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Prepartum and postpartum glycogen accumulation in bovine uterine arteries
Animal Reproduction Science, 16 (1988) 191-198
191
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Prepartum and Postpartum Glycogen Accumulation in B o v i n e Uterine Arteries S. KAMIYA ~and M. DAIGO
Department o[ Veterinary Anatomy, Nippon Veterinary and Zootechnical College, 1-7-1 Kyonan-cho, Musashino, Tokyo 180 (Japan) (Accepted 12 January 1988 )
ABSTRACT Kamiya, S. and Daigo, M., 1988. Prepartum and postpartum glycogen accumulation in bovine uterine arteries. Anim. Reprod. Sci., 16: 191-198. Histochemical examination of bovine uterine arteries revealed that massive accumulation of glycogen occurred in the tunica media of the arteries at the late gestational and early puerperal stages. The amount of glycogen appeared to increase progressively during pregnancy and then decreased gradually after parturition. In contrast, little glycogen was found in cycling cows. There was no direct relationship between glycogen deposition and frequency of parturition. These results suggest that massive storage of glycogen in the uterine artery may serve as an important source of energy for the recovery of the uterus and its arteries to their nonpregnant states.
INTRODUCTION
The uterus and its blood vessels have been studied by many investigators. Most emphasis has been focused on the morphological and histochemical modifications in the epithelial and supporting tissue components of the uterus during various stages of the reproductive cycle (Klinge, 1959; Likar and Likar, 1964; Hafez, 1980; Jansen et al., 1985). In turn, morphological investigations on the blood vessels of the uterus have revealed that intimal thickenings and elastosis of the uterine arteries are caused by pregnancy and estrus (Pankow, 1906; HSfliger, 1954; Miyagi, 1966; Albert and Bhussry, 1967). There have been few detailed histochemical studies on the blood vessels supplying the uterus in cattle and this paper describes massive accumulation of glycogen in the arteries. ~To whom correspondence should be addressed.
0378-4320/88/$03.50
© 1988 Elsevier Science Publishers B.V.
192 MATERIALS AND METHODS
Female genital organs and their blood vessels were obtained from 24 Holstein cows at various stages of reproduction (Table 1 ). The stage was determined by gross and histological examination of the ovaries and uterus in each cow (Klinge, 1959 ). Gestational stage was estimated by the crown-rump length of the fetus (Evans and Sack, 1973), and the days postpartum was based on the history of parturition. Frequency of parturition was also estimated from the number of the corpus albicans found in a pair of ovaries (Miyagi, 1966 ). Trunci of the uterine artery were bilaterally taken from the 1 cm proximal portion of the first bifurcation of the vessel. Tissues were fixed in 2% calcium acetate-10% formalin or in 10% buffered neutral formalin, and routinely processed into paraffin blocks. Paraffin sections (3-6 z m ) were stained with hematoxylin and eosin for histological observations. For the demonstration of glycogen, the following histochemical techniques were employed: periodic acid-Schiff (PAS), dimedone-PAS, Best's carmine, Lugol's iodine and Grocott's methenamine silver method (Bulmer, 1959; Lillie, 1965; Pearse, 1985). To substantiate the presence of glycogen in tissues, some sections were first digested for 1 h at 37°C with 1 m g / m l a-amylase (Sigma type IV-A) in buffered neutral saline; as a control, other sections were incubated without enzyme. TABLE1 Stages of the estrous or reproductive cycle for the cows used Cow number
Stage of estrous cycle*
Cow number
Stage of reproductive cycle
1 2 3 4 5 6 7 8 9 10 11 12
Proestrus Proestrus Proestrus Estrus Estrus Metestrus Metestrus Metestrus Metestrus Diestrus Diestrus Diestrus
13 14 15 16 17 18 19 20 21 22 23 24
Pregnant 4 weeks Pregnant 5 weeks Pregnant 20 weeks Pregnant 28 weeks Pregnatn 34 weeks Pregnant 40 weeks Day 2 postpartum Day 3 postpartum Day 19 postpartum Day 21 postpartum Day 29 postpartum Day 35 postpartum
*Proestrus: One or more developing follicles were visible, and the corpus luteum was regressing. Estrus: A single, large follicle was the dominant structure on the ovaries. Metestrus: A corpus hemorrhagicum was present. Diestrus: A corpus luteum was the dominant structure on the ovaries.
193 RESULTS
Varying amounts of PAS-positive substances could be demonstrated in smooth muscle fibers of the uterine arteries in the present study (Fig. 1 ). These substances, also positive with dimedone-PAS, carmine, iodine and methenamine silver, were completely digested with amylase (Figs. 2a, b), indicating glycogen. Small amounts of glycogen were observed on both sides (two cases) and on one side (one case) at different stages of the estrous cycle (Table 2). Little glycogen was seen in the tunica media or in the longitudinal muscle bundles of the tunica adventitia in these cases. At 4 or 5 weeks of gestation, glycogen was present in the tunica media of the arteries on the pregnant side (Fig. 3 ). The amount seemed to be slightly more than that in nonpregnant cows. At 20 and 28 weeks of gestation, glycogen content seemed to increase moderately (Fig. 4). At 34 and 40 weeks of gestation, large amounts of glycogen were found in the tunica media on both sides of the arteries (Fig. 5). On 2 and 3 days postpartum, marked glycogen accumulation was found throughout the tunica media on both sides of the uterine arteries (Figs. 2, 6). The amount of glycogen seemed to decrease on 19 and 21 days after parturition (Fig. 7). On 29 and 35 days postpartum, the amount decreased significantly (Fig. 8). These findings clearly show that glycogen accumulation in uterine arteries increased at late pregnancy and decreased after parturition (Fig. 1, and Table 3 ), but there was no distinct relationship between the glycogen deposition and frequency of parturition (Table 2, 3).
A
B
E
C
F
D
G
Fig. 1. Various depositions of PAS-positive substances (GL) on the bovine uterine arteries at the stages: A, estrous cycle; B, early pregnancy; C, mid-pregnancy; D, late pregnancy; E, early puerperium; F, mid-puerperium; G, late puerperium. I: tunica intima, L: lamina elastica interna, M: tunica media.
194
Fig. 2. Pregnant side of the uterine artery at 3 days after parturition, showing the large amount of dimedone-PAS-positive substances (a). These substances were completely digested with a-amylase (b). I: tunica intima, M: tunica media, A: tunica adventitia. (Dimedone-PAS; × 30 ). TABLE 2 Glycogen in uterine arteries of nonpregnant cows Cow number
Glycogen*
NP
R
L
1
--
--
1
2
-
-
4
3
-
-
7
4
-
-
3
5
+
+
5
6
-
-
2
7
-
-
3
8
-
-
5
9
+
+
7
10
-
+
1
11
-
-
5
12
-
-
8
*Amounts of glycogen are graded by the dimedone-PAS stain as follows: - , absent; + , small. Abbreviations: R, right uterine artery; L, left uterine artery; NP, number of parturition estimated from number of corpus albicans (Miyagi, 1966).
195
5
.........
M
Fig. 3. Glycogen was found in the tunica media of the pregnant-side artery at 5 weeks of gestation. I: tunica intima, M: tunica media, A: tunica adventitia. (Grocott's methenamine silver; X 75). Fig. 4. Moderate amounts of glycogen were present in the pregnant-side artery at 28 weeks of gestation (PAS; X30). Fig. 5. Marked accumulation of glycogen was found in the tunica media of pregnant-side artery at 40 weeks of gestation. (Dimedone-PAS; X 30). Insert: Pregnant-side (P) and nonpregnant-side (N) artery. (Dimedone-PAS; X !.2).
196
Fig. 6. Large amounts of glycogen were present on the pregnant-side artery at 2 days after parturition. (PAS; X30). Fig. 7. Moderate amounts of glycogen occurred diffusely in the tunica media of the pregnant-side artery at 19 days after parturition. I: tunica intima, M: tunica media, A: tunica adventitia. (Grocott's methenamine silver; × 75). Fig. 8. Small amounts of glycogen were found in the tunica media of the pregnant-side artery at 35 days after parturition. Arrows: double-layered lamina elastica interna. (PAS; ×30).
197 TABLE 3 Glycogenin uterine arteries of pregnant and postpartal cows Cow n u m b e r
13 14 15 16 17 18 19 20 21 22 23 24
NP
Glycogen* I
C
+ + ++ ++ +++ +++ +++ +++ ++ ++ + +
+ + ++ +++ +++ +++ ++ + -
4 4 6 5 7 2 2 3 3 10 7 3
*Amounts of glycogenare graded by the dimedone-PAS stain as follows: - , absent; +, small; + +, moderate; + + +, large. Abbreviations: I, ipsilateral and C, contralateral uterine artery of gravid uterine horn; NP, number of parturition. DISCUSSION In spite of numerous reports of glycogen in the uterus (Brody, 1958; Chew and Rinard, 1979; Carrington and Bailey, 1985), there have been few descriptions of glycogen in the uterine arteries (Hall, 1965 ). The present study clearly demonstrates a massive accumulation of glycogen in the bovine uterine arteries and shows t h a t it increased during pregnancy and decreased gradually after parturition. Myometrial glycogen has been reported to increase prior to parturition and decrease immediately after parturition (Brody, 1958; Chew and Rinard, 1979 ), and Chew and Rinard (1979) suggest t h a t myometrial glycogen may serve as an important source of energy in the maintenance of uterine contractions during labour. In the present study, however, rapid reduction of arterial glycogen was not confirmed, and glycogen gradually decreased after parturition. It is concluded from the previous and present results t h a t the massive glycogen stores in the uterine artery may be consumed as an energy source during recovery of the uterus and its arteries to the nonpregnant state. There have been a number of reports t h a t estrogen stimulates glycogen deposition in the uteri of mice, rats and women (Brody, 1958; Hall, 1965; Carrington and Bailey, 1985 ). To our knowledge, there is no experimental evidence to indicate direct effects of estrogen on the accumulation of glycogen in uterine arteries of the cow. In serum of the pregnant cow, however, progesterone levels fall and estrogen rises at about 200 days of gestation (Hafez, 1980). Thus, the
198 i n c r e a s e d e s t r o g e n s e c r e t i o n n e a r t e r m m i g h t c o n t r i b u t e to t h e increase in arterial glycogen. ACKNOWLEDGEMENTS T h e a u t h o r s wish to t h a n k Dr. K. Suzuki, of the D e p a r t m e n t of V e t e r i n a r y P h y s i o l o g y , N i p p o n V e t e r i n a r y a n d Z o o t e c h n i c a l College, for his critical readings of t h e m a n u s c r i p t . T h e a u t h o r s are also deeply i n d e b t e d to Miss M. T s u k u s h i for her excellent t e c h n i c a l assistance.
REFERENCES Albert, E.N. and Bhussry, B.R., 1967. The effects of multiple pregnancies and age on the elastic tissue of uterine arteries in the guinea pig. Am. J. Anat., 121: 259-270. Brody, S., 1958. Hormonal influence on the glycogen content of the human myometrium. Acta Endocrinol. (Copenhagen), 27: 377-384. Bulmer, D., 1959. Dimedone as an aldehyde blocking reagent to facilitate the histochemicai demonstration of glycogen. Stain Technol., 34: 95-98. Carrington, L.J. and Bailey, C.J., 1985. Effects of natural and synthetic estrogen and progestins on glycogen deposition in female mice. Horm. Res., 21: 199-203. Chew, C.S. and Rinard, G.A., 1979. Glycogen levels in the rat myometrium at the end of pregnancy and immediately postpartum. Biol. Reprod., 20:1111-1114. Evans, H.E. and Sack, W.O., 1973. Prenatal development of domestic and laboratory mammals: growth curves, external features and selected references. Anat. Histol. Embryol., 2:11-45. Hafez, E.S.E., 1980. Reproduction in Farm Animals, 4th edition. Lea and Febiger, Philadelphia, U.S.A., pp. 63-82,267-278. Hall, K., 1965. Histochemical investigation of the effects of oestrogen, progesterone and relaxin on glycogen, amylophosphorelase, transglycosylase and uridine diphosphate glucose-glycogen transferase in uteri of mice. J. Endocrinol. (London }, 32: 245-257. Htifliger, H., 1954. Der Nachweis stattgehabter Tr~ichtigkeit bei Rind und Schwein. Schweiz. Arch. Tierheilkd; 96: 635-641. Jansen, R.P.S., Turner, M., Johannisson, E., Landgren, B.-M. and Diszfalusy, E., 1985. Cyclic changes in human endometrial surface glycoproteins: a quantitative histochemical study. Fertil. Steril., 44: 85-91. Klinge, A., 1959. Zum zyklischen Verhalten vornehmlich der HShe des Endometriums beim Rind. Zentralbl. Veterinaermed., 6: 742-780. Likar, I.N. and Likar, L.J., 1964. Acid mucopolysaccharides and mast cells in the bovine uterus at different stages of the sexual cycle. Acta Endocrinol. (Copenhagen), 46: 493-506. Lillie, R.D., 1965. Histopathologic Technic and Practical Histochemistry, 3rd edition. McGrawHill, New York, Toronto, Sydney, London., pp. 493-525. Miyagi, M., 1966. Studies on changes in arteria uterina media of cows caused by their pregnancy. Sci. Bull. Coll. Agric. Univ. Ryukyus, 13:1-99 {in Japanese with English abstract). Pankow, 1906. Gravidit~its-, Menstruations- und Ovulatinssklerose der Uterus- und Ovarialgef~isse. Arch. Gyn~ikol., 80: 271-282. Pearse, A.G.E., 1985. Histochemistry, Theoretical and Applied, Vol. 2, 4th edition. Churchill Livingstone, Edinburgh, London, Melbourne, New York., pp. 675-753.
191
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Prepartum and Postpartum Glycogen Accumulation in B o v i n e Uterine Arteries S. KAMIYA ~and M. DAIGO
Department o[ Veterinary Anatomy, Nippon Veterinary and Zootechnical College, 1-7-1 Kyonan-cho, Musashino, Tokyo 180 (Japan) (Accepted 12 January 1988 )
ABSTRACT Kamiya, S. and Daigo, M., 1988. Prepartum and postpartum glycogen accumulation in bovine uterine arteries. Anim. Reprod. Sci., 16: 191-198. Histochemical examination of bovine uterine arteries revealed that massive accumulation of glycogen occurred in the tunica media of the arteries at the late gestational and early puerperal stages. The amount of glycogen appeared to increase progressively during pregnancy and then decreased gradually after parturition. In contrast, little glycogen was found in cycling cows. There was no direct relationship between glycogen deposition and frequency of parturition. These results suggest that massive storage of glycogen in the uterine artery may serve as an important source of energy for the recovery of the uterus and its arteries to their nonpregnant states.
INTRODUCTION
The uterus and its blood vessels have been studied by many investigators. Most emphasis has been focused on the morphological and histochemical modifications in the epithelial and supporting tissue components of the uterus during various stages of the reproductive cycle (Klinge, 1959; Likar and Likar, 1964; Hafez, 1980; Jansen et al., 1985). In turn, morphological investigations on the blood vessels of the uterus have revealed that intimal thickenings and elastosis of the uterine arteries are caused by pregnancy and estrus (Pankow, 1906; HSfliger, 1954; Miyagi, 1966; Albert and Bhussry, 1967). There have been few detailed histochemical studies on the blood vessels supplying the uterus in cattle and this paper describes massive accumulation of glycogen in the arteries. ~To whom correspondence should be addressed.
0378-4320/88/$03.50
© 1988 Elsevier Science Publishers B.V.
192 MATERIALS AND METHODS
Female genital organs and their blood vessels were obtained from 24 Holstein cows at various stages of reproduction (Table 1 ). The stage was determined by gross and histological examination of the ovaries and uterus in each cow (Klinge, 1959 ). Gestational stage was estimated by the crown-rump length of the fetus (Evans and Sack, 1973), and the days postpartum was based on the history of parturition. Frequency of parturition was also estimated from the number of the corpus albicans found in a pair of ovaries (Miyagi, 1966 ). Trunci of the uterine artery were bilaterally taken from the 1 cm proximal portion of the first bifurcation of the vessel. Tissues were fixed in 2% calcium acetate-10% formalin or in 10% buffered neutral formalin, and routinely processed into paraffin blocks. Paraffin sections (3-6 z m ) were stained with hematoxylin and eosin for histological observations. For the demonstration of glycogen, the following histochemical techniques were employed: periodic acid-Schiff (PAS), dimedone-PAS, Best's carmine, Lugol's iodine and Grocott's methenamine silver method (Bulmer, 1959; Lillie, 1965; Pearse, 1985). To substantiate the presence of glycogen in tissues, some sections were first digested for 1 h at 37°C with 1 m g / m l a-amylase (Sigma type IV-A) in buffered neutral saline; as a control, other sections were incubated without enzyme. TABLE1 Stages of the estrous or reproductive cycle for the cows used Cow number
Stage of estrous cycle*
Cow number
Stage of reproductive cycle
1 2 3 4 5 6 7 8 9 10 11 12
Proestrus Proestrus Proestrus Estrus Estrus Metestrus Metestrus Metestrus Metestrus Diestrus Diestrus Diestrus
13 14 15 16 17 18 19 20 21 22 23 24
Pregnant 4 weeks Pregnant 5 weeks Pregnant 20 weeks Pregnant 28 weeks Pregnatn 34 weeks Pregnant 40 weeks Day 2 postpartum Day 3 postpartum Day 19 postpartum Day 21 postpartum Day 29 postpartum Day 35 postpartum
*Proestrus: One or more developing follicles were visible, and the corpus luteum was regressing. Estrus: A single, large follicle was the dominant structure on the ovaries. Metestrus: A corpus hemorrhagicum was present. Diestrus: A corpus luteum was the dominant structure on the ovaries.
193 RESULTS
Varying amounts of PAS-positive substances could be demonstrated in smooth muscle fibers of the uterine arteries in the present study (Fig. 1 ). These substances, also positive with dimedone-PAS, carmine, iodine and methenamine silver, were completely digested with amylase (Figs. 2a, b), indicating glycogen. Small amounts of glycogen were observed on both sides (two cases) and on one side (one case) at different stages of the estrous cycle (Table 2). Little glycogen was seen in the tunica media or in the longitudinal muscle bundles of the tunica adventitia in these cases. At 4 or 5 weeks of gestation, glycogen was present in the tunica media of the arteries on the pregnant side (Fig. 3 ). The amount seemed to be slightly more than that in nonpregnant cows. At 20 and 28 weeks of gestation, glycogen content seemed to increase moderately (Fig. 4). At 34 and 40 weeks of gestation, large amounts of glycogen were found in the tunica media on both sides of the arteries (Fig. 5). On 2 and 3 days postpartum, marked glycogen accumulation was found throughout the tunica media on both sides of the uterine arteries (Figs. 2, 6). The amount of glycogen seemed to decrease on 19 and 21 days after parturition (Fig. 7). On 29 and 35 days postpartum, the amount decreased significantly (Fig. 8). These findings clearly show that glycogen accumulation in uterine arteries increased at late pregnancy and decreased after parturition (Fig. 1, and Table 3 ), but there was no distinct relationship between the glycogen deposition and frequency of parturition (Table 2, 3).
A
B
E
C
F
D
G
Fig. 1. Various depositions of PAS-positive substances (GL) on the bovine uterine arteries at the stages: A, estrous cycle; B, early pregnancy; C, mid-pregnancy; D, late pregnancy; E, early puerperium; F, mid-puerperium; G, late puerperium. I: tunica intima, L: lamina elastica interna, M: tunica media.
194
Fig. 2. Pregnant side of the uterine artery at 3 days after parturition, showing the large amount of dimedone-PAS-positive substances (a). These substances were completely digested with a-amylase (b). I: tunica intima, M: tunica media, A: tunica adventitia. (Dimedone-PAS; × 30 ). TABLE 2 Glycogen in uterine arteries of nonpregnant cows Cow number
Glycogen*
NP
R
L
1
--
--
1
2
-
-
4
3
-
-
7
4
-
-
3
5
+
+
5
6
-
-
2
7
-
-
3
8
-
-
5
9
+
+
7
10
-
+
1
11
-
-
5
12
-
-
8
*Amounts of glycogen are graded by the dimedone-PAS stain as follows: - , absent; + , small. Abbreviations: R, right uterine artery; L, left uterine artery; NP, number of parturition estimated from number of corpus albicans (Miyagi, 1966).
195
5
.........
M
Fig. 3. Glycogen was found in the tunica media of the pregnant-side artery at 5 weeks of gestation. I: tunica intima, M: tunica media, A: tunica adventitia. (Grocott's methenamine silver; X 75). Fig. 4. Moderate amounts of glycogen were present in the pregnant-side artery at 28 weeks of gestation (PAS; X30). Fig. 5. Marked accumulation of glycogen was found in the tunica media of pregnant-side artery at 40 weeks of gestation. (Dimedone-PAS; X 30). Insert: Pregnant-side (P) and nonpregnant-side (N) artery. (Dimedone-PAS; X !.2).
196
Fig. 6. Large amounts of glycogen were present on the pregnant-side artery at 2 days after parturition. (PAS; X30). Fig. 7. Moderate amounts of glycogen occurred diffusely in the tunica media of the pregnant-side artery at 19 days after parturition. I: tunica intima, M: tunica media, A: tunica adventitia. (Grocott's methenamine silver; × 75). Fig. 8. Small amounts of glycogen were found in the tunica media of the pregnant-side artery at 35 days after parturition. Arrows: double-layered lamina elastica interna. (PAS; ×30).
197 TABLE 3 Glycogenin uterine arteries of pregnant and postpartal cows Cow n u m b e r
13 14 15 16 17 18 19 20 21 22 23 24
NP
Glycogen* I
C
+ + ++ ++ +++ +++ +++ +++ ++ ++ + +
+ + ++ +++ +++ +++ ++ + -
4 4 6 5 7 2 2 3 3 10 7 3
*Amounts of glycogenare graded by the dimedone-PAS stain as follows: - , absent; +, small; + +, moderate; + + +, large. Abbreviations: I, ipsilateral and C, contralateral uterine artery of gravid uterine horn; NP, number of parturition. DISCUSSION In spite of numerous reports of glycogen in the uterus (Brody, 1958; Chew and Rinard, 1979; Carrington and Bailey, 1985), there have been few descriptions of glycogen in the uterine arteries (Hall, 1965 ). The present study clearly demonstrates a massive accumulation of glycogen in the bovine uterine arteries and shows t h a t it increased during pregnancy and decreased gradually after parturition. Myometrial glycogen has been reported to increase prior to parturition and decrease immediately after parturition (Brody, 1958; Chew and Rinard, 1979 ), and Chew and Rinard (1979) suggest t h a t myometrial glycogen may serve as an important source of energy in the maintenance of uterine contractions during labour. In the present study, however, rapid reduction of arterial glycogen was not confirmed, and glycogen gradually decreased after parturition. It is concluded from the previous and present results t h a t the massive glycogen stores in the uterine artery may be consumed as an energy source during recovery of the uterus and its arteries to the nonpregnant state. There have been a number of reports t h a t estrogen stimulates glycogen deposition in the uteri of mice, rats and women (Brody, 1958; Hall, 1965; Carrington and Bailey, 1985 ). To our knowledge, there is no experimental evidence to indicate direct effects of estrogen on the accumulation of glycogen in uterine arteries of the cow. In serum of the pregnant cow, however, progesterone levels fall and estrogen rises at about 200 days of gestation (Hafez, 1980). Thus, the
198 i n c r e a s e d e s t r o g e n s e c r e t i o n n e a r t e r m m i g h t c o n t r i b u t e to t h e increase in arterial glycogen. ACKNOWLEDGEMENTS T h e a u t h o r s wish to t h a n k Dr. K. Suzuki, of the D e p a r t m e n t of V e t e r i n a r y P h y s i o l o g y , N i p p o n V e t e r i n a r y a n d Z o o t e c h n i c a l College, for his critical readings of t h e m a n u s c r i p t . T h e a u t h o r s are also deeply i n d e b t e d to Miss M. T s u k u s h i for her excellent t e c h n i c a l assistance.
REFERENCES Albert, E.N. and Bhussry, B.R., 1967. The effects of multiple pregnancies and age on the elastic tissue of uterine arteries in the guinea pig. Am. J. Anat., 121: 259-270. Brody, S., 1958. Hormonal influence on the glycogen content of the human myometrium. Acta Endocrinol. (Copenhagen), 27: 377-384. Bulmer, D., 1959. Dimedone as an aldehyde blocking reagent to facilitate the histochemicai demonstration of glycogen. Stain Technol., 34: 95-98. Carrington, L.J. and Bailey, C.J., 1985. Effects of natural and synthetic estrogen and progestins on glycogen deposition in female mice. Horm. Res., 21: 199-203. Chew, C.S. and Rinard, G.A., 1979. Glycogen levels in the rat myometrium at the end of pregnancy and immediately postpartum. Biol. Reprod., 20:1111-1114. Evans, H.E. and Sack, W.O., 1973. Prenatal development of domestic and laboratory mammals: growth curves, external features and selected references. Anat. Histol. Embryol., 2:11-45. Hafez, E.S.E., 1980. Reproduction in Farm Animals, 4th edition. Lea and Febiger, Philadelphia, U.S.A., pp. 63-82,267-278. Hall, K., 1965. Histochemical investigation of the effects of oestrogen, progesterone and relaxin on glycogen, amylophosphorelase, transglycosylase and uridine diphosphate glucose-glycogen transferase in uteri of mice. J. Endocrinol. (London }, 32: 245-257. Htifliger, H., 1954. Der Nachweis stattgehabter Tr~ichtigkeit bei Rind und Schwein. Schweiz. Arch. Tierheilkd; 96: 635-641. Jansen, R.P.S., Turner, M., Johannisson, E., Landgren, B.-M. and Diszfalusy, E., 1985. Cyclic changes in human endometrial surface glycoproteins: a quantitative histochemical study. Fertil. Steril., 44: 85-91. Klinge, A., 1959. Zum zyklischen Verhalten vornehmlich der HShe des Endometriums beim Rind. Zentralbl. Veterinaermed., 6: 742-780. Likar, I.N. and Likar, L.J., 1964. Acid mucopolysaccharides and mast cells in the bovine uterus at different stages of the sexual cycle. Acta Endocrinol. (Copenhagen), 46: 493-506. Lillie, R.D., 1965. Histopathologic Technic and Practical Histochemistry, 3rd edition. McGrawHill, New York, Toronto, Sydney, London., pp. 493-525. Miyagi, M., 1966. Studies on changes in arteria uterina media of cows caused by their pregnancy. Sci. Bull. Coll. Agric. Univ. Ryukyus, 13:1-99 {in Japanese with English abstract). Pankow, 1906. Gravidit~its-, Menstruations- und Ovulatinssklerose der Uterus- und Ovarialgef~isse. Arch. Gyn~ikol., 80: 271-282. Pearse, A.G.E., 1985. Histochemistry, Theoretical and Applied, Vol. 2, 4th edition. Churchill Livingstone, Edinburgh, London, Melbourne, New York., pp. 675-753.