Are blastocyst prostaglandins produced endogenously?

This study was undertaken to determine whether preimplantation mouse and rabbit blastocysts possess cyclooxygenase activity and therefore are able to metabolize arachidonic acid (AA) to prostaglandins (PGs) and thromboxane B2. Single rabbit blastocysts or groups of 100 mouse blastocysts were incubated for 4 h in the presence of 5 muCi/ml [3H] AA (sp. ac. 61 Ci/mmol) or for 24 h and 18 h with 0.2 and 0.1 muCi/ml [14C] AA (sp. ac. 56.5 Ci/mmol), respectively. Incubated blastocysts were subsequently exposed for 10 min to 24 h to the Ca2+ specific ionophore, X-537A (10 microM), to stimulate release of radiolabeled AA from the phospholipid pool. After incubation, incorporation of AA into the phospholipid and neutral lipid pools, and metabolism of the fatty acid to PGs and thromboxane B2, were determined using thin-layer chromatographic (TLC) and liquid scintillation spectroscopic techniques. In addition, spent incubation media were analyzed for radiolabeled PG content. In blastocysts of both species, incorporation of AA into phospholipids was greater than that into neutral lipids (mouse: 1.0 vs. 0.7 pmol/blastocyst; rabbit: 159.7 vs. 56.9 pmol/blastocyst). The ionophore stimulated the release of AA from the phospholipid, and to a lesser extent, from the neutral lipid pool, of both blastocysts. No newly synthesized PGs were detected in either mouse blastocysts or their spent incubation media after stimulation with X-537A. Radiolabeled PGs (PGE2, PGF2 alpha, PGD2, PGA2) and thromboxane B2 were present in the media of rabbit blastocyst incubations, however, but were undetectable in the tissue extracts. Increases in metabolism of AA into each compound were observed with an increase in time of exposure to ionophore, and meclofenamic acid (2 microM) partially inhibited the synthesis of all compounds during a 24-h incubation. The results are discussed with regard to the role of blastocyst PGs in the events of implantation.     

Protein synthesis and degradation in non-cultured and in-vitro cultured rabbit blastocysts

In 'pulse-chase' experiments synthesis and half-lives of leucine-labelled proteins were determined in rabbit blastocysts. Embryos were either non-cultured controls or were cultured for 24 h or 48 h in Ham's F-10 medium supplemented with homologous serum or uterine flushings. In control blastocysts protein synthesis increased by a factor of 10 between Day 4 and Day 5. Half-lives of newly synthesized proteins were 32 h in Day-4 and 99 h in Day-5 control blastocysts. In-vitro culture of Day-4 blastocysts led to dramatically shortened half-lives, amounting to 6-10 h. Blastocysts developing in uterine flushing-supplemented media differed significantly from those cultured in serum-supplemented media. Protein synthesis was enhanced and protein degradation was normal for culture times up to 24 h. These results demonstrate (1) that half-lives of proteins in rabbit blastocysts increase with advancing embryonic age, and (2) that a characteristic feature of the altered metabolism of cultured blastocysts is a dramatically accelerated protein degradation, which (3) can be prevented for some time by supplementation of the culture medium with uterine secretions.     

Oxidative phosphorylation-dependent and -independent oxygen consumption by individual preimplantation mouse embryos

The self-referencing electrode technique was employed to noninvasively measure gradients of dissolved oxygen in the medium immediately surrounding developing mouse embryos and, thereby, characterized changes in oxygen consumption and utilization during development. A gradient of depleted oxygen surrounded each embryo and could be detected >50 microm from the embryo. Blastocysts depleted the surrounding medium of 0.6+/-0.1 microM of oxygen, whereas early cleavage stage embryos depleted the medium of only 0.3+/-0.1 microM of oxygen, suggesting a twofold increase in oxygen consumption at the blastocyst stage. Mitochondrial oxidative phosphorylation (OXPHOS) accounted for 60-70% of the oxygen consumed by blastocysts, while it accounted for only 30% of the total oxygen consumed by cleavage-stage embryos. The amount of oxygen consumed by non-OXPHOS mechanisms remained relatively constant throughout preimplantation development. By contrast, the amount of oxygen consumed by OXPHOS in blastocysts is greater than that consumed by OXPHOS in cleavage-stage embryos. The amount of oxygen consumed by one-cell embryos was modulated by the absence of pyruvate from the culture medium. Treatment of one-cell embryos and blastocysts with diamide, an agent known to induce cell death in embryos, resulted in a decline in oxygen consumption, such that the medium surrounding dying embryos was not as depleted of oxygen as that surrounding untreated control embryos. Together these results validate the self-referencing electrode technique for analyzing oxygen consumption and utilization by preimplantation embryos and demonstrate that changes in oxygen consumption accompany important physiological events, such as development, response to medium metabolites, or cell death.     

Effect of RU486 on development and implantation of rat embryos

This study evaluated the effects of postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0, 2.0, or 3.0 mg/rat of RU486 were injected subcutaneously on days 1, 1 + 2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose-related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12). Treatment on day 4 was the least effective. Blastocysts recovered from RU486-treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486-treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486-treated pseudopregnant recipients failed to implant completely when the dose was greater than or equal to 1.0 mg. The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.     

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.     

Differentiation of embryonic haemopoietic stem cells from mouse blastocysts grown in vitro

Embryonic haemopoietic stem cells can differentiate from mouse blastocysts grown in vitro. Mouse blastocysts were cultured for 3 or 4 days and the resultant cells were injected intravenously into lethally X-irradiated or genetically anaemic recipient mice. Blastocysts grown in vitro did not maintain normal embryonic morphology. The presence of donor haemoglobin and donor lymphocytic glucose phosphate isomerase in grafted recipients, demonstrates the presence of embryonic haemopoietic stem cells. Recipients of embryonic haemopoietic stem cells, obtained from growth in vitro, were haematologically stable with no evidence of neoplasia. Pluripotent embryonic cells, maintained on fibroblast feeder layers, were unable to colonize X-irradiated or genetically anaemic mice. Recipients of pluripotent cells died at the same time as saline-injected controls.     

Effect of inhibiting prostaglandin synthesis on edema formation and albumin leakage during thermal trauma in the rat

Prostaglandins (PGs) participate in the inflammatory response, but the contribution of endogenously synthesized PGs to edema formation and increased vascular permeability is not known. Using a 10% scald burn in the rat, we measured water content (as percent, wet minus dry/wet weight) and ¹³¹I-RISA leakage (counts/g dry tissue) in scalded and normal skin at 30 minutes and 3 hr post injury. Four groups (10 rats/group) in each time period studied: control; scald; scald, 5 mg/kg indomethacin; scald, 10 mg/kg indomethacin. Indomethacin was administered intravenously 30 minutes before the scald; RISA was injected intravenously 30 min before termination of the study. In all indomethacin-treated groups immunoreactive plasma PGA was significantly lower (p < 0.05) than in scalded, untreated groups. All scalded groups showed significantly higher RISA counts and water content than did the control group (p < 0.01). At 30 min post-injury the indomethacin-treated groups did not differ from the untreated scald group (p> 0.20). In the 3 hour study all scalded groups had significantly higher content and RISA counts than control (p < 0.01). Thus PGs produced during thermal trauma do not greatly contribute to the edema formation and increase in vascular permeability.     

Morphometric estimation of stromal edema during delayed implantation in the rat

Summary Morphometric methods were used at the light microscopic level to investigate the appearance of edema in the endometrial stroma of rats during estradiol-induced implantation after an experimental delay. Comparisons between blastocyst-free and blastocyst-containing sites were made at 8, 12, 16 and 24 h after injection of estradiol (h.a.e.). The development of stromal edema during implantation was found to be diphasic. First, during the initial 8–12 h.a.e., a generalized edema developed all along the uterine horns. Later, from 16 h.a.e. onwards, a local edema was present around the blastocysts. The Pontamine Blue Reaction (PBR) became visible between 20 and 24 h.a.e. The results indicate that the blastocyst is recognized by the stroma considerably before the PBR. The appearance of a local edema around the blastocysts before the PBR might be related to a slow increase in vascular permeability and/or to the increased stromal cell synthetic activity that is known to precede the PBR during early implantation.     

Cold culture of different stage mouse embryos in bicarbonated and bicarbonate-free media

Mouse embryos of different stages of development were cultured to expanded blastocysts following storage (1 to 8 d) at 4 degrees C in the presence or absence of HCO(3)(-). The effect of oxygen tension on the cold storage of one- and two-cell mouse embryos at 4 degrees C was evaluated by 37 degrees C culture and transfer to pseudopregnant recipients. Survival at 4 degrees C of early, one- to four-cell mouse embryos was improved with HCO(3)(-) in the medium. The presence of HCO(3)(-) was not of benefit for morulae or blastocyst survival following cold storage. Reducing the oxygen atmosphere from 20 to 5% O(2) improved survival of one-cell mouse embryos stored at 4 degrees C. The survival of two- and four-cell embryos, morulae and blastocysts at 4 degrees C was similar in 90% N(2), 5% CO(2) and 5% CO(2) in air, but it was significantly poorer in air alone. The collapse of morulae and blastocysts during cold storage up to 5 d was reduced with HCO(3)(-) in the storage medium. Blastocysts stored for 6 d at 4 degrees C failed to survive following immediate transfer to pseudopregnant recipients. Blastocyst survival was improved compared to controls (direct transfer of unstored blastocysts to recipients) when cultured for 36 h at 37 degrees C following 6 d of cold storage. This result suggests that cold-stored mouse blastocysts may require a metabolic period of readjustment to survive following transfer to synchronized recipients.     

Bovine blastocyst development in vitro: timing, sex, and viability following vitrification

Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.     

Amino acid requirements for attachment and outgrowth of the mouse blastocyst in vitro

Arginine, cystine, histidine, leucine and threonine were needed for outgrowth of the mouse blastocyst in vitro. Omission of lysine, methionine, phenylalanine, tryptophane and tyrosine from the culture medium markedly reduced blastocyst outgrowth, but did not inhibit it completely; while omission of isolecine and valine reduced the extent of outgrowth only slightly. Blastocysts kept for seven days in a free-floating condition by omitting arginine and leucine from the medium, grew out when these amino acids were added. Such behavior may be analogous to delayed implantation in utero and suggests that the free amino acid content of the uterus could be an important factor in the control of implantation. Blastocysts delayed from implanting in the uterus by ovariectomy were activated to outgrowth in a complete medium, but the intracellular changes associated with outgrowth occurred more slowly than in undelayed blastocysts.     

Effects of indomethacin on uterine capillary permeability and blastocyst development in rabbits

Increased capillary permeability at implantation sites was demonstrated in rabbits by extravasation of intravascular blue dye on day 7 of pregnancy. Subcutaneous administration of indomethacin (Id, 8 mg/kg twice daily) on days 4–6 of pregnancy inhibited this uterine blueing response and appeared to reduce the size of implantation swellings. To test the latter observation blastocyst diameter and development of the embryonic disk were assessed at 144 hr . In females receiving indomethacin at the dose level which inhibited uterine blueing, blastocysts were significantly smaller than those from control females. Developmental staging of embryonic disks revealed only slight differences between the smaller (Id-treated) blastocysts and control blastocysts. No effect of Id was seen on ovarian function as judged by luteal weights and plasma progesterone and estradiol levels. Since the major biological effects of indomethacin are due to its inhibition of prostaglandin synthesis, it appears that prostaglandins may play a role in the uterine response to blastocyst stimulation and in the expansion of blastocysts in the rabbit.     

Production of mice derived entirely from embryonic stem cells after injecting the cells into heat treated blastocysts

The sensitivity of the inner cell mass (ICM) and trophectoderm (TE) of mouse blastocysts to high temperatures was examined. When blastocysts with a diameter of 100 to 120 microm treated for 15 to 20 min at 45 degrees C were cultured in vitro, the cell number in the ICM did not increase, although that in the TE did increase. After transfer of treated blastocysts to recipients, implantation was not drastically inhibited but no live fetuses were obtained. These results demonstrated that the ICM at the blastocyst stage was more sensitive to high temperature than the TE. ICM clumps or ES cells were injected into blastocysts treated for 20 min at 45 degrees C. After transfer of injected blastocysts to recipients, we obtained mice derived completely from ICM or ES cells as judged by GPI analysis. Since 4 of 7 ES-cell derived mice, but none of the 6 mice derived from the ICM died after birth, an as yet unidentified epigenetic alteration might have occurred during the establishment and/or culture of ES cells.     

Estrogen and uterine sensitization for the decidual cell reaction in the rat: role of prostaglandin E2 and adenosine 3':5'-cyclic monophosphate

The possibility that estrogen affects uterine sensitization for decidualization by altering the ability of E-series prostaglandins (PGs) to increase adenosine 3':5'-cyclic monophosphate (cAMP) concentrations was investigated. To determine if increased endometrial vascular permeability, a response which precedes decidualization, could be obtained in nonsensitized uteri by treatments designed to increase endometrial intracellular cAMP concentrations, cholera toxin, an activator of adenylate cyclase, was injected into the uterine lumen of immature rats pretreated with progesterone and either 0, 0.5 or 10 micrograms estrone with indomethacin to inhibit endogenous PG synthesis. Endometrial vascular permeability, determined using 125I-labeled bovine serum albumin, was assessed 8 h later. Cholera toxin produced a dose-dependent increase in endometrial vascular permeability in all groups; the uteri of rats pretreated with the optimal hormone regimen (0.5 micrograms estrone plus 2 mg progesterone) responded to a lower dose of the toxin. As determined by uterine weights and histologic examination 5 days after the intrauterine administration of cholera toxin or its vehicle, the toxin induced decidualization in rats pretreated with progesterone and 0 or 0.5 micrograms estrone, but not in those receiving 10 micrograms estrone. Cholera toxin had no detectable effect on uterine cAMP concentrations in animals sacrificed 15 min or 3 h after intrauterine treatment. The intrauterine injection of 8-Br-cAMP, with or without 3-isobutyl-1-methyl-xanthine, did not increase endometrial vascular permeability in indomethacin-treated animals pretreated with the different hormone regimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction

We present a polymerase chain reaction (PCR)-based procedure for rapid bovine embryo sexing and classifying embryos for the presence of exogenous DNA. Fourteen bovine blastocysts microinjected with gene construct DNA at the pronuclear stage were divided into quarters and subjected to amplification with construct-specific and sex gene-specific (ZFY/ZFX) primers in the same initial PCR reaction. Blastocysts carrying microinjected construct DNA could be identified by the presence of construct-specific PCR product in approximately 4 h. Approximately half of the microinjected and two of 16 non-microinjected blastocysts typed PCR-positive for the construct DNA. Owing to erroneous amplifications in the two non-microinjected control blastocysts, and the inability of the system to distinguish integrated from non-integrated copies of the microinjected construct, the number of construct-positive blastocysts determined in our assay most likely overestimates the number of true transgenic embryos. Nevertheless, using this assay, we were able to determine that approximately half of the microinjected embryos were negative for the transgene construct and thus could be eliminated from transfer to a recipient cow. Embryo sexing was achieved in less than 6 h by restriction fragment length polymorphism analysis of nestedZFY/ZFXPCR products reamplified from initial PCR reactions. In 11/14 microinjected blastocysts all sections assayed unambiguously as the same sex. In one embryo, only one section was analysed, while two other blastocysts whowed some discrepancies of sexing results between the sections analysed. The approach employed here to determine the sex and presence of microinjected construct DNA in bovine preimplantation embryos is rapid, accurate among different sections of an embryo and can be used to increase the efficiency of current transgenic cattle production procedures.     

Prostaglandins Stimulate Serotonin Acetylation in Chick Pineal Cells: Involvement of Cyclic AMP-Dependent and Calcium/Calmodulin-Dependent Mechanisms

Abstract: The effects of prostaglandins (PGs) on the activity of the rate-limiting enzyme of melatonin biosynthesis, aryl-alkylamine- N -acetyltransferase (NAT) were investigated on primary cultures of dispersed chick pineal cells. In indomethacin-treated cells, PGs caused a four-fold increase in NAT activity. This response was associated with an eightfold increase in cyclic AMP (cAMP) levels. The potency order of PGs was the same for NAT and for cAMP responses (PGE¹ > PGE² > PGF^2α≫ cloprostenol). However, each PG tested was 30- to 200-fold more potent to increase NAT activity than to stimulate cAMP accumulation. As a result, half-maximal stimulation of NAT by PGs was not associated with an increase in cAMP levels. Half-maximal stimulation of NAT by PGE¹ was highly sensitive to inhibition by a calcium/calmodulin antagonist (W-7). In contrast, maximal stimulation of NAT by PGE¹ as well as stimulations evoked by either forskolin or 8-bromo-cAMP were poorly sensitive to inhibition by W-7. These results indicate that an increase in cAMP levels may be responsible for the maximal stimulation of NAT evoked by PGs, whereas half-maximal stimulation of NAT by PGs would rely principally on a calcium/calmodulin-dependent mechanism.     

Antagonists of embryo-derived platelet-activating factor act by inhibiting the ability of the mouse embryo to implant.

This study utilized the transfer of preimplantation embryos to pseudo-pregnant mice to determine whether PAF-antagonists act primarily on the maternal or embryonic components of implantation. The first experiment used reciprocal embryo transfers, in which blastocysts from mice treated with PAF antagonist (SRI 63-441) or saline (controls), from Days 1 to 4 of pregnancy, were transferred to Day-3 pseudo-pregnant recipients which were also treated with SRI 63-441 or saline on Days 1-4 of pregnancy. The antagonist (40 micrograms) was administered at 16:00 h on Day 1 and at 09:00 h on Days 2-4 of pregnancy. The percentage of the transferred embryos which implanted was determined on Day 8 of pregnancy. Treatment of the recipient or the donor female with SRI 63-441 resulted in a reduction in implantation rate, from a control level of 45% to 33.8% or 34.7% (P less than 0.0002, P less than 0.007) respectively. These results suggest that the PAF antagonist affected implantation at the embryonic and maternal levels. However, when the blastocysts were transferred to Day-4 pseudopregnant recipients, treatment of the donor female had a dramatic effect on the implantation rate, resulting in a reduction of 64% (from 40% to 14.3%, P less than 0.04), while treatment of the recipient female had no significant effect. In this later experiment the transferred embryos were exposed to the recipient uterine environment for a shorter period before implantation. These results suggest that PAF antagonists affected implantation at the embryonic level and did not adversely affect maternal physiology.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.     

An ultrastructural study of in-vitro interaction of guinea-pig and mouse blastocysts with extracellular matrices.

Guinea-pig (intrusive) and mouse (displacement) blastocysts display different cellular mechanisms of implantation. Blastocysts were placed in CMRL-1066 supplemented with either 10 or 20% fetal calf serum, 0.1M L-glutamine and antibiotics and then transferred to dishes previously coated with either Matrigel or type I collagen. After culture for 48 or 72 h, the dishes were processed for transmission electron microscopy. Blastocysts had attached to both extracellular matrices by 48 h. Matrigel elicited minimal trophoblast cell activity. Trophoblast cell projections were oriented parallel to the Matrigel and displayed little invasive activity, but trophoblast cells displayed active interaction with type I collagen. By 72 h, trophoblast cells exhibited slender, anastomosing projections which extended into the collagen matrix. Bundles of microfilaments running parallel with the long axis of the projections were observed. The morphology of type I collagen was altered in the immediate vicinity of the trophoblast projections. The projections interdigitated and desmosomes developed between processes. Projections appeared to meet, fuse and entrap matrix. These results suggest that trophoblast cells do not significantly interact with Matrigel, but penetrate into type I collagen.     

Changes in endometrial vascular permeability during the periimplantation period in the ferret (Mustela putorius)

A highly localized increase in permeability of uterine blood vessels in the immediate vicinity of implanting blastocysts was first detected on the morning of the 12th day of pregnancy (290 h post coitum). The amount of extravasated dye which accumulated at implantation sites continued to increase through the evening of Day 13 (321 h p.c.). Blastocyst expansion, as indicated by small uterine swellings, preceded a detectable change in vascular permeability by about 10 h, suggesting that the timing of increased permeability is closely associated with initial blastocyst attachment to the uterine epithelium. The results do not support the hypothesis that prostaglandins are required for increased uterine vascular permeability as two doses of indomethacin (4 and 8 mg/kg body wt) administered 5 times/day failed to decrease endometrial vascular permeability. However, the 8 mg dose did cause a significant reduction in size and number of uterine swellings and delayed or inhibited attachment of the trophoblast to the uterine epithelium in 2 of 5 ferrets. These findings suggest that prostaglandins play an important role in the process of implantation that is unrelated to decidual formation as the ferret is an adeciduate species.