Luteal control of endometrial receptivity and its modification by progesterone antagonists.

The objective of this study was to determine preimplantational effects of progesterone antagonists (PA) on the cell biology of the endometrium, on corpus luteum (CL) function and on the complex interactions between these two organs. The PA onapristone (ZK 98.299) or lilopristone (ZK 98.734) was given to pseudopregnant rabbits at days 5, 6, and 7 p.hCG. Three treatment protocols were investigated: Exp I, onapristone or lilopristone treatment only; Exp II, onapristone treatment after hysterectomy at day 1 p.hCG; Exp III, onapristone treatment together with 17 beta-estradiol, which represents the ultimate luteotropic hormone in the rabbit. In Exp I, onapristone and lilopristone gave rise to endometrial regression (inhibition of epithelial proliferation and differentiation, increase of apoptosis). Simultaneous addition of 17 beta-estradiol in Exp III did not alter these findings. A rapid luteolysis was found in Exp I. In Exp II and III, however, onapristone was unable to impair luteal development and function. Due to the unaffected CL in Exp III and due to the 17 beta-estradiol substitution, the endometrium was capable of starting a new transformation, which met all requirements for receptivity at day 12 p.hCG. Transfers of day 4 p.c. blastocysts from untreated donors into such delayed secretion recipient rabbits at days 12 p.hCG resulted in normal implantations and normal embryonic development. Contrary to Exp III, the missing of any luteotropic substitutions in Exp I resulted in a complete inhibition of further uterine transformation. The present findings suggest that PA can exert a direct inhibitory effect on the endometrium, which is followed by an indirect luteolytic effect via endometrial mediators. The simultaneous addition of a proper luteotropic signal to the PA protocol results in survival of CL. Furthermore, this prolongation of the CL life span can be interpreted as a functional dissociation of the endometrium from the CL.

Effect of clomiphene on the functional morphology of oviductal and uterine mucosa.

The effect of clomiphene on the functional morphology of the uterine and oviductal mucosa was studied in rabbits by means of light and electron microscopy (SEM and TEM). Tissues were obtained from mature nulliparous animals receiving subcutaneous doses ranging from 0.01-10 mg/kg per day. In all cases the effects were evaluated 2 days after termination of treatment. With 2 and 10 mg, effects were studied up to 12 and 7 days, respectively. Normally appearing oviductal and endometrial tissues, corresponding to various stages of the cycle, were observed with doses up to 5 mg. However, a burst of cellular secretory activity becomes evident with the administration of higher doses. Apical protrusions or cytoplasmic portions seem to be extruded, and draw attention. These cytologic events are concentrated near gland openings in the endometrium and can be seen abundantly among cilia of oviductal cells. Other ultrastructural changes are evident as well. These histologic changes may reflect nonsynchronous cellular activities which in turn interfere with oviductal and endometrial functions before implantation.

The dynamic structure of rabbit blastocyst coverings. III. Transformation of coverings under non-physiological developmental conditions.

Under physiological conditions the zona pellucida disappears in the rabbit between Day 3 and early Day 4 post coitum (p.c.) and is replaced by a new layer, the neozona. The dissolution of the zona pellucida and the formation of the neozona was investigated in three different experimental approaches, all of them characterized by non-physiological developmental conditions for the embryo: Prevention of embryo migration from the oviduct into the uterus by postcoital (48 h p.c.) tubal ligation, in vitro culture, and asynchronous embryo transfer into uteri of recipient rabbits. Embryos of age 2 1/2, 3, 4 and 4 1/2 days p.c. were cultured for 12 to 72 h. The media used for in vitro culture were supplemented with BSA, serum or with uterine secretions that were collected either synchronously or asynchronously to the developmental stage of the cultured embryos. Three-day-old embryos were transferred into uteri of pseudopregnant foster rabbits of either synchronous (Day 3) or asynchronous stages (Day 0, 2, 4, 5, 6) and were recovered 24 to 72 h after transfer. The transformation of the coverings was evaluated by light and transmission electron microscopy. The dissolution of the zona pellucida was greatly disturbed in tube-locked embryos, and in cultured embryos if standard protein supplements (BSA or serum) had been used for in vitro culture. In many cases the zona was still completely preserved after 2 or 3 days in culture, at a time when it normally would have already been replaced by the neozona in vivo. The dissolution in vitro, however, progressed incomparably better if the culture medium had been substituted with synchronous or asynchronous uterine secretions. The formation of the neozona could not be verified in cultured blastocysts. After embryo transfer, the dissolution of the zona pellucida was completed in most cases by 2 days after transfer, irrespective of the recipients' progestational stage. Present results indicate that uterine components are essential for the dissolution of the rabbit zona pellucida. These components appear to be present in the uterine cavity constitutively, i.e. independently of the uterine progestational transformation, and need not be in synchrony with the embryo's developmental stage for dissolution of the zona. Normal formation of the neozona does not take place under the non-physiological developmental conditions of in vitro culture.

[Analysis on x-ray-sensitivity of rabbit embryos (author's transl)]. / Analysen der Röntgenstrahlensensibilität von Kaninchen-embryonen.

The sensitivity of Day-4-blastocysts to total x-ray irradiation at a dosage of 130r is studied at developmental stages 10, 11, 12 and 17 days p. c., after the treated blastocysts could develop in synchronized foster mothers. The effects by 130r are compared to normal controls and other dosages of 90r and 250 r. A maximum of developmental failure is recorded particularly in 130 r --irradiated embryos. The following reasons are considered to being causally involved: 1. Reduced mitotic activity between Days 5 and 7. 2. Reduced growth of the embryonic disk at Days 6 and 7, such inhibiting differentiation and further formation of the embryo. 3. Increasing imbalances in the essential synchronisation demands of the embryonic and maternal systems. 4. Developmental retardation accompanied by a failure in the dissolution of the blastocyst coverings, by this resulting in a considerable number of degenerating blastocysts at Day 8 p.c. 5. Increased imbalances in the ratio of the differentiation and growth, specific for each embryonic stage. Growth of the embryo, in general, occurs more slowly than the process of differentiation. 6. Failure of adaptation to the chorionic-allantoic-placenta supply at Day 11. This investigation may be considered as a basis for further causal relationship studies to find out the reason for the more pronounced sensitivity to 130 r than to 250 r of Day 4 rabbit blastocysts.

Significance of maternal uterine proteins in the establishment of pregnancy.

Mammalian embryonic development depends on extrinsic support by the maternal organism. During the preimplantation period the growing blastocyst needs favourable environmental conditions, which are provided by endometrial transformations and secretion. The macromolecular composition of uterine secretion at different times before implantation is characterized by a spectrum of protein patterns which change daily. In an attempt to shed light on the significance of the uterine secretion proteins, particularly uteroglobin in the rabbit, in the establishment of early pregnancy, analyses are reported which demonstrate the origin and endocrine control of the synthesis and release of these proteins. In a comparative study of blastocyst development in vivo and in vitro the significance and consequences of asynchrony between the embryonic and maternal reproductive phases are investigated. Particular attention is drawn to uterine influences on the rabbit blastocyst coverings, and how embryonic development can be retarded by abnormal structural transformation of the zona pellucida and the mucin coat. Finally, the covenient experimental systems of pseudopregnancy and of delayed uterine secretion in the rabbit permit an approach the question of whether the uterine macromolecular components constitute a maternal response to the presence of the as yet unimplanted blastocyst.

Development of rabbit preimplantation blastocysts cultured with precultured endometrial tissue.

Endometrial fragments were explanted from pseudopregnant rabbits 4.5 days after injecting with human chorionic gonadotrophin and were precultured for 2 days in suspension culture in the presence of oestradiol and progesterone equivalent to concentrations in rabbit serum at that stage. Preimplantation blastocysts were obtained at day 6.5 of pregnancy and cultured in the presence or absence of precultured endometrial fragments. Attachment of the trophoblast to the endometrium was prevented by continuous agitation. After 2 and 3 days, specimens were monitored for development in vitro using light and scanning electron microscopy. Although the development of blastocysts was slower in vitro than in vivo in both groups, development was clearly superior in the presence of precultured synchronous endometrial fragments. In the absence of endometrium, the embryonic anlage appeared disordered, particularly in the caudal region, but in the presence of uterine tissue the blastocysts developed much better. Up to nine somites were differentiated; the neural tube had started to close and the various parts of the brain anlage showed incipient differentiation. Syncytiotrophoblast differentiated in the presence or absence of endometrium in the embryonic and abembryonic hemispheres, but typical patterns were maintained better and cell degeneration was less frequent during co-culture. Although the culture model described here has not been optimized using criteria of blastocyst differentiation, the results suggest that culture of blastocysts with precultured synchronous endometrial fragments is advantageous.

The effect of clomiphene citrate on blastocyst development and implantation in the rabbit.

The induction of ovulation with clomiphene citrate (CC) in human patients results in a high ovulation rate but achieves a relatively low pregnancy rate. To clarify the possible role of CC in interfering with the normal reproductive physiology and embryology, we have used our rabbit model and transferred 4-day-old blastocysts from untreated donors to CC-treated pseudopregnant recipients and from CC-treated donors to untreated pseudopregnant recipients to study embryonic development and implantation. Each group was further subdivided into two subgroups, one receiving CC before and the other after ovulation. CC was administered subcutaneously in three consecutive doses of 10 mg/kg body weight. Ovulation was induced with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG). The implantation rate of the control group, evaluated on day 8 of pregnancy, reached 62.0%. When recipients were treated with CC before ovulation, implantation rate was reduced to 18.8% (P less than 0.0002), and to 20.0% (P less than 0.003) when CC was administered after ovulation. The implantation rate of blastocysts transferred from donors, treated before ovulation, is 22.2% (P less than 0.0055), however, reached 70.8% when treatment was started after ovulation. All implantations were analysed microscopically and showed normal morphological features. Our results demonstrate a potential multiple effect of CC, first on the endometrium by altering its receptivity for the implanting conceptus, second, on tubal physiology by altering egg transport, and finally on ovum maturation before ovulation interfering with development of blastocysts. These parameters may all result in rapid decrease in establishment of implantations and in turn in very low pregnancy rates.

Uteroglobin-like antigen in the pulmonary epithelium and secretion of the lung.

The immunological identity of a uteroglobin-like protein, occurring in respiratory tract secretions and tissue, with uteroglobin from rabbit endometrial secretion is demonstrated. A uteroglobin-like antigen has been localized in bronchial epithelial cells and in bronchioles by immunofluorescence. This secretory protein is, in contrast to the authentic uteroglobin, hormone-independent, as far as estrogens and progesterone are concerned. The possible significance of comparative studies on uteroglobin and the uteroglobin-like antigen is discussed, taking into account cytological, endocrinological, and molecularbiological aspects.

Modification of endometrial cell biology using progesterone antagonists to manipulate the implantation window.

The preimplantation effects of progesterone antagonists on the cell biology of the endometrium, corpus luteum function and interactions between these two organs have been studied. The antagonists lilopristone (ZK 98.734) and onapristone (ZK 98.299) were initially given per os to rabbits early or late in pseudopregnancy in combination with human chorionic gonadotrophin (HCG). These protocols were then modified to include hysterectomy or luteotrophic support with 17 beta-oestradiol. Given alone, the antagonists gave rise to endometrial regression (inhibition of epithelial proliferation and differentiation, increase of apoptosis). The simultaneous addition of oestradiol did not alter these findings. A rapid luteolysis occurred when the antagonists were given in late pseudopregnancy, but not if combined with oestradiol or hysterectomy. The endometrium was capable of renewal and of sustaining implantation if the corpora lutea survived or oestradiol was administered, and transferred blastocysts displayed normal implantation and normal embryonic development. These events did not occur when the antagonists were given during late pseudopregnancy without any steroid supplement. Progesterone antagonists can evidently exert a direct inhibitory effect on the endometrium, possibly with a later indirect luteolytic effect via endometrial mediators. Simultaneous addition of a proper luteotrophic signal results in corpora lutea which are refractory to lysis, so revealing a potential functional dissociation between endometrium and corpus luteum. The endometrium has the capacity to differentiate normally after an interrupted transformation and becomes receptive and sustains normal pregnancy, due to an expanded lifespan of the corpora lutea and a transposition of the implantation window. Uterine secretions from patients undergoing in-vitro fertilization, collected at the onset of the luteal phase, were evaluated by SDS-PAGE densitometry. The protein profiles gave indications of an adequate luteal phase pattern and of a receptive preimplantation phase. These results open the prospect of manipulating the human implantation window.

Dissociation of corpus luteum, endometrium and blastocyst in human implantation research.

We describe a well-established approach for studying the parameters and mechanisms of synchronization or desynchronization between the maternal and embryonic systems before implantation. It is useful for inducing 'delayed secretion' of the endometrium by different endocrine interventions, which dissociate the endometrial transformation from its control by the corpus luteum. The technique has been achieved by means of direct progesterone antagonists which competitively bind to the progesterone receptor and, in turn, inhibit the physiological effects of progesterone. During the luteal phase, secretory protein patterns indicate the receptive stage of the endometrium. Evidence is presented to show that these patterns, analysed by electrophoresis and densitometry, define the time at which an embryo transfer is promising for implantation and establishment of pregnancy.