Endometrial and embryonic enzymes in relation to implantation of the rabbit blastocyst.код для вставкиСкачать
Endometrial and Embryonic Enzymes in Relation to Implantation of the Rabbit Blastocyst E. s. E. HAFEZ AND I. G . WHITE Department of Veterinury Physiology, University of Sydney, Sydney, N. S. W. Australia ABSTRACT The activity of alkaline phosphatase, acid phosphatase, plutamic-oxdacetic transaminase (GOT),lactic dehydrogenase (LDH), amylase, succinic dehydrogenase (SDH), glucose-&phosphate dehydrogenase (GD) has been determined in the rabbit endometrium at estrus, in pseudopregnancy, and at days 5-10 of pregnancy. Enzyme analyses were also made on the placenta and embryo during early pregnancy. The most striking changes were increases in the SDH and GDH levels of the endometrium and a decrease in amylase when the does became pregnant or pseudopregnant. Amylase did not rise significantly from its lower level between five and ten days of pregnancy; SDH and GDH activity, however, fell away after reaching a maximum prior to implantation. At implantation there was some evidence of an increase in LDH and phosphatase activity. All enzymes had lower activities following implantation (10 days pregnancy). The endometrial enzyme activities of prepubertal does were usually similar to those of adult females in estrus. However, alkaline phosphatase activity of the prepubertal endometrium was particularly low. The activity of enzymes in the placental areas were, in general, similar to those found in the interplacental areas of the endometrium. However, from 7-10 days of pregnancy the activity of SDH was lower in the placental area than in the interplacental areas; whereas, amylase was higher on the eighth and ninth days of pregnancy. Changes in the activities of the phosphatases, GOT and SDH occurred in the blastocyst and trophoblast on eight to ten days of pregnancy. Enzyme activities in blastocoelic fluid were much less than in the trophoblast with the exception of amylase which was higher. The female reproductive tract possesses a wide and characteristic distribution of enzymes with varying activity depending on the endocrine condition and the stage of the reproductive cycle (cf. Hafez, '64). In the rabbit endometrium, dipeptidase activity increases rapidly from estrus to fourth day of pseudopregnancy. This is followed by four days of a much slower rise, four days of a maintained plateau and five days of a steady decline until a level slightly lower than that present during estrus is reached (Albers, Bedford and Chang, '61). The carbonic anhydrase activity of the rabbit endometrium has also been shown to increase during early or pseudopregnancy (Lutwak-Mann and Laser, '54; Lutwak-Mann, '55) and Boving ('62) has suggested that it causes local alkalinity which makes both the membranes and the trophoblast sticky and thus promotes implantation. According to Delgado and Fridhandler ('64) the lactic dehydrogenase activity of the rabbit endometrium, particularly in the interplacental region, is decreased in early pregnancy whilst malic ANAT. REC..159: 273-2230. - dehydrogenase and glutamic oxalacetic transaminase activity is increased from five to seven days of pregnancy. In the rat, uterine p-glucuronidase (Prahald, '62) and endometrial peptidase have been shown to increase at implantation (Albers et al., '61). The occurrence of proteolytic enzymes in the uterus capable of digesting collagens as well as muscle protein (Morione and Swifter, '62; Woessner and Brewer, '63) may also be important for implantation. This report describes fluctuations in the activity of a number of enzymes - alkaline phosphatase, acid phosphatase, glutamicoxalacetic transaminase (GOT), lactic dehydrogenase (LDH), amylase, succinic dehydrogenase (SDH) , glucose-&phosphate dehydrogenase (GDH) - in the rabbit ~~ XPresent address: Department of Animal Sciences, Washington State Univmsity, Pullman Washington U. S. A. College of Agriculture Scientdc Paper 2958: Project 1695. 2 We wish to thank Professor C. W. Emmens for providing laborato facilities and for his. interest and advice, and x e U. S. National Insbtutes of Child Health and Human Development for a Special Fellowship (IF3 HD-24. 411-01) while one of us (E.S.E.H.) was on sabbatical leave, 1965/66. 2 73 274 E. S. E. IIAFEZ AND I. G . WHITE endometrium and embryo during early pregnancy. MATERIAL AND iMETHODS 1. Experimental animals Rabbits of mixed breeds, mostly New Zealand White, were obtained from the University of Sydney animal house at Castle Hill. All does were primiparous, aged 7-10 months, except the prepubertal (nonpregnant) group which were 3-4 months old and the aged (pregnant) group which were 2-3 years old and had over five previous pregnancies. Does were made pseudopregnant by intravenous injection 20 I.U. of Human Chorionic Gonadotropin (HCG) (Schering) and pregnant by simultaneous insemination with 0.1 ml of pooled semen freshly collected with an artificial vagina (White, '55). 2. Preparation of tissues The does were killed at the reproductive stages indicated in table 2 and the uteri removed immediately and immersed in crushed ice. All subsequent manipulation of the tissue was performed at 4°C. Blastocysts 5 to 6 days of pregnancy (post coitum) were flushed from the uterine horns with calcium-free Krebs-Ringer buffered to pH = 7.0 with Tris (122 mM NaC1, 5 mM KCl, 1 mM MgS04, 25 mM Tris) and the healthy ones (as judged by stereoscopic examination) transferred to a homogenizing tube. Seven-day old blastocysts were removed after making three incisions i n the uterine wall near each blastocyst; a strip of uterine wall was then peeled off using fine forceps, releasing each blastocyst. The maternal placenta, trophoblast and blastocoelic fluid were collected from eight, nine and ten days of pregnancy. After three incisions were made in the uterus 2 mm from the conceptus, the uterine wall was teased around the conceptus using two fine forceps. The blastocoelic fluid (ca. 0.5 ml/conceptus) was collected in a small pre-cooled petri dish. The antimesometrial side of the uterus was turned inside out over a finger and the trophoblast scraped from the uterine wall using the edge of fine forceps. The uterine wall surrounding the blastocyst was dissected to determine if any trophoblastic tissue remained after cleaning with tissue paper or cheese cloth. The uterine horn was then stretched on a piece of filter paper and opened at the antimesometrial side. The exposed endometrium was blotted with filter paper to remove any traces of the flushing fluid or the endometrial secretions. Tissues from the placental fold and the interconceptus endometrial areas were dissected using fine forceps. 3. Enzyme analyses Samples of tissue ( 0 . 5 to 1 gm) were homogenized in ten parts of calcium-free Krebs-Ringer-Tris except in the case of blastocysts when two parts were used. A teflon Potter-Elvehjem homogenizer was found to be satisfactory and after filtering through cheese cloth enzymes were assayed either directly or after appropriate dilution in calcium-free Krebs-Tris (table 1) by assay procedures which, except for succinic dehydrogenase, are set out in Bergmeyer ('63). TABLE 1 The dilution of rabbit tissue homogenates and fluid for enzyme analyses One volume of the homogenate (see Materials and Methods for preparation) or fluid was diluted to the following volumes with calcium free Krebs-Ringer-Tris. N.D. indicates that the homogenate or fluid was not diluted. Alkaline Homogenate Endometrium Placenta Blastocyst Trophoblast Fluid &%&e 20 20 10 20 2 Acid phosphatase 10 10 10 10 5 GOT 10 10 5 10 N.D. Lactic dehydrogenase 10 10 10 10 N.D. Amylase N.D. 2 10 2 20 Succinic dehydrogenase 2 N.D. N.D. N.D. N.D. Glucose-6phosphate dehydrogrrnnse _ - - --- 20 20 N.D. 10 N.D. ENZYME ACTIVITIES DURING IMPLANTATION pNitrophenylphosphate was used as the substrate for determining acid and alkaline phosphatase activity (Bessey, Lowry and Brock, '46; Andersch and Szcypinski, '47). One phosphatase unit is the amount of enzyme contained in 1,000 ml of homogenate which liberates 1 m.mole (139.11 mg) p-nitrophenyl at 37°C. Glutamic-oxalacetic transaminase (GOT) activity was measured by determining the rate of formation of oxalacetate colorimetrically as the 2,4-&nitrophenylhydrazine (Reitman and Frankel, '56). The optical density at 546 mv was converted to GOT units from the table in Bergmeyer ('63). A GOT unit is the amount of transaminase in 1 ml of homogenate which decreases the optical density of DPNH at 340 mu by 0.001 in one minute, in a 3 ml assay mixture at 25°C (La Due, Wroblewski and Karmen, '54 ) . Lactic dehydrogenase (LDH) activity was measured by the decrease of optical density at 340 my due to oxidation of DPNH in the prcsence of pyruvatc. A unit is the amount of LDH which changes the optical density of DPNH at 340 my by 0.001 in one minute; in a 3 ml assay mixture at 24-27°C (Wroblewski and La Due, '55). Amylase was estimated by the change in iodine color of an amylase solution (Street and Close, '56) using one tenth of the volumes set out in Bergmeyer ('63) for the micro method. Under our conditions one Street-Close unit would be contained in 10 ml of homogenate when 0.1 ml hydrolysed 0.2 mg of amylase in 15 minutes at pH 7.0 and 37°C. Succinic dehydrogenase (SDH) was determined by the reduction of neotetrazolium chloride with sodium succinate as substrate (Eckstein, Kahan and Borut, '57; Sobel and Eckstein, '59). The succinic dehydrogenase activity of the homogenate was expressed as micrograms of neotetrazolium reduced during 20 minutes at 37°C. Spectrophotometric determination of the rate of TPNH formation was used for the assay of glucose-6-phosphate dehydrogenase (GDH) (Warburg, Christian and Griese, '35). One unit of activity is the amount of enzyme in 1 ml of sample which at 25°C in a 3 ml assay mixture changes the optical density of TPNH at 340 my by 275 0.001 in one minute (La Due, Wroblewski and Karmen, '54). The protein concentration of homogenates was determined by the biuret method (Wales, Scott and White, '61) and dry weight determined after three hours at 105°C. Analyses of variance were done using the SILLIAC electronic computer and the interaction mean square used to calculate standard errors of the difference between means, for t-tests. RESULTS 1. Endometrium Table 2 shows the activity of alkaline phosphatase, acid phosphatase, GOT, LDH, amylase, SDH and GDH per mg of endometrial protein in the rabbit at estrus, in pseudopregnancy and at days 5 to 10 of pregnancy. Samples from prepubertal (nonpregnant) does are also included for comparison. The most striking changes are the significant increases in SDH and GDH and the decrease in amylase when the doe becomes pregnant or pseudopregnant. Implantation in the rabbit occurs at nine days of pregnancy as judged from histological evidence and studies on the vascular architecture of the endometrium in relation to the blastocyst. Prior to this time, the endometrium undergoes morphological changes and proliferation (cf. Hafez and Tsutsumi, ' 6 6 ) . Table 2 shows the variations in enzyme activity that occur during this stage of development. Amylase did not rise significantly from its lower level between 5 and 10 days of pregnancy. SDH and GDH activity, however, fell away after reaching a maximum prior to implantation. At implantation there is the suggestion of an increase in the activity of LDH and the two phosphatases. All the enzymes assayed had lower activities following implantation (10 days of pregnancy) than before. The endometrial enzyme activities of old-multiparous does compared to youngprimiparous does, seven days of pregnancy, were similar except for the higher activity of alkaline phosphatase (733 and 550 X lo+ units per mg of protein, respectively) and lower activity of SDH (498 and 971 276 E. S. g 8 & !i2lg; 89%;: b U -e 6 f - a52& ob - s+g; * + * + 0 0) + m + rl Q m + + H I3 E. HAFEZ AND I. G. WHITE X lo-* units per mg of protein, respectively) in the older animals (table 2). In prepubertal does, most enzyme activities were similar to adults in estrus, and with the exception of amylase, lower than in pregnant does. The alkaline phosphatase level of the prepubertal endometrium was particularly low. * * + m w Q) zagg?ggaaGg * ** ** daaX8GSeN-R * * * * I * * m ccyomL n m * d o 2. Placental areas The activity of the enzymes in the placenta 7 to 10 days of pregnancy are shown in table 3. There was a statistically significant increase in alkaline phosphatase and amylase at or near implantation, i.e. 8-9 days of pregnancy. No large differences were found between multiparous and primiparous does seven days of pregnancy. Values for SDH were higher than for the comparable stages of development. As with the endometrium, the activity of all enzymes was lower on day 10 than 9. mcc~w rn5W" ?! 5 g f * + + * * +* ** * 66 $ 1 ff: * G m 3c or )ommmc vl m ) wo Yc F- rb roc( -o c v c r ) r ( r ( d r ( r l d t X r l $5 2 H $4: sax3 d$M mmmt-t-m oav) naG3%3zxaa E 2 $ m-3 srl $$g:%%gF?g$ 3 $ :. b 2 *sf?' '-0Ug clam& a o * + o m a a b c E - t - b ~ N b w u l c o m b A 8 E 3 3 3* z z$7 S 3-&5 * YF %832 a * PI $ ?i L; $ 2 ; i 2 3 v 41 Ln xrn 3 \ g 8 2 2 Z 5 b .* .q n v) L 0 ff- c 3 .la 8 V Ln 0 0 nn bag W r J 0 ' a 9 E~=-yyyu~u : T ;45 ZaJ a:&gggigSg 32 "&f g8E 3g2 ,g" 228 ,62" 68," g $$2 7 r n R h R h h R d =2 3r 0 a a 2 ad W a d ad a a n 4 vJ 0i g ~ z ~n ~ n n n w n r n - tn- m m 2 3. Blastocyst and t r q h o b l a s t Alkaline and acid phosphatase were detected in one pooled sample of blastocyst as early as five days of pregnancy, the values being 10 and 8 X units mg protein respectively. The small amount of tissue available, however, did not permit detection of the other enzymes at this stage of development. The activity of acid phosphatase, GOT and SDH in the blastocysts was significantly higher on the eighth day compared to the sixth. The results also suggest that the seven-day blastocysts from multiparous animals have higher GOT and amylase activity and lower LDH activity than comparable blastocysts from primiparous animals. Insufficient material was available, however, to prove this point statistically. In the trophoblast, the activity of the two phosphatases and possibly also SDH decreased gradually from eight to ten days of pregnancy. The activity of GOT and probably also LDH increased markedly at implantation (9 days of pregnancy) and then declined. The enzyme activities in the blastocoelic fluid were generally 10 to 100-fold less than in the trophoblast; the only exception was amylase, which was 2 to 5-fold higher. The activity of LDH increased from eight days to ten days of pregnancy, whereas the ENZYME ACTIVITIES DURING IMPLANTATION 277 activity of alkaline phosphatase and amylase showed a tendency to decline. SDH and GDH could not be detected with any certainty. DISCUSSION -% * -% * During the early stages of development, the blastocyst must become attached to the endometrium but little is known about the changes of the enzyme in the endometrium and blastocyst that allow this to occur. The main episodes in nidation are the preparation of a uterine zone of receptiveness in which the blastocyst may become embedded and the active penetration of that zone by the blastocyst or the attachments of the blastocyst by the uterine tissue. The present results suggest that under the influence of progesterone the SDH and GDH activity of the rabbit endometrium increases and the amylase levels fall. Telfer and Hisaw ('57) report a similar progestational response in endometrial succinoxidase, an enzyme which plays such an important part in the tricaboxylic acid cycle. GDH is the enzyme giving access to the pentose shunt and an increase in both oxidative pathways may be a prerequisite for implantation. Our results could be interpreted to mean that at implantation the blastocyst has a differential effect on the enzyme levels of the interplacental and placental areas. From seven to ten days of pregnancy, the activity of SDH was lower in the placental area than in the interplacental areas, whereas amylase was higher on days 8 and 9 of pregnancy. There were slight variations, but no significant trends between the activity of the other enzyme in these two areas. A decline, however, occurred in the activity of all the enzymes in both areas following implantation. These differences in enzyme levels, as well as differences in the placental tissues at different stages of pregnancy, may, of course, be partly due to differences in the degree of the vascularity of the placenta and the amount of blood engorged in the tissues. The vascularity of the placental tissues increases markedly at nine days of pregnancy (Hafez and Tsutsumi, '66). Individual differences in enzyme activity at the same stage of pregnancy may be 278 E. S. E. HAFEZ AND I. G . WHITE related to differences in the degree of physiological maturity of the uterus. The determinations of LDH and SDH in the blastocyst and trophoblast are consistent with Fridhandler's ('61) finding that after six days the rabbit blastocyst is capable of metabolizing glucose via the Embden-Meyerhof pathway and the tricarboxylic acid cycle. The switch from the hexose monophosphate oxidation pathway at this stage of blastocyst development is presumably not due to loss of GDH activity since it could still be detected in the blastocyst and trophoblast on the tenth day after mating. The changes in the activities of acid phosphatase, GOT and SDH in the blastocyst and trophoblast on days 8-10 of pregnancy may in some way be related to the implantation process. Lutwak-Mann ('59) has shown that the concentration of potassium, bicarbonate, protein and glucose in the blastocyst fluid of the rabbit change at implantation in the direction of the concentration in the maternal blood. This seems unIikely to be the explanation for the increase in the LDH of the blastocyst fluid, however, since the concentration is about three times that of rabbit plasma on day 10 of pregnancy. It is known that the giant cells of the trophoblast perform several functions including invasiveness and synthesis of luteotrophic and/or steroid hormones. The histological appearances at the margin of the growing trophoblastic shell of the human placenta in the early months of gestation suggest that the cells and matrix of the decidua are attacked and slowly destroyed by the action of the advancing growing cytotrophoblast (Wislocki and Padykula, '61). In fact, fragmentation of collagen fibers in the vicinity of the trophoblast has been notcd in the placenta of rodents (Wislocki, Dean and Dempsey, '46). The previous history of the female may have an effect on the enzyme distribution. Thus the endometrium of aged animals had a lower activity of succinic dehydrogenase and possibly higher activity of alkaline phosphatase than that of primiparous animals which may be related to the effect of aging on the rate of placental development and rate of implantation. ** cy* d * Q ** 4- * ** ENZYME ACTIVITIES I JRING IMPLANTATION 279 tribution and hormonal dependence. J. EndoThe enzymes present in prepubertal and crinol., 13: 26-38. mature (non-pregnant) endometrium were 1959 Biochemical approach to the study also found to differ in some respects. Thus of ovum implantation in the rabbit. Memoirs SOC.Endocrinol., 6: 35-49. the prepubertal does had a low activity Qf C., and H. Laser 1954 Bicarboalkaline phosphatase while the levels of Lutwak-Mann, nate content of the blastocyst fluid and carbonic LDH, amylase, SDH and GDH tended to be anhydrase in the pregnant rabbit uterus. Nahigher. The significance of these findings ture, 173: 268-269. is not clear, but may be correlated with the Morione, T., and S. Swifter 1962 Alteration in the collagen content of the human uterus durmaturation of the female reproductive ing pregnancy and postpartum involution. J. tract. Exp. Med., 115: 357-365. LITERATURE CITED Albers, H. J., J. M. Bedford and M. C. Chang 1961 Uterine peptidase activity in the rat and rabbit during pseudoprcgnancy. Amer. J. Physiol., 201: 554-556. 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Fridhandler, L. 1961 Pathways of glucose metabolism in fertilized rabbit ova a t various pre-implantation stages. Exp. Cell. Res., 303: 303-316. Hafez, E. S. E. 1964 Uterine and placental enzymes. Acta Endocrinologica, 46: 217-229. Hafez, E. S. E., and E. S. Tsutsumi 1966 Endometrial vascularity during pseudopregnancy in the rabbit. J. Morph., 118: 43-56. La Due, T. S., F. Wroblewski and A. Karmen 1954 Serum glutamic oxalacetic transaminase activity i n human acute transmural myocardial infection. Science, 120: 497-499. Lutwak-Mann, C. 1955 Carbonic anhydrase in the female reproductive tract. Occurrence, dis- Prahald, K. V. 1962 A study of the r a t pglucuronidase prior to implantation of the ovum. Acta Endocrin., 39: 407-410. Reitman, S., and S. Frankel 1956 A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Amer. J. Clin. Pathology, 28: 56-63. Sobel, H., and B. Eckstein 1959 Succinic dehydrogenase activity in brain tissue of rats after ovariectomy and steroid administration. Nature (Lond.), 183: 54-55. Street, H. V., and J. R. Close 1956 Determination of amylase activity in biological fluids. Clin. Chim. Acta, I: 256-268. Tclfer, M. A., and F. L. Hisaw 1957 Biochemical resuonses of the rabbit endometrium and myome&ium to oestradiol and progesterone. Acta Endocrin. (Kbh.), 25: 390404. Wales, R. G.,T. W. Scott and I. G. White 1961 Biuret reactive materials in semen. Aust. J. Exp. Biol. Med. Sci., 39: 455-462. Warburg, O., W. Christian and A. Griese 1935 Wasserstoffubertragendes Co-Ferment seine zusanimensetzung und Wirkingsweisi. Biochem. Zeits., 282: 157-205. White, I. G. 1955 The collection of rabbit semen, Aust. J. Biol. Sci., 33: 367-370. Wislocki, G. B., H. W. Deane and E. W. Dempsey 1916 The histochemistry of the rodent's placenta. Am. J. Anat., 78: 281-345. Wislocki, G. B., and H. A. Padykula 1961 Histochemistry and electron microscopy of thc placenta. Chap. 15. In: Sex and I n t e r n d Secretions, (ed. by W. C. Young). Bailliere, Tindall & Cox, Ltd., London, 2: 883-957. Woessner, J. F., Jr., and T. H. Brewer 1963 Formation and breakdown of collagen and elastin in the human uterus during pregnancy and postpartum involution. Biochem. J., 89: 75-82. Wroblewski, F., and J. S. La Due 1955 Lactic dehydrogenase activity in blood. Proc. SOC. exp. Biol. Med., 90: 210-213.