Chorionic gonadotrophin and embryo-maternal recognition during the peri-implantation period in primates.

Genes for chorionic gonadotrophin (CG) are transcribed by the 16-cell embryo stage in humans, but there is no clear evidence of CG secretion as a bioactive dimer before attachment and trophoblast outgrowth stages of implantation. The studies summarized question the timing of CG expression and secretion, the possible roles of CG for intraembryonic differentiation and at the implantation site, and the recognition of this primate embryo-derived signal in support of the corpus luteum. The data suggest that the implantation window in primates may be broader than in non-primate species, where a closer synchrony between embryonic, tubal and uterine events appears to be necessary for embryonic survival. Some preliminary data concerning an association between peripheral thrombocytopenia, ovarian inhibin secretion and peri-implantation stages of embryo development indicate that an unknown embryonic signal may be secreted before bioactive CG can be detected.

Embryonic signals during the peri-implantation period in primates.

During the peri-implantation period of pregnancy in primates, chorionic gonadotrophin (CG) is the first clear signal of the embryo's presence and viability. In the marmoset (Callithrix jacchus) implantation begins on Day 11-12 after ovulation and CG is secreted by the embryo from this time. The inner cell mass is necessary for the normal secretion of CG by the trophoblast. Implantation can be disrupted both in vivo and in vitro by antisera to the hCG-beta subunit. The secretion of a platelet activating factor by the preimplantation embryo has yet to be confirmed, as has the physiological function of this and other preimplantation signals. Local sampling by perfusion of the corpus luteum allows a direct measurement of the interactions between luteotrophins and luteolysins, as well as a method of screening potential new agents for regulating the function of the corpus luteum.

Use of a bioassay for embryo-derived platelet-activating factor as a means of assessing quality and pregnancy potential of human embryos.

The production of an embryo-derived platelet-activating factor (PAF) was recently shown to have a correlation with embryo quality and viability. The detection of this factor was used as a means of examining the effect of various aspects of the in vitro fertilization and embryo transfer procedure on human preimplantation embryo quality. Embryos that resulted in pregnancy produced significantly higher levels of embryo-derived PAF in vitro than embryos that failed to result in pregnancy. Of a further 85 embryos, 43% had a level of embryo-derived PAF that fell in the same range as the embryos that resulted in pregnancy. The production of embryo-derived PAF was related to the type of treatment used to induce follicular development (with clomiphene citrate and human menopausal gonadotropin commencing on day 5 giving best results); the size and estradiol production of the follicles producing the embryo; the age of the embryo culture medium; and the morphology and cell number of the embryos.

Effects of growth hormone on pregnancy-associated murine protein-1.

In male mice which normally do not synthesize measurable amounts of the pregnancy-associated murine protein-1 (PAMP-1), synthesis occurred when there was continuous infusion of hGH but not by repeated subcutaneous injections. The decrease in PAMP-1 values after hypophysectomy in female mice was rapidly restored by continuous infusion of hGH, 80 micrograms daily. PAMP-1 has generally been regarded as an 'oestrogen-inducible' protein regulated by the oestrogen/androgen balance. Our results suggest that the apparent effects of sex steroids are mediated via the pituitary and possibly growth hormone secretion.

Initiation and control of ovulation in the mouse luteal phases. Effects of oestrogen, progesterone and prolactin.

The aim of the present study was to examine the effect of exogenous oestrogen and progesterone on ovulation in early pregnant mice and at the end of pseudopregnancy. The results demonstrate that there are mature follicles during early pregnancy which were induced to ovulate by administration of large doses of oestrogen (greater than or equal to 10 micrograms) on day 4 of pregnancy. Ovulation was also induced by inhibition of progesterone secretion with bromocriptine. If progesterone or prolactin was given in conjunction with bromocriptine, the ovulation effect was abolished. At the end of pseudopregnancy, the spontaneous ovulation was inhibited by injections of progesterone (1.0 mg). Oestrogen given by itself at the end of pseudopregnancy caused the animals to mate earlier than the control animals. This confirms the presence of mature follicles in mice during pregnancy and suggests that the LH peak needed for ovulation can either be provoked by administration of oestrogen or by blocking of progesterone.

Initiation and control of ovulation in the mouse luteal phases. Effects of gonadotropins and gonadotropin releasing hormone.

The aim of the present study was to examine the induction of ovulation during pregnancy, pseudopregnancy, and suckling-delayed pregnancy in mice using exogenous gonadotropins. The present results demonstrate that there are mature follicles in the ovary which can be induced to ovulate with administration of either exogenous human chorionic gonadotropin (hCG) or luteinizing hormone (LH) during pregnancy (Days 1-12) and pseudopregnancy (Days 4-8) in the mouse. hCG was relatively ineffective in initiating ovulation during suckling-delayed pregnancy, and hCG could not induce ovulation on Days 3-6 in any animals, suggesting that follicular growth is not continuous during suckling-delayed pregnancy in the mouse. Ovulation occurred in pregnant and pseudopregnant mice following injection of gonadotropin releasing hormone (GnRH) in a gelatin delay vehicle. Injection of GnRH in saline did not initiate ovulation in pregnant or pseudopregnant mice. A large release of LH was shown to occur following injection of GnRH in gelatin, but no release occurred after the same dose of GnRH in saline. In conclusion, the experiments demonstrate the existence of mature follicles during murine pregnancy and pseudopregnancy, and the lack of inductable follicles during suckling-delayed pregnancy.

Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of altered progesterone/estrogen ratios in humans and mice.

Daily blood samples were taken for progesterone (P) and estradiol (E2) measurements from women who showed a platelet response consistent with the presence of viable embryos after in vitro fertilization and embryo transfer procedures. A comparison of steroid levels between those women who became pregnant and those who did not revealed the following: at and after the time of transfer, women who failed to become pregnant had significantly higher E2 levels and a lower ratio of P/E2 than women who became pregnant. The P/E2 ratio was a better predictor of implantation failure than was the absolute level of either hormone. Experiments were done in mice to test the hypothesis that P could protect implantation of the embryo against the inhibitory effects of high E2. In mice, implantation was inhibited by relatively high levels of E2. This effect was overcome by concomitant administration of P. There was a significant dose-response-related interaction of P with the E2.

Maternal blood platelet physiology and luteal-phase endocrinology as a means of monitoring pre- and postimplantation embryo viability following in vitro fertilization.

The discovery that the fertilized mouse ovum triggers an increased demand for platelets and results in thrombocytopenia during the preimplantation phase of pregnancy provides a monitor for embryo survival and viability. This paper reports a study in which the platelet count was significantly reduced throughout the human preimplantation phase of pregnancy and returned to normal following embryo implantation. The human embryo was shown to produce a platelet activating factor in vitro which caused the reduction in platelet count after embryo transfer. This factor in the embryo culture medium could be measured using a bioassay which provided a means of assessing embryo viability prior to transfer. Some women showed no reduction in platelets after transfer. These embryos failed to produce a platelet activating factor in vitro and pregnancy was not established. Other women displayed a reduction in platelets following transfer but failed to become pregnant. All of these women had elevated luteal-phase plasma E2 levels compared to pregnant patients, which may have interfered with the implantation process. Our observations provide a possible rapid and simple means for monitoring the viability of human embryos cultured in vitro and the survival of embryos in utero.

The effect on pregnancy of intrauterine administration of antibodies against two pregnancy-associated murine proteins: murine pregnancy-specific beta 1-glycoprotein and murine pregnancy-associated alpha 2-glycoprotein.

Intrauterine administration of 100 arbitrary units (AU) of purified monospecific rabbit anti murine pregnancy specific beta 1-glycoprotein antibodies resulted in the loss of the fetuses in pregnant mice (14 out of 14). By contrast, a similar treatment with 100 AU of rabbit anti murine pregnancy-associated alpha 2-glycoprotein had an effect similar to saline on the outcome of pregnancy. Intravenous administration of 1000 AU of rabbit anti murine pregnancy specific beta 1-glycoprotein during pregnancy in mice had no effect on the outcome of pregnancy.

The influence of pituitary and gonadal hormones on serum levels of pregnancy-associated murine protein-1.

Hypophysectomy of female mice resulted in the disappearance of pregnancy-associated murine protein-1 (PAMP-1) from the circulation within a week. Maintenance of physiological levels of oestrogen, progesterone, testosterone, LH, FSH or prolactin was not sufficient to maintain a normal PAMP-1 level in the hypophysectomized animals. However, administration of oestrogen in large doses to adult male mice with undectatable levels of circulating PAMP-1 caused PAMP-1 to appear in the blood. Testosterone treatment of females inhibited the PAMP-1 synthesis.

Immunochemical demonstration of a new pregnancy protein in the mare.

An antiserum against the serum of a pregnant mare was absorbed with stallion serum. This antiserum then gave two precipitates in crossed immunoelectrophoresis with serum from pregnant mares as the antigen. The two precipitates exhibited beta-1 and alpha-2 electrophoretic mobility. Identity was demonstrated between the alpha-2 mobile protein and PMSG. The absorbed antiserum inhibited the biological action of the PMSG preparation when tested in mouse ovarian weight assays. The beta-1 mobile protein was not detected in the serum from non-pregnant mares, stallions or geldings and was detected earlier in pregnancy (Day 30) than was PMSG (Day 42).

Ovum factor: a first signal of pregnancy?

Previous studies have shown that, in the mouse, a factor is produced by the fertilized ovum within 24 h of mating. It cooperates with prolactin to stimulate ovarian production of component B or early pregnancy factor (EPF). This paper presents an initial characterization of the substance, termed ovum factor (OF). An indirect assay based on the rosette inhibition test for EPF has shown that OF is first released upon penetration of the ovum by the fertilizing spermatozoon. OF continues to be produced at least until blastulation. Processes which parthenogenetically activate the ovum are also capable of stimulating OF release from unfertilized ova. Gel filtration studies reveal that OF exists in multiple MW forms of approximately 160,000; 2,800; and 1,500. A substance with these characteristics has not been described previously; it may represent the first embryonic signal to the mother.

Endocrine control of implantation and delayed implantation in rats and mice.

Progesterone and oestrogen are required for implantation in the mouse and rat. In both animals prolactin and LH stimulate ovarian progesterone production oestrogen is stimulated by FSH in the mouse but both LH and FSH appear to be required in the rat. There is no evidence that preimplantation embryonic gonadotrophins, if they exist in these species, are involved in the initiation of implantation. The delay of implantation in suckling mice and rats is due to a lack of oestrogenic stimulus. This is caused by an inhibition of FSH release from the pituitary gland. The inhibition of FSH release appears to be caused by lack of a competent hypothalamic releasing factor. High circulating levels of prolactin and/or LH and/or progesterone do not seem to be responsible for the FSH inhibition during suckling-induced delayed implantation. Increased or decreased prolactin release does not affect FSH release during early pregnancy in the mouse.

Effects of adrenalectomy on reproduction in the mouse.

Survival of mice treated with sesame seed oil after adrenalectomy was very low and suggested no beneficial effect, whereas treatment with progesterone improved the chances of survival. Treatment with desoxycorticosterone acetate (DOCA) and methyl prednisolone acetate also increased the number of animals surviving after adrenalectomy. The corticosteroids were significantly more effective in ensuring survival than was progesterone. There was no significant difference in survival between mice receiving a single injection of 1.0 mg DOCA and those being given an injection of 1.0 mg DOCA per day for 3 days after the operation. To ensure minimum interference of exogenous corticosteroid with the experimental investigation, animals routinely received only a single injection of 1.0 mg DOCA after the operation. The chance of survival after adrenalectomy was higher in pregnant than in non-pregnant mice. There was a significant linear increase in survival during the first 5 days of pregnancy. Progesterone and prolactin both appeared to be involved in increasing the chance of survival in adrenalectomized pregnant mice. Adrenalectomy had no effect on the number of mice mating and ovulating. Adrenalectomized mice were apparently having normal cycles and 4 weeks after adrenalectomy they were able to mate and ovulate. Compensatory ovulation was seen in hemi-ovariectomized mice and was not abolished by adrenalectomy. Implantation was also unaffected by the operation.

Pituitary hormone control of implantation in the mouse.

In hypophysectomized pregnant mice replacement therapy designed to mimic the normal physiological situation showed that FSH in combination with either prolactin or LH, or prolactin plus LH, could initiate implantation in the absence of the pituitary gland. No pituitary hormone was by itself capable of achieving this result. The combination of prolactin with FSH gave better results than a combination of LH with FSH. Prolactin from sheep, cattle or rats was equally effective in combination with rat FSH in initiating implantation. In mice exhibiting suckling-induced delay of implantation this delay was terminated by injection of FSH. GH by itself or in conjunction with other hormones had no significant effect on implantation or on any of the other parameters associated with implantation that were measured. On the basis of these experimental results it is suggested that prolactin and LH are involved with progesterone production and FSH with oestrogen production, both of which are required for implantation in the mouse.

Plasma progesterone, FSH and LH levels associated with implantation in the mouse.

Measurement of plasma progesterone, LH and FSH were made every 6 h during the first 6 days of pregnancy in the mouse. Plasma progesterone and LH were low on day 1, minimum values being recorded at 24 h post coitus. Concentrations of both these hormones started rising during the second half on day 2 with the rise continuing during day 3 to a progesterone peak of 25 ng/ml early on day 4 and an LH peak of 37 ng/ml late on day 4. Levels of progesterone fell during day 4 and LH during day 5 to approximately half their respective peak values and then remained relatively constant over the remainder of the measurement period. Levels of FSH, which were high early on day 1 (180 ng/ml), fell sharply by midday with a small rise late in the day followed by a decline during day 2 to a minimum level of 2 ng/ml at 48 h post coitus. Early on day 3 FSH values rose to 120 ng/ml then fell to 50-60 ng/ml during the next 6 h and remained relatively stable at this level during days 4 and 5. It is suggested that LH is concerned with progesterone production and maintenance of the corpus luteum whilst FSH is concerned with the production of oestrogen required for implantation in this species.

Solid-phase assay for luteinizing hormone in mouse plasma.

A solid-phase tube assay for measuring LH levels in mouse plasma is described. The assay utilizes an antiserum to ovine LH and ovine LH standards and it measures LH levels in 20 mul of plasma with a sensitivity of less than 0.6 ng/ml. Various parameters affecting the sensitivity and specificity of the assay were investigated. Serial dilutions of plasma from pregnant mice, a pituitary homogenate from mice and plasma from hypophysectomized mice, injected subcutaneously with ovine LH, ran parallel with ovine LH standards in plasma from hypophysectomized mice and plasma with low LH levels from intact mice. Ovine TSH showed about 12% cross reaction in the assay system, whilst rat FSH and prolactin and also ovine FSH, prolactin and GH showed practically no cross reaction. Measurements of plasma LH levels have been made in hypophysectomzied mice after injection with different vehicles containing 10 or 50 mug LH or 50 mug FSH per animal. Daily measurements of LH levels throughout pregnancy in the mouse show a rise in LH level prior to implantationand a further rise around mid-pregnancy which drops off sharply to levels which remain fairly constant until parturition when there is another rise.

Extra-ovarian effects of bovine prolactin and growth hormone on the vagina of the mouse.

Hypophysectomized-ovariectomized mice receiving oestrogen and progesterone were treated with and without bovine prolactin and growth hormone (GH). Prolactin significantly increased vaginal weight and vaginal sialic acid content, but had no effect on vaginal sialic acid concentration, an indicator of vaginal mucification. Growth hormone significantly increased all three vaginal parameters. For vaginal sialic acid content and concentration, the synergism between oestrogen and progesterone was found to be greater in the presence than in the absence of GH and the maximum response to GH was obtained when between 20 and 100 mug GH was administered daily. It was concluded that GH but not prolactin has an extra-ovarian effect on vaginal mucification in the mouse.