Changes in the pattern of expression of alkaline phosphatase in the mouse uterus and placenta during gestation.

The development of the rodent chorio-allantoic placenta is a complicated process that results in the formation of a transport system capable of sustaining embryonic and fetal growth and development. Intimately linked to this process is alkaline phosphatase (AP), a cell-surface glycoprotein that possibly functions as a transport protein. In the present study, we have mapped the location of AP-expressing cells in the mouse utero-placental unit during the development of the chorio-allantoic placenta by use of enzyme histochemistry and in situ hybridization histochemistry. We found that at implantation the expression of the tissue non-specific AP (TNAP) gene is located exclusively in the decidua and that most of this decidual expression ceases as the placenta starts to form. One exception is a mesometrially located marginal zone of the decidua, which continues to express the TNAP gene until day 12 and the active protein until at least day 16. Trophoblasts of the chorion already express AP before the time of fusion with the ectoplacental cone, after which AP is expressed by trophoblasts of the resulting ectoplacental plate. AP expression in the mature chorio-allantoic placenta is localized in the placental labyrinth and spongy zones. In the latter zone, expression ceases on about day 14. Giant trophoblasts start to express AP on about day 10, with some cells still positive for AP at day 16. The yolk sac does not express AP at any developmental stage. The results show that AP expression during placental development is neither restricted to cells known to be involved in transport, nor expressed in all cells thought to be involved in this transport. This may indicate that AP is not merely a transport protein but has additional functions.

Expression of alkaline phosphatase in the mature mouse placenta visualized by in situ hybridization and enzyme histochemistry.

Alkaline phosphatases (APs) are a family of cell surface glycoproteins that are expressed in a variety of tissues. Their physiological functions are still unclear. Three different AP genes have been found to be expressed in mice, and AP cloned from the placenta is of the tissue non-specific (TNAP) type. We have in investigated the location of TNAP mRNA and active AP in mature mouse placenta, using in situ hybridization and enzyme histochemistry on serial sections. Digital image analysis was used to estimate relative amounts of TNAP mRNA. Tissue non-specific alkaline phosphatase messenger was detected only in the placental labyrinth, whereas active AP was present both in the labyrinth and in a zone of cells at the margin of the decidua basalis, bordering the myometrium and the metrial gland. This latter location of AP activity has not been described previously. The AP-positive zone of the decidua had a condensed appearance and a central defect in the zone was visible on sections taken from the middle of the placenta. No TNAP messenger was found in the zone of AP-positive decidual cells.

Electron microscopy and X-ray microanalyses of uterine epithelium from lead-injected mice in an experimental delay of implantation.

Mice in experimental delay of implantation were injected intravenously with 75 micrograms.g-1 body weight of lead chloride, corresponding to a dose of lead of about 56 micrograms.g-1 body weight. Delay of implantation was obtained by ovariectomy 3 days after mating followed by a depot dose of progesterone every fifth day. Electron microscopy showed that the uterine lumen, which was closed in control mice, was opened in lead-injected mice. This morphology suggested that lead caused an increase in uterine secretion. X-ray microanalysis of pyroantimonate precipitates in the uterine epithelium of injected mice demonstrated lead in the precipitates, suggesting that lead could have a direct effect on the function of the uterine epithelium and that lead also could be secreted into the uterine lumen and affect the blastocysts.

Particle-induced X-ray emission (PIXE) for detection of lead in mouse blastocysts.

Mice were injected intravenously with lead chloride, 75 micrograms/g body weight. The mice were in an experimental delay of implantation, which offered the functionally steady conditions required for developing the testing procedures. One day after the administration of lead, blastocysts were flushed from the uterine cavity, placed on thin foils of Kapton, and air-dried. The dried blastocysts were analyzed in a nuclear microprobe with a particle intensity of 10 nanoampere (nA) for 300 s using a spatial resolution of 3 microns. The average lead concentration of blastocysts at implantation was 3.90 micrograms/g dry weight. We judge this technique to be useful for evaluating the transport of heavy metals from mother to preembryos, not only under the present experimental conditions but also in normal pregnancies and in other species.

Comparative embryotoxicity of selenite and selenate: uptake in murine embryonal and fetal tissues and effects on blastocysts and embryonic cells in vitro.

The placental transfer and distribution pattern of selenium (Se) in embryonic and fetal tissues after intravenous injection of Na2(75)SeO3 (Se4+) or Na2(75)SeO4 (Se6+) has been investigated in mice in early, mid, and late pregnancy by use of whole-body autoradiography combined with gamma-counting. The given doses correspond to 45 micrograms Se/kg. In order to compare the direct toxicity in early embryonic stages, the two valence forms were studied by using in vitro systems with (1) mouse blastocysts and (2) chick embryo fibroblasts, differentiating into chondrocytes. The embryonic and fetal uptake after administration of Se4+ and Se6+ was almost identical. The placental transfer of Se was restricted but, nevertheless, increased with time after dosing and with progression from embryonic through fetal stages thereby suggesting active placental transfer. The highest uptake in the embryo was observed in the neuro-epithelium while the eye, liver and skeleton dominated the distribution pattern in the fetal period. Selenite (Se4+) was more toxic than selenate (Se6+) in the in vitro system, in which embryonic mesenchymal limb bud cells differentiated into chondrocytes, as well as in the blastocyst culture system. The results may suggest a direct effect of Se in the embryo as explanation to reported malformations in experimental studies.

High frequency of in situ hybridization on thin plastic sections of human placenta with a cDNA probe for beta hCG.

We describe two different techniques with plastic embedding in in situ hybridization histochemistry (ISHH). Their applicability was demonstrated by use of human placenta of the tenth gestational week and a tritium-labeled cDNA probe for the beta-subunit of hCG. In the first method, ISHH was performed on whole pieces of tissue (en bloc ISHH) pretreated with a weak acid solution, embedded in methacrylate, and sectioned at 3 microns for autoradiography. In the second technique, en bloc ISHH was carried out on tissue pre-treated with the weak acid and thereafter with detergent to further facilitate probe penetration. An acrylic resin was used for embedding, and section thickness was reduced to 1 microns. With both techniques, beta hCG cDNA/mRNA hybrids were localized exclusively to the syncytiotrophoblast (ST), in agreement with a previous study using sections of frozen placentas for hybridization to the same probe. However, owing to the higher resolution of the plastic sections the reliability of this localization was greatly increased. The number of autoradiographic grains over the acrylic resin 1-microns sections was found to be considerably higher than that over the methacrylate 3-microns sections. This study showed that treatment of tissue with detergent before en bloc ISHH, with subsequent embedding in acrylic resin and sectioning at 1 microns, gives high resolution in combination with a high signal-to-noise ratio after autoradiography. As the acrylic resin permits cutting of ultrathin sections, the results suggest that the technique may become useful for ISHH studies at the subcellular level.

Localization of mRNA for the beta-subunit of placental hCG by in situ hybridization.

The cellular origin of placental hCG is not yet completely established. Depending on the method used, the syncytium, cytotrophoblast or both types of tissue have been claimed to synthesize hCG. In the present study in situ hybridization was used on sections of chorionic villi in order to detect expression of the gene for the beta-subunit of hCG (beta-hCG). Placental tissue was obtained from the 8th to the 11th weeks of pregnancy, when the concentration of hCG is high. A cDNA clone, encoding the entire amino acid sequence of beta-hCG, was used as a probe. Hybrids of beta-hCG cDNA/mRNA were found only over the syncytiotrophoblast. Background noise was extremely low and no signals above that were detected in the cytotrophoblast cells. It is concluded that at this stage of pregnancy the gene for beta-hCG is activated in the syncytial parts of chorionic villi and not in the cytotrophoblast.

Effects of lead on the male mouse as investigated by in vitro fertilization and blastocyst culture.

Long-term exposure of male mice to inorganic lead (lead chloride, 1 g/liter) in the drinking water reduces their fertility. The cause of this reduction, expressed as a decrease in the number of mated females showing implantations, was investigated, using an in vitro fertilization method. It was found that spermatozoa from lead-exposed males had a significantly lower ability to fertilize mouse eggs than those from unexposed males. Preimplantation embryos, isolated from uterine horns of mice mated with lead-exposed males, were examined. No morphologically abnormal embryos were found. However, when cultured in vitro over the implantation period, blastocysts of the group mated with lead-exposed males showed an increased frequency of delayed hatching from the zona pellucida or an inability to hatch. Among blastocysts from this group a decreased frequency of inner cell mass development was also found.

Long-term exposure of the male mouse to lead: effects on fertility.

Sexually mature male mice, exposed to lead in the drinking water for 3 months, attained a mean blood level of 32 micrograms/100 ml. In control mice, given deionized water, blood lead levels were less than 0.5 microgram/100 ml. At the end of the exposure period, each male was caged with three untreated females. On Day 17 after mating, the frequency of pregnant females and the number of fetuses and resorptions were recorded. Females without implantations were significantly more numerous (P less than 0.05) in the group that had been mated with the lead-exposed males. The frequencies of resorptions and fetal malformations were similar in the two groups. The two groups of males were compared with respect to body weight, plasma level of testosterone, number of epididymal sperms, and weight of reproductive organs. No statistically significant differences were found. Increased tissue concentrations of lead were noted for all the male reproductive organs, as well as the hypothalamus. It cannot be decided at present whether the decrease in the number of pregnant females was due to a reduction in the ability of spermatozoa from the lead-exposed males to fertilize or to a preimplantation loss of fertilized ova.

Effect of inorganic lead on the primordial germ cells in the mouse embryo.

Embryos from mice exposed to lead chloride (20 micrograms/gm body weight) by a single intravenous injection on day 8 of gestation were examined regarding the number and distribution of their primordial germ cells on 4 consecutive days of development. The cells, visualized by histochemical staining for alkaline phosphatase, showed a normal body distribution but were significantly fewer at all four stages compared with those of control embryos of corresponding age. Furthermore, the staining of the primordial germ cells was much weaker in the embryos from the lead-treated dams than in those from control dams, suggesting that the lead had interfered with the production or activity of alkaline phosphatase. It is assumed that these effects could have contributed to the fertility reduction previously observed in female offspring of mice exposed to lead at an early stage of pregnancy.

Distribution of tungstate in pregnant mice and effects on embryonic cells in vitro.

Whole-body autoradiography and impulse counting experiments were used to study the distribution and retention of radioactivity in the pregnant mouse after administration of [185W]tungstate. A rapid uptake was found in a number of tissues--skeleton, red pulp of the spleen, adrenal, liver, thyroid, pituitary, and ovary--and in the intestine and kidneys, through which it was rapidly excreted. 185W was also readily transported from mother to fetus, although more in late than in early gestation. The largest metal retention was found in the maternal skeleton, kidneys, and spleen and in the visceral yolk sac epithelium and the skeleton of the fetus. Furthermore, in vitro cytotoxicity experiments showed inhibition by tungstate of cartilage production in limb bud mesenchymal cultures at concentrations similar to those found in vivo.

Lead exposure on critical days of fetal life affects fertility in the female mouse.

Female mice were exposed to lead in utero by intravenous injection of lead chloride into the mothers at different stages of pregnancy. At a mature age the mice exposed as fetuses (F1 generation) conceived at a normal rate, but the litter size and fetal survival varied significantly. Small litters and increased numbers of fetal deaths were observed in mice exposed to lead on day 8 of intrauterine life. The live fetuses in this group were normal with respect to weight and morphological appearance. Serum levels of estradiol and progesterone, measured on day 17 of pregnancy, did not differ significantly between F1 mice of a control, unexposed group and of the group exposed to lead on day 8 of intrauterine life. Ovarian follicle counts revealed a significantly smaller number of primordial follicles in the latter group. It is suggested that the exposure to lead at a time of early organogenesis caused the observed fertility decrease by interfering with the development of the female germ cells.

Higher plasma disappearance rate in the mouse for pituitary follicle-stimulating hormone of young women compared to that of men and elderly women.

Female mice were injected iv with extracts of different human pituitaries and the recovery of hFSH in plasma and the disappearance rates of the hormone from the circulation were measured. In the first samples taken 5 min after injection, the recovery of pituitary FSH of young women (52%) was significantly lower (P less than 0.001) than that of men (64%) and elderly women (63%). The plasma concentration of human FSH was measured in the mice up to 242 min after injection. During this period the disappearance rate of the FSH of a young woman continued to be higher than that of the man and the elderly woman. The plasma disappearance curves were multiexponential and gave a poor fit in a two-component exponential model, probably due both to distribution into more than one compartment and to heterogeneity of the material investigated. The MCR and the t1/2 of irreversible loss of human FSH from the circulation were therefore also calculated from graphic integration of the areas under the disappearance curves extrapolated to 480 min. The values obtained in this way for MCR of FSH of a young and an elderly woman and a man were 2.4, 1.4, and 1.7 ml/h, respectively, and the values for t1/2 were 19, 33, and 28 min, respectively. The differences between these values for FSH of the three individuals were highly significant (P less than 0.001). The results indicate that the relatively low in vivo biological activity reported for pituitary FSH of young women compared to that of men and elderly women is due to more rapid clearance of the hormone from the circulation of the test animal.

Effect of short-term exposure to five industrial metals on the embryonic and fetal development of the mouse.

An increase is expected in the world's industrial use of several metals, Al, Co, Mo, V, and W, among others. Very little is known about their possible effects on early mammalian development. Groups of mice were injected with compounds of these metals either before implantation or at early organogenesis. None of the metal compounds showed any interference with implantation, but all of them significantly affected fetal development: Al caused an increased frequency of fetal internal hemorrhage, Mo inhibited fetal normal weight gain, W increased the frequency of resorptions, and Al, Co, Mo, and V all interfered with fetal skeletal ossification.

Retained developmental capacity of blastocysts transferred from lead-intoxicated mice.

Blastocysts from mice injected with an implantation-inhibiting dose of lead were transferred nonsurgically to pregnant foster mothers. This was done to obtain information about the ability of these blastocysts to implant and to develop into late fetal stages. There was no difference in the ability of lead-treated blastocysts to implant as compared with the transferred nontreated blastocysts. Nor was there any effect of the exposure to lead before implantation upon the development of the blastocysts into normal fetuses. The results indicate that the preimplantation blastocysts are protected from possible harmful influences from the lead treatment of the mothers, and that the implantation failure in lead-treated mice is not due to the inability of the blastocysts to implant. A significant increase in the frequency of exencephaly was observed in fetuses derived from transplanted blastocysts as compared with those from normal pregnant mice.

Estradiol receptor activity in uteri of pregnant mice given lead before implantation.

Lead chloride given intravenously to mice on day 4 of pregnancy has been shown previously to inhibit implantation that normally occurs on the following day. The effect of such lead treatment on the estradiol receptor activity in uterine cytosol from day 5 of pregnancy was investigated. The quantity of estradiol receptors, expressed per uterus, per wet weight of uterine tissue, and per amount of protein, was significantly (P < 0.001) larger in lead-treated animals than in control animals. The estradiol receptor binding affinities were similar for both groups. A possible relationship between the increased estradiol receptor activity and implantation failure in the lead-treated mice is discussed. In vitro addition of 1 and 5 mmoles of lead chloride/liter of cytosol resulted in reduced estradiol binding, which is in accord with previous reports. However, our study indicates that this reduction is due to co-precipitation of lead and receptor proteins.

Interference of lead with implantation in the mouse: effect of exogenous oestradiol and progesterone.

The implantation inhibiting effect of an intravenous injection of 75 ppm of lead chloride, giving blood levels of about 32 mumol/l, could be counteracted by exogenous steroid hormones. Subcutaneous injections of 0.1 microgram of oestradiol-17 beta and 1 mg of progesterone were given together with the lead on day 4 of pregnancy, and the same hormone doses were given also on day 5. The frequency of animals having implantations on day 6 of pregnancy in this group was the same as in the control group (75%), whereas in animals given lead and no hormones, the frequency of pregnancies was only 20%. Morphological examination by light microscopy of implantation sites on day 6 of pregnancy in the lead + hormone-treated group showed that the decidual swellings contained implanted embryos of a normal appearance. Serum levels of oestradiol-17 beta and progesterone in nontreated and lead-treated mice were not significantly different between the two groups on day 5 of pregnancy. It is suggested that the implantation failure may be due to an effect of lead on uterine responsiveness to ovarian steroids.

Chorionic gonadotrophin in the mouse from implantation to term.

Chorionic gonadotrophin (CG) in implantation sites from Days 5 to 12 and placentae from Days 13 to 19 of pregnancy in the mouse was determined by utilizing a cross-reaction with human CG in a radioimmunoassay. Significant amounts of CG could be detected throughout the period of gestation investigated. The amount of CG per implantation site increased steadily from Day 5 to a peak on Day 11. In the second part of pregnancy a second peak was observed with the highest amount of CG per placenta found on Day 16. No gonadotrophic activity could be detected in blastocysts flushed from the uteri on Day 5 of pregnancy or in uteri of non-pregnant mice.

Interference of lead with implantation in the mouse: a study of the surface ultrastructure of blastocysts and endometrium.

Implantation chambers, trophoblast and uterine luminal surfaces were examined on days 5 and 6 of pregnancy by electron microscopy in mice with implantation failure due to an intravenous injection of 75 ppm of lead chloride on day 4. Attachment of the trophoblast cells to the surface of the endometrium and closure of the uterine lumina had failed to occur. Uterine epithelial cells in implantation chambers and along the lumina were covered with abundant microvilli. This appearance is similar to that seen in mice in experimental delay of implantation before the oestrogen-induced attachment of the blastocyst has occurred. It may therefore be assumed that lead has in some way interfered with the activity of ovarian steroid hormones on the endometrium. No significant changes were observed in surface ultrastructure of the blastocysts from the lead-treated and control groups.

Effect of inorganic lead on the mouse blastocyst in vitro.

In vitro culture of mouse blastocysts over the implantation period was used (1) to find out whether embryos recovered from uteri of mice given an injection of lead, which has been shown in a previous study to inhibit implantation in vivo, were capable of attachment and outgrowth when transferred to a lead free culture medium; (2) to study the effect of different amounts of inorganic lead in the culture medium on blastocyst viability and ability to attach and grow out. The levels of lead used in the medium were comparable to those found in uterine tissue of animals given an implantation inhibiting dose of lead. It was found that the preimplantation blastocyst was unaffected by the administration of lead to the mother as regards the ability to attach and grow out. It was also shown that normal blastocysts were adversely affected by the presence of lead in the culture medium. Addition of increasing amounts of inorganic lead (5, 10 and 20 mumol/l) caused a corresponding decrease in the ability of the blastocysts to attach and grow out, the highest concentration also causing abnormal appearance of the embryos within 48 hours of culture.