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WV) mice during experimentally induced deciduoma formation
Animal Reproduction Science, 14 (1987) 75-82
75
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Uterine Histamine Content in Mast-Cell Deficient (W/W v) Mice During Experimentally Induced Deciduoma Formation FRED L. JACKSON, EDWARD L. ORR, ALAN S. MORRILL and ROBERT J. WORDINGER
Department of Anatomy, Texas College of Osteopathic Medicine, North Texas State University, Fort Worth, TX 76107 (U.S.A.) (Accepted 11 February 1987)
ABSTRACT Jackson, F.L., Orr, E.L., Morrill, A.S. and Wordinger, R.J., 1987. Uterine histamine content in mast-cell deficient (W/W v) mice during experimentally induced deciduoma formation. Anim. Reprod. Sci., 14: 75-82. This study examines the changes in uterine histamine content in mast-cell normal and deficient mice during deciduoma formation. Surgical trauma (e.g., sutures) and intraluminal injection of oil produce deciduoma formation in ovariectomized mast-cell normal ( + / + ) and mast-cell deficient (W/W v) pseudopregnant mice that had previously received a single control ovary transplanted under the kidney capsule. Mast-cell normal ( + / + ) mice exhibit significant elevation of total uterine histamine content during both trauma and oil induced deciduoma formation. Mastcell deficient ( W/W v) mice under conditions of similiar deciduoma formation did not show any change in total uterine histamine content. These results indicate that the change in histamine observed in mast-cell normal mice during deciduoma formation is most likely of mast cell origin. The lack of any measurable change in uterine histamine in mast-cell deficient mice indicates that an increase of non-mast cell histamine is not a prerequisite for deciduoma formation.
INTRODUCTION
The role of histamine in deciduoma formation has been the subject of controversy for many years. Shelesnyak (1960) and Nalbandov (1971) have implicated uterine mast cell histamine in deciduoma formation and implantation. The number of uterine mast cells changes at the time of implantation (Shelesnyak, 1957; Defeo, 1967; Brandon and Bibby, 1979) as does the total uterine histamine content (Marcus et al., 1964). In addition, the effects of various histamine receptor antagonists have suggested a role for uterine histamine during implantation (Brandon and Wallis, 1977; Brandon and Raval, 1979). 0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
76 The WBB6FI-W/W v ( W / W v) inbred mouse strain provides an excellent model for the examination of the role of uterine histamine since it has been demonstrated that W / W v mice are deficient in stainable mast cells (Kitamura and Hatanaka, 1978; Wordinger et al., 1985 ). Our laboratory has reported the existence of a small pool of non-mast cell uterine histamine in W / W v mice (Wordinger et al., 1983). Subsequently, we have demonstrated the ability of mast-cell deficient ( W / W v) mice to undergo deciduoma formation, implantation, and successful live births (Wordinger et al., 1986). This latter finding indicates that mast cells are not required for these events. However, the possibility that the non-mast cell pool of histamine may be involved in deciduoma formation has not been examined. The objectives of this study were to (1) examine the total uterine histamine content in mast-cell normal ( + / + ) mice in response to oil or trauma induced deciduoma formation, and ( 2 ) compare the same parameters in mast-cell deficient ( W / W v) mice to evaluate the relative contribution of mast cells or the non-mast cell pool to the changes in uterine histamine content. MATERIALSAND METHODS
Animals Female WBB6FI-W/W v mast-cell deficient and mast-cell normal congenic female W B B 6 F 1 - + / + litter mates were purchased at 4-6 weeks of age from Jackson Laboratories, Bar Harbor, ME. Animals were housed five to six per cage in polycarbonate cages with a light:dark cycle of 14 h light:10 h dark per day. Food and water were provided ad libitum.
Ovariectomy and ovary transplantation W / W v mice are sterile due to a defect in germ cell migration (Russell, 1954). The ovaries are atrophic thus requiring ovariectomy and the use of exogenous steroids to maintain estrous cycles and responsiveness to deciduoma stimulation (Wordinger et al., 1985). An alternative method to re-establish hormone levels sufficient for deciduoma formation was recently reported by our laboratory {Wordinger et al., 1986). It consists of transplanting a normal ( + / + ) ovary under the kidney capsule of a W / W v female mouse. Two weeks prior to experimental deciduoma induction, + / + and W / W v mice underwent bilateral ovariectomy using the inhalant anesthesia metofane ( methoxyflurane; Pitman Moore, Washington Crossing, NJ). At the same time each animal received a single ÷ / ÷ ovary transplanted under the kidney capsule. The transplantation involved a small incision in the fibrous capsule surrounding the kidney with the ovary being inserted under this capsule. Vaginal smears were used to confirm the re-establishment of estrous cycles in ovariec-
77 tomized animals containing a transplanted ovary prior to their use for deciduoma induction.
Deciduoma induction Ovariectomized, ovary-transplanted + / + and W / W v mice were stimulated to undergo an artificial decidual cell response 3 days following successful mating, to induce a state ofpseudopregnancy. Two methods were utilized to induce deciduoma: (1) physical trauma, and (2) injection of oil. For induction of experimental decidual cell response by trauma, animals were anesthetized and the uterus exposed via an abdominal incision. Three sutures (4-0 silk; Ethicon, Somerville, NJ) were placed at equal distances along one uterine horn. For induction by injection of oil, animals were anesthetized, one uterine horn was exposed, and 10 pl of arachis oil was then injected into the lumen of the exposed uterine horn. For both trauma and oil injection, the contralateral uterine horn served as a non-stimulated control horn. Animals were killed 72 h following deciduoma induction and uterine horns were weighed and collected for measurement of uterine histamine content.
Histamine assay Uterine horns to be assayed for total histamine content were homogenized in 500 pl of ice-cold distilled water using a polytron homogenizer (Brinkmann Instruments, Westburg, NY). The homogenate was heated for 10 min in a boiling-water bath to destroy endogenous S-adenosyl-L-methionine and precipitate proteins. Samples were cooled on ice, centrifuged at 50 000g for 10 min, and 10-pl samples of the resulting supernatant were assayed for histamine. Histamine was assayed radioenzymically using a partially purified preparation of rat kidney histamine-N-methyltransferase to transfer a tritiumlabelled methyl group from 3H-adenosylmethionine onto histamine (Orr, 1984). The radioactive product of the reaction, 3H-methylhistamine was separated from 3H-adenosylmethionine by ascending thin-layer chromatography using a chloroform:methanol:ammonium hydroxide (12:7:1 by volume) solvent system ( Orr and Pace, 1984 ). 3H-methylhistamine was localized with ninhydrin, scraped from the plate, eluted with 500 pl of absolute ethanol and the radioactivity determined by liquid scintillation counting. Internal standards of known quantities of histamine were used to quantitate the total histamine in the samples.
Light microscopy Tissue for light microscopy (uteri and kidneys with transplanted ovaries) was placed in 10% neutral-buffered formalin for 48 h, processed in a tissue
78 TABLE 1 Uterine horn weights (rag) and histamine content (ng/horn) of + / + and W/Wv mice following trauma (suture} or oil induced decidual cell response Treatment
Tissue
(N)
Weight (rag)
Histamine (ng/horn)
Suture + / +
Stimulated horn Unstimulated horn
(7) (7)
131.3+20.8"* 31.7 +__ 1.3
177.1_+32.3" 59.7 _+ 7.9
Suture W / W v
Stimulated horn Unstimulatedhorn
(5) (5)
157.6_+40.1" 22.8_+ 1.5
Oil + / +
Stimulated horn Unstimulatedhorn
(6) (6)
342.6 _+23.2" * 35.1+_ 2.8
55.6 _+ 6.9* 24.5_+ 5.5
Oil W / W v
Stimulated horn Unstimulated horn
(4) (4)
288.4_+ 18.9"* 26.2_+ 7.1
3.6 _+ 1.4 3.1 _+ 1.3
4.1_+ 0.9 3.9_+ 0.3
Values represent the mean _+SEM. * = P < 0.05, ** = P < 0.01 compared to unstimulated horn
processor, embedded in paraffin and sectioned at 6 p m on a rotary microtome. Sections were stained with acidified ( p H 3.0) toluidine blue for mast cell identification. M a s t cells were identified by their large ovoid size (50 # m ) and a central nucleus which was often obscured by a dense accumulation of purple metachromatic cytoplasmic granules.
Statistical analysis All data were analyzed by one-way analysis of variance (ANOVA) with multiple range testing to determine differences between individual groups. Differences were considered statistically significant at P < 0.05. RESULTS
Trauma induced deciduoma induction As shown in Table 1, there was a significant ( P < 0.01) increase in weights of stimulated uterine horns of both + / + and W / W v mice in comparison to unstimulated contralateral control uterine horns. There was a significant ( P < 0.05) increase in total histamine content in stimulated horns of + / + mice when compared to unstimulated contralateral uterine horns. In contrast, the stimulated horns of W / W v mice failed to exhibit an increase in uterine histamine as a result of a t r a u m a induced deciduoma.
79
Oil induced deciduoma formation As shown in Table 1, there was a significant ( P < 0.01) increase in weights of stimulated uterine horns of both + / + and W/W v mice in comparison to unstimulated contralateral control horns. The increase in weights of uterine horns produced by the intraluminal injection ofoil was greater than the increase due to trauma. There was a significant {P < 0.05 ) increase in total histamine content in stimulated horns of + / + mice as compared to unstimulated contralateral control horns. The total histamine content of stimulated uterine horns of W / W v mice was not altered by oil induced deciduoma.
Light microscopy Histological examination of both the stimulated and unstimulated uterine horns from + / + and W / W v mice confirmed the abscence of mast cells in W/W v uteri. In addition, histological examination of the kidney-ovary interface and surrounding connective tissue capsule of W/W v mice which had received a + / + ovary transplanted under the kidney capsule failed to show any stainable mast cells. These results indicate that the transplantation of a + / + ovary does not establish a mast cell population in W/W v mice. Qualitative examination of + / + uterine horns revealed normal or slightly elevated numbers of mast cells. DISCUSSION The ability of mast-cell deficient mice (W/W v) to exhibit a trauma induced deciduoma was first demonstrated by Hatanaka et al. (1982). A trauma induced deciduoma is progesterone dependent but is estrogen independent (Finn and Hinchliffe, 1964). Formation of deciduoma in response to intraluminal injection of oil is both progesterone and estrogen dependent as is normal implantation ( Mayer, 1963 ). Wordinger et al. (1986) reported that W/W v mice would form a deciduoma with both trauma and also with intra-uterine injection of oil. Our results confirm the ability of W/W v mice to form a deciduoma as a result of either trauma or intra-uterine oil injection. These results indicate that mast cells and/or mast cell products are not required for this critical step in implantation. Both Hatanaka et al. (1982) and Wordinger et al. (1983) have reported the existence of a non-mast cell pool of uterine histamine. This pool is small and is not significantly altered by ovariectomy or exogenous steroid administration (Wordinger et al., 1985). The current results confirm the existence of this nonmast cell pool and also demonstrate that this pool is not significantly altered during the formation of either a trauma induced deciduoma or a deciduoma induced by intraluminal injection of oil. These results may indicate that the
80 non-mast cell pool of histamine is not critically involved in deciduoma formation. There is an increase in total uterine histamine content in the mast-cell normal ( + / + ) mice associated with the experimentally induced deciduoma formation. Since W/W v mice do not exhibit a similiar increase, it is likely that the source of this histamine is the uterine mast cells. This increase in histamine may be due to an increase in mast cell number or may reflect changes in histamine content and/or turnover in the uterine mast cells of the + / + animals. Increases in total uterine histamine during the peri-implantation period have been reported in the hamster (Hine et al., 1985) and the rat (Marcus et al., 1964 ). Both studies reported a subsequent decrease to non-pregnant levels of total uterine histamine following implantation. These studies were performed using pregnant animals whereas our results are on pseudopregnant animals with artificially induced deciduoma. These differences make direct comparison of data from the various studies difficult. The dramatic increase in uterine histamine levels in + / + animals may indicate a response of uterine mast cells to the process of deciduoma formation. Deciduoma formation is hormonally dependent and sufficient levels of progesterone and estrogen are required for decidualization to occur. The response of uterine mast cells may be due to a mast cell reaction to the changes in the hormonal milieu which are associated with deciduoma formation. The process of deciduoma formation involves the dramatic hypertrophy of stromal tissue with breakdown of the basement membrane resulting in large increases in uterine size which involve stretching of the muscle layers of the uterus. The uterine mast cells may be responding to this disruption of the physical organization of the uterus during deciduoma formation. One or both of these possibilities could account for the increase in uterine histamine observed in mast-cell normal + / + animals. W/W v mice produce a deciduoma in response to either trauma or oil stimuli without any alteration in total uterine histamine content. These results argue against a requirement for histamine of either mast cell origin or from the nonmast cell pool in deciduoma formation. This reasoning is further supported by the observation by Bartholeyns and Bouclier (1982) that implantation and embryonic development were unaffected by a-fluoromethylhistidine, an irreversible inhibitor of histamine synthesis (Kollonitsch et al., 1978). Since this study examined only total uterine histamine levels, there is a possibility that localized changes in histamine levels occur at the site of deciduoma formation. These localized changes in non-mast cell histamine may not be measurable relative to total uterine histamine levels. In addition, Dey and Johnson (1980) have proposed a role for embryonic histamine during the initial stages of implantation and the decidual cell response. The role of embryonic histamine may involve endometrial vascular changes or stromal cell differentiation. Experiments in our laboratory are currently being conducted to address the role (s) of embryonic histamine.
81
I n c o n c l u s i o n , we h a v e c o n f i r m e d t h e ability of W / W v m i c e to f o r m a decid u o m a w i t h s t i m u l u s b y t r a u m a or i n t r a l u m i n a l i n j e c t i o n o f oil. O u r r e s u l t s d e m o n s t r a t e a n i n c r e a s e in u t e r i n e h i s t a m i n e in m a s t - c e l l n o r m a l ( ÷ / ÷ ) anim a l s d u r i n g d e c i d u o m a f o r m a t i o n . W / W v m i c e failed to d e m o n s t r a t e a n y c h a n g e in t o t a l u t e r i n e h i s t a m i n e levels d u r i n g d e c i d u o m a f o r m a t i o n . T h e s e r e s u l t s w o u l d i n d i c a t e t h a t u t e r i n e m a s t cells are t h e p r o b a b l e source o f t h e i n c r e a s e d u t e r i n e h i s t a m i n e . T h e ability o f W / W v m i c e to f o r m a d e c i d u o m a w i t h no m e a s u r a b l e c h a n g e in t o t a l u t e r i n e h i s t a m i n e i n d i c a t e s t h a t i n c r e a s e d u t e r i n e h i s t a m i n e c o n t e n t is n o t r e q u i r e d for d e c i d u o m a f o r m a t i o n . I t would a p p e a r m o r e likely t h a t t h e m a s t cell r e s p o n s e a s s o c i a t e d w i t h d e c i d u o m a form a t i o n in ÷ / ÷ a n i m a l s is a r e s p o n s e to r a t h e r t h a n a c a u s e o f decidualization. W h e t h e r t h i s m a s t cell r e s p o n s e m a y be i n v o l v e d in s o m e o t h e r a s p e c t of t h e m a t e r n a l r e s p o n s e to p r e g n a n c y w o u l d be speculative. ACKNOWLEDGEMENT T h e w o r k p r e s e n t e d h e r e w a s s u p p o r t e d b y T e x a s College o f O s t e o p a t h i c M e d i c i n e g r a n t s # 34111 a n d # 34119.
REFERENCES Bartholeyns, J. and Bouclier, M., 1982. Effect of t~-monofluoromethyl histidine, an irreversible inhibitor of histidine decarboxylase, on gestation in mice. Contraception, 26: 535-542. Brandon, J.M. and Bibby, M.C., 1979. A study of changes in uterine mast cells during early pregnancy in the rat. Biol. Reprod., 20: 977-980. Brandon, J.M. and Raval, R.M., 1979. Interaction of estrogen and histamine during ovum implantation in the rat. Eur. J. Pharmacol., 57: 171-177. Brandon, J.M. and Wallis, R.M., 1977. Effect of mepyramine, a histamine Hi-, and buramamide, a histamine H2-receptor antagonist, on ovum implantation in the rat. J. Reprod. Fertil., 50: 251-254. Defeo, V.J., 1967. Decidualization. In: R.M. Wynn (Editor), Cellular Biology of the Uterus. Appleton-Century Crofts, New York, NY, pp. 191-290. Dey, S.K. and Johnson, D.C., 1980. Reevaluation of histamine in implantation. In: F. Kimball (Editor), The Endometrium. Spectrum Publications, New York, NY, pp. 269-283. Finn, C.A. and Hinchliffe, J.R., 1964. The reaction of the mouse uterus during implantation and deciduoma formation as demonstrated by changes in the distribution of alkaline phosphatase. J. Reprod. Fertil., 8: 331-338. Hatanaka, K., Kitamura, Y., Maeyma, K., Watanabe, T. and Matsumoto, K., 1982. Deciduoma formation in uterus of genetically mast cell-deficient W/W v mice. Biol. Reprod., 27: 25-28. Hine, R.J., Orsini, M.W. and Hegstrand, L.R., 1985. Changes in tissue histamine during the estrous cycle, pregnancy and pseudopregnancy in the golden hamster (42095). Proc. Soc. Exp. Biol. Med., 179: 271-278. Kitamura, Y., Go, A. and Hatanaka, K., 1978. Decrease of mast cells in W/W v mice and their increase by bone marrow transplantation. Blood, 52: 447-452. Kollonitsch, J., Patchett, A.A., Marburg, S. Maycock, A.L., Perkins, L.M., Doldouras, G.A., Dug-
82 gan, D.E. and Aster, S.D., 1978. Selective inhibitors of biosynthesis of aminergic neurotransmitters. Nature (London), 274: 906-908. Marcus, G.J., Shelesnyak, M.C. and Kracier, P.F., 1964. Studies on the mechanism of nidation. X. The oestrogen surge, histamine-release and decidual induction in the rat.Acta Endocrinol., (Copenhagen), 47: 255-264. Mayer, G., 1963. Delayed nidation in rats: method of exploring the mechanisms of ovo-implanration. In: A.C. Enders (Editor), Delayed Implantation. University of Chicago Press, Chicago, IL, pp. 213-231. Nalbandov. A.V., 1971. Endocrine control of implantation. In: R.J. Blandau (Editor), Biology of the Blastocyst. University of Chicago Press, Chicago, IL, pp. 383-392. Orr, E.L., 1984. Dural mast cells:source of contaminating histamine in analyses of mouse brain histamine levels.J. Neurochem., 43: 1497-1499. Orr, E.L. and Pace, K.R., 1984. The significanceof mast cellsas a source of histamine in the mouse brain. J. Neurochem., 42: 727-732. Russell, E.S., 1954. Review of the pleiotropic effectsof W-series genes on growth and differentiation. In: D. Rudnick (Editor), Aspects of Synthesis and Order in Growth. Princeton University Press, Princeton, NJ, pp. 113-126. Shelesnyak, M.C., 1957. Some experimental studies on the mechanism of ova implantation in the rat. Recent Prog. Horm. Res., 13: 269-322. Shelesnyak, M.C., 1960. Nidation of the fertilizedovum. Endeavour, 19: 81-86. Wordinger, R.J., Orr, E., Pace, K. and Brown, D., 1983. Evidence for a steroid-responsive nonmast cellpool of uterine histamine in the mouse. Anat. Rec., 205:220 (abstract). Wordinger, R.J., Orr, E.L., Pace, K., Oakford, L. and Morrill, A., 1985. An assessment of mastcelldeficientmice ( W / W v) as a model system to study the role of histamine in implantation and deciduoma formation. J. Reprod. Fertil.,73: 451-456. Wordinger, R.J.,Jackson, F.L. and Morrill,A., 1986. Implantation, deciduoma formation and live births in mast-cell deficientmice (W/WV). J. Reprod. Fertil.,77:471-476.
75
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Uterine Histamine Content in Mast-Cell Deficient (W/W v) Mice During Experimentally Induced Deciduoma Formation FRED L. JACKSON, EDWARD L. ORR, ALAN S. MORRILL and ROBERT J. WORDINGER
Department of Anatomy, Texas College of Osteopathic Medicine, North Texas State University, Fort Worth, TX 76107 (U.S.A.) (Accepted 11 February 1987)
ABSTRACT Jackson, F.L., Orr, E.L., Morrill, A.S. and Wordinger, R.J., 1987. Uterine histamine content in mast-cell deficient (W/W v) mice during experimentally induced deciduoma formation. Anim. Reprod. Sci., 14: 75-82. This study examines the changes in uterine histamine content in mast-cell normal and deficient mice during deciduoma formation. Surgical trauma (e.g., sutures) and intraluminal injection of oil produce deciduoma formation in ovariectomized mast-cell normal ( + / + ) and mast-cell deficient (W/W v) pseudopregnant mice that had previously received a single control ovary transplanted under the kidney capsule. Mast-cell normal ( + / + ) mice exhibit significant elevation of total uterine histamine content during both trauma and oil induced deciduoma formation. Mastcell deficient ( W/W v) mice under conditions of similiar deciduoma formation did not show any change in total uterine histamine content. These results indicate that the change in histamine observed in mast-cell normal mice during deciduoma formation is most likely of mast cell origin. The lack of any measurable change in uterine histamine in mast-cell deficient mice indicates that an increase of non-mast cell histamine is not a prerequisite for deciduoma formation.
INTRODUCTION
The role of histamine in deciduoma formation has been the subject of controversy for many years. Shelesnyak (1960) and Nalbandov (1971) have implicated uterine mast cell histamine in deciduoma formation and implantation. The number of uterine mast cells changes at the time of implantation (Shelesnyak, 1957; Defeo, 1967; Brandon and Bibby, 1979) as does the total uterine histamine content (Marcus et al., 1964). In addition, the effects of various histamine receptor antagonists have suggested a role for uterine histamine during implantation (Brandon and Wallis, 1977; Brandon and Raval, 1979). 0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
76 The WBB6FI-W/W v ( W / W v) inbred mouse strain provides an excellent model for the examination of the role of uterine histamine since it has been demonstrated that W / W v mice are deficient in stainable mast cells (Kitamura and Hatanaka, 1978; Wordinger et al., 1985 ). Our laboratory has reported the existence of a small pool of non-mast cell uterine histamine in W / W v mice (Wordinger et al., 1983). Subsequently, we have demonstrated the ability of mast-cell deficient ( W / W v) mice to undergo deciduoma formation, implantation, and successful live births (Wordinger et al., 1986). This latter finding indicates that mast cells are not required for these events. However, the possibility that the non-mast cell pool of histamine may be involved in deciduoma formation has not been examined. The objectives of this study were to (1) examine the total uterine histamine content in mast-cell normal ( + / + ) mice in response to oil or trauma induced deciduoma formation, and ( 2 ) compare the same parameters in mast-cell deficient ( W / W v) mice to evaluate the relative contribution of mast cells or the non-mast cell pool to the changes in uterine histamine content. MATERIALSAND METHODS
Animals Female WBB6FI-W/W v mast-cell deficient and mast-cell normal congenic female W B B 6 F 1 - + / + litter mates were purchased at 4-6 weeks of age from Jackson Laboratories, Bar Harbor, ME. Animals were housed five to six per cage in polycarbonate cages with a light:dark cycle of 14 h light:10 h dark per day. Food and water were provided ad libitum.
Ovariectomy and ovary transplantation W / W v mice are sterile due to a defect in germ cell migration (Russell, 1954). The ovaries are atrophic thus requiring ovariectomy and the use of exogenous steroids to maintain estrous cycles and responsiveness to deciduoma stimulation (Wordinger et al., 1985). An alternative method to re-establish hormone levels sufficient for deciduoma formation was recently reported by our laboratory {Wordinger et al., 1986). It consists of transplanting a normal ( + / + ) ovary under the kidney capsule of a W / W v female mouse. Two weeks prior to experimental deciduoma induction, + / + and W / W v mice underwent bilateral ovariectomy using the inhalant anesthesia metofane ( methoxyflurane; Pitman Moore, Washington Crossing, NJ). At the same time each animal received a single ÷ / ÷ ovary transplanted under the kidney capsule. The transplantation involved a small incision in the fibrous capsule surrounding the kidney with the ovary being inserted under this capsule. Vaginal smears were used to confirm the re-establishment of estrous cycles in ovariec-
77 tomized animals containing a transplanted ovary prior to their use for deciduoma induction.
Deciduoma induction Ovariectomized, ovary-transplanted + / + and W / W v mice were stimulated to undergo an artificial decidual cell response 3 days following successful mating, to induce a state ofpseudopregnancy. Two methods were utilized to induce deciduoma: (1) physical trauma, and (2) injection of oil. For induction of experimental decidual cell response by trauma, animals were anesthetized and the uterus exposed via an abdominal incision. Three sutures (4-0 silk; Ethicon, Somerville, NJ) were placed at equal distances along one uterine horn. For induction by injection of oil, animals were anesthetized, one uterine horn was exposed, and 10 pl of arachis oil was then injected into the lumen of the exposed uterine horn. For both trauma and oil injection, the contralateral uterine horn served as a non-stimulated control horn. Animals were killed 72 h following deciduoma induction and uterine horns were weighed and collected for measurement of uterine histamine content.
Histamine assay Uterine horns to be assayed for total histamine content were homogenized in 500 pl of ice-cold distilled water using a polytron homogenizer (Brinkmann Instruments, Westburg, NY). The homogenate was heated for 10 min in a boiling-water bath to destroy endogenous S-adenosyl-L-methionine and precipitate proteins. Samples were cooled on ice, centrifuged at 50 000g for 10 min, and 10-pl samples of the resulting supernatant were assayed for histamine. Histamine was assayed radioenzymically using a partially purified preparation of rat kidney histamine-N-methyltransferase to transfer a tritiumlabelled methyl group from 3H-adenosylmethionine onto histamine (Orr, 1984). The radioactive product of the reaction, 3H-methylhistamine was separated from 3H-adenosylmethionine by ascending thin-layer chromatography using a chloroform:methanol:ammonium hydroxide (12:7:1 by volume) solvent system ( Orr and Pace, 1984 ). 3H-methylhistamine was localized with ninhydrin, scraped from the plate, eluted with 500 pl of absolute ethanol and the radioactivity determined by liquid scintillation counting. Internal standards of known quantities of histamine were used to quantitate the total histamine in the samples.
Light microscopy Tissue for light microscopy (uteri and kidneys with transplanted ovaries) was placed in 10% neutral-buffered formalin for 48 h, processed in a tissue
78 TABLE 1 Uterine horn weights (rag) and histamine content (ng/horn) of + / + and W/Wv mice following trauma (suture} or oil induced decidual cell response Treatment
Tissue
(N)
Weight (rag)
Histamine (ng/horn)
Suture + / +
Stimulated horn Unstimulated horn
(7) (7)
131.3+20.8"* 31.7 +__ 1.3
177.1_+32.3" 59.7 _+ 7.9
Suture W / W v
Stimulated horn Unstimulatedhorn
(5) (5)
157.6_+40.1" 22.8_+ 1.5
Oil + / +
Stimulated horn Unstimulatedhorn
(6) (6)
342.6 _+23.2" * 35.1+_ 2.8
55.6 _+ 6.9* 24.5_+ 5.5
Oil W / W v
Stimulated horn Unstimulated horn
(4) (4)
288.4_+ 18.9"* 26.2_+ 7.1
3.6 _+ 1.4 3.1 _+ 1.3
4.1_+ 0.9 3.9_+ 0.3
Values represent the mean _+SEM. * = P < 0.05, ** = P < 0.01 compared to unstimulated horn
processor, embedded in paraffin and sectioned at 6 p m on a rotary microtome. Sections were stained with acidified ( p H 3.0) toluidine blue for mast cell identification. M a s t cells were identified by their large ovoid size (50 # m ) and a central nucleus which was often obscured by a dense accumulation of purple metachromatic cytoplasmic granules.
Statistical analysis All data were analyzed by one-way analysis of variance (ANOVA) with multiple range testing to determine differences between individual groups. Differences were considered statistically significant at P < 0.05. RESULTS
Trauma induced deciduoma induction As shown in Table 1, there was a significant ( P < 0.01) increase in weights of stimulated uterine horns of both + / + and W / W v mice in comparison to unstimulated contralateral control uterine horns. There was a significant ( P < 0.05) increase in total histamine content in stimulated horns of + / + mice when compared to unstimulated contralateral uterine horns. In contrast, the stimulated horns of W / W v mice failed to exhibit an increase in uterine histamine as a result of a t r a u m a induced deciduoma.
79
Oil induced deciduoma formation As shown in Table 1, there was a significant ( P < 0.01) increase in weights of stimulated uterine horns of both + / + and W/W v mice in comparison to unstimulated contralateral control horns. The increase in weights of uterine horns produced by the intraluminal injection ofoil was greater than the increase due to trauma. There was a significant {P < 0.05 ) increase in total histamine content in stimulated horns of + / + mice as compared to unstimulated contralateral control horns. The total histamine content of stimulated uterine horns of W / W v mice was not altered by oil induced deciduoma.
Light microscopy Histological examination of both the stimulated and unstimulated uterine horns from + / + and W / W v mice confirmed the abscence of mast cells in W/W v uteri. In addition, histological examination of the kidney-ovary interface and surrounding connective tissue capsule of W/W v mice which had received a + / + ovary transplanted under the kidney capsule failed to show any stainable mast cells. These results indicate that the transplantation of a + / + ovary does not establish a mast cell population in W/W v mice. Qualitative examination of + / + uterine horns revealed normal or slightly elevated numbers of mast cells. DISCUSSION The ability of mast-cell deficient mice (W/W v) to exhibit a trauma induced deciduoma was first demonstrated by Hatanaka et al. (1982). A trauma induced deciduoma is progesterone dependent but is estrogen independent (Finn and Hinchliffe, 1964). Formation of deciduoma in response to intraluminal injection of oil is both progesterone and estrogen dependent as is normal implantation ( Mayer, 1963 ). Wordinger et al. (1986) reported that W/W v mice would form a deciduoma with both trauma and also with intra-uterine injection of oil. Our results confirm the ability of W/W v mice to form a deciduoma as a result of either trauma or intra-uterine oil injection. These results indicate that mast cells and/or mast cell products are not required for this critical step in implantation. Both Hatanaka et al. (1982) and Wordinger et al. (1983) have reported the existence of a non-mast cell pool of uterine histamine. This pool is small and is not significantly altered by ovariectomy or exogenous steroid administration (Wordinger et al., 1985). The current results confirm the existence of this nonmast cell pool and also demonstrate that this pool is not significantly altered during the formation of either a trauma induced deciduoma or a deciduoma induced by intraluminal injection of oil. These results may indicate that the
80 non-mast cell pool of histamine is not critically involved in deciduoma formation. There is an increase in total uterine histamine content in the mast-cell normal ( + / + ) mice associated with the experimentally induced deciduoma formation. Since W/W v mice do not exhibit a similiar increase, it is likely that the source of this histamine is the uterine mast cells. This increase in histamine may be due to an increase in mast cell number or may reflect changes in histamine content and/or turnover in the uterine mast cells of the + / + animals. Increases in total uterine histamine during the peri-implantation period have been reported in the hamster (Hine et al., 1985) and the rat (Marcus et al., 1964 ). Both studies reported a subsequent decrease to non-pregnant levels of total uterine histamine following implantation. These studies were performed using pregnant animals whereas our results are on pseudopregnant animals with artificially induced deciduoma. These differences make direct comparison of data from the various studies difficult. The dramatic increase in uterine histamine levels in + / + animals may indicate a response of uterine mast cells to the process of deciduoma formation. Deciduoma formation is hormonally dependent and sufficient levels of progesterone and estrogen are required for decidualization to occur. The response of uterine mast cells may be due to a mast cell reaction to the changes in the hormonal milieu which are associated with deciduoma formation. The process of deciduoma formation involves the dramatic hypertrophy of stromal tissue with breakdown of the basement membrane resulting in large increases in uterine size which involve stretching of the muscle layers of the uterus. The uterine mast cells may be responding to this disruption of the physical organization of the uterus during deciduoma formation. One or both of these possibilities could account for the increase in uterine histamine observed in mast-cell normal + / + animals. W/W v mice produce a deciduoma in response to either trauma or oil stimuli without any alteration in total uterine histamine content. These results argue against a requirement for histamine of either mast cell origin or from the nonmast cell pool in deciduoma formation. This reasoning is further supported by the observation by Bartholeyns and Bouclier (1982) that implantation and embryonic development were unaffected by a-fluoromethylhistidine, an irreversible inhibitor of histamine synthesis (Kollonitsch et al., 1978). Since this study examined only total uterine histamine levels, there is a possibility that localized changes in histamine levels occur at the site of deciduoma formation. These localized changes in non-mast cell histamine may not be measurable relative to total uterine histamine levels. In addition, Dey and Johnson (1980) have proposed a role for embryonic histamine during the initial stages of implantation and the decidual cell response. The role of embryonic histamine may involve endometrial vascular changes or stromal cell differentiation. Experiments in our laboratory are currently being conducted to address the role (s) of embryonic histamine.
81
I n c o n c l u s i o n , we h a v e c o n f i r m e d t h e ability of W / W v m i c e to f o r m a decid u o m a w i t h s t i m u l u s b y t r a u m a or i n t r a l u m i n a l i n j e c t i o n o f oil. O u r r e s u l t s d e m o n s t r a t e a n i n c r e a s e in u t e r i n e h i s t a m i n e in m a s t - c e l l n o r m a l ( ÷ / ÷ ) anim a l s d u r i n g d e c i d u o m a f o r m a t i o n . W / W v m i c e failed to d e m o n s t r a t e a n y c h a n g e in t o t a l u t e r i n e h i s t a m i n e levels d u r i n g d e c i d u o m a f o r m a t i o n . T h e s e r e s u l t s w o u l d i n d i c a t e t h a t u t e r i n e m a s t cells are t h e p r o b a b l e source o f t h e i n c r e a s e d u t e r i n e h i s t a m i n e . T h e ability o f W / W v m i c e to f o r m a d e c i d u o m a w i t h no m e a s u r a b l e c h a n g e in t o t a l u t e r i n e h i s t a m i n e i n d i c a t e s t h a t i n c r e a s e d u t e r i n e h i s t a m i n e c o n t e n t is n o t r e q u i r e d for d e c i d u o m a f o r m a t i o n . I t would a p p e a r m o r e likely t h a t t h e m a s t cell r e s p o n s e a s s o c i a t e d w i t h d e c i d u o m a form a t i o n in ÷ / ÷ a n i m a l s is a r e s p o n s e to r a t h e r t h a n a c a u s e o f decidualization. W h e t h e r t h i s m a s t cell r e s p o n s e m a y be i n v o l v e d in s o m e o t h e r a s p e c t of t h e m a t e r n a l r e s p o n s e to p r e g n a n c y w o u l d be speculative. ACKNOWLEDGEMENT T h e w o r k p r e s e n t e d h e r e w a s s u p p o r t e d b y T e x a s College o f O s t e o p a t h i c M e d i c i n e g r a n t s # 34111 a n d # 34119.
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