Autoradiographic studies of the mouse Fallopian tube

Autoradiographic

studies of the mouse

Fallopian tube LAWRENCE UWE

E.

DEVOE, FREESE,

GEORGIA Chicago,

M.D. M.D.

PAULOS,

H.T.(A.S.C.P.)

Illinois

Cyclic histologic changes in the mouse Fallopian tube corresponding to a specific changing pattern of sH-thymidine uptake into tubal epithelium suggest a direct correlation between the pattern of nuclear labeling and the cycle-dependent alterations in hormone titers. Human chorionic gonadotropin (HCG)-treated animals showed a peak uptake in reserve cells comparable to that of late proestrus/early estrus. Estradiol-treated animals showed an uptake pattern into secretory cells comparable to the pattern seen in late estrus/metestrus-I. Progesterone-treated animals showed a return to predominantly reserve cell uptake similar to that of the late cycle, metestru.r-?/diestrus. These cyclic changes of uptake of “H-thymidine are interpreted as a growth-maturation-function cycle designed to sustain the egg or zygote in the tube and to replenish the tubal epithelium at the end of a cycle or pregnancy. The intercalary cells are recognized as end-stage e1ement.c of predominantly degranulated secretory cellr.

I‘) UR I N G o U R s T U LI Y of preimplantation pregnancy, it soon became apparent that certain areas in the basic knowledge of the Fallopian tube needed further elucidation. Thus, it was necessary to consider and to investigate several fundamental concepts. 1. Events in the development from conception to hlastocyst formation should 1~ accompanied by some corollary changes in thr> Fallopian tube epithelium that comes into contact with the transported zygote. 2. As preparation for possible conception

and subsequent zygote transport, there should be an observable response in the tubal epithelium during a normal estrus cycle. 3. A reproducible, sequential pattern of these tubal responses should occur from one cycle to the next. 4. Observed sequential changes in the tubal epith4ium must be controlled by both intraand extra-tubal influence. Thus, it would be equally important to focus on specific cellular events and possibly general mediation by hormones. The significance of this consideration is underscored by the obvious histologic differences in Fallopian tuhe epithelium between pregnant and nonpregnant aninlals, as well as animals in differing phases of the estrus cycle. The above hypotheses form the rationale for our first sc,ries of experiments. studies’, I’, 6. ‘8 have examined Previous tubal function via detailed analyses of tubal secretions for enzymes, polysaccharides, lipid contents. and other substances. The importance of anatomic localization is suggested

From the Department of Obstetrics and Gynecology, Chicago Lying-in Hospital, University of Chicago. Supported by Ford Grant 690-0108. Received Revised Accepted

for

publication

April

3, 1973.

April

Foundation

March 5, 1973.

6, 1973.

Reprint requests: Dr. Uwe E. Freese, Dept. of Ob./Gyn:, Chicago Lying-in Hospital, Universrty of Chicago, 5841 S. Maryland Ave., Chicago, Illinois. 490

Autoradiographic

by Glass” in a study which shows that, from early life, the Fallopian tube consists of antigenically discrete portions, all of which will, at some time, be in contact with the fertilized egg. Preimplantation pregnancy has been studied with radiotracer “H-thymidine by Prasad and associates.” The blastocysts which were labeled in this study, however, were obtained by flushing the oviducts, and the autoradiographs therefore do not represent in situ specimens. In addition, radioisotope investigation of the Fallopian tube has been performed by Pauerstein and Woodruff.’ Their study has indicated several areas for our current research, especially with reference to the role of the “stem” or “indifferent” cell in epithelial histodynamics-’ Material

and

methods

I Basic autoradiography of Fallopian tube ( Experiment 1) . One hundred and sixty female mice, 8 weeks of age, were accommodated to a light/dark cycle, i.e., 10 hours dark/l4 hours light, for one week. Each animal was then followed with daily vaginal smears and assigned to one of the 5 stages of the rstrus cycle. Individual animals were then selected from each phase of the cycle and injected with a dose of “H-thymidine of 27.7 IIIC. per millimole or approximately 70 /I,(: per animal. The injected animals were killed within 3 hours after injection, and thr Fallopian tubes, ovaries, and uterine cornua were removed. Freeze-dried!’ and paraffin sections of the specimens were then prepared. From these sections, autoradiographs were developed over a period of 2 weeks. The completed autoradiographs were stained with methyl green-pyronin (MGP) stain and reviewed for labeling characteristics, adequacy of histology, amount of artifact formation, etc. The “H-thymidine uptake of the acceptable autoradiographs was then correlated with the specific estrus stage of the appropriate animal. Final autoradiographic data were then evaluated as to reproducibility, consistency, and pattern of labeling within each stage. Hormonal factors in Fallopian tube and

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Fig. 1. Cross-section of mouse Fallopian tube. sHthymidine label confined mostly to fimbria. (Methyl greenPpyronin. Original magnification X88.) uptake of “H-thymidine (Experiment 2). The basic investigative techniques remain the same for this study with one major change. The animals were divided into 4 basic populations: ( 1) control (normal mice followed through the cycle and labeled at each of the 5 stages as in Experiment 1 [proestrus, estrus, etc.] ) ; (2) estradiol-treated (these mice were selected at random from the general population and injected daily over a period of 10 days with 1.0 pg of 17p-hydroxy-estradiol subcutaneously prior to administration of isotope and killing) ; (3) human chorionic gonadotropin (HCG) -treated (another random population treated with HCG, 1,000 U. subcutaneously for 10 days prior to injection and killing) ; (4) progesterone-treated (a final random group treated daily for 10 days with 4 mg. of progesterone subcutaneously before final preparation) ; the dosage of isotope, length of exposure, duration of development of the autoradiographs, and histologir techniques remained as before, Results

Experiment 1. In sections where the experimental criteria previously noted were met, the following patterns of isotope uptake were noted (Fig. 1). Stromal cells were those which could not be assigned to mature groups and were located at or below the basement membrane. Mature cells were those belonging to readily identifiable histologic groups and were, for

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view of fimbria folds during Uptake of SW-thymidine label is prcinto stromal cells of the tuhe. Arrows stromal cells. (Methyl greenPpyronin. magnification r200.j

the most part, secretory cells. Mixed cells were those showing essentially equal numbers of cells from each of the above groups. The above patterns were next correlated with the 5 stages of the estrus cycle: ( 1) Proestrus : A stromal pattern of uptake is predominant with occasional secretory cells labeled. There is less over-all uptake than in successive stages (Fig. 2 i . (2) Estrus: A mixed pattern of isotope uptake is seen with approximately equal numbers of secretory and basal cells labeled. There is more intense over-all uptake than in the preceding stage. (3) Metestrus-1 : Secretory cell labeling is predominant in this stage with very few reserve cells showing uptake. This stage of the cycle exhibited the greatest over-all uptake of “H-thymidine during all of estrus (Fig. 3 ) (1) Metestrus-2 : Increasing numbers of reserve cells were labeled with decreasing secretory cell predominance. The general uptake is less than that in metestrus-1 but marginally greater than that of estrus. (5) Diestrus: A return to stromal predominance is seen in this stage with an over-all labeling intensity equal to that of proestrus. Comparisons of each phase of the cycle in Figs. 4 and 5 are based on measurements relative to metestrus-1 (100 per cent intensity) with the other phases expressed as a percentage of this figure. Intensity per se was calculated by taking the number of cells which were labeled in corresponding sections

Fig. 3. View of tube during metestrus-1. Note that “H-thymidine uptake is prodominantly into secretory cells. Arrow indicate ciliated and secretory cells. (Methyl grernPp)mnin. Original magnifiration X400.1 of each stage and dividing the result by the total number of epithelial cells present in each section. Experiment 2. Estradiol-treated group. In addition to striking changes in gross morphology of the tube, i.e., swelling and hyperemia, the microscopic picture was equally impressive. The pattern resembled that of late estrus/ metestrus-1 in its general distribution of label. However, the intensity of labeling as well as the actual number of labeled cells was appreciably increased to at least threefold that of the control for this phase of the cycle. There also appeared to be a markedly increased number of secretory cells (Fig. 6). HCG-treated group. The typical appearance of this group was similar but slightly more intense in its uptake than that of earl) estrus. The pattern corresponded to what has been designated the mixed category, i.e., essentially equal percentages of reserve and secretory cells were labeled, but the distribution of :‘H-thymidine was more diffuse than expected normally at the estrus stage. Progesterone-treated group. The nature of isotope uptake here was strikingly different from either of the above groups. The sections closely resembled the postovulatory phase of the reproductive cycle (metestrus-2 and diestrus), in both histology and the scant labeling, predominantly in reseme cells. For a graphic summary of these data, see Fig. 7.

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Fig. 4. The phenomenon of sH-thymidine uptake during the estrus cycle is observed (x-x) with reference to the nuclear uptake of isotope during metestrus-1 as the maximum intensity point. All other stages are graphed as percentages of the total isotope uptake in metestrus-1. In addition, the trend of nuclear labeling is viewed vis-a-vis relative titers of estrogen and progesterone. %

Fig. 5. The relative intensity of isotope uptake during the estrus cycle is divided into the nuclear uptake among stromal (basal) cells ( 0-0 ) and secretory cells (x-x). The percentage of cells labeled is calculated by dividing the number of specific cells, stromal or secretory, labeled in any one phase by the total number of epithelial cells laheled during that same phase of the cycle.

which represents a composite. of the three series of hormonally treated animals and their comparison with representative stages of the normal estrus cycle. Comment Our principal investigative tool, “Hthymidine, is used on the assumption that the nuclear activity of these cells is related to their general biological activity. This activity, gauged by the relative uptake of the isotope in each cell’s nucleus, may reflect

Fig. 6. Cross-section of tube from mice. Pattern of uptake resembles metestrus-1. (Methyl green-pyronin. nification X400.)

estradiol-treated that of estrus/ Original mag-

synthesis of nuclear deoxyribonucleic acid for replication, maturation, or coding for synthesis of proteins active in maintenance of structure and function. It is our contention that changes in 3H-thymidine uptake represent at least some qualitative measure of changes in the activities of these cells. However, little or no isotope uptake could not be construed as indicating absence of function and quantitative labeling could not be used as more than an approximate guide to the degree of cellular activity. From the first run of autoradiographs, several trends in isotope uptake were apparent. Maximal uptake of 3H-thymidine occurred primarily in two cell lines, the reserve and the secretory. The ciliated and intercalary lines incorporated little or no isotope at any phase of the estrus cycle. Of the two labeled cell types, the greatest uptake was noted at different but complementary stages of the cycle. Reserve (stromal) cell labeling was at its peak in proestrus and again in late diestrus. Secretory cells were most heavily labeled in late estrus/ metestrus-I. Nuclear uptake of isotope was not only more intense but more extensive in terms of numbers of nuclei labeled during the above-mentioned stages. In general, the intensity of label uptake showed a cyclic pattern, increasing from proestrus to metestrus-1 and then returning to base-line levels by diestrus.’ This pattern

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Fig. 7. With labeling intensity of metestrus-1 as 100 per cent, the amount of nuclear uptake isotope is compared among the various phases of the estrus cycle and the groups of hormonally treated animals. Note similarities between the progesterone-treated group and proestrus/diestrus and between HCG and metestrus-1.

appeared to correspond to the changes in hormone titers which took place during the course of the estrus cycle. Peak labeling intensity coincides with the maximum level of circulating estrogen with the decrease in intensity of uptake conforming to the fall of estrogen levels and the rise of progesterone levels. Finally, it was apparent that quantitative laheling of the Fallopian tube was at least one order of magnitude less than that of either the ovarian granulosa cell or the rpithelial cells of the endometrial glands. We infer from these experiments that there exists the distinct possibility that at least a portion of the tubal epitheliurn is under cyclic hormonal influence, not unlike other end organs. We also note the probability of a developmental relationship, stem cell to daughter cell, hetween the reserve cell and the secretory cell. The first inference led LIS to the second series of experiments. From the data of the second experiment. it is apparent that a distinct pattern of “Hthymidine uptake exists for each group of hormonally treated animals. In general, each group resembles a particular stage of the estrus cycle during which one of the gi\.en

of

hormones is most influential. The exaggerated response seen in at least two of the groups, estrogen- and HCG-treated, is most probably a function of the greater than physiologic dose administered. By effectively overwhelming the animals’ autoregulatory mechanism with large doses of each hormone, we attempted to establish which of these compounds is most influential in the uptake of :‘H-thymidine. By comparing the histologic picture of sections from our treated groups with those of our control animals, it hecomes clearer that the influence each hormone exerts on isotope uptake exists during a discrctr portion of the reproductive cycle. As an example of this phenomenon, the sections from the estradiol-treated group strongly resembled those corresponding to late estrus/metestrus1 in our control group. Fig. 8 illustrates isotope uptake patterns of both reserve and secretory cells during the normal cycle and the points at which our study hormones are effective. From the foregoing data, as we expected, there is a strong suggestion of a positive correlation between the nuclear labeling of the tubal epithelium and changes in the hormone titers during the reproductive cycle.

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Fig. 8. The labeling intensity of specific cell types is charted in relation to the relative hormone level during the normal estrus cycle. Note the increase in secretory cell uptake relative to the rise in HCG and the estrogen level and the increase in reserve cell uptake relative to the rise in the progesterone level. Conclusions In these two studies, our primary goal has been to formulate a different approach of viewing the Fallopian tube as a milieu for preimplantation pregnancy. Our basic assumption is that there are observable changes in the tubal epithelium which reflect both extratubal influences and intratubal dynamics. These alterations presumably act to accommodate the tube for supporting passage of egg and/or early pregnancy, We feel that in situ alterations in function and composition at a cellular level are best studied by correlative investigations, with the uptake of an appropriate radioisotope used as a principal parameter. From our first study, there resulted evidence for a positive correlation between different phases of the reproductive cycle and the pattern of nuclear labeling in reserve and secretory cells. The pattern of this relationship strongly suggested to us that, among other fatcors, we would need to consider the influence of one or more of the hormones active in the reproductive cycle. Results from our second experiment indicate that there is an obvious relationship between the type of hormone used and the

nature of the “H-thymidine uptake pattern seen. HCG-treated animals showed a peak uptake in reserve cells comparable to but slightly more intense than that of late proestrus/early estrus. Estradiol-treated animals showed an uptake pattern comparable to but noticeably greater than that of late estrus/metestrus1. Progesterone-treated animals showed an uptake pattern similar to that of the late cycle, metestrus-2/diestrus. Therefore, we felt that our data from the group of specially treated animals helped to confirm our impression from the first study of normal, control animals, i.e., that these patterns of isotope uptake are indeed mediated by endogenous hormones of the reproductive system. Based upon these observations, we are proposing the following theoretical model as an explanation of how events in the Fallopian tube relate chronologically to ovulation and the passage of eggs prior to implantaion: Initially, in the preovulatory cycle, there is a relatively greater activity among the precursor elements, i.e., reserve cells. Most likely, this occurs under the influence of human pituitary extract (follicle-stimulating hormone) directly or through its mediation

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of the cstrogenic hormone. Logically, this would reflect the replenishing of mature elrrnents needed for egg transport and sustenance. In the next stage of the cycle, estrus, during whirh ovulation occurs, the secretory cell uptake is markedly increased, presumably by the effect of the rising estrogen level, and this may mark the priming of these cells fat their function of egg support. The postovulatory stages of the cycle, characterized by the increasing dominance of progestational hormones, exhibited a general decline in mature cell uptake of isotope and a gradual increase in the labeling of reserve cells once more. This may represent a final shift in emphasis from cells actively engaged in support of early pregnancy to the preparation of cells needed for the next cycle in the event that no pregnancy is achieved. Kegarding the virtual nonparticipation of

ciliated cells and intercalary cells in the uptake of “H-thymidine, \W must make the following inferences: Intercalary cells are most likely to be end-stage elements. eithri degranulated secretory cells or old ciliated cells, and therrfore would not be expected to engage in significant nuclear synthetic activity. Ciliated cells are mature elements with a low turnover rate and synthesize primarily structural protein. It is probable that their intrinsic level of nuclear function is rather low and that their ultimate course is one of slow degradation rather than rapid replacement, also grounds for a lower degree of nuclear uptake. It is appropriate to point out, in conclusion, that the Fallopian tube. which is the literal link in pregnancy between the ovary and uterus, is also the weakest link in terms of the status of our current knowledge about it.

REFERENCES

1. 2.

3.

4.

Bertalanfly, F. D., and Lau, C.: Acta Anat. 54: 39, 1963. Glass, L. E.: Immunocytological studies of the mouse oviduct, in Hafez, E. S. E., and Blandau, R. J., editors: The Mammalian Oviduct, Chicago, 1969, The University of Chicago Press, pp. 459-476. Greenwald, G. S.: Endocrinology of oviductal secretions, in Hafez, E. S. E., and Blandau, R. J,, editors: The Mammalian Oviduct, Chicago, 1969, The University of Chicago Press, pp. 183-202. Lehto, L.: Acta Obstet. Gynecol. Stand. 42: 1, 1963. (Suppl. 4.)

5.

Mashimoto, M., et al.: Sot. 9: 200, 1962, 11: Mastroianni. L.. Forest Proc. Sot. Exp: Biol. Pauerstein, C. J., and

J. Jap. Obstet. Gynecol. 92,i964. 6. W.. and Winter. W. W.: Med. 107: 86, 1961. 7. Woodruff, J. D.: AM. J. OBSTET.GYNECOL.Q& 121,1967. 8. Prasad, M. P. N., Dass, C. M. S., and Mohla, S.: J. Reprod. Fertil. 16: 97, 1968. 9. Stumnf. W. E.. and Roth. L. I.: High resolution autoradiography and’its applica?ion to in vitro experiments, in Radioisotopes in Medicine: In Vitro Studies, Proceedings of the Oak Ridge Symposium in Medicine, 1967. 1

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