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Δ5-3β-Hydroxysteroid dehydrogenase and estradiol-17β-hydroxysteroid dehydrogenase activity in preimplantation hamster embryos
DEVELOPMENTAL
BIOLOGY
40, 196-198 (1974)
BRIEF
A5-3@-Hydroxysteroid 17@hydroxysteroid
Dehydrogenase
and Estradiol-
Dehydrogenase
Activity
Preimplantation Z. DICKMANN Departmertt
NOTE
Hamster AND JAYASREE
of Gynecology and Obstetrics, University Accepted
Embryos SEN GUFTA
of Kansas Medical
April
in
Center, Kansas City, Kansas, 66103
29, 1974
Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. AS-3@Hydroxysteroid dehydrogenase (So-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00-09:OO hr on day 3, the activity then increased, peaked at 01: 00-03 : 00 hr on day 4, considerably declined by 08 : 00-09 : 00 hr (day 4)) and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster. In addition to 3PHSD, estradiol-17&hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen. MATERIALS
INTRODUCTION
Previous studies in our laboratory have demonstrated A5-3/I-hydroxysteroid dehydrogenase (3&HSD) and estradiol-17Phydroxysteroid dehydrogenase (17@HSD) activity in rat morulae and blastocysts (Dickmann and Dey, 1973, 1974; Dey and Dickmann, 1974a,b). These results indicated that preimplantation embryos have the capacity to synthesize steroid hormones. Based on these and other findings (Dickmann, 1970, 1973), it was postulated that steroids produced by the preimplantation embryo play an important role in regulating the phenomena of morula to blastocyst transformation and implantation of the blastocyst. The current study investigated 3/3-HSD activity in the preimplantation and early postimplantation hamster embryo; 17&HSD activity was studied in day-3 embryos. 0 1974 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Golden hamsters (Mesocricetus aurutus) were housed in a windowless, air-conditioned room in which the lights were on from 05: 00 to 19 : 00 hr. Young adult females were mated with fertile males. The morning of finding spermatozoa in the vagina was designated day 1 of pregnancy. On days 1 through 4, preimplantation embryos were obtained by flushing the appropriate section of the reproductive tract. In the postimplantation period, on days 5 and 6, gestation sacs were excised, frozen, and sectioned at 20 pm in a cryostat at -20°C. In whole preimplantation embryos and in sectioned postimplantation embryos, 3&HSD activity was determined histochemically as previously described (Dickmann and Dey, 1974). 17/3-HSD activity in day 3 embryos was also determined histochemically (Dey and Dick196
Copyright All rights
AND
197
BRIEF NOTE TABLE SD-HSD Group No.
1
ACTIVITV IN PREIMPLANTATION HAMSTER EMBRYOS RECOVERED ON DAYS 1 THROUGH 4 OF PREONANC~ Embryo
3&HSD
recovery time Hr
Day 1
1
15:00-16:00
2 3 4 5 6 7
2 3 3 4 4 4
15:00-16:00 08:00-09:OO 15:00-16:OO
Negative 33 41 0 0
Ol:OO-03:oo
0
04:00-05:oo 08:00-09:OO
0 4
mann, 1974b). A positive reaction of both 3/3-HSD and 17P-HSD is manifested by the presence of blue formazan granules which are deposited at the site of the enzymes’ activity. The intensity of the enzymes’ activity was scored as negative, weak, moderate, or strong. RESULTS
AND
DISCUSSION
In Table 1 are presented the results for 3P-HSD activity in preimplantation embryos recovered on days 1 through 4. There was no enzyme activity in day-l and -2 embryos. Weak activity was first observed in embryos recovered at 08 : 00-09 : 00 hr on day 3; the activity subsequently increased, reached a peak at 01: 00-03 : 00 hr on day 4, declined considerably by 08 : 00-09 : 00 hr on day 4, and was negative on days 5 (10 randomly selected embryos obtained from 6 hamsters) and 6 (6 embryos from 3 hamsters). Concurrent with each of the experimental groups (groups 1-7, Table l), control embryos were incubated in substrate-free media. A total of 167 control embryos were studied, and the reaction in all of them was negative. In both control and experimental day-l and day-2 embryos, there was a light purple color reaction but no deposition of formazan granules. Since the color reaction was similar in controls and experimentals, and since no formazan granules were present, the embryos were scored negative. There was no color reaction in day-3 and -4 control embryos.
activity
Weak 0 0 28 26 0 2 8
in embryos Moderate 0 0 0 25 16 16 12
Strong 0 0 0 0 Ii
Total number of embryos 33 41 28 51 33
11
29
0
24
At 08:00-09: 00 hr on day 3, hamster embryos are morulae (4 to 8 cells), blastocoel formation begins at 20 : 00-24 : 00 hr on day 3, and by 1O:OO hr on day 4 most blastocysts are firmly attached to the uterine epithelium so that they can no longer be dislodged by flushing the uterus. The developmental stages of the hamster embryo at which 3P-HSD first appeared, subsequently increased, peaked, declined, and disappeared were very similar to those observed in the rat (Dickmann and Dey, 1973, 1974; Dey and Dickmann, 1974a). In the hamster, however, development is faster, so that, for example, the morning day-4 stage is equivalent to the morning day-6 stage in the rat. 17/3-HSD determinations were done on 41 day 3 (15 : 00-16: 00 hr) embryos: all were positive, and the intensity of the enzyme’s activity was weak to moderate. In 24 control embryos, incubated in substrate-free media, the reaction was negative. 17@-HSD is the enzyme which catalyzes the interconversion of estrone and estradiol. Its presence in conjunction with 3P-HSD suggests synthesis of estrogen. The rat was the first (Dickmann and Dey, 1973, 1974; Dey and Dickmann, 1974b), and the hamster the second, mammalian species in which 3@-HSD and 17/3HSD have been demonstrated in the preimplantation embryos. Because the times at which 3P-HSD first appears, peaks, and disappears in the hamster embryos are very similar to those in rat
198
DEVELOPMENTAL BIOLOGY
embrvos. the hvnothesis formulated for the rit iDickma;n and Dey, 1973, 1974) is applicable to the hamster-namely, steroids produced by the preimplantation embryo play a major role in controlling morula to blastocyst transformation and implantation of the blastocyst. This study was supported in part by the Ford Foundation. J.S.G. is a postdoctoral Ford Foundation Fellow. REFERENCES DEY, S. K., ~~~DICKMANN, Z. (1974a). As-3P-Hydroxysteroid dehydrogenase activity in rat embryos on days 1 through 7 of pregnancy. &zdocrinology 95, 321-322.
VOLUME 40, 1974
DEY, S. K., and DICKMANN, Z. (197413). Estradiol-17@hydroxysteroid dehydrogenase activity in preimplantation rat embryos. Steroids in press. DICKMANN, Z. (1970). Effects of progesterone on the develoument of the rat morula. Fert. Steril. 21. 541-54’8. DICKMANN, Z. (1973). Postcoital contraceptive effects of medroxyprogesterone acetate and oestrone in rats. J. Reprod. Fert. 32, 65-69. DICKMANN, Z., and DEY, S. K. (1973). Two therories: the preimplantation embryo is a source of steroid hormones controlling (1) morula-blastocyst transformation, and 12)
BIOLOGY
40, 196-198 (1974)
BRIEF
A5-3@-Hydroxysteroid 17@hydroxysteroid
Dehydrogenase
and Estradiol-
Dehydrogenase
Activity
Preimplantation Z. DICKMANN Departmertt
NOTE
Hamster AND JAYASREE
of Gynecology and Obstetrics, University Accepted
Embryos SEN GUFTA
of Kansas Medical
April
in
Center, Kansas City, Kansas, 66103
29, 1974
Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. AS-3@Hydroxysteroid dehydrogenase (So-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00-09:OO hr on day 3, the activity then increased, peaked at 01: 00-03 : 00 hr on day 4, considerably declined by 08 : 00-09 : 00 hr (day 4)) and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster. In addition to 3PHSD, estradiol-17&hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen. MATERIALS
INTRODUCTION
Previous studies in our laboratory have demonstrated A5-3/I-hydroxysteroid dehydrogenase (3&HSD) and estradiol-17Phydroxysteroid dehydrogenase (17@HSD) activity in rat morulae and blastocysts (Dickmann and Dey, 1973, 1974; Dey and Dickmann, 1974a,b). These results indicated that preimplantation embryos have the capacity to synthesize steroid hormones. Based on these and other findings (Dickmann, 1970, 1973), it was postulated that steroids produced by the preimplantation embryo play an important role in regulating the phenomena of morula to blastocyst transformation and implantation of the blastocyst. The current study investigated 3/3-HSD activity in the preimplantation and early postimplantation hamster embryo; 17&HSD activity was studied in day-3 embryos. 0 1974 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Golden hamsters (Mesocricetus aurutus) were housed in a windowless, air-conditioned room in which the lights were on from 05: 00 to 19 : 00 hr. Young adult females were mated with fertile males. The morning of finding spermatozoa in the vagina was designated day 1 of pregnancy. On days 1 through 4, preimplantation embryos were obtained by flushing the appropriate section of the reproductive tract. In the postimplantation period, on days 5 and 6, gestation sacs were excised, frozen, and sectioned at 20 pm in a cryostat at -20°C. In whole preimplantation embryos and in sectioned postimplantation embryos, 3&HSD activity was determined histochemically as previously described (Dickmann and Dey, 1974). 17/3-HSD activity in day 3 embryos was also determined histochemically (Dey and Dick196
Copyright All rights
AND
197
BRIEF NOTE TABLE SD-HSD Group No.
1
ACTIVITV IN PREIMPLANTATION HAMSTER EMBRYOS RECOVERED ON DAYS 1 THROUGH 4 OF PREONANC~ Embryo
3&HSD
recovery time Hr
Day 1
1
15:00-16:00
2 3 4 5 6 7
2 3 3 4 4 4
15:00-16:00 08:00-09:OO 15:00-16:OO
Negative 33 41 0 0
Ol:OO-03:oo
0
04:00-05:oo 08:00-09:OO
0 4
mann, 1974b). A positive reaction of both 3/3-HSD and 17P-HSD is manifested by the presence of blue formazan granules which are deposited at the site of the enzymes’ activity. The intensity of the enzymes’ activity was scored as negative, weak, moderate, or strong. RESULTS
AND
DISCUSSION
In Table 1 are presented the results for 3P-HSD activity in preimplantation embryos recovered on days 1 through 4. There was no enzyme activity in day-l and -2 embryos. Weak activity was first observed in embryos recovered at 08 : 00-09 : 00 hr on day 3; the activity subsequently increased, reached a peak at 01: 00-03 : 00 hr on day 4, declined considerably by 08 : 00-09 : 00 hr on day 4, and was negative on days 5 (10 randomly selected embryos obtained from 6 hamsters) and 6 (6 embryos from 3 hamsters). Concurrent with each of the experimental groups (groups 1-7, Table l), control embryos were incubated in substrate-free media. A total of 167 control embryos were studied, and the reaction in all of them was negative. In both control and experimental day-l and day-2 embryos, there was a light purple color reaction but no deposition of formazan granules. Since the color reaction was similar in controls and experimentals, and since no formazan granules were present, the embryos were scored negative. There was no color reaction in day-3 and -4 control embryos.
activity
Weak 0 0 28 26 0 2 8
in embryos Moderate 0 0 0 25 16 16 12
Strong 0 0 0 0 Ii
Total number of embryos 33 41 28 51 33
11
29
0
24
At 08:00-09: 00 hr on day 3, hamster embryos are morulae (4 to 8 cells), blastocoel formation begins at 20 : 00-24 : 00 hr on day 3, and by 1O:OO hr on day 4 most blastocysts are firmly attached to the uterine epithelium so that they can no longer be dislodged by flushing the uterus. The developmental stages of the hamster embryo at which 3P-HSD first appeared, subsequently increased, peaked, declined, and disappeared were very similar to those observed in the rat (Dickmann and Dey, 1973, 1974; Dey and Dickmann, 1974a). In the hamster, however, development is faster, so that, for example, the morning day-4 stage is equivalent to the morning day-6 stage in the rat. 17/3-HSD determinations were done on 41 day 3 (15 : 00-16: 00 hr) embryos: all were positive, and the intensity of the enzyme’s activity was weak to moderate. In 24 control embryos, incubated in substrate-free media, the reaction was negative. 17@-HSD is the enzyme which catalyzes the interconversion of estrone and estradiol. Its presence in conjunction with 3P-HSD suggests synthesis of estrogen. The rat was the first (Dickmann and Dey, 1973, 1974; Dey and Dickmann, 1974b), and the hamster the second, mammalian species in which 3@-HSD and 17/3HSD have been demonstrated in the preimplantation embryos. Because the times at which 3P-HSD first appears, peaks, and disappears in the hamster embryos are very similar to those in rat
198
DEVELOPMENTAL BIOLOGY
embrvos. the hvnothesis formulated for the rit iDickma;n and Dey, 1973, 1974) is applicable to the hamster-namely, steroids produced by the preimplantation embryo play a major role in controlling morula to blastocyst transformation and implantation of the blastocyst. This study was supported in part by the Ford Foundation. J.S.G. is a postdoctoral Ford Foundation Fellow. REFERENCES DEY, S. K., ~~~DICKMANN, Z. (1974a). As-3P-Hydroxysteroid dehydrogenase activity in rat embryos on days 1 through 7 of pregnancy. &zdocrinology 95, 321-322.
VOLUME 40, 1974
DEY, S. K., and DICKMANN, Z. (197413). Estradiol-17@hydroxysteroid dehydrogenase activity in preimplantation rat embryos. Steroids in press. DICKMANN, Z. (1970). Effects of progesterone on the develoument of the rat morula. Fert. Steril. 21. 541-54’8. DICKMANN, Z. (1973). Postcoital contraceptive effects of medroxyprogesterone acetate and oestrone in rats. J. Reprod. Fert. 32, 65-69. DICKMANN, Z., and DEY, S. K. (1973). Two therories: the preimplantation embryo is a source of steroid hormones controlling (1) morula-blastocyst transformation, and 12)