Organ culture of human endometrium

Organ culture of human endometrium Effect of ovarian

EDWARD

C.

LAURENCE TIIU DAVID Syracuse,

steroids

HUGHES, M.

CSERMELY, B. New

JONES,

M.D.

DEMERS,

M.S

PH.D. M.D.

York

Glycogen turnover was determined in 86 normal and 52 sterility patients through the chemical analysis of endometrial glycogen content, glycogen synthetase and glycogen phosphorylase activities. Specimens from 3% of these normal patients as well as 31 sterility patients were also subjected to the organ culture process. These studies suggest to us that (1) an organ culture system is an ideal avenue of approach for the study of this glandular epithelial tissue and (2)~ that estradiol is not solely responsible for the increase in glycogen deposition in human endometrium, but that progesterone following estrogen priming is essential for a complete response of glycogenesis. This study further demonstrates in organ culture that estradiol-induced proliferative changes in the tissue, while Progesterone not only caused cellular alterations but also induced an increase in glycogen deposition. Preliminary studies with the enzyme systems in organ culture suggest a role of progesterone in phosphorylase activity, particularly during the secretory phase where a controlled breakdown of glycogen is desired.

0 NE OF THE MO s T iIllpOrtaIlt fUIlCtions of the endometrium in respect to the process of reproduction is the initiation and completion of the processes, glycogenesis and glycogenolysis. These processes are inherent in the growth, proliferation, and secretory activity of the glandular epitheliurn and are requisite for successful implantation of the blastocyst, its early growth, and development. The nature of control of glycogen turnover appears to be exerted at the level of the glycogen metabolic reguladiphosphoglucosetory enzymes, uridine glycogen transglucosylase (glycogen synthe-

tase) and glycogen phosphorylase through a well-integrated hormonal response system involving estrogen and progesterone. Glycogen synthetase exists in two interconvertible forms (independent and dependent of the cofactor glucose-6-phosphate) while glycogen phosphorylase has two interconvertible forms (independent and dependent of the cofactor 5’ adenosine monophosphate) with the active or independent form predominating. Glycogen synthetase activity, which is responsible for the initiation of glycogenesis, is believed to fall under the influence of estrogen, while glycogenolysis appears to be the result of a progestational influence on glycogen phosphorylase activity. The cyclic nature of these two enzyme systems support these concepts where the glycogen synthetase. enzyme reaches maximal activity during the sixteenth to twentieth days of the human menstrual cycle, a time coincident with maximal glycogen deposition while glycogen phosphorylase reaches its maximum activity

From the Department of Obstetrics and Gynecology, State University of New York Upstate Medical Center. This work was supported by a research grant from the Life Insurance Medical Research Fund and a grant from the Ayerst Luboratories. Presented at the Ninety-second Annual Meeting of the American Gynecological Society, New Orleans, Louisiana, May 5-7, 1969.

707

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et al

during the t\\entieth to twenty-fifth clays of the cycle, xvhen the tissw is under the influence of progesterone. The activities of thew rnzy~Jr~s xc irwgular in some sterility patients, particularly those xvith hormonal imhalancr. In most. the activities of both enzylnes are decreased, and in some either one or the other wqlilc system is depressed. All, holvrvrr. fail to provide adequate ,glycoSgcn tlux~o\w essential for the nutritional demands of thfx cndometrial tissue. Figs. 1 through 7 show sow comparative findings in 86 normal and 52 strsrility patients in the \xrioris phnscs of the, menstrual cvcl~. Organ

culture

Materials

and

methods

Source of specimens. Endtrmctrial

tissw‘ \vas obtainwl ;rt \.arious stagea of t11c* IIICJ~5trual cycle t)v c~ndo~n~~tr~i~~l ciiwtte in 118 (I! the original CJJOU~ of Jl~rIJla~ ]Xlti~Il~S ;HJCi in 31 women of thca group witll ;I llistrbq. c;: sterility. A portion of this tissue xvx placed immediatelv in qowth rtlctliunr for organ culture: anothw portiorl ~\‘;Ls prepared for histologic a~itl histoch~~riical StId\ and some were studied by clcctron microscopic waluation. and the third porticm was plawti in cracked kc and used for ~l~w~grn content. ,glycogen syntlietasta. ilTlt1 ]‘]loS]‘ll~)t’)‘laS(’ activity measureiiwnts.

experimentation

The present investigation was initiated in an attempt to study thr relationship of o\,arian steroids to thrw enzyme systems. The organ culture was used so that the tissue could be isolated from the complexitirs of other organ and endocrine intrractions. It \vas an attempt to provide 3 srmi in viva environment lvhere histologic studies as 11VII biochcmicnl analysis could he coml~letcd on a routine basis and \vherc, the presence of

Organ culture. An

or,qarJ

c~rl1tuJ.t~

s)rtcv~:

irlitially lt\ was prepared as described ‘lYro~cl1.~~ ” Endometrial tissue was slictxi into 2 mm.” csplants and piaccd in a Falter! polystyrene organ culture dish on a Gclman Metricel filter port six, 0.80 1-1. which \va‘ \ct on a stainless steel grid wsting over thr, r’enter well of thcx culture dish. The centc,r wail vcxltainrvi I tttl. of ymwth nwtlirlJrl.

lOoo.-

12--15 DAYS

Fig.

1. The

comparative

lb-20 CYCLE

in viva glycogen levels during the various phases of the menstrual patients and 52 sterility patients. The values are expressed in milliper 100 Gm. of wet weight of tissue. It can be noted that maximum occurs between the sixteenth and twentirth days of the cycle. l’hrrr~ i5 in the glycogen levels of the stwility ucql.

cycle from 87 normal grams of glycogen glycogen deposition a marked decrease

OF

Volume Number

105 5

Organ

I

Cl2

12-15

>20

16-20

DAYS

OF

culture of endometrium

CYCLE

Fig. 2. The activity of the glycogen synthetase enzyme during the various phases of the menstrual cycle. Values incorporated into glycogen per gram of wet tissue per during the sixteenth and twentieth days of the cycle.

(I and D form) in normal are expressed as micromoles hour. The maximal activity

patients glucose occurs

80

60

40-d?$Iy ;G;/

20--

y---

<12

.

f

‘I

12-15 DAYS

.

16-20 OF

>20

CYCLE

Fig. 3. The activity of the glycogen synthetase enzyme in the sterility group at various phases of the menstrual decreased.

(I and D form) in human endometrium cycle. Note that values are statistically

709

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Mt.%

L <12

12-15

w-96 DAYS

Of

204s

>H

CYCLE

Fig. 4. The activity of glycogen phosphorylase enzyme (t and a form) in the normal patients during the various phases of the menstrual cycle, Units of enqme activity are ex@csed aa micromoks glucose incorporated into~ glycogen per gram of wet tissue per hour. Note &at peak activity occurred between the twentieth and twenty-fifth days of the cycie with the a form predominating.

QAYJ

Fig. 5. The activity of tke &cog metrium from the sten”Hty gro&. the normal group.

OF

CYCLE

Volume Number

105 5

Fig. 6. The deposition of glycogen at the base and urn. The endometrium was dated as 14 day. (Best’s globules from the Fig. 7. The release of glycogen endometrium. (P.A.S. glycogen. x430.) 24
Organ

culture

of endometrium

within the cells of the glandular epithe :licarmine stain for glycogen. x900.) glandular surface of an epithelial cell in

71

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Hughes et al,

while the peripheral portion of the dish contained sterile distilled water to maintain a moist atmosphere. All cultures were maintained at 37%. in an incubator bubbled with 95 per cent 0, and 5 per cent CO, during the culture period. Culture medium. The medium used in this organ culture system was Trowell’s T8 medium supplemented with 10 per cent fetal calf serum and containing antibiotics. When hormones were used, they were dissolved in 95 per cent ethanol and added to the medium to give the desired concentration. The progesterone concentration was 0.5 Fg per milliliter, while the estradiol concentration was 0.1 pg per milliliter. The control medium contained the same percentage of alcohol, 1 per cent by volume, but lacked the hormone supplement.

Biochemical studies. Glycogen content. Tissue glycogen content was quantitated by a phenol-sulfuric acid assay as described by Montgomery.” Results are expressed as milligrams of glycogen/lOO Gm. wet weight of tissue. Glucose determination. The growth medium was assayed for glucose using a modified calorimetric method as described by Dubowski.4 Glycogen synthetase activity (I and D form). Synthetase activity was determined by following the incorporation of Cl4 glucose into glycogen from uridine diphospho-Cl4 glucose in the presence (D) and absence (I) of the cofactor glucose-6-P. A unit of enzyme activity was defined as 1 pmole glucose incorporated into glycogen per gram wet tissue per hour. Glycogen phosphorylase activity (a an-d t form]. Phosphorylase activity was assayed by following the incorporation of Cl4 glucose into glycogen from Cl4 glucose- 1 -phosphate in the presence (t) and absence (a) of the cofactor 5’ AMP. A unit of enzyme activity was expressed as 1 kmole glucose incorporated into glycogen per gram wet tissue per hour. Histobgic evahation. Explants for histology were fixed in absolute alcohol, embedded in paraffin, and sectioned at 6 p thickness. Slides were stained with hematoxyjin

:?iicrverr!brr I) 1w Am. J. Obst. Pr Gynrr.

and eosin, periodic acid-Schiff reagent (for Gomori stain r&w-n i , and a modified (for alkaline phosphatase activity). Index of viabUy of tissue. The index of viability of the explants was based on histologic appearance, glucose uptake from the growth medium, and glycogen content of the explants themselves. Initial biochemical values and histologic observations were made on the tissue at the time of biopsy to establish a base line. Control specimens were maintained throughout the culture period in hormone-free medium to detect nonphysiologic changes induced by the culture process itself. Resuik This study indicates that human endometrium placed in organ culture will maintain its differentiation and function for extended periods of time. Some explants survived as long as 18 days, while others became necrotic in 4 to 5 days. The average length of viability was 10 days, although central necrosis occurred in some of these explants. It was observed that the addition of steroids prolonged the survival time of the tissue. Normal group. The biochemical analysis of the specimens presented some interesting results. There was a slight decrease in the glycogen content between the initial tisstic value and the control explants after 3 days in culture at all stages of the cycle in the normal patients. This lower value could be expected inasmuch as it would take some. time for the tissue to become active in itsnew environment. The addition of 0.1 & per milliliter of estradiol to the medium did not alter significantly the glycogen 1eveI over the control samples after 3 days in culture. The addition of 0.5 pg per milliliter of progesterone to the culture medium, however, had a marked effect upon the glycogen content and function of the endometrium in culture. There was an increase in the glycogen content at all phases of the cycle and this increase reached statistically significant proportions in the 8 to 12 day specimens. Histochemical studies of the same

Volume Number

105 5

Organ

Table I. Glycogen No. of patients

content

in normal

Cycle period

endometrium-3

Initial value

< 12 12-15 16-20 > 20

12 6 13 7

human

of endometrium

713

day culture

Control

187 + 20 550 2 43 903 -I 23 633+51

culture

Progesterone

Estrogen

1712 25 474r 9 764 2 102 590? 110

147+ 42 3722 60 5032 32 627 t 140

690 f 149 566+ 42 975 k 158 833 f 238

38

Table II.

Glycogen

content

in

human

endometrium

from

sterility

patients-3

day culture No. of patients

Cycle period

7 6 12 6

I

12 12-15 16-20 20

Initial value 162 282 284 367

+ + + f

Control 20 43 51 46

Estrogen

199 + 54 318 + 87 212 + 28 297 + 70

Progesterone

239 f 46 224 + 170 257 + 16 594 + 84

561 767 745 499

+ 110 2 114 + 123 + 84

31

------INITIAL

of hormones endometrium

CONTROL

m

ESTRADIOL

0

PROGESTE’RONE

----~INITIAL

VALUE

DAYS OF CYCLE

Fig. 8. Effect tent of human specimens.

m

upon glycogen conin vitro in normal

tissue supported these findings. Table I and Fig. 8 demonstrate these differences. Sterility group. In the sterility group there was a slight change in glycogen levels between initial tissue values and the explants in the control medium after 3 days in cul-

VALUE

DAYS

OF CYCLE

Fig. 9. Effect of hormones upon of human endometrium in vitro mens.

glycogen in sterility

content speci-

ture. The addition of 0.1 pg per milliliter of estradiol to the culture medium did not alter the glycogen levels until the twentieth day of the cycle, when an increase was observed. The addition of 0.5 pg per milliliter of

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progesterone to the culture medium produced significant increases in the glycogrn content in every phase of the rycle, particuIarly in midcycle specimens where levefs reached the values of the normal tissue. ‘Table II and Fig. 9 demonstrate these results. Glucose uptake from the culture medium. ‘I’he glucose content of the culture medium was measured at the conclusion of the incubation period with the control specimens as xvell as with those specimens whcrc hormone supplementation was used. ‘There \vas an increase in uptake of glucose from the meditml both in the normal and sterility specimcnq after the addition of progestcronr. This increased uptake \vas most prono~mcrd durirx

Volume Number

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culture of endometrium

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Fig. 11. A 12 day normal endometrium after 3 days in culture in control medium. This is a cross section of an endometrial gland. Note some stratification of the cells. No subnuclear vacuolization. (Hematoxylin and eosin. x720. j

Fig. 12. An endometrial gland after the explant had been in culture for 3 days to which 0.1 pg per milliliter of estradiol had been added. Note some slight increase in stratification of the glandular epithelium. Nuclei increased in size. No evidence of secretory changes. (x720.)

I

/

1 1

Fig. 13. A cross section of an endometrial gland of the same specimen as demonstrated in Figs. 11 and 12. Explant was in culture 3 days to which 0.5 pg per milliliter of progesterone had been added. Note the increased size of the gland, elevation of the nuclei to the upper half of the cell, and marked vacuolization beneath the nuclei. These vacuoles contained large globules of glycogen. (Hematoxylin and eosin. x720.)

716

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Fig. 14. A cross section of two endometrial glands of initial 18 day endometrium from a sterility patient before culture. Note secretory activity. P.A.S. stain showed the usual amounts of glycogen found in 18 day endometrium in sterility patients. (Hematoxylin and eosin. ~720.)

Fig. 15. A cross section ~of endometrial gland of same specimen in 3 day control culture. Note increased stratification of the cells. No increase in the secretory activity of the gland. Glycogen levels were not increased. (x720.)

Fig. 16. A cross section of an endometrial gland of the same specimen as shown in Figs. 14 and 15. Three days in culture to which 0.5 &g per millikr of progesterone was added. Note the markad secretory change in the gIand. Marked increase in the vacuoIiaation. Nuclei elevated to the upper third of the cells. PAS, s&h markad increase in gi* content vf thti ce$ls. ay~og@t is dsrmm passing from the celr surface. (~1,270.)

November 1, 1969 Am. .I. Obst. & Gyncc.

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culture of endometrium

717

Fig. 17. Electron micrograph of the normal cell structure with glycogen deposition in several cells. Two days in culture with progesterone supplementation. The open space represents the accumulation of glycogen elevating the nucleus toward the periphery. (~4,250.)

The stroma became more compact with some mitosis. The glands increased in size slightly and the glandular epithelium became more stratified. The dating patterns generally remained the same, although in some specimens some progression did develop. Secretory changes did not occur in any of the control explants. In the estradiol supplemented cultures, the explants showed stimulatory growth changes in the stroma and glandular epithelium in both groups. There was more stratification and crowding of the glandular epithelium, change in cell polarity, and in some specimens the nuclei became larger. The glands were also smaller in size in some instances. The deposition of glycogen in the glandular epithelium increased only slightly.

The cellular changes in the progesteronesupplemented specimens were interesting and provocative in both groups. The glands became larger; the glandular epithelium more columnar. The nuclei were elevated to the upper third of the columnar cells and the marginal surface of the cells became frayed. Cell membranes were practically eliminated in some cells. Glycogen distribution also was altered with large subnuclear globules of glycogen developing at the base of the epithelial cells. These globules were increased in size and droplet forms of glycogen were observed to be eliminated from the cell surface into the gland lumen. There is evidence that membrane permeability was enhanced by the increase of alkaline phosphatase. These changes indicated that

718

Hughes et al.

secretory changes had occurred in these explants after the addition of progesterone to the medium (Figs. 11-16). Studies of the endometrium in culture by the electron microscope indicated that during the midcycle, glycogen appearing as open spaces was observed at the base of the epithelial cells. The mitochondria, Golgi, apparatus, and lysosomes were prominent in these specimens. During the late secretory phase (26 days) glycogen was noted to be present at the periphery of the glandular epithelium. The cell membrane with its many microvilli was exceedingly thin, allowing the substance to pass from the cell surface. The mitochondria, Golgi apparatus, and other subnuclear particles were not as prominent (Figs. 17-18).

Comment This study indicates that endometrium placed in organ culture will continue to function for varying periods of time. This function occurred in tissue from all phases of the cycle and was viable enough for study. Ehrmann, McKelvey, and Hertig5 together with FiggeG failed to demonstrate discernible morphologic and histochemical changes in the tissue in their experiments which could be related to hormonal stimulation. This study has revealed biochemical, histologic, and histochemical changes that did result from the addition of estradiol and progesterone to the culture medium. It has been demonstrated for the first time that these hormones do have a specific action upon this tissue. There are indications that

rig. 18. Electron micrograph showing glycugen reacSy to be released from the cell surface with many microvilli Yhe open space represents glycogen. Note the thin cell membrane ~4,250.)

Volume 105 Number 5

estradiol causes proliferation or growth changes in the tissue and perhaps initiates the process of glycogenesis through its action upon gIycogen synthetase. The study, however, also indicates that estradiol alone does not complete the process of glycogenesis but that progesterone following estrogen priming is essential for maximum deposition of glycogen between the sixteenth and twentieth days of the cycle. Evidence is presented that progesterone not only has specific action upon the tissue, creating secretory changes, but also appears to have a stimulatory effect upon glycogen phosphorylase

Organ

culture of endometrium

719

activity. Although estradiol was not added to these cultures, it was assumed that the tissue was already primed by the endogenous estrogen. Progesterone also, through its action on alkaline phosphatase, presumably induced increased permeability of the cell membrane, allowing glucose as well as globular glycogen to leak out through the cell membranes. This disengorgement was further demonstrated by the electron microscope. The leakage provides an optimum glucose content of the uterine fluid beneficial for the early blastocyst.

REFERENCES

1. Trowel& 0. A.: Exper. Cell Res. 16: 118, 1959. 2. Trowell, 0. A.: Ann. New York Acad. SC. 95: 847, 1961. 3. Montgomery, R.: Arch. Biochem. 67: 378, 1957. Discussion

W. MITCHELL, JR., Boston, Massachusetts. In this latest chapter in his longterm study of the endometrium, Dr. Hughes, together with his co-workers, has accumulated data sufficient for three ordinary papers. I have found the task of sifting this material a formidable one. It is clear that a significant contribution has been made in the development of a model system for the in vitro study of the endometrium. The success of Dr. Hughes in reproducing in endometrial explants the changes occurring in the cycles of normal women has, to my knowledge, not previously been achieved by others. The failure of Ehrmann, McKelvey, and Hertigl and FiggeZ to accomplish similar goals led them to postulate that the in vivo effects of ovarian steroids on the endometrium must he mediated indirectly. This report proves that the effect must be direct, although there exists a definite possibility that the culture medium, containing as it does both insulin and fetal calf serum, enhances the action of estradiol and progesterone. In previous studies, the explanted endometrium underwent secretory change in culture without the addition of steroids to the substrate. One question concerns the specificity of the reactions. Are they necessarily relatecl to the type and dosage of steroids used DR.

GEORGE

4. Dubowski, 5. Ehrmann, tig, A. T.: 6. Figge, D.

K. M.: Clin. Chem. 8: 215, 1962. R. L., McKelvey, H. A., and HerObst. & Gynec. 17: 416, 1961. C.: Acta cytol. 7: 245, 1963.

in these experiments? Would, for instance, estriol behave like estradiol, or testosterone like progesterone? The use of a group of sterility patients as controls for those ostensibly normal, I found somewhat confusing, especially since the cause of sterility is not specified. Presumably, these were oligoovulatory patients. If so, the day of biopsy is not comparable to the day of biopsy in patients with normal cycles, since, in the sterility group, the time of ovulation may be indefinitely postponed. This relative immaturity may account for the failure of the endometrium obtained from sterile patients to utilize progesterone to form glycogen in the same manner as that from normal patients after ovulation. A very fine quantitative balance must, therefore, be essential to the effect of ovarian steroids on the endometrium, both in vitro and in vivo. The importance of this quantitation has recently been shown by Kohorn and Tehao” in their studies of organ cultures of endometrial carcinomas. Small concentrations of progesterone enhanced the tumor, and larger concentrations caused necrosis. The biochemical and histochemical studies which comprise an integral part of this paper corroborate the morphology. Two interesting hypotheses are introduced : (1) That estrogen initiates glycogenesis by stimulating

720

Hughes et al.

Ilycogen synthetase in the proliferative phase Mhich is in contradistinction to the view that Ilycogen is produced by the differentiated progestational endometrium and (2) That the effect of alkaline phosphatase is to increase zell membrane permeability, although the conzentration of this enzyme, along with glycogen, at the cell membrane, and their simultaneous Sscharge on the seventeenth or eighteenth day af a normal cycle does not imply a cause-andeffect relationship. I am impressed with this work. Whether Dr. Hughes is fiddling, or skiing, dabbling in tissue culture, or lining up insurance companies to support his research, he excites my admiration. REFERENCES

1. Ehrmann, R. L., McKelvey, H. A., and He&, A. T.: Obst. & Gynec. 17: 416, 1961. 2. Figge, D. C.: Acta cytol. 7: 245, 1963. 3. Kohorn, E. I. and Tehao, R.: J. Obst. & Gynaec. Brit. Comm. 75: 1262, 1968. DR. HOWARD W. JONES, JR., Baltimore, Maryland. I arise simply to pursue a question which Dr. Mitchell asked because it seemed to me as we saw these interesting data of Dr. Rughes that perhaps we had an indication that we had a test for or measure of luteal function. As Dr. Mitchell said, the lumping together of the infertility patients was somewhat confusing. If I understood the data correctly, the addition of progesterone to the specimens of abnormal endometrium raised the biochemically determined glycogen level to the level obtained in the normal cycle. This would presume, therefore, that these patients were deficient in progesterone. I think it would be very helpful if you could tell us whether the infertility patients had been studied by other techniques to determine their luteal function. Dad they, indeed, ovulated? And were these endometrial samples from a portion of the cycle which should have had a normal amount of glycogen for the luteal share of the cycle? One other point-is the glycogen level doserelated? Have you tried other amounts of progesterone which would raise the glycogen level to something short of normal or, indeed, above normal? As Dr. Mitchell said, Dr. Hughes always has exciting information. He has, indeed, taken the endometrium as his own unique organ.

DR. HUGEIES (Closing). CUSS the dose levels first.

I

would

lik,: to dis-

We have tried all levels of dosage both fat estradiol and progesterone. An entirely different action was obtained if a dose over 0.5 Pg per milliliter was used. It did not make much difference between 0.2 and 0.5 pg, but when 10, 25, or 50 pg was used, the endometrium actually was depressed instead of stimulated. Others have also found this in their studies. There was a paper on organ culture at The American College of Obstetricians and Gynecologists last week where they had quite similar data. In order to arrive at this dose level, we have tried varying doses of both estradiol and progesterone. The dose reported seemed to be the best do* range to get a stimulative effect upon endometrium in vitro and upon glycogen metabolism. All sterility patients had a complete sterility work-up consisting of total goaadotropins, total estrogens, and pregnanediol, measured at the same day of the cycle that the tissue was taken by biopsy. They all had basal body temperature charts, and most tests were completed between the sixteenth and twentieth days of the cycle. Most of them were menstruating quite regularly, and if they were not, the tests and biopsy were not completed until after the temperature had risen and ovulation had occurred. -All normal cases were tested at the same times of the cycle as the sterility group. It was interesting to us to find the endometrium in sterility patients did respond even better than the normal patients. In other words, I think it is a luteal phase failure in these patients, and the tissue in the culture was very responsive to the progesterone, but not particularly to the estradiol. I think the organ culture opens a new channel for us to work with. We have many more studies concerning endometrium and organ culture which we could not report. I think it is encouraging to know that we do have an experimental tool that we can use to test the action of various drugs, and hormones. We have also tested out some of the contraceptive hormones. We get a different effect with practically every one of them, depending upon the dose levels of the progesterone in the drug. Since the presentation of this paper a ,more detailed description of the effects of progesterone has been published by the same authors (Obst. & Gynec. 34: 252, 1969).