanov, I., 1992. Transdifferentiation of murine squamous vaginal epithelium in proestrus is associated with changes in the expression of keratin polypeptides. Exp Cell Res 199, pp. 234–239. Abstract | Article | PDF (5817 K) | View Record in Scopus | Cited By in Scopus (13)

Long, J.A. and Evans, H.M., 1922. The oestrous cycle in the rat and its associated phenomena vol. 6, University of California Press, Berkeley, CA p. 1 .

Mercer, E.H., 1961. Keratin and keratinization. , Pergamon Press, New York p. 230 .

Parakkal, P.F., 1974. Cyclical changes in the vaginal epithelium of the rat seen by scanning electron microscopy. Anat Rec 178, pp. 529–538.

Roomans, G.M., 1979. Standards for X-ray microanalysis of biological specimens. Scanning Electron Microsc II, pp. 649–657.

Russ, J.C., 1977. Principles of EDAX analysis on the electron microscope. , EDAX International, Prairie View, IL.

Sato, T., Fukazawa, Y., Kojima, H., Enari, M., Iguchi, T. and Ohta, Y., 1997. Apoptotic cell death during the oestrous cycle in the rat uterus and vagina. Anat Rec 248, pp. 76–83. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (50)

Von Zglinicki, T., Lindberg, M., Roomans, G.M. and Forslind, B., 1993. Water and ion distribution profiles in human skin. Acta Derm Venereol (Stockh) 73, pp. 340–343. View Record in Scopus | Cited By in Scopus (48)

Warner, R.R., Myers, B.S. and Taylor, D.A., 1988. Electron probe analysis of human skin: element concentration profiles. J Invest Dermatol 90, pp. 78–85. Full Text via CrossRef

Wei, X., Roomans, G.M. and Forslind, B., 1982. Elemental distribution in guinea-pig skin as revealed by x-ray microanalysis in the scanning transmission microscope. J Invest Dermatol 79, pp. 167–169. View Record in Scopus | Cited By in Scopus (7)

Corresponding Author Contact InformationCorresponding author. Tel.: +1-312-996-7551; fax: +1-312-996-6044.


Cell Biology International
Volume 27, Issue 4, April 2003, Pages 355-359
Result list | previous < 7 of 13 > next 



The most striking finding is the low phosphorous value for cornified epithelial cells at estrus as compared to pro- and post-estrus cells. Highest sulfur levels were observed in estrus cells with lower values in pro-estrus cells and the lowest concentration in post-estrus cells. The concentrations of the cations Mg, K, and Ca were lowest in the estrus cells. The elevated values for Na and Cl in stages 2 and 3 are due to confounding by the secretion overlay of the cycle itself, and will be considered in the discussion. The levels reported for pro-estrus cells appear to be more representative of the intracellular concentrations of Na and Cl.

A statistical analysis is presented in Table 2. Element levels were tested for equality of means between stages (two-sample t-test with unequal variances) and for equality of variance (two-sample F-test). Analysis of variance (ANOVA) was performed. The more conservative two-sided tests were used. Differences in means show significant changes (bold type) in progressing from pro-estrus to estrus in ion and element levels based on dry weight of cell contents in Mg, P, and Ca. The ratios of variance differ significantly between these stages for Mg and P. A one-way ANOVA was performed for each element, grouping the values according to stages, yielding significant p-values for Mg, P, and S, again in terms of dry weight; differences for K were not significant.


Table 2. Statistical significance (p-value) between stages

Image

Significance beyond 0.01 is shown in bold type.

4. Discussion

Major morphological changes occur in the epithelial cells shed into the vagina during the estrous cycle of the rat. These cells progress from the nucleated cells of pro-estrus to the fully keratinized stage of late estrus with dissolution of the cell nucleus prior to complete cell loss. At the same time, progressive changes in composition occur in cell macromolecules (Barbee, 1962 and Bern et al., 1957).

The synthesis and accumulation of keratins in the cells of the stratum corneum described by Gimenez-Conti et al., 1994 and Horvat et al., 1992 is consistent with the increase in sulfur in stage 2 which we have reported. The marked drop in phosphorous follows the nuclear dissolution ( Bern et al., 1957 and Sato et al., 1997). The phosphorus level is regained by cells of stage 3. While the values for these two major elements apply to a cornifying mucosa, some interesting comparisons canbe made with skin epithelium. In earlier studies on skin keratinization ( Engström and Lundström, 1947 and Mercer, 1961) an increase in sulfur concentration in the stratum corneum was reported. More recent microprobe investigations on human skin ( Von Zglinicki et al., 1993 and Warner et al., 1988) and guinea pig skin ( Wei et al., 1982) confirm an increase of sulfur in passing from the inner epidermal layer through the stratum corneum. These authors also confirm the sharp drop in phosphorus level in the cornified surface. The element profiles described by Warner et al. are especiallystriking.

Barrington (1968) found that keratinized cells of the palatal epithelium had a higher dry cell mass than lower layers and the microprobe data of Von Zglinicki et al. (1993) further indicates that keratinizing cells are losing water. These authors found that the stratum corneum of human skin contains one-third less water than more proximal epithelial cell layers (54 vs. 78%). If this value were applicable to rat vaginal epithelial cells, stages 1 and 2, and the results of Table 1 were expressed in terms of cell water, then the sulfur value at estrus would be even higher, and cations Mg, K, and Ca would be increased over pre- and post-estrus levels. The phosphorus level would remain significantly low.

We need to comment on the effect of vaginal tract secretion, especially in stages 2 and 3. This confounds measurements of Na and Cl by forming an overlay to the cells during processing. Studies in cell-free areas of the preparations show their composition to be largely Na and Cl, with other ions negligible. Consistently lower values at stage 1 may more reasonably reflect the true intracellular Na and Cl values at this stage of proestrus.

While the methodology and materials of Cameron et al. (1980) come closest to ours as a hormonal study of ions and elements of rat vaginal epithelial cells, their results apply specifically to the cytoplasm of the vaginal basal cell layer of previously castrated female rats, treated once with estrogen and examined at 2, 17, and 24 h. In terms of ions and elements there was a fall at 2 h in Na, P, and Cl and subsequent increases by 24 h, most pronounced for P. There were highly significant increases in K and Mg by 24 h. Estrogen clearly affects major cell ions and elements. The behavior, especially that of K in relation to Na, was discussed in relation to prevalent theories of ionic distribution in cells. These authors also considered the possibility that ionic changes may reflect, secondarily, changes in the growth state of vaginal epithelial cells in response, in this case, to hormones directing the cycle. We support and generalize this latter concept as applicable to all cell ions and elements included in the present study. Differences in a range of elements and ions in all of the quoted experiments would find their basis in measurable physiochemical properties of the cells in question (Engel and Catchpole, 1989 and Catchpole and Engel, 1996). Among them we have considered composition and fixed charge of cell macromolecules, the content and state of the cell water, and intrinsic properties of the individual elements.

Acknowledgements

The microprobe analysis was done at the Electron Microscope Facility of the Research Resource Center, University of Illinois at Chicago. We thank Kristina Jarosius and Linda Juarez of the Facility for their technical assistance.

References

Barbee FE. Histochemical demonstration of some protein substances in mucosal epithelia of rat and man. MS thesis. Chicago, IL: University of Illinois; 1962.

Barrington EP. Dry residue density of cheek and palate mucosa of the rat. PhD thesis. Chicago, IL: University of Illinois; 1968.

Bern, H.A., Alpert, M. and Blair, S.M., 1957. Cytochemical studies of keratin formation and of epithelial metaplasia in the rodent vagina and prostate. J Histochem 5, pp. 105–119.

Cameron, I.L., Pool, T.B. and Smith, N.K.R., 1980. Intracellular concentration of potassium and other elements in vaginal epithelial cells stimulated by estradiol administration. J Cell Physiol 104, pp. 121–125. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1)

Catchpole, H.R. and Engel, M.B., 1996. Microprobe analysis of element distribution in rabbit and dog erythrocytes as examples of ‘high’ and ‘low’ potassium cells. Scanning Microsc 10 3, pp. 745–752. View Record in Scopus | Cited By in Scopus (3)

Centola, G.M., 1978. Surface features of exfoliated vaginal epithelial cells during the oestrous cycle of the rat examined by scanning electron microscopy. J Anat 127 3, pp. 553–561. View Record in Scopus | Cited By in Scopus (5)

Engel, M.B. and Catchpole, H.R., 1989. Microprobe analysis of element distribution in bovine extracellular matrices and muscle. Scanning Microsc 3, pp. 387–394.

Engström, A.J. and Lundström, B., 1947. Histochemical analysis by X-rays of long wave lengths. Experientia 3, pp. 191–193. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (1)

Gimenez-Conti, I.B., Lynch, M., Roop, D., Bhowmik, S., Majeski, P. and Conti, C.J., 1994. Expression of keratins in mouse vaginal epithelium. Differentiation 56, pp. 143–151. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (10)

Horvat, B., Vrcic, H. and Amjanov, I., 1992. Transdifferentiation of murine squamous vaginal epithelium in proestrus is associated with changes in the expression of keratin polypeptides. Exp Cell Res 199, pp. 234–239. Abstract | Article | PDF (5817 K) | View Record in Scopus | Cited By in Scopus (13)

Long, J.A. and Evans, H.M., 1922. The oestrous cycle in the rat and its associated phenomena vol. 6, University of California Press, Berkeley, CA p. 1 .

Mercer, E.H., 1961. Keratin and keratinization. , Pergamon Press, New York p. 230 .

Parakkal, P.F., 1974. Cyclical changes in the vaginal epithelium of the rat seen by scanning electron microscopy. Anat Rec 178, pp. 529–538.

Roomans, G.M., 1979. Standards for X-ray microanalysis of biological specimens. Scanning Electron Microsc II, pp. 649–657.

Russ, J.C., 1977. Principles of EDAX analysis on the electron microscope. , EDAX International, Prairie View, IL.

Sato, T., Fukazawa, Y., Kojima, H., Enari, M., Iguchi, T. and Ohta, Y., 1997. Apoptotic cell death during the oestrous cycle in the rat uterus and vagina. Anat Rec 248, pp. 76–83. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (50)

Von Zglinicki, T., Lindberg, M., Roomans, G.M. and Forslind, B., 1993. Water and ion distribution profiles in human skin. Acta Derm Venereol (Stockh) 73, pp. 340–343. View Record in Scopus | Cited By in Scopus (48)

Warner, R.R., Myers, B.S. and Taylor, D.A., 1988. Electron probe analysis of human skin: element concentration profiles. J Invest Dermatol 90, pp. 78–85. Full Text via CrossRef

Wei, X., Roomans, G.M. and Forslind, B., 1982. Elemental distribution in guinea-pig skin as revealed by x-ray microanalysis in the scanning transmission microscope. J Invest Dermatol 79, pp. 167–169. View Record in Scopus | Cited By in Scopus (7)

Corresponding Author Contact InformationCorresponding author. Tel.: +1-312-996-7551; fax: +1-312-996-6044.


Cell Biology International
Volume 27, Issue 4, April 2003, Pages 355-359
Result list | previous < 7 of 13 > next 


4 Discussion

Major morphological changes occur in the epithelial cells shed into the vagina during the estrous cycle of the rat. These cells progress from the nucleated cells of pro-estrus to the fully keratinized stage of late estrus with dissolution of the cell nucleus prior to complete cell loss. At the same time, progressive changes in composition occur in cell macromolecules (Barbee, 1962; Bern et al., 1957).

The synthesis and accumulation of keratins in the cells of the stratum corneum described by Gimenez-Conti et al. (1994) and Horvat et al. (1992) is consistent with the increase in sulfur in stage 2 which we have reported. The marked drop in phosphorous follows the nuclear dissolution (Bern et al., 1957; Sato et al., 1997). The phosphorus level is regained by cells of stage 3. While the values for these two major elements apply to a cornifying mucosa, some interesting comparisons canbe made with skin epithelium. In earlier studies on skin keratinization (Engström and Lundström, 1947; Mercer, 1961) an increase in sulfur concentration in the stratum corneum was reported. More recent microprobe investigations on human skin (Von Zglinicki et al., 1993; Warner et al., 1988) and guinea pig skin (Wei et al., 1982) confirm an increase of sulfur in passing from the inner epidermal layer through the stratum corneum. These authors also confirm the sharp drop in phosphorus level in the cornified surface. The element profiles described by Warner et al. are especiallystriking.

Barrington (1968) found that keratinized cells of the palatal epithelium had a higher dry cell mass than lower layers and the microprobe data of Von Zglinicki et al. (1993) further indicates that keratinizing cells are losing water. These authors found that the stratum corneum of human skin contains one-third less water than more proximal epithelial cell layers (54 vs. 78%). If this value were applicable to rat vaginal epithelial cells, stages 1 and 2, and the results of Table 1 were expressed in terms of cell water, then the sulfur value at estrus would be even higher, and cations Mg, K, and Ca would be increased over pre- and post-estrus levels. The phosphorus level would remain significantly low.

We need to comment on the effect of vaginal tract secretion, especially in stages 2 and 3. This confounds measurements of Na and Cl by forming an overlay to the cells during processing. Studies in cell-free areas of the preparations show their composition to be largely Na and Cl, with other ions negligible. Consistently lower values at stage 1 may more reasonably reflect the true intracellular Na and Cl values at this stage of proestrus.

While the methodology and materials of Cameron et al. (1980) come closest to ours as a hormonal study of ions and elements of rat vaginal epithelial cells, their results apply specifically to the cytoplasm of the vaginal basal cell layer of previously castrated female rats, treated once with estrogen and examined at 2, 17, and 24h. In terms of ions and elements there was a fall at 2h in Na, P, and Cl and subsequent increases by 24h, most pronounced for P. There were highly significant increases in K and Mg by 24h. Estrogen clearly affects major cell ions and elements. The behavior, especially that of K in relation to Na, was discussed in relation to prevalent theories of ionic distribution in cells. These authors also considered the possibility that ionic changes may reflect, secondarily, changes in the growth state of vaginal epithelial cells in response, in this case, to hormones directing the cycle. We support and generalize this latter concept as applicable to all cell ions and elements included in the present study. Differences in a range of elements and ions in all of the quoted experiments would find their basis in measurable physiochemical properties of the cells in question (Catchpole and Engel, 1996; Engel and Catchpole, 1989). Among them we have considered composition and fixed charge of cell macromolecules, the content and state of the cell water, and intrinsic properties of the individual elements.

Acknowledgements

The microprobe analysis was done at the Electron Microscope Facility of the Research Resource Center, University of Illinois at Chicago. We thank Kristina Jarosius and Linda Juarez of the Facility for their technical assistance.

References

. . Barbee FE. Histochemical demonstration of some protein substances in mucosal epithelia of rat and man. MS thesis. Chicago, IL: University of Illinois; 1962.

. . Barrington EP. Dry residue density of cheek and palate mucosa of the rat. PhD thesis. Chicago, IL: University of Illinois; 1968.

Bern HA, Alpert, M, Blair, SM. Cytochemical studies of keratin formation and of epithelial metaplasia in the rodent vagina and prostate. J Histochem 1957:5:105-19

Cameron IL, Pool, TB, Smith, NKR. Intracellular concentration of potassium and other elements in vaginal epithelial cells stimulated by estradiol administration. J Cell Physiol 1980:104:121-5
Crossref   Medline   

Catchpole HR, Engel, MB. Microprobe analysis of element distribution in rabbit and dog erythrocytes as examples of ‘high’ and ‘low’ potassium cells. Scanning Microsc 1996:10:3:745-52
Medline   

Centola GM. Surface features of exfoliated vaginal epithelial cells during the oestrous cycle of the rat examined by scanning electron microscopy. J Anat 1978:127:3:553-61
Medline   

Engel MB, Catchpole, HR. Microprobe analysis of element distribution in bovine extracellular matrices and muscle. Scanning Microsc 1989:3:387-94

Engström AJ, Lundström, B. Histochemical analysis by X-rays of long wave lengths. Experientia 1947:3:191-3
Crossref   

Gimenez-Conti IB, Lynch, M, Roop, D, Bhowmik, S, Majeski, P, Conti, CJ. Expression of keratins in mouse vaginal epithelium. Differentiation 1994:56:143-51
Crossref   Medline   

Horvat B, Vrcic, H, Amjanov, I. Transdifferentiation of murine squamous vaginal epithelium in proestrus is associated with changes in the expression of keratin polypeptides. Exp Cell Res 1992:199:234-9
Crossref   Medline   

Long JA, Evans, HM. . The oestrous cycle in the rat and its associated phenomena 1922:vol. 6:

Mercer EH. Keratin and keratinization. 1961:

Parakkal PF. Cyclical changes in the vaginal epithelium of the rat seen by scanning electron microscopy. Anat Rec 1974:178:529-38
Crossref   Medline   

Roomans GM. Standards for X-ray microanalysis of biological specimens. Scanning Electron Microsc 1979:II:649-57

Russ JC. Principles of EDAX analysis on the electron microscope. 1977:

Sato T, Fukazawa, Y, Kojima, H, Enari, M, Iguchi, T, Ohta, Y. Apoptotic cell death during the oestrous cycle in the rat uterus and vagina. Anat Rec 1997:248:76-83
Crossref   Medline   

Von Zglinicki T, Lindberg, M, Roomans, GM, Forslind, B. Water and ion distribution profiles in human skin. Acta Derm Venereol (Stockh) 1993:73:340-3

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Received 19 June 2002/18 October 2002; accepted 2 December 2002

doi:10.1016/S1065-6995(02)00354-2


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)