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Early pregnancy factor: Tissues involved in its production in the mouse
Journal of Reproductive Immunology. 2 (1980) 73-82
73
© Elsevier/North-Holland Biomedical Press
EARLY PREGNANCY FACTOR: TISSUES INVOLVED IN ITS PRODUCTION
IN THE MOUSE
H. MORTON, B.E. ROLFE, L. McNEILL, P. CLARKE, F.M. CLARKE and G.J.A. CLUNIE *
Department of Surgery, University of Queensland, Princess Alexandra Hospital, Brisbane, and School of Science, Griffith University, Nathan, Qld., Australia (Received 7 November 1979; accepted 7 January 1980)
The mouse, 24 h after mating, has been used as an experimental model to determine the site of production of early pregnancy factor (EPF). Results have shown that EPF is formed as two separate components, which appear similar to component A and component B formed after dissociation of EPF by 40% ammonium sulphate. Component A is produced by the oviduct during oestrus and pregnancy. It is present in serum during oestrus in an inactive form, that is, not capable of binding with lymphocytes. Component B can be produced in culture from the ovaries of 24-b pregnant mice. Incubation experiments described in this paper show that the production of component B can also be initiated from non-pregnant mouse ovaries, during oestrus or dioestrus, in the presence of both fertilised ovum and non-pregnancy pituitary but not in the presence of fertilised ovum alone. The pituitary from a 24-h pregnant mouse alone can also stimulate the production of component B from oestrous or dioestrous mice. To summarize, component A is derived from the oviduct and.is oestrus-dependent. Component B, the pregnancy-dependent component of EPF, is produced by the ovary; its production can be initiated from non-pregnancy ovaries by the fertilised ovum in cooperation with the pituitary.
INTRODUCTION Early Pregnancy Factor (EPF) can be detected initially in the serum of mice, humans and sheep within the first 24 h after fertilisation (Morton et al., 1976, 1977, 1979), indicating that its production at this stage is not dependent on the presence of tissues of the placenta or of the foetus. This previous work has demonstrated that the development of EPF is not initiated by the act of mating or the presence of sperm or semen in the reproductive tract; the conditions necessary for the production of EPF are that the animal has ovulated and that the ovum has been fertilised. In mice, EPF has been detected from 6 h after mating, that is, before the fusion of the male and female pronuclei completes fertilisation and cell division commences. Levinson et al. (1978) have shown that from the single fertilised cell stage there are numerous stage-specific polypeptides synthesised during murine pre-implantation development and several groups have found specific functions for embryo-derived substances. Kent (1975) described a polypeptide from the 2- to 4-cell ova of the hamster which prevents further
* Present address: University of Melbourne, Royal Melbourne Hospital, ParkviUe,Vic., Australia.
74 ovulation of the.pregnant animal. Godkin et al. (1978) and Martal et al. t.1979) hay,.~ described a substance produced by the sheep embryo which directly stimulates progeste: one synthesis and contributes to the maintenance of pregnancy; the presence of a simila~ substance in rabbits has been suggested by Channing et al. (1978) and in humans b?;~ Yoshimi et al. (1969). Channing and his group have compared the substance the) identified with hCG but Sundaram et al. (1975), using a bioassay, found no hCG-likc activity in the blastocyst of the rabbit between 4 and 7 days gestation. These substance;, originating from the embryo are thought to act directly on the tissues of the ovary. Although the production of EPF is dependent on the presence of a fertilised o v u ~ it seemed unlikely that this would be the source of production; more probably Oat:. embryo acts as a stimulus to produce EPF from some other source. In this mvestigatio~ the mouse was used as the experimental model to determine the site of EPF productio~a The different phases oi oestrus in the mouse can readily be identified by vaginal smear:. and the formation of a vaginal plug allows for fairly precise timing of mating and subse quent fertilisation (Rugh, 1968). This precise timing in the mouse, compared with tha~ in humans and sheep, allows for correlation of the formation of regulatory substance~ with the established data on hormonal events associated with various stages of pregnancy METHODS Outbred Quackenbush mice, aged 8-- 16 wk, were used in this study.
Stages of oestrus In the experiments involving oestrous and dioestrous mice, the stage o! oestrus was determined by vaginal smear (Rugh, t968). Mating schedule Groups of three virgin females and one male were caged together between 8.30 and 9.30 a.m. and those females showing vaginal plugs as evidence of mating were segregated After 24 h, unless otherwise stated, the mated female mice were anaesthetized with ethel exsanguinated by cardiac puncture and the tissues required for experiment removed Dissection was done under sterile conditions. Tissue samples Serum. Serum was collected from the blood sample obtained by cardiac puncture. heat inactivated and frozen. Serum from each mated female mouse was tested for the presence of EPF. Ovary and oviduct. The ovaries and oviducts were removed into Hank's balanced salt solution (BSS; C.S.L. Australia). In some experiments the oviduct was incubated with the fertilised ova in situ (oviduct + ova); in other experiments the fertilised ova were flushed from the oviduct before incubation. Fertilised ova. Ova were flushed from the oviducts of mice with Ovum Flushing Medium (Flow Laboratories) using a syringe fitted with a 30 gauge needle. The ova weft" examined to establish the stage of division (Rugh, 1968), then transferred with a Pasteur pipette to an incubation chamber. From 3 to 7 ova were incubated in each experiment.
75
Uterus. A section ( 3 - 5 mm) of uterus, adjacent to the oviduct, was removed from a 24 h pregnant mouse. This section was placed in BSS in an incubation chamber. Pituitary gland. The pituitary was removed from the mouse and placed whole in an incubation chamber. Incubation protocol Tissues needed for the particular experiment were placed in a dialysis bag containing 0.5 ml BSS or, in the experiments involving fertilised ova, Ovum Flushing Medium. The bag was sealed, placed in a tube containing 10 ml BSS (to remove the products of metabolism) and incubated at 37°C for 24 h. After incubation, the material from inside the dialysis bag was removed, centrifuged and either frozen prior to testing or added to the next incubation stage of the experiment. Assay o f EPF EPF in mouse serum and incubation medium was assayed by the rosette inhibition test using non-pregnant mouse spleen ceils and rabbit anti-mouse lymphocyte serum (ALS) (Morton et al., 1976). Results of this test were recorded as the rosette inhibition titre (RIT); this titre is the reciprocal of the highest dilution of ALS in which the number of rosettes formed was less than 75% of the control value. The RIT was expressed as the logarithm (to base 2) of the reciprocal dilution of ALS × 10 -3. The RIT obtained with spleen cells after incubation in non-pregnant mouse serum (or BSS)was 10-14. An RIT greater than 14 was positive evidence of the presence of EPF. Components A and B of EPF were prepared from 24-h pregnant mouse serum by differential precipitation with 40% ammonium sulphate. To test for the presence of EPF components in medium from the incubation experiments, 0.1 ml of medium was added to 0.1 ml component A (40% ammonium sulphate supernatant) or 0.1 ml component B (40% ammonium sulphate precipitate), the mixture incubated at 37°C for 0.5 h, then tested for EPF (Morton et al., 1976). RESULTS
R I T with control non-pregnant mouse spleen cells The RIT of the ALS with non-pregnant mouse spleen cells was estimated. In a group of 16 mice, the mean RIT was 10.9, S.E. 0.4. RIT results obtained from the following experiments were assessed on the significance of their variation from this value.
In vitro incubation o f ovary, ova and oviduct The ovary and oviduct + ova were removed from 24-h pregnant mice and incubated as described in Methods. The organs from the left side of the mouse were incubated together and those from the right side of the mouse were incubated separately. After incubation, the three samples of medium obtained from each mouse experiment were tested for EPF. There was no change in the RIT with non-pregnant mouse spleen cells after incubation in the medium from the ovary alone or the oviduct + ova alone. However, when these two
76
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Fig. 1. The RIT with medium from incubation of 24-h pregnancy ovary and oviduct + ova incubated separately and together. The media from separate incubation of ovary and oviduct + ova were recombined and tested. The RIT with serum from these mice is also shown. An RIT above 14 indicated the presence of EPF; oviduct + ova indicates oviduct containing fertilised ova.
Fig. 2. The RIT with media from incubation of the ovary, ova and oviduct of 24 h pregnant mice, incubated separately and together. The RIT with serum from these mice is also shown. An RIT above 14 indicated the presence of EPF.
organs were incubated together and the medium tested, EPF was detected (Fig. 1), the mean RIT with these samples betng significantly different (n = 13, P < 0 . 0 0 t ) from the control value. EPF was detected in serum from these pregnant mice (Fig. I; n = 13,
P < 0.001). These results suggest two possibilities for the production of EPF. Firstly, EPF could bc produced as two components, one from the oviduct + ova and one from the ovary: secondly, EPF could be produced from either the ovary or the oviduct + ova in response to a stimulation from the other organ. To clarify this point, medium from the separate incubations of pregnancy ovary and pregnancy oviduct + ova were mixed after incubation, incubated for a further 0.5 h at 37°C and then tested for EPF. An increased RIT was obtained with normal spleen ceils after incubation in this recombinant (Fig. 1 ; n = ~. P < 0.001), similar to the results obtained after testing the medium from the ovary and the oviduct + ova incubated together. These results suggest that two components are present, one from the ovary and one from the oviduct + ova, which can combine in vitro to form EPF. Fertilised ova were flushed from the oviducts of 24-h pregnant mice to determine whether the ovum, the oviduct or both were involved in the production of EPF. These
77
fertilised ova from 24-h pregnant mice were at the one or two cell stage of division (Rugh,
1968). In some experiments, the ova were examined after 24 h incubation in Ovum Flushing Medium; ova were seen at the 2-cell, 4-cell and 8-cell stages. Medium from the ovary, ova or flushed oviduct, incubated alone, gave no EPF activity, neither did that from the ova incubated with the ovary. However, the ovary incubated with the oviduct did produce EPF, the mean RIT being significantly increased above that of the nonpregnant group (Fig. 2; n = 6, P < 0.001). Similar experiments were performed using the ovaries and oviducts from oestrous and dioestrous mice. The results showed no increase in RIT with medium from the incubation of ovaries and oviducts, separately or together. These was no increase in RIT obtained when testing the serum from these mice.
Incubation medium tested with components A and B The medium, collected from the separate incubations of the ovary and oviduct + ova, were tested to determine if they would combine in vitro with EPF components A or B to give EPF activity. Compqnents A and B were each mixed with medium collected after the separate incubations of ovary and oviduct + ova obtained from 24-h pregnant, oestrous and dioestrous mice. The serum from oestrous and dioestrous mice was also tested with components A and B. In testing samples from 6 pregnant mice, the recombinant of medium from the ovary
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Fig. 3. The RIT with medium from incubation of ovary and oviduct + ova of 24-h pregnant mice combined with either component A or component B. An RIT above 14 indicated the presence of EPF; oviduct + ova indicates oviduct containing fertilised ova.
78 with component B and medium from the oviduct + ova with component A gave rL~ increase in RIT; by comparison, the recombinant of ovary medium with component A and of oviduct + ova medium with component B both gave a significant increase in RI't (Fig. 3; P < 0.001). The medium obtained from the incubation of a section of uterus wa,~ tested for the presence of a component A-like substance by the addition of component 13: no EPF was detected in this recombinant. The medium from the oestrous ovary gave no increase in activity after the addition ~,~ component A or component B. rhe medium from the oestrous oviduct gave no increase in activity after the addition of component A but did give a significant increase after the' addition of component B (mean RIT 23.3, S.E. 1.2, n = 6, P < 0.001). Activity was n ~ detected in oestrous serum after the addition of component A but was detected after lht' addition of component B (mean RIT 19.6, S.E. 1.0, n = 6, P < 0.001). Component A separated from sheep serum EPF by ammonium sulphate wilt combine with lymphocytes in the absence of component B (unpublished observationst. As the sub stance produced from the oviduct during oestrus resembles component A, this substance:: was tested for its ability to combine with lymphocytes. Lymphocytes were incubated with medium from oestrous oviduct, or oestrous serum, then washed before the addition of component B. No EPF activity was detected on the lymphocytes, the RIT not being significantly different from that of the control cells. After the addition of components A or B to the medium from the incubation of dioestrous mouse organs or h~ dioestrous serum, there was no EPF activity observed. The conclusions drawn from ~hese experiments were that the oviduct, during oestrt~: and pregnancy, produced a substance similar in reaction to component A and that the ovary, only during pregnancy, produced a substance similar to component B. tloweve~. the component A produced during oestrus appeared to exist in an inactive form as i~ would not adhere to lymphocytes in the absence of component B. Incubation o f fertilised ~n,a with ~estrous ovarr
Oestrous ovaries were incubated with fertilised ova in vitro to determine whether the presence of the fertilised o~;a could initiate the production of the pregnancy-dependent component of EPF. No EPF was detected after addition of component A lo the incubation medium, the RIT in all 6 experiments not being significantly different from the control value. Incubation o f pituitary (24 tt pregnant) with oestrous or dioestrous ovary
Similar experiments t o those described above were set up using pituitaries from 24-h pregnant mice incubated with tile ovaries from oestrous or dioestrous mice. After incuba. tion, component A was added to the medium, which was then tested for EPF. In thi.~: case, the production of EPF was stimulated, the RIT with the medium tiom bt)ti~ oestrous and dioestrous ovaries being significantly increased (Fig. 4; n = 6; P < 0.00t ). There was no activity detected in the control experiments which contained pituitaries but no ovaries.
79 24h
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Fig. 4. Medium from incubation of 24-h pregnant mouse pituitary with oestrous or dioestrous ovary was tested for EPF activity after the addition of component A. The results are expressed as the RIT; values above 14 indicated the presence of EPF. Fig. 5. Fertilised ova tested for their ability to stimulate the production of the pregnancy-dependent component of EPF from non-pregnancy ovary in the presence of pituitary. Pituitaries were taken from mice during pregnancy, oestrus and dioestrus; ovaries were taken during oestrus and dioestrus. The control tests were: (1) fertilised ova with oestrous ovary; (ii) unfertilised ova with oestrous pituitary and ovary. The medium from the incubations was tested for EPF after addition of component A and the results were expressed as the RIT; values above 14 indicated the presence of EPF.
Incubation o f pituitary (non-pregnant) with fertilised ova and oestrous or dioestrous ovary The pituitary appears to be involved in the production of EPF. The fertilised ovum, while unable to stimulate a non-pregnancy ovary directly, may do so in the presence of a non-pregnancy pituitary. Pituitaries were removed from oestrous and dioestrous mice and each incubated with fertilised ova from 24-h pregnant mice. Pituitaries from 24-h pregnant mice were used as a positive control. After 24 h, the incubation medium was removed and incubated for a further 24 h as follows: (i) Medium from fertilised ova + pregnancy pituitary with oestrous ovary (ii) Medium from fertilised ova + oestrous pituitary with oestrous ovary (iii) Medium from fertilised ova + dioestrous pituitary with dioestrous ovary. The medium obtained from these incubations was tested for the presence of EPF after the addition of component A. The negative control tests consisted of fertilised ova which were incubated with oestrous ovary and of unfertilised ova which were incubated with oestrous pituitary and oestrous ovary.
80 EPF was detected using the incubation medium from fertilised ova with (i) 24-h pregnancy pituitary and oestrous ovary, (ii) oestrous pituitary and ovary and (iii) dioestrous pituitary and ovary; the RIT values obtained after addition of component A to these samples are shown in Fig. 5 (n = 6, P < 0.001). No activity was detected after incubatiot~ of fertilised ova with oestrous ovary or of unfertilised ova with oestrous pituitary and oestrous ovary. DISCUSSION During oestrus, the oviduct is stimulated to produce a substance, an oestrus-dependem component of EPF, similar in its reactions to component A separated from 24 h pregnant mouse serum. The putative component A, present in serum during oestrus and produced by the pregnancy oviduct in culture, combines with component B as does the componem A separated from 24-h pregnant mouse serum by ammonium sulphate. Until the structure of the substances produced by different methods is determined biochemically, it will nor be known whether or not they are identical; however, on the basis of their reaction with component B to produce EPF activity, they have all been designated component A. Component A is the unit of EPF which combines with lymphocytes. However, component A, detected in serum during oestrus, appears to be in an inactive form and will n~.~, combine with lymphocytes unless in the presence of component B. The production of component A by the oviduct is probably controlled by hormones, associated with the oestrous cycle. It has been noted that in cycling animals the rapid upsurge in secretion of oviductal fluid occurs during oestrus and metoestrus (Perkins. 1974). Mastroianni (1970) has shown in the monkey that following ovulation a new protein occurred immediately in oviductal fluid and remained for at least 5 days; this protein was absent when ovulation did not occur. The necessity of a high-molecularweight component of oviductal fluid for development through the blastocyst stage has been suggested by the work of Cole and Paul (1965) and Gwatkin (1966). A comparison could be made between this protein and component A, also a high-molecular-weight protein, which is detected during oestrus and may indicate that EPF is necessary tbr the normal development of the blastocyst. It seems unlikely that component A is the same substance as blastokinin (Beier, 1968), otherwise called utero-globulin (Daniel, 1968), which is a glycoprotein of M.W. 25 000 produced in the rabbit from uterine tissue 2 days post-coitum. Although the molecular weight of this substance is similar to that of component A (unpublished observations), the kinetics differ as blastokinin is first detected 2 days after mating; component A was not detected in the medium from the cultured uterine tissue taken from the mouse 24 h after mating. Component B, produced in culture from 24-h pregnancy ovary, is initiated by the presence of a fertilised ovum as there is no detectable EPF present in pseudopregnant mice (Morton et al., 1976). Incubation experiments described here have shown that ~ stimulation from the fertilised ovum cannot directly initiate production of component B from either oestrous or dioestrous ovaries but that stimulation can occur in cooperation with the pituitary. Once stimulated the pituitary will initiate the production of compo. nent B from oestrous or dioestrous ovaries in culture. This pathway differs from tha! proposed by Godkin et al. (1978) for the maintenance of the corpus luteum of early
81 pregnancy; their proposal involves a direct stimulation of the luteal cells from the preimplantation embryo. Follicle stimulating hormone, luteinizing hormone and prolactin are produced by the pituitary and control the secretion of oestrogen and progesterone. A cooperative action seems to exist between these hormones as prolactin and luteinizing hormone, in combination, do not initiate implantation in hypophysectomized mice, although either can do so when administered with follicle-stimulating hormone (Gidley-Baird and Emmens, 1978). The mode of action of the factor from the fertilised ovum could thus be either to initiate the production by the pituitary of an ovary-stimulating factor or to work in cooperation with one or more of the known pituitary hormones. Hypophysectomised women and ani.. mals can be induced to ovulate by the administration of purified pituitary gonado. trophins with hCG and pregnant mare's serum gonadotrophin (Marshall et al., 1973)and the resulting ova to be fertilised. This would appear to favour the theory of a cooperative action between the factor produced by the fertilised ovum and known pituitary hormones. However, two circumstances must be taken into consideration, the first that: removal of the pituitary in humans is very often incomplete and the second that there is a possibility of an unidentified impurity being present in the administered hormones. In summary, component A appears to be produced by the oviduct during oestrus and early pregnancy. It is present in the serum in an inactive form, that is, not capable of reacting with lymphocytes. Component B, the pregnancy-dependent component of EPF, has been produced in culture from non-pregnant mouse ovaries in the presence of fertilised ova and non-pregnant mouse pituitary. The factor produced from the fertilised ovum may stimulate the production of component B from the ovary either by means of a second factor produced by the pituitary or by synergism with the known pituitary hot. mones. A substance has not previously been described which is produced as soon after fertilisation and which is directly involved with the pituitary; this ovum factor may be the initial embryonic signal in the maternal recognition of pregnancy. ACKNOWLEDGEMENTS The autors wish to thank Mr. Walter Dammasch for his excellent care of the experimental animals. This investigation received financial support from the World Health Organisation, N.H. and M.R.C. REFERENCES Beier, H.M. (1968) Uteroglobin: a hormone-sensitive endometrial protein involved in blastocyst development. Biochim. Biophys. Acta (Aust.) 160, 289-291. Channing, C.P., Stone, S.L., Sakai, C.N., Haour, F. and Saxena, B.B. (1978) A stimulatory effect of the fluid from preimplantation rabbit btastocysts upon luteinization of monkey granulose cell cultures. J. Reprod. Fert. 54,215-220. Cole, R.J. and Paul, J. (1965) Properties of cultural preimplantation mouse and rabbit embryos and cell strain derived from them. In Preimplantation Stages of Pregnancy (Wolstenhome, G.E.W. and O'Connor, M., eds.), pp. 82-112. J. and A. Churchill, London. Daniel Jr., J.C. (1968) Comparison of electrophoretic patterns of uterine fluid from rabbits and mammals having delayed implantation. Comp. Biochem. Physiol. 24, 297-300.
82 Gidley-Baird, A.A. and Emmens, C.W. (1978) Pituitary hormone control of implantation in the' mouse. Aust. J. Biol. Sci. 31,657--666. Godkin, J.D., Cot6, C. and Duby, R.T. (1978) Embryonic stimulation of ovine and bovine corporea lutea. J. Reprod, Fert. 54,375-378. Gwatkin, R.B.L. (1966) Defined media and development of mammalian eggs in vitro. Ann. N.Y. Acad. Sci. 139, 79-90. Kent, Jr., H.A. (1975) Contraceptive polypeptide from hamster embryos; sequence of amino acids i~ the compound. Biol. Reprod. 12,504-507. Levinson, J., Goodfellow, P., Vadeboncoeur, M. and McDevitt, H. (1978) Identification of stagespecific polypeptides synthesized during murine preimplantation development. Prec. Natl. Acad Sci. U.S.A. 75, 3332-3336. Marshall, J.R., Jacobsen, A. and CargiUe, C.M. (1973) Effects of estrogen on FSH, LH and ovulator3 response rate to gonadotropin therapy in women with normal sellae turcicae or following hypo. physectomy. In Gonadotropin Therapy in Female Infertility (Rosemberg, E., ed.), pp. 145-149. Excerpta Medica, Amsterdam. Martal, J., Lacroix, M.C., Loudes, C., Samaier, M. and Wintenberger-Torr6s, S. (1979) Trophoblastin. an antiluteolytic protein present in early pregnancy in sheep. J. Reprod. Fert. 56, 63-73. Mastroianni, L.J., Urzua, M. and St. Ambaugh, R. (1970) Protein patterns in monkey oviductal fluid before and after ovulation. Fert. Steril. 21,817-820. Morton, H., Hegh, V. and Clunie, G.J.A. (1976) Studies of the rosette inhibition test in pregnant mice evidence of immunosuppression? Prec. R. See. Lend. B. 193, 413-419. Morton, H., Nancarrow, C.D., Searamuzzi, R.J., Evison, B.M. and Clunie, G.J.A. (1979) Detection ~l early pregnancy in sheep by the rosette inhibition test. J. Reprod. Fert. 56, 75-80. Morton, H., Rolfe, B.E., Clunie, G.J.A., Anderson, M.J. and Morrison, J. (1977) An early pregnancy factor detected in human serum by the rosette inhibition test. Lancet i, 394-397. Perkins, J. (1974) Fluid flow of the oviduct. In The Oviduct and its Function (Johnson, A.D. and Foley, C.W, eds.), pp. 119-132, Academic Press, New York. Rugh, R. (1968) The Mouse: Its Reproduction and Development (Roberts Rught, ed.), pp. 3 6 - 6 6 Burgess Publ. Co., Minneapolis. Sundaram, K., Cormell, K.G. and Passantino, T. (1975) Implication of absence of HCG-like gonadotro. phin in the blastocyst for control of corpus luteum function in pregnant rabbit. Nature (London) 256,739-741. Yoshimi, T., Strott, C.A. Marshall, J.R., Lipsett, M.B. (1969) Corpus luteum function in early pre~* nancy. J. Clin. Endocrinol. Metab. 29, 225-230.
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© Elsevier/North-Holland Biomedical Press
EARLY PREGNANCY FACTOR: TISSUES INVOLVED IN ITS PRODUCTION
IN THE MOUSE
H. MORTON, B.E. ROLFE, L. McNEILL, P. CLARKE, F.M. CLARKE and G.J.A. CLUNIE *
Department of Surgery, University of Queensland, Princess Alexandra Hospital, Brisbane, and School of Science, Griffith University, Nathan, Qld., Australia (Received 7 November 1979; accepted 7 January 1980)
The mouse, 24 h after mating, has been used as an experimental model to determine the site of production of early pregnancy factor (EPF). Results have shown that EPF is formed as two separate components, which appear similar to component A and component B formed after dissociation of EPF by 40% ammonium sulphate. Component A is produced by the oviduct during oestrus and pregnancy. It is present in serum during oestrus in an inactive form, that is, not capable of binding with lymphocytes. Component B can be produced in culture from the ovaries of 24-b pregnant mice. Incubation experiments described in this paper show that the production of component B can also be initiated from non-pregnant mouse ovaries, during oestrus or dioestrus, in the presence of both fertilised ovum and non-pregnancy pituitary but not in the presence of fertilised ovum alone. The pituitary from a 24-h pregnant mouse alone can also stimulate the production of component B from oestrous or dioestrous mice. To summarize, component A is derived from the oviduct and.is oestrus-dependent. Component B, the pregnancy-dependent component of EPF, is produced by the ovary; its production can be initiated from non-pregnancy ovaries by the fertilised ovum in cooperation with the pituitary.
INTRODUCTION Early Pregnancy Factor (EPF) can be detected initially in the serum of mice, humans and sheep within the first 24 h after fertilisation (Morton et al., 1976, 1977, 1979), indicating that its production at this stage is not dependent on the presence of tissues of the placenta or of the foetus. This previous work has demonstrated that the development of EPF is not initiated by the act of mating or the presence of sperm or semen in the reproductive tract; the conditions necessary for the production of EPF are that the animal has ovulated and that the ovum has been fertilised. In mice, EPF has been detected from 6 h after mating, that is, before the fusion of the male and female pronuclei completes fertilisation and cell division commences. Levinson et al. (1978) have shown that from the single fertilised cell stage there are numerous stage-specific polypeptides synthesised during murine pre-implantation development and several groups have found specific functions for embryo-derived substances. Kent (1975) described a polypeptide from the 2- to 4-cell ova of the hamster which prevents further
* Present address: University of Melbourne, Royal Melbourne Hospital, ParkviUe,Vic., Australia.
74 ovulation of the.pregnant animal. Godkin et al. (1978) and Martal et al. t.1979) hay,.~ described a substance produced by the sheep embryo which directly stimulates progeste: one synthesis and contributes to the maintenance of pregnancy; the presence of a simila~ substance in rabbits has been suggested by Channing et al. (1978) and in humans b?;~ Yoshimi et al. (1969). Channing and his group have compared the substance the) identified with hCG but Sundaram et al. (1975), using a bioassay, found no hCG-likc activity in the blastocyst of the rabbit between 4 and 7 days gestation. These substance;, originating from the embryo are thought to act directly on the tissues of the ovary. Although the production of EPF is dependent on the presence of a fertilised o v u ~ it seemed unlikely that this would be the source of production; more probably Oat:. embryo acts as a stimulus to produce EPF from some other source. In this mvestigatio~ the mouse was used as the experimental model to determine the site of EPF productio~a The different phases oi oestrus in the mouse can readily be identified by vaginal smear:. and the formation of a vaginal plug allows for fairly precise timing of mating and subse quent fertilisation (Rugh, 1968). This precise timing in the mouse, compared with tha~ in humans and sheep, allows for correlation of the formation of regulatory substance~ with the established data on hormonal events associated with various stages of pregnancy METHODS Outbred Quackenbush mice, aged 8-- 16 wk, were used in this study.
Stages of oestrus In the experiments involving oestrous and dioestrous mice, the stage o! oestrus was determined by vaginal smear (Rugh, t968). Mating schedule Groups of three virgin females and one male were caged together between 8.30 and 9.30 a.m. and those females showing vaginal plugs as evidence of mating were segregated After 24 h, unless otherwise stated, the mated female mice were anaesthetized with ethel exsanguinated by cardiac puncture and the tissues required for experiment removed Dissection was done under sterile conditions. Tissue samples Serum. Serum was collected from the blood sample obtained by cardiac puncture. heat inactivated and frozen. Serum from each mated female mouse was tested for the presence of EPF. Ovary and oviduct. The ovaries and oviducts were removed into Hank's balanced salt solution (BSS; C.S.L. Australia). In some experiments the oviduct was incubated with the fertilised ova in situ (oviduct + ova); in other experiments the fertilised ova were flushed from the oviduct before incubation. Fertilised ova. Ova were flushed from the oviducts of mice with Ovum Flushing Medium (Flow Laboratories) using a syringe fitted with a 30 gauge needle. The ova weft" examined to establish the stage of division (Rugh, 1968), then transferred with a Pasteur pipette to an incubation chamber. From 3 to 7 ova were incubated in each experiment.
75
Uterus. A section ( 3 - 5 mm) of uterus, adjacent to the oviduct, was removed from a 24 h pregnant mouse. This section was placed in BSS in an incubation chamber. Pituitary gland. The pituitary was removed from the mouse and placed whole in an incubation chamber. Incubation protocol Tissues needed for the particular experiment were placed in a dialysis bag containing 0.5 ml BSS or, in the experiments involving fertilised ova, Ovum Flushing Medium. The bag was sealed, placed in a tube containing 10 ml BSS (to remove the products of metabolism) and incubated at 37°C for 24 h. After incubation, the material from inside the dialysis bag was removed, centrifuged and either frozen prior to testing or added to the next incubation stage of the experiment. Assay o f EPF EPF in mouse serum and incubation medium was assayed by the rosette inhibition test using non-pregnant mouse spleen ceils and rabbit anti-mouse lymphocyte serum (ALS) (Morton et al., 1976). Results of this test were recorded as the rosette inhibition titre (RIT); this titre is the reciprocal of the highest dilution of ALS in which the number of rosettes formed was less than 75% of the control value. The RIT was expressed as the logarithm (to base 2) of the reciprocal dilution of ALS × 10 -3. The RIT obtained with spleen cells after incubation in non-pregnant mouse serum (or BSS)was 10-14. An RIT greater than 14 was positive evidence of the presence of EPF. Components A and B of EPF were prepared from 24-h pregnant mouse serum by differential precipitation with 40% ammonium sulphate. To test for the presence of EPF components in medium from the incubation experiments, 0.1 ml of medium was added to 0.1 ml component A (40% ammonium sulphate supernatant) or 0.1 ml component B (40% ammonium sulphate precipitate), the mixture incubated at 37°C for 0.5 h, then tested for EPF (Morton et al., 1976). RESULTS
R I T with control non-pregnant mouse spleen cells The RIT of the ALS with non-pregnant mouse spleen cells was estimated. In a group of 16 mice, the mean RIT was 10.9, S.E. 0.4. RIT results obtained from the following experiments were assessed on the significance of their variation from this value.
In vitro incubation o f ovary, ova and oviduct The ovary and oviduct + ova were removed from 24-h pregnant mice and incubated as described in Methods. The organs from the left side of the mouse were incubated together and those from the right side of the mouse were incubated separately. After incubation, the three samples of medium obtained from each mouse experiment were tested for EPF. There was no change in the RIT with non-pregnant mouse spleen cells after incubation in the medium from the ovary alone or the oviduct + ova alone. However, when these two
76
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Fig. 1. The RIT with medium from incubation of 24-h pregnancy ovary and oviduct + ova incubated separately and together. The media from separate incubation of ovary and oviduct + ova were recombined and tested. The RIT with serum from these mice is also shown. An RIT above 14 indicated the presence of EPF; oviduct + ova indicates oviduct containing fertilised ova.
Fig. 2. The RIT with media from incubation of the ovary, ova and oviduct of 24 h pregnant mice, incubated separately and together. The RIT with serum from these mice is also shown. An RIT above 14 indicated the presence of EPF.
organs were incubated together and the medium tested, EPF was detected (Fig. 1), the mean RIT with these samples betng significantly different (n = 13, P < 0 . 0 0 t ) from the control value. EPF was detected in serum from these pregnant mice (Fig. I; n = 13,
P < 0.001). These results suggest two possibilities for the production of EPF. Firstly, EPF could bc produced as two components, one from the oviduct + ova and one from the ovary: secondly, EPF could be produced from either the ovary or the oviduct + ova in response to a stimulation from the other organ. To clarify this point, medium from the separate incubations of pregnancy ovary and pregnancy oviduct + ova were mixed after incubation, incubated for a further 0.5 h at 37°C and then tested for EPF. An increased RIT was obtained with normal spleen ceils after incubation in this recombinant (Fig. 1 ; n = ~. P < 0.001), similar to the results obtained after testing the medium from the ovary and the oviduct + ova incubated together. These results suggest that two components are present, one from the ovary and one from the oviduct + ova, which can combine in vitro to form EPF. Fertilised ova were flushed from the oviducts of 24-h pregnant mice to determine whether the ovum, the oviduct or both were involved in the production of EPF. These
77
fertilised ova from 24-h pregnant mice were at the one or two cell stage of division (Rugh,
1968). In some experiments, the ova were examined after 24 h incubation in Ovum Flushing Medium; ova were seen at the 2-cell, 4-cell and 8-cell stages. Medium from the ovary, ova or flushed oviduct, incubated alone, gave no EPF activity, neither did that from the ova incubated with the ovary. However, the ovary incubated with the oviduct did produce EPF, the mean RIT being significantly increased above that of the nonpregnant group (Fig. 2; n = 6, P < 0.001). Similar experiments were performed using the ovaries and oviducts from oestrous and dioestrous mice. The results showed no increase in RIT with medium from the incubation of ovaries and oviducts, separately or together. These was no increase in RIT obtained when testing the serum from these mice.
Incubation medium tested with components A and B The medium, collected from the separate incubations of the ovary and oviduct + ova, were tested to determine if they would combine in vitro with EPF components A or B to give EPF activity. Compqnents A and B were each mixed with medium collected after the separate incubations of ovary and oviduct + ova obtained from 24-h pregnant, oestrous and dioestrous mice. The serum from oestrous and dioestrous mice was also tested with components A and B. In testing samples from 6 pregnant mice, the recombinant of medium from the ovary
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78 with component B and medium from the oviduct + ova with component A gave rL~ increase in RIT; by comparison, the recombinant of ovary medium with component A and of oviduct + ova medium with component B both gave a significant increase in RI't (Fig. 3; P < 0.001). The medium obtained from the incubation of a section of uterus wa,~ tested for the presence of a component A-like substance by the addition of component 13: no EPF was detected in this recombinant. The medium from the oestrous ovary gave no increase in activity after the addition ~,~ component A or component B. rhe medium from the oestrous oviduct gave no increase in activity after the addition of component A but did give a significant increase after the' addition of component B (mean RIT 23.3, S.E. 1.2, n = 6, P < 0.001). Activity was n ~ detected in oestrous serum after the addition of component A but was detected after lht' addition of component B (mean RIT 19.6, S.E. 1.0, n = 6, P < 0.001). Component A separated from sheep serum EPF by ammonium sulphate wilt combine with lymphocytes in the absence of component B (unpublished observationst. As the sub stance produced from the oviduct during oestrus resembles component A, this substance:: was tested for its ability to combine with lymphocytes. Lymphocytes were incubated with medium from oestrous oviduct, or oestrous serum, then washed before the addition of component B. No EPF activity was detected on the lymphocytes, the RIT not being significantly different from that of the control cells. After the addition of components A or B to the medium from the incubation of dioestrous mouse organs or h~ dioestrous serum, there was no EPF activity observed. The conclusions drawn from ~hese experiments were that the oviduct, during oestrt~: and pregnancy, produced a substance similar in reaction to component A and that the ovary, only during pregnancy, produced a substance similar to component B. tloweve~. the component A produced during oestrus appeared to exist in an inactive form as i~ would not adhere to lymphocytes in the absence of component B. Incubation o f fertilised ~n,a with ~estrous ovarr
Oestrous ovaries were incubated with fertilised ova in vitro to determine whether the presence of the fertilised o~;a could initiate the production of the pregnancy-dependent component of EPF. No EPF was detected after addition of component A lo the incubation medium, the RIT in all 6 experiments not being significantly different from the control value. Incubation o f pituitary (24 tt pregnant) with oestrous or dioestrous ovary
Similar experiments t o those described above were set up using pituitaries from 24-h pregnant mice incubated with tile ovaries from oestrous or dioestrous mice. After incuba. tion, component A was added to the medium, which was then tested for EPF. In thi.~: case, the production of EPF was stimulated, the RIT with the medium tiom bt)ti~ oestrous and dioestrous ovaries being significantly increased (Fig. 4; n = 6; P < 0.00t ). There was no activity detected in the control experiments which contained pituitaries but no ovaries.
79 24h
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Fig. 4. Medium from incubation of 24-h pregnant mouse pituitary with oestrous or dioestrous ovary was tested for EPF activity after the addition of component A. The results are expressed as the RIT; values above 14 indicated the presence of EPF. Fig. 5. Fertilised ova tested for their ability to stimulate the production of the pregnancy-dependent component of EPF from non-pregnancy ovary in the presence of pituitary. Pituitaries were taken from mice during pregnancy, oestrus and dioestrus; ovaries were taken during oestrus and dioestrus. The control tests were: (1) fertilised ova with oestrous ovary; (ii) unfertilised ova with oestrous pituitary and ovary. The medium from the incubations was tested for EPF after addition of component A and the results were expressed as the RIT; values above 14 indicated the presence of EPF.
Incubation o f pituitary (non-pregnant) with fertilised ova and oestrous or dioestrous ovary The pituitary appears to be involved in the production of EPF. The fertilised ovum, while unable to stimulate a non-pregnancy ovary directly, may do so in the presence of a non-pregnancy pituitary. Pituitaries were removed from oestrous and dioestrous mice and each incubated with fertilised ova from 24-h pregnant mice. Pituitaries from 24-h pregnant mice were used as a positive control. After 24 h, the incubation medium was removed and incubated for a further 24 h as follows: (i) Medium from fertilised ova + pregnancy pituitary with oestrous ovary (ii) Medium from fertilised ova + oestrous pituitary with oestrous ovary (iii) Medium from fertilised ova + dioestrous pituitary with dioestrous ovary. The medium obtained from these incubations was tested for the presence of EPF after the addition of component A. The negative control tests consisted of fertilised ova which were incubated with oestrous ovary and of unfertilised ova which were incubated with oestrous pituitary and oestrous ovary.
80 EPF was detected using the incubation medium from fertilised ova with (i) 24-h pregnancy pituitary and oestrous ovary, (ii) oestrous pituitary and ovary and (iii) dioestrous pituitary and ovary; the RIT values obtained after addition of component A to these samples are shown in Fig. 5 (n = 6, P < 0.001). No activity was detected after incubatiot~ of fertilised ova with oestrous ovary or of unfertilised ova with oestrous pituitary and oestrous ovary. DISCUSSION During oestrus, the oviduct is stimulated to produce a substance, an oestrus-dependem component of EPF, similar in its reactions to component A separated from 24 h pregnant mouse serum. The putative component A, present in serum during oestrus and produced by the pregnancy oviduct in culture, combines with component B as does the componem A separated from 24-h pregnant mouse serum by ammonium sulphate. Until the structure of the substances produced by different methods is determined biochemically, it will nor be known whether or not they are identical; however, on the basis of their reaction with component B to produce EPF activity, they have all been designated component A. Component A is the unit of EPF which combines with lymphocytes. However, component A, detected in serum during oestrus, appears to be in an inactive form and will n~.~, combine with lymphocytes unless in the presence of component B. The production of component A by the oviduct is probably controlled by hormones, associated with the oestrous cycle. It has been noted that in cycling animals the rapid upsurge in secretion of oviductal fluid occurs during oestrus and metoestrus (Perkins. 1974). Mastroianni (1970) has shown in the monkey that following ovulation a new protein occurred immediately in oviductal fluid and remained for at least 5 days; this protein was absent when ovulation did not occur. The necessity of a high-molecularweight component of oviductal fluid for development through the blastocyst stage has been suggested by the work of Cole and Paul (1965) and Gwatkin (1966). A comparison could be made between this protein and component A, also a high-molecular-weight protein, which is detected during oestrus and may indicate that EPF is necessary tbr the normal development of the blastocyst. It seems unlikely that component A is the same substance as blastokinin (Beier, 1968), otherwise called utero-globulin (Daniel, 1968), which is a glycoprotein of M.W. 25 000 produced in the rabbit from uterine tissue 2 days post-coitum. Although the molecular weight of this substance is similar to that of component A (unpublished observations), the kinetics differ as blastokinin is first detected 2 days after mating; component A was not detected in the medium from the cultured uterine tissue taken from the mouse 24 h after mating. Component B, produced in culture from 24-h pregnancy ovary, is initiated by the presence of a fertilised ovum as there is no detectable EPF present in pseudopregnant mice (Morton et al., 1976). Incubation experiments described here have shown that ~ stimulation from the fertilised ovum cannot directly initiate production of component B from either oestrous or dioestrous ovaries but that stimulation can occur in cooperation with the pituitary. Once stimulated the pituitary will initiate the production of compo. nent B from oestrous or dioestrous ovaries in culture. This pathway differs from tha! proposed by Godkin et al. (1978) for the maintenance of the corpus luteum of early
81 pregnancy; their proposal involves a direct stimulation of the luteal cells from the preimplantation embryo. Follicle stimulating hormone, luteinizing hormone and prolactin are produced by the pituitary and control the secretion of oestrogen and progesterone. A cooperative action seems to exist between these hormones as prolactin and luteinizing hormone, in combination, do not initiate implantation in hypophysectomized mice, although either can do so when administered with follicle-stimulating hormone (Gidley-Baird and Emmens, 1978). The mode of action of the factor from the fertilised ovum could thus be either to initiate the production by the pituitary of an ovary-stimulating factor or to work in cooperation with one or more of the known pituitary hormones. Hypophysectomised women and ani.. mals can be induced to ovulate by the administration of purified pituitary gonado. trophins with hCG and pregnant mare's serum gonadotrophin (Marshall et al., 1973)and the resulting ova to be fertilised. This would appear to favour the theory of a cooperative action between the factor produced by the fertilised ovum and known pituitary hormones. However, two circumstances must be taken into consideration, the first that: removal of the pituitary in humans is very often incomplete and the second that there is a possibility of an unidentified impurity being present in the administered hormones. In summary, component A appears to be produced by the oviduct during oestrus and early pregnancy. It is present in the serum in an inactive form, that is, not capable of reacting with lymphocytes. Component B, the pregnancy-dependent component of EPF, has been produced in culture from non-pregnant mouse ovaries in the presence of fertilised ova and non-pregnant mouse pituitary. The factor produced from the fertilised ovum may stimulate the production of component B from the ovary either by means of a second factor produced by the pituitary or by synergism with the known pituitary hot. mones. A substance has not previously been described which is produced as soon after fertilisation and which is directly involved with the pituitary; this ovum factor may be the initial embryonic signal in the maternal recognition of pregnancy. ACKNOWLEDGEMENTS The autors wish to thank Mr. Walter Dammasch for his excellent care of the experimental animals. This investigation received financial support from the World Health Organisation, N.H. and M.R.C. REFERENCES Beier, H.M. (1968) Uteroglobin: a hormone-sensitive endometrial protein involved in blastocyst development. Biochim. Biophys. Acta (Aust.) 160, 289-291. Channing, C.P., Stone, S.L., Sakai, C.N., Haour, F. and Saxena, B.B. (1978) A stimulatory effect of the fluid from preimplantation rabbit btastocysts upon luteinization of monkey granulose cell cultures. J. Reprod. Fert. 54,215-220. Cole, R.J. and Paul, J. (1965) Properties of cultural preimplantation mouse and rabbit embryos and cell strain derived from them. In Preimplantation Stages of Pregnancy (Wolstenhome, G.E.W. and O'Connor, M., eds.), pp. 82-112. J. and A. Churchill, London. Daniel Jr., J.C. (1968) Comparison of electrophoretic patterns of uterine fluid from rabbits and mammals having delayed implantation. Comp. Biochem. Physiol. 24, 297-300.
82 Gidley-Baird, A.A. and Emmens, C.W. (1978) Pituitary hormone control of implantation in the' mouse. Aust. J. Biol. Sci. 31,657--666. Godkin, J.D., Cot6, C. and Duby, R.T. (1978) Embryonic stimulation of ovine and bovine corporea lutea. J. Reprod, Fert. 54,375-378. Gwatkin, R.B.L. (1966) Defined media and development of mammalian eggs in vitro. Ann. N.Y. Acad. Sci. 139, 79-90. Kent, Jr., H.A. (1975) Contraceptive polypeptide from hamster embryos; sequence of amino acids i~ the compound. Biol. Reprod. 12,504-507. Levinson, J., Goodfellow, P., Vadeboncoeur, M. and McDevitt, H. (1978) Identification of stagespecific polypeptides synthesized during murine preimplantation development. Prec. Natl. Acad Sci. U.S.A. 75, 3332-3336. Marshall, J.R., Jacobsen, A. and CargiUe, C.M. (1973) Effects of estrogen on FSH, LH and ovulator3 response rate to gonadotropin therapy in women with normal sellae turcicae or following hypo. physectomy. In Gonadotropin Therapy in Female Infertility (Rosemberg, E., ed.), pp. 145-149. Excerpta Medica, Amsterdam. Martal, J., Lacroix, M.C., Loudes, C., Samaier, M. and Wintenberger-Torr6s, S. (1979) Trophoblastin. an antiluteolytic protein present in early pregnancy in sheep. J. Reprod. Fert. 56, 63-73. Mastroianni, L.J., Urzua, M. and St. Ambaugh, R. (1970) Protein patterns in monkey oviductal fluid before and after ovulation. Fert. Steril. 21,817-820. Morton, H., Hegh, V. and Clunie, G.J.A. (1976) Studies of the rosette inhibition test in pregnant mice evidence of immunosuppression? Prec. R. See. Lend. B. 193, 413-419. Morton, H., Nancarrow, C.D., Searamuzzi, R.J., Evison, B.M. and Clunie, G.J.A. (1979) Detection ~l early pregnancy in sheep by the rosette inhibition test. J. Reprod. Fert. 56, 75-80. Morton, H., Rolfe, B.E., Clunie, G.J.A., Anderson, M.J. and Morrison, J. (1977) An early pregnancy factor detected in human serum by the rosette inhibition test. Lancet i, 394-397. Perkins, J. (1974) Fluid flow of the oviduct. In The Oviduct and its Function (Johnson, A.D. and Foley, C.W, eds.), pp. 119-132, Academic Press, New York. Rugh, R. (1968) The Mouse: Its Reproduction and Development (Roberts Rught, ed.), pp. 3 6 - 6 6 Burgess Publ. Co., Minneapolis. Sundaram, K., Cormell, K.G. and Passantino, T. (1975) Implication of absence of HCG-like gonadotro. phin in the blastocyst for control of corpus luteum function in pregnant rabbit. Nature (London) 256,739-741. Yoshimi, T., Strott, C.A. Marshall, J.R., Lipsett, M.B. (1969) Corpus luteum function in early pre~* nancy. J. Clin. Endocrinol. Metab. 29, 225-230.