1I. pp,
Jaurnul of Sferoid Biwhumisrry. Vol. 771 to 773 Pergamon Prsss Ltd 1979. Printed in Great Britain
BLASTOCYST OESTROGEN: AN ESSENTIAL FACTOR FOR THE CONTROL OF IMPLANTATION Z. DICKMANN Department
of Gynaecology and Obstetrics, and Ralph L. Smith Human Development Research Centre, University of Kansas Medical Centre, Kansas City, Kansas 66103, U.S.A. SUMMARY
A new hypothesis is proposed concerning the hormonal control of blastocyst implantation. In preparation for implantation, the uterus has first to come under the dominance of progesterone, derived from the ovaries. As the time of implantation approaches, a distinction is made between the conditioning of the prospective implantation sites and the remainder of the uterus. While progestero~e~ominance has to be maintained in the interimplantation areas, the implantation sites have to be relieved temporarily from the dominance of this hormone (an anti-inflammatory agent), in order to permit a local inflammatory-like reaction which is a prerequisite for implantation. The local relief from progesteronedominance is accomplished through the action of oestrogen released by the blastocyst. In some species, like rat and mouse, blastocyst oestrogen by itself is inadequate to overcome the progesteronedominance. Therefore, the action of systemic oestrogen (the “oestrogen surge”) has to precede the action of blastocyst oestrogen.
INTRODUCTION It is well established that the morphological and biochemical events occurring during the process of bl~tocyst-implantation vary considerably among different mammalian species. However, regardless of the diversity of implantation, hormones play the key role in its regulation. The various features of the hormonal control of implantation have been adequately reviewed [l, 21. In the present paper, certain aspects of these hormonal controls will be compiled so as to form the background for presenting a new hypothesis. The hormonal control of implantation has been studied more extensively in rat and mouse than in other mammalian species. For this reason, the rat will be used as a primary model. Two basic components are required to accomplish implantation: a mature, zona-pellucida-free blastocyst and a properly conditioned uterus. The conditioning of the uterus results from stimulations with progesterone followed by oestrogen (the “oestrogen surge”). If a pregnant rat is ovariectomized before the occurrence of the oestrogen surge and is treated .with progesterone, implantation does not take place; the blastocysts enter a phase of low metabolic activity, but they remain viable in the uterine lumen. Under such conditions, known as “delayed implantation”, when a female is given a single injection of oestrogen, implantation begins about 24 hr later. During regular pregnancy as well as during delayed-implantation progesterone is the key hormone for preparing the uterus for implantation. However, a relatively mild oestrogen intervention is necessary for the induction of implantation. Thus, during delayed-implantation a subcutaneous injection of 5Ong of oestradioL17fi suffices to induce implantation [2]. It may be surmised that the oestrogen intervention could cause
a slight weakening of the progesterone-dominance, but by and large the progesterone-dominance is retained. The oestrogen intervention is obligatory in rat and mouse, but not in rabbit [3,4], hamster [5,6] or sheep [7J Thus, implantation does occur in pregnant ovariectomized-adrenalectomized rabbits and hamsters and in ovariectomized sheep (in which ovariectomy-a~enaie~tomy studies have not been done) treated with progesterone alone. Due to a lack of large-scale comparative studies, it is not known whether the requirement for an oestrogen intervention is common or uncommon among mammalian species in general. However, the hypothesis proposed here is applicable regardless of whether or not a species requires an oestrogen intervention.
771
THEORETlCAL CONSIDERATIONS
A distinction should be made between the preparation of the uterus as a whole and the preparation of the actual site of impIantation. At the prospective implantation site, an inflammatory-like reaction (e.g., increase in capillary permeability) has to precede implantation 181. Since it has been demonstrated that progesterone can act as an anti-inflammatory agent [9-123, it may be postulated that while progesterone-dominance is obligatory for the uterus as a whole, it is inhibitory for the local inflammatory-like reaction. Hence, it is compulsory to nullify, or sufficiently reduce, the local progesterone-dominance to permit a local inflammatory-like response. A substance to counteract progesterone locally would have to come from a local source, because delivery to the uterus via the general circulation would affect the entire uterus. The obvious local source is the blastocyst. What is this substance that emanates from the
2. DICKMANN
172
blastocyst? The proposition put forward here is that it is oestrogen, for the following reasons. (a) It is well established that oestrogen can nullify or reduce the effects of progesterone. (b) It has been shown that rabbit blastocysts contain [13-153 and can synthesize[l6] oestrogen. (c) There is evidence that in the rat, a few hours before implantation commences, the oestrogen concentration is considerably higher in the uterine segments comprising the implantation sites than in the segments comprising the interimplantation areas [17]. (d) It has been demonstrated that a local application of a minute quantity of oestrogen, to a properly primed rat uterus, results in a local increase in capillary permeability [18]. (e) My unpublished preliminary results show that instilling the antioestrogen Cl-628 into the uterine lumens of pregnant rabbits inhibits implantation, and that this inhibition can be neutralized by concurrent instillation of oestradiol-17/I. Since implantation in the rabbit is independent of systemic oestrogen, these experiments provide strong evidence that blastocyst oestrogen is an indispensable factor for the control of implantation in rabbits. DISCUSSION
Direct evidence for steroidogenic activity by blastocysts has been obtained in rabbits [19], pigs [20] and cows[21]. So far, the specific synthesis of oestrogzr has been demonstrated only in pig blastocysts [20]. There is strong indirect evidence for oestrogen synthesis in rabbit blastocysts [13, 14, the present paper], but direct evidence is not yet available. George and Wilson [16] were able to demonstrate oestrogen synthesis in day 7 rabbit embryos, but not in day 6 blastocysts. In a given experiment, they incubated three day 6 blastocysts for one hour, in the presence of a precursor (testosterone). Later, they assayed the oestrogen formed. Since the total amount of oestradiol-17jl per blastocyst is quite small (about 1 pg) [13], it is not surprising these authors were unable to detect oestrogen, as the amounts of the hormone synthesized under their experimental conditions must have been far below the sensitivity of the assay used. It may be mentioned parenthetically that other authors have used inadequate amounts of blastocyst tissue, and then erroneously concluded that mouse blastocysts do not exhibit steroidogenesis [22]. Although in the opinion of the author this is unlikely, it cannot be ruled out that in some species the oestrogen contained in blastocysts is not synthesized by the blastocyst. Thus, if oestrogen was available in the uterine lumen, blastocysts could absorb and possibly concentrate it. Also, the oocyte, while residing in the follicle, could absorb oestrogen, retain it through the preimplantation period, and release it at the appropriate time to affect implantation. The question may be asked that, if blastocyst oestrogen controls implantation, why should there be a need for systemic oestrogen in some species? An
answer is that in a species like the rat, which requires systemic oestrogen, the blastocyst does not have the capacity to secrete sufficient amounts of oestrogen to overcome the progesterone-dominance. Hence, a function of the systemic oestrogen is to reduce the progesterone action to a level at which blastocyst oestrogen can carry out its local function. This explanation is supported by the findings that, at the time of implantation, the progesterone concentration in the serum of rats is relatively high, 90 ng/ml [23], whereas in rabbits, which do not require systemic oestrogen, the concentration is only 5 ng/ml [24]. However, the serum progesterone level is not the only factor determining the balance of oestrogen/progesterone action at the site of implantation. Other factors are the rate of blood flow to the uterus, oestrogen concentration in the blood, concentration of oestrogen and progesterone receptors at the implantation site, and the rates of production and release of oestrogen by the blastocyst. If systemic oestrogen is available, why can’t it regulate implantation by itself? It is well established that an excess of systemic oestrogen prevents implantation by causing changes such as abnormal orientation of the blastocyst in the uterine lumen, and expulsion of the blastocyst from the uterus [25]. Perhaps, in the course of evolution, the body found it difficult to regulate the levels of systemic oestrogen with a degree of precision so as to satisfy the needs of the implantation site, and at the same time not overstimulate the remainder of the uterus. To overcome this difficulty, the body developed a second source of oestrogen-the blastocyst. On the other hand, the blastocyst may have been the sole source of oestrogen for regulating implantation, and only later in evolution when this source became inadequate, the ovaries made up for the deficiency. In conclusion, the contention here is that blastocyst oestrogen is a key factor for the regulation of implantation. There are other key factors, some of which are known while others have yet to be discovered. Acknowledgements-This study was supported in part by grants from the NIH (lROl-HD08644) and from the National Foundation (l-406). I wish to thank Ms. Pat Brahl for typing the manuscript. REFERENCES
1. McLaren
A. Endocrinology of implantation.: J. Reprod. Fert. Suppl. 18, (1973) 159-166. 2. Psychoyos A.: Hormonal control of ovoimplantation. Warn. Harm. 31 (1973) 201-256. 3. Wu D. H. and Allen W. M.: Maintenance of pregnancy in castrated rabbits by 17-alpha-hydroxy-progesterone caproate and by progesterone. Fert. Steril. 10 (1959) 439460. 4. Kwun J. K. and Emmens C. W.: Hormonal
requirements for implantation and pregnancy in the ovariectomized rabbit. Amt. J. Biol. Sci. 27 (1974) 275-283. 5. Orsini M. W. and Meyer R. K.: Implantation of the castrate hamster in the absence of exogenous estrogen. Anat. Rec. 134 (1959) 619-620.
Control
of implantation:
6. Harper M. J. K., Prostkoff B. T. and Reeve R. J.: Implantation and embryonic development in the ovariectomized hamster. Acta Endow. Copenh. 52 (1966) 465-470. 7. Bindon B. M.: The role of progesterone in implantation in the sheep. Amt. J. Biol. Sci. 24 (1971) 149-158. 8. Psychoyos A.: The hormonal interplay controlling eggimplantation in the rat. Adtlan. Reprod. Physiol. 2 (1967) 251-271. 9. Black W. G.. Ulberg L. C.. Kidder H. E.. Simon J.. McNutt S. H. and Casida L. E.: Inflammatory response of the bovine endometrium. Am. J. Vet. Res. 14 (1953) 179-183. IO. Black W. G.. Simon J., McNutt S. H. and Casida L. E.: Investigations on the physiological basis for the differential response of estrous and pseudopregnant rabbit uteri to induced infection. Am. J. Vet. Res. l4 (1953) 318-323. 11. Broome A. W. J., Lamming G. E. and Smith W.: Studies on the relationship between ovarian hormones and uterine infection. IV.-The role of the granulocyte system in uterine defence. .!. Endocr. 19 (1959) 274-281. 12. Siiteri, P. K.. Febres F., Clemens L. E., Chang R. J., Gondos B. and Stites D.: Progesterone and maintenance of pregnancy: Is progesterone nature’s immunosuppressant? Ann. N.Y. Acad. Sci. 286 (1977) 384-397. 13. Dickmann Z., Dey S. K. and Sen Gupta J.: Steroidogenesis in rabbit preimplantation embryos. Proc. Nat. Acad. Sci., U.S.A. 72 (1975) 298-300. 14. Dickmann Z.. Dey S. K. and Sen Gupta J.: A new concept: Control of early pregnancy by steroid hormones originating in the preimplantation embryo. Vitam. Horm. 34 (1976) 211-242. IS. Borland R. M., Erickson G. F. and Ducibella T.: Ac-
blastocyst
16. 17.
,l8.
19.
20.
21.
22.
23.
24.
25.
oestrogen
113
cumulation of steroids in rabbit preimplantation hlastocvsts. J. Reorod. Fert. 49 (1977) 21%224. Gebrge F. Wl and Wilson j. D.: Estrogen formation in the early rabbit embryo. Science 199 (1978) 200-201. Ward W. F.. Frost A. G. and Orsini M. W.: Estrogen binding by gmbryonic and interembryonic segments of the rat uterus prior to implantation. Biol. Reprod. 18 (1978) 598-601. Dickmann Z., Sen Gupta J. and Dey S. K.: Does “blastocyst estrogen” initiate implantation’? Science 195 (1977) 681-688. Huff R. L. and Eik-Nes K. B.: Metabolism in ritro of acetate and certain steroids by six-day-old rabbit blastocysts. J. Reprod. Fert. 11 (1966) 57-63. Perry J. S.. Heap R. B. and Amoroso E. C.: Steroid hormone production by pig blastocysts. Nature 245 (1973) 45-47. Shemesh M., Melaguir F.. Lavi S. and Ayalon N.: Steroidogenesis and prostaglandin synthesis by cultured bovine blastocysts. Eleventh Ann. Meet. Sot. Study of Reprod. (1978) Abstr. 61. Sherman M. I. and Atienza S. B.: Production and metabolism of progesterone and androstenedione by cultured mouse blastocysts. Biol. Reprod. 16 (1977) 19cb199. Morishige W. K., Pepe G. J. and Rothchild I.: Serum luteinizing hormone, prolactin and progesterone levels during pregnancy in the rat. Endocrinology 92 (1973) 1527-l 530. Harrington F. E. and Rothermel J. D.: Daily changes in peripheral plasma progesterone concentrations in pregnant and pseudopregnant rabbits. Lljce Sci. 20 (1977) 1333-1340. Harper M. J. K.: Interference with early pregnancy in rats by estrogen: mechanism of action of dienestrol. Anar. Rec. 162 (1968) 433-452.
Jaurnul of Sferoid Biwhumisrry. Vol. 771 to 773 Pergamon Prsss Ltd 1979. Printed in Great Britain
BLASTOCYST OESTROGEN: AN ESSENTIAL FACTOR FOR THE CONTROL OF IMPLANTATION Z. DICKMANN Department
of Gynaecology and Obstetrics, and Ralph L. Smith Human Development Research Centre, University of Kansas Medical Centre, Kansas City, Kansas 66103, U.S.A. SUMMARY
A new hypothesis is proposed concerning the hormonal control of blastocyst implantation. In preparation for implantation, the uterus has first to come under the dominance of progesterone, derived from the ovaries. As the time of implantation approaches, a distinction is made between the conditioning of the prospective implantation sites and the remainder of the uterus. While progestero~e~ominance has to be maintained in the interimplantation areas, the implantation sites have to be relieved temporarily from the dominance of this hormone (an anti-inflammatory agent), in order to permit a local inflammatory-like reaction which is a prerequisite for implantation. The local relief from progesteronedominance is accomplished through the action of oestrogen released by the blastocyst. In some species, like rat and mouse, blastocyst oestrogen by itself is inadequate to overcome the progesteronedominance. Therefore, the action of systemic oestrogen (the “oestrogen surge”) has to precede the action of blastocyst oestrogen.
INTRODUCTION It is well established that the morphological and biochemical events occurring during the process of bl~tocyst-implantation vary considerably among different mammalian species. However, regardless of the diversity of implantation, hormones play the key role in its regulation. The various features of the hormonal control of implantation have been adequately reviewed [l, 21. In the present paper, certain aspects of these hormonal controls will be compiled so as to form the background for presenting a new hypothesis. The hormonal control of implantation has been studied more extensively in rat and mouse than in other mammalian species. For this reason, the rat will be used as a primary model. Two basic components are required to accomplish implantation: a mature, zona-pellucida-free blastocyst and a properly conditioned uterus. The conditioning of the uterus results from stimulations with progesterone followed by oestrogen (the “oestrogen surge”). If a pregnant rat is ovariectomized before the occurrence of the oestrogen surge and is treated .with progesterone, implantation does not take place; the blastocysts enter a phase of low metabolic activity, but they remain viable in the uterine lumen. Under such conditions, known as “delayed implantation”, when a female is given a single injection of oestrogen, implantation begins about 24 hr later. During regular pregnancy as well as during delayed-implantation progesterone is the key hormone for preparing the uterus for implantation. However, a relatively mild oestrogen intervention is necessary for the induction of implantation. Thus, during delayed-implantation a subcutaneous injection of 5Ong of oestradioL17fi suffices to induce implantation [2]. It may be surmised that the oestrogen intervention could cause
a slight weakening of the progesterone-dominance, but by and large the progesterone-dominance is retained. The oestrogen intervention is obligatory in rat and mouse, but not in rabbit [3,4], hamster [5,6] or sheep [7J Thus, implantation does occur in pregnant ovariectomized-adrenalectomized rabbits and hamsters and in ovariectomized sheep (in which ovariectomy-a~enaie~tomy studies have not been done) treated with progesterone alone. Due to a lack of large-scale comparative studies, it is not known whether the requirement for an oestrogen intervention is common or uncommon among mammalian species in general. However, the hypothesis proposed here is applicable regardless of whether or not a species requires an oestrogen intervention.
771
THEORETlCAL CONSIDERATIONS
A distinction should be made between the preparation of the uterus as a whole and the preparation of the actual site of impIantation. At the prospective implantation site, an inflammatory-like reaction (e.g., increase in capillary permeability) has to precede implantation 181. Since it has been demonstrated that progesterone can act as an anti-inflammatory agent [9-123, it may be postulated that while progesterone-dominance is obligatory for the uterus as a whole, it is inhibitory for the local inflammatory-like reaction. Hence, it is compulsory to nullify, or sufficiently reduce, the local progesterone-dominance to permit a local inflammatory-like response. A substance to counteract progesterone locally would have to come from a local source, because delivery to the uterus via the general circulation would affect the entire uterus. The obvious local source is the blastocyst. What is this substance that emanates from the
2. DICKMANN
172
blastocyst? The proposition put forward here is that it is oestrogen, for the following reasons. (a) It is well established that oestrogen can nullify or reduce the effects of progesterone. (b) It has been shown that rabbit blastocysts contain [13-153 and can synthesize[l6] oestrogen. (c) There is evidence that in the rat, a few hours before implantation commences, the oestrogen concentration is considerably higher in the uterine segments comprising the implantation sites than in the segments comprising the interimplantation areas [17]. (d) It has been demonstrated that a local application of a minute quantity of oestrogen, to a properly primed rat uterus, results in a local increase in capillary permeability [18]. (e) My unpublished preliminary results show that instilling the antioestrogen Cl-628 into the uterine lumens of pregnant rabbits inhibits implantation, and that this inhibition can be neutralized by concurrent instillation of oestradiol-17/I. Since implantation in the rabbit is independent of systemic oestrogen, these experiments provide strong evidence that blastocyst oestrogen is an indispensable factor for the control of implantation in rabbits. DISCUSSION
Direct evidence for steroidogenic activity by blastocysts has been obtained in rabbits [19], pigs [20] and cows[21]. So far, the specific synthesis of oestrogzr has been demonstrated only in pig blastocysts [20]. There is strong indirect evidence for oestrogen synthesis in rabbit blastocysts [13, 14, the present paper], but direct evidence is not yet available. George and Wilson [16] were able to demonstrate oestrogen synthesis in day 7 rabbit embryos, but not in day 6 blastocysts. In a given experiment, they incubated three day 6 blastocysts for one hour, in the presence of a precursor (testosterone). Later, they assayed the oestrogen formed. Since the total amount of oestradiol-17jl per blastocyst is quite small (about 1 pg) [13], it is not surprising these authors were unable to detect oestrogen, as the amounts of the hormone synthesized under their experimental conditions must have been far below the sensitivity of the assay used. It may be mentioned parenthetically that other authors have used inadequate amounts of blastocyst tissue, and then erroneously concluded that mouse blastocysts do not exhibit steroidogenesis [22]. Although in the opinion of the author this is unlikely, it cannot be ruled out that in some species the oestrogen contained in blastocysts is not synthesized by the blastocyst. Thus, if oestrogen was available in the uterine lumen, blastocysts could absorb and possibly concentrate it. Also, the oocyte, while residing in the follicle, could absorb oestrogen, retain it through the preimplantation period, and release it at the appropriate time to affect implantation. The question may be asked that, if blastocyst oestrogen controls implantation, why should there be a need for systemic oestrogen in some species? An
answer is that in a species like the rat, which requires systemic oestrogen, the blastocyst does not have the capacity to secrete sufficient amounts of oestrogen to overcome the progesterone-dominance. Hence, a function of the systemic oestrogen is to reduce the progesterone action to a level at which blastocyst oestrogen can carry out its local function. This explanation is supported by the findings that, at the time of implantation, the progesterone concentration in the serum of rats is relatively high, 90 ng/ml [23], whereas in rabbits, which do not require systemic oestrogen, the concentration is only 5 ng/ml [24]. However, the serum progesterone level is not the only factor determining the balance of oestrogen/progesterone action at the site of implantation. Other factors are the rate of blood flow to the uterus, oestrogen concentration in the blood, concentration of oestrogen and progesterone receptors at the implantation site, and the rates of production and release of oestrogen by the blastocyst. If systemic oestrogen is available, why can’t it regulate implantation by itself? It is well established that an excess of systemic oestrogen prevents implantation by causing changes such as abnormal orientation of the blastocyst in the uterine lumen, and expulsion of the blastocyst from the uterus [25]. Perhaps, in the course of evolution, the body found it difficult to regulate the levels of systemic oestrogen with a degree of precision so as to satisfy the needs of the implantation site, and at the same time not overstimulate the remainder of the uterus. To overcome this difficulty, the body developed a second source of oestrogen-the blastocyst. On the other hand, the blastocyst may have been the sole source of oestrogen for regulating implantation, and only later in evolution when this source became inadequate, the ovaries made up for the deficiency. In conclusion, the contention here is that blastocyst oestrogen is a key factor for the regulation of implantation. There are other key factors, some of which are known while others have yet to be discovered. Acknowledgements-This study was supported in part by grants from the NIH (lROl-HD08644) and from the National Foundation (l-406). I wish to thank Ms. Pat Brahl for typing the manuscript. REFERENCES
1. McLaren
A. Endocrinology of implantation.: J. Reprod. Fert. Suppl. 18, (1973) 159-166. 2. Psychoyos A.: Hormonal control of ovoimplantation. Warn. Harm. 31 (1973) 201-256. 3. Wu D. H. and Allen W. M.: Maintenance of pregnancy in castrated rabbits by 17-alpha-hydroxy-progesterone caproate and by progesterone. Fert. Steril. 10 (1959) 439460. 4. Kwun J. K. and Emmens C. W.: Hormonal
requirements for implantation and pregnancy in the ovariectomized rabbit. Amt. J. Biol. Sci. 27 (1974) 275-283. 5. Orsini M. W. and Meyer R. K.: Implantation of the castrate hamster in the absence of exogenous estrogen. Anat. Rec. 134 (1959) 619-620.
Control
of implantation:
6. Harper M. J. K., Prostkoff B. T. and Reeve R. J.: Implantation and embryonic development in the ovariectomized hamster. Acta Endow. Copenh. 52 (1966) 465-470. 7. Bindon B. M.: The role of progesterone in implantation in the sheep. Amt. J. Biol. Sci. 24 (1971) 149-158. 8. Psychoyos A.: The hormonal interplay controlling eggimplantation in the rat. Adtlan. Reprod. Physiol. 2 (1967) 251-271. 9. Black W. G.. Ulberg L. C.. Kidder H. E.. Simon J.. McNutt S. H. and Casida L. E.: Inflammatory response of the bovine endometrium. Am. J. Vet. Res. 14 (1953) 179-183. IO. Black W. G.. Simon J., McNutt S. H. and Casida L. E.: Investigations on the physiological basis for the differential response of estrous and pseudopregnant rabbit uteri to induced infection. Am. J. Vet. Res. l4 (1953) 318-323. 11. Broome A. W. J., Lamming G. E. and Smith W.: Studies on the relationship between ovarian hormones and uterine infection. IV.-The role of the granulocyte system in uterine defence. .!. Endocr. 19 (1959) 274-281. 12. Siiteri, P. K.. Febres F., Clemens L. E., Chang R. J., Gondos B. and Stites D.: Progesterone and maintenance of pregnancy: Is progesterone nature’s immunosuppressant? Ann. N.Y. Acad. Sci. 286 (1977) 384-397. 13. Dickmann Z., Dey S. K. and Sen Gupta J.: Steroidogenesis in rabbit preimplantation embryos. Proc. Nat. Acad. Sci., U.S.A. 72 (1975) 298-300. 14. Dickmann Z.. Dey S. K. and Sen Gupta J.: A new concept: Control of early pregnancy by steroid hormones originating in the preimplantation embryo. Vitam. Horm. 34 (1976) 211-242. IS. Borland R. M., Erickson G. F. and Ducibella T.: Ac-
blastocyst
16. 17.
,l8.
19.
20.
21.
22.
23.
24.
25.
oestrogen
113
cumulation of steroids in rabbit preimplantation hlastocvsts. J. Reorod. Fert. 49 (1977) 21%224. Gebrge F. Wl and Wilson j. D.: Estrogen formation in the early rabbit embryo. Science 199 (1978) 200-201. Ward W. F.. Frost A. G. and Orsini M. W.: Estrogen binding by gmbryonic and interembryonic segments of the rat uterus prior to implantation. Biol. Reprod. 18 (1978) 598-601. Dickmann Z., Sen Gupta J. and Dey S. K.: Does “blastocyst estrogen” initiate implantation’? Science 195 (1977) 681-688. Huff R. L. and Eik-Nes K. B.: Metabolism in ritro of acetate and certain steroids by six-day-old rabbit blastocysts. J. Reprod. Fert. 11 (1966) 57-63. Perry J. S.. Heap R. B. and Amoroso E. C.: Steroid hormone production by pig blastocysts. Nature 245 (1973) 45-47. Shemesh M., Melaguir F.. Lavi S. and Ayalon N.: Steroidogenesis and prostaglandin synthesis by cultured bovine blastocysts. Eleventh Ann. Meet. Sot. Study of Reprod. (1978) Abstr. 61. Sherman M. I. and Atienza S. B.: Production and metabolism of progesterone and androstenedione by cultured mouse blastocysts. Biol. Reprod. 16 (1977) 19cb199. Morishige W. K., Pepe G. J. and Rothchild I.: Serum luteinizing hormone, prolactin and progesterone levels during pregnancy in the rat. Endocrinology 92 (1973) 1527-l 530. Harrington F. E. and Rothermel J. D.: Daily changes in peripheral plasma progesterone concentrations in pregnant and pseudopregnant rabbits. Lljce Sci. 20 (1977) 1333-1340. Harper M. J. K.: Interference with early pregnancy in rats by estrogen: mechanism of action of dienestrol. Anar. Rec. 162 (1968) 433-452.
