- Email: [email protected]
Hormonal response of intact rodent uterus in organ culture
Q 1968 by Academic Press Inc.
32
Experimental
Cell Research 50, 32-36 (1968)
HORMONAL RESPONSE OF INTACT RODENT UTERUS IN ORGAN CULTURE CAROLEE
Special Laboratory Veterans Administration
M. LENAHAN
of Nuclear Medicine and Biology, Hospital,
Omaha, Nebr. 68105, USA
Received May 31, 1967
CULTURING cells, tissues, and organs outside the body has afforded the researcher the opportunity to study cellular behavior in greater detail. One area in which these techniques can be effectively utilized is in the study of uterine tissue, and the nature of the effects caused by estrogen and progesterone. Everett [2] tested the effect of these hormones on endometrium in culture and concluded that the presence of both progesterone and estrogen resulted in a synergism: well preserved epithelium and secreting glands, as well as an antogonism: fewer stromal cells of smaller size than with either hormone alone. These results are in accord with the work of Biggers et al. [l] which confirmed that, contrary to previous theory that the estrogenic effect occurs only after passage of the hormone through the body, the action in culture can indeed be direct. This study was undertaken to establish the optimal hormonal background for in vitro preparation of intact rodent uterus.
METHODS
AND
MATERIALS
Procedure for obtaining and preparing tissue.-Mature female Syrian golden hamsters were anesthetized with ether and the bicornuate uterus and associated oviducts and ovaries were surgically removed when the animal was known to be in early estrus. Determination of this stage was made by vaginal smears [6].’ Following surgical removal, the procedure consisted of stripping the uterus of fat and blood vessels in sterile BSS, removing the ovaries and oviducts, cutting pieces of the uterine tube into 8.0 mm lengths. Each piece of the tube was cut open using blunt-tipped scissors which are easily inserted into the lumen of the tube. The opened tube was then laid into the culture dish on the grid and gently flattened into the open position so that the endometrial surface was exposed. Culture procedure.-Culture dishes were disposable plastic presterilized organ 1 Smears were taken each day for eight days (two complete Experimental
Cell Research 50
cycles).
Hormonal
response of infacf rodent uterus in organ culture
33
culture dishes (Falcon Plastic Organ Culture Dishes). One piece of uterine tissue was placed in each dish on the grid. Tissue was incubated at 37°C in a humid environment (saturated) and in an atmosphere of 95 per cent O,, 5 per cent CO,. Procedure for medium preparation.-Medium used was Puck’s N-16 supplemented with 15 per cent fetal calf serum and 100 units each of streptomycin and penicillin/ml [7]. The capacity of the organ culture dishes is 1 ml of medium. Medium was changed every 48 h during the culture interval. Incubations were carried out with various concentrations of crystalline progesterone and (178) estradiol-acetate (see Table 1). The hormone, in the correct dose level, was dissolved in one or two drops of abTABLE Medium no.
a/ml estrogen
A B C D E F G H I J K L M N 0 P
1.0 2.0 4.0 6.0 1.0 2.0 4.0 6.0 1.0 2.0 4.0 6.0 1.0 2.0 4.0 6.0 1.0 2.0 4.0 6.0 0.0 0.0 0.0 0.0 0.0 0.0
Q R s T U V w X Y za
i4ml
progesterone 1.0 1.0 1.0 1.0 2.0 2.0 2.0 2.0 4.0 4.0 4.0 4.0 6.0 6.0 6.0 6.0 0.0 0.0 0.0 0.0 1.0 2.0 4.0 6.0 0.0 0.0
1
N
Endometrial mucosai cell height in p
48 36 56 44 60 52 52 44 52 20 41 32 28 32 44 36 36 28 28 32 20 20 20 20 44 28
7.12 7.28 8.29 7.99 7.02 9.38 9.76 10.50 8.32 11.25 14.08 7.80 7.98 8.51 9.38 8.17 7.05 8.66 9.08 6.09 9.11 8.93 7.16 7.90 7.37 6.04
S.E.
.65* .50* .54* .56* .44* .55* .48* .61* .54* 1.22 .54 .51* .58* .72* .72* .59* .39* .73* .s1* .51* 1.11* .93* .52* .71* .4s* .49*
* P value ( ~0.001) was computed by comparing the high-valued medium (K) with others. See text for complete explanation. a Alcohol was added to half the controls (l-2 drops/200 ml medium) to counteract the possible effect of the alcohol used to dissolve the hormone(s) in the experimentals. 3 - 681806
Experimental Cell Research 50
34
Carolee M. Lenahan
solute ethyl alcohol and added to 200 ml of the medium. Sections of the uterus were then randomly placed on the different types of medium so that the individual animal variations could not influence the findings. Responses of the tissues incubated on these media, in terms of endometrial mucosal epithelial cell height and general histology, were compared to those of tissues incubated in medium without the hormonal supplement. Selection of the medium having the optimal hormonal supplement was based on the two criteria mentioned. Procedure for studying the tissue.--Following the seven-day culture interval, the tissue was fixed in 10 per cent neutral formalin for from 24 to 48 hr and prepared for sectioning at 7 ,u. Slides were stained according to the procedure of Papanicolaou for cervical smears [3]. RESULTS
AND DISCUSSION
Following the routine culture and sacrifice of the tissue, sections were studied for the selection of the medium which preserved the best histology of the tissue and optimal cell height of the endometrical mucosa. Determination of the endometrical epithelial cell height was done by means of micrometer measurements of the endometrium using slides prepared from tissue cultured on all 26 types of medium (see Table 1). Four measurements were taken of each tissue section. Approximately 15 uterine sections were cultured in each medium. Variance in N is due to the discarding of any histological preparations which, due to the embedding angle, would not permit accurate endometrial measurements. A minimum of five acceptable sections resulted in at least 20 measurements for each type of medium. Studies of these tissues indicated Medium K to preserve histology and maintain endometrial cytology in a phase similar to the characteristic estrus pattern. Medium K (4.0 pg/ml each of progesterone and estradiol), gave a mean endometrial cell height of 14.08 ,U as contrasted to the control without hormone (medium Z), for which the measurement was 6.04 ,u. Although histology does not represent a quantitative measurement, general appearance was easily observed, and medium K provided the best histological integrity (see Figs 1 and 2). Cell heights were plotted against hormonal concentrations for each medium; each was then compared to medium K on the basis of the Student t test. Analysis of these figures shows a significant difference (P
Cell Research 50
Hormonal
response of intact rodent uterus in organ culture
35
Fig. I.--Section of tissue incubated on medium K indicating healthy endometrial mucosal cells. Papanicolaou stain. x 129. Fig. 2.-Section of tissue incubated on medium Y indicating poor endometrial proliferation. Papanicolaou stain. x 129. Experimental Cell Research 50
Carolee M. Lenahan
36
certain culture conditions in causing keratinization of the epithelium. It was concluded in this experiment that concentrations of 4.0 ,ug each of progesterone and estrogen per milliliter of medium afforded the best histological maintenance of the intact hamster uterine tissue slices and increased endometrial cell height. This represents a moderate dosage in light of those used in this experiment: (from 1.0 to 6.0 pg/ml). Lower doses were not adequate to give the desired effect; higher doses appeared to provide a toxicity. Comparison of results in this report with those of others [l, 21 shows variance in the level of hormonal supplement used in each laboratory. The variance in optimal maintenance hormone levels can probably be attributed to differences in technique, tissue, and species. Everett’s work utilized guinea pig endometrium which was manually stripped of underlying stroma and hence was exposed to direct contact with the medium. Biggers also maintained the epithelial surface of mouse vagina in contact with the medium. The technique presented here utilizes the entire uterine section; a high diffusion gradient involving muscular and stromal elements may explain the higher hormonal values for endometrial maintenance reported here. This procedure, while limited to small animals (where total uterine thickness is minimal), has the advantage of simplicity of preparation. It also affords minimal trauma to the tissue and maximal available endometrial surface area. SUMMARY
Organ culture of hamster uterus was undertaken to test uterine tissue response to hormonal stimulus. Graded doses of progesterone and estradiol supplements to standard liquid medium permitted choice of optimal hormonal conditions for maintaining the endometrium. The selection of the most ideally supplemented medium was based on measurements of endometrial cell heights and histological appearance of the tissue sections; this medium, containing 4.0 ,ug/ml each of progesterone and estradiol, permitted maintenance of 8.0 mm lengths of hamster uterine tube (longitudinally opened) for seven days without necrosis. REFERENCES 1. BIGGERS, 2. EVERETT, 3. HUMASO~, 4. KAHN, R. 5. LASNITZKI, 6. PECZENIK, 7. PUCK, T.
Experimental
J. D., CLARINGBOLD, P. J. and HARDY, J., J. Endocrinol. 24, 491 (1962). 6. H., I., O., T.,
M. H.,
J. Physiol.
L., Animal Tissue Techniques, pp. 357-9. Freeman, Nature 174, 317 (1954). Int. Reu. CytoZ. 7, 79 (1958). J. Endocrinol. 3, 157 (1942). CIECIURA, S. J. and ROBINSON, A., J. Exptl Med.
Cell Research 50
131, 497 (1956).
San Francisco,
108, 945 (1958).
1962.
32
Experimental
Cell Research 50, 32-36 (1968)
HORMONAL RESPONSE OF INTACT RODENT UTERUS IN ORGAN CULTURE CAROLEE
Special Laboratory Veterans Administration
M. LENAHAN
of Nuclear Medicine and Biology, Hospital,
Omaha, Nebr. 68105, USA
Received May 31, 1967
CULTURING cells, tissues, and organs outside the body has afforded the researcher the opportunity to study cellular behavior in greater detail. One area in which these techniques can be effectively utilized is in the study of uterine tissue, and the nature of the effects caused by estrogen and progesterone. Everett [2] tested the effect of these hormones on endometrium in culture and concluded that the presence of both progesterone and estrogen resulted in a synergism: well preserved epithelium and secreting glands, as well as an antogonism: fewer stromal cells of smaller size than with either hormone alone. These results are in accord with the work of Biggers et al. [l] which confirmed that, contrary to previous theory that the estrogenic effect occurs only after passage of the hormone through the body, the action in culture can indeed be direct. This study was undertaken to establish the optimal hormonal background for in vitro preparation of intact rodent uterus.
METHODS
AND
MATERIALS
Procedure for obtaining and preparing tissue.-Mature female Syrian golden hamsters were anesthetized with ether and the bicornuate uterus and associated oviducts and ovaries were surgically removed when the animal was known to be in early estrus. Determination of this stage was made by vaginal smears [6].’ Following surgical removal, the procedure consisted of stripping the uterus of fat and blood vessels in sterile BSS, removing the ovaries and oviducts, cutting pieces of the uterine tube into 8.0 mm lengths. Each piece of the tube was cut open using blunt-tipped scissors which are easily inserted into the lumen of the tube. The opened tube was then laid into the culture dish on the grid and gently flattened into the open position so that the endometrial surface was exposed. Culture procedure.-Culture dishes were disposable plastic presterilized organ 1 Smears were taken each day for eight days (two complete Experimental
Cell Research 50
cycles).
Hormonal
response of infacf rodent uterus in organ culture
33
culture dishes (Falcon Plastic Organ Culture Dishes). One piece of uterine tissue was placed in each dish on the grid. Tissue was incubated at 37°C in a humid environment (saturated) and in an atmosphere of 95 per cent O,, 5 per cent CO,. Procedure for medium preparation.-Medium used was Puck’s N-16 supplemented with 15 per cent fetal calf serum and 100 units each of streptomycin and penicillin/ml [7]. The capacity of the organ culture dishes is 1 ml of medium. Medium was changed every 48 h during the culture interval. Incubations were carried out with various concentrations of crystalline progesterone and (178) estradiol-acetate (see Table 1). The hormone, in the correct dose level, was dissolved in one or two drops of abTABLE Medium no.
a/ml estrogen
A B C D E F G H I J K L M N 0 P
1.0 2.0 4.0 6.0 1.0 2.0 4.0 6.0 1.0 2.0 4.0 6.0 1.0 2.0 4.0 6.0 1.0 2.0 4.0 6.0 0.0 0.0 0.0 0.0 0.0 0.0
Q R s T U V w X Y za
i4ml
progesterone 1.0 1.0 1.0 1.0 2.0 2.0 2.0 2.0 4.0 4.0 4.0 4.0 6.0 6.0 6.0 6.0 0.0 0.0 0.0 0.0 1.0 2.0 4.0 6.0 0.0 0.0
1
N
Endometrial mucosai cell height in p
48 36 56 44 60 52 52 44 52 20 41 32 28 32 44 36 36 28 28 32 20 20 20 20 44 28
7.12 7.28 8.29 7.99 7.02 9.38 9.76 10.50 8.32 11.25 14.08 7.80 7.98 8.51 9.38 8.17 7.05 8.66 9.08 6.09 9.11 8.93 7.16 7.90 7.37 6.04
S.E.
.65* .50* .54* .56* .44* .55* .48* .61* .54* 1.22 .54 .51* .58* .72* .72* .59* .39* .73* .s1* .51* 1.11* .93* .52* .71* .4s* .49*
* P value ( ~0.001) was computed by comparing the high-valued medium (K) with others. See text for complete explanation. a Alcohol was added to half the controls (l-2 drops/200 ml medium) to counteract the possible effect of the alcohol used to dissolve the hormone(s) in the experimentals. 3 - 681806
Experimental Cell Research 50
34
Carolee M. Lenahan
solute ethyl alcohol and added to 200 ml of the medium. Sections of the uterus were then randomly placed on the different types of medium so that the individual animal variations could not influence the findings. Responses of the tissues incubated on these media, in terms of endometrial mucosal epithelial cell height and general histology, were compared to those of tissues incubated in medium without the hormonal supplement. Selection of the medium having the optimal hormonal supplement was based on the two criteria mentioned. Procedure for studying the tissue.--Following the seven-day culture interval, the tissue was fixed in 10 per cent neutral formalin for from 24 to 48 hr and prepared for sectioning at 7 ,u. Slides were stained according to the procedure of Papanicolaou for cervical smears [3]. RESULTS
AND DISCUSSION
Following the routine culture and sacrifice of the tissue, sections were studied for the selection of the medium which preserved the best histology of the tissue and optimal cell height of the endometrical mucosa. Determination of the endometrical epithelial cell height was done by means of micrometer measurements of the endometrium using slides prepared from tissue cultured on all 26 types of medium (see Table 1). Four measurements were taken of each tissue section. Approximately 15 uterine sections were cultured in each medium. Variance in N is due to the discarding of any histological preparations which, due to the embedding angle, would not permit accurate endometrial measurements. A minimum of five acceptable sections resulted in at least 20 measurements for each type of medium. Studies of these tissues indicated Medium K to preserve histology and maintain endometrial cytology in a phase similar to the characteristic estrus pattern. Medium K (4.0 pg/ml each of progesterone and estradiol), gave a mean endometrial cell height of 14.08 ,U as contrasted to the control without hormone (medium Z), for which the measurement was 6.04 ,u. Although histology does not represent a quantitative measurement, general appearance was easily observed, and medium K provided the best histological integrity (see Figs 1 and 2). Cell heights were plotted against hormonal concentrations for each medium; each was then compared to medium K on the basis of the Student t test. Analysis of these figures shows a significant difference (P
Cell Research 50
Hormonal
response of intact rodent uterus in organ culture
35
Fig. I.--Section of tissue incubated on medium K indicating healthy endometrial mucosal cells. Papanicolaou stain. x 129. Fig. 2.-Section of tissue incubated on medium Y indicating poor endometrial proliferation. Papanicolaou stain. x 129. Experimental Cell Research 50
Carolee M. Lenahan
36
certain culture conditions in causing keratinization of the epithelium. It was concluded in this experiment that concentrations of 4.0 ,ug each of progesterone and estrogen per milliliter of medium afforded the best histological maintenance of the intact hamster uterine tissue slices and increased endometrial cell height. This represents a moderate dosage in light of those used in this experiment: (from 1.0 to 6.0 pg/ml). Lower doses were not adequate to give the desired effect; higher doses appeared to provide a toxicity. Comparison of results in this report with those of others [l, 21 shows variance in the level of hormonal supplement used in each laboratory. The variance in optimal maintenance hormone levels can probably be attributed to differences in technique, tissue, and species. Everett’s work utilized guinea pig endometrium which was manually stripped of underlying stroma and hence was exposed to direct contact with the medium. Biggers also maintained the epithelial surface of mouse vagina in contact with the medium. The technique presented here utilizes the entire uterine section; a high diffusion gradient involving muscular and stromal elements may explain the higher hormonal values for endometrial maintenance reported here. This procedure, while limited to small animals (where total uterine thickness is minimal), has the advantage of simplicity of preparation. It also affords minimal trauma to the tissue and maximal available endometrial surface area. SUMMARY
Organ culture of hamster uterus was undertaken to test uterine tissue response to hormonal stimulus. Graded doses of progesterone and estradiol supplements to standard liquid medium permitted choice of optimal hormonal conditions for maintaining the endometrium. The selection of the most ideally supplemented medium was based on measurements of endometrial cell heights and histological appearance of the tissue sections; this medium, containing 4.0 ,ug/ml each of progesterone and estradiol, permitted maintenance of 8.0 mm lengths of hamster uterine tube (longitudinally opened) for seven days without necrosis. REFERENCES 1. BIGGERS, 2. EVERETT, 3. HUMASO~, 4. KAHN, R. 5. LASNITZKI, 6. PECZENIK, 7. PUCK, T.
Experimental
J. D., CLARINGBOLD, P. J. and HARDY, J., J. Endocrinol. 24, 491 (1962). 6. H., I., O., T.,
M. H.,
J. Physiol.
L., Animal Tissue Techniques, pp. 357-9. Freeman, Nature 174, 317 (1954). Int. Reu. CytoZ. 7, 79 (1958). J. Endocrinol. 3, 157 (1942). CIECIURA, S. J. and ROBINSON, A., J. Exptl Med.
Cell Research 50
131, 497 (1956).
San Francisco,
108, 945 (1958).
1962.