Rat Blastocysts Research Articles

The Influence of Extracellular Matrix Components on the Proliferation and Migration of Inner Cell Mass-Derived Parietal Endodermal Cells1

Reichert's membrane is a basement membrane deposited on the inner surfaces of rat and mouse trophectodermal (TE) cells beginning at the blastocyst stage of embryonic development that may play a role in the migration of the parietal endodermal (PE) cells to form an inner lining to the TE. The abilities of various glycoproteins present in Reichert's membrane to support PE cell migration and replication in vitro were examined by isolating inner cell masses (ICMs) from Day 5 rat blastocysts (Day 1 = day of vaginal plug) and culturing them (24-72 h) either on surfaces that had been precoated with collagen IV, fibronectin, or laminin or on thin (1-2-mm) gels of Matrigel (a tumor cell-derived basement membrane preparation) or type I collagen. Time-dependent changes in the area occupied by each ICM on the culture surface and the number of migrating cells per ICM were quantified by morphometric analysis. Type IV collagen, the basement membrane-specific collagen, supported ICM attachment and the outward migration (overall increase of approx. 60-fold in mean ICM area occupied on the culture surface) and proliferation (cell doublings following every 24 h of culture) of laminin-containing PE-like cells. These effects were not altered by the inclusion of exogenous fibronectin or laminin in the culture medium. Collagen IV coating concentrations as low as 0.16 micrograms/ml supported PE cell attachment and migration, and maximal responses were seen with a coating concentration of 0.63 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory and Inhibitory Effects of Glucose and Insulin on Rat Blastocyst Development in Vitro

The effect of glucose and insulin on the in vitro development of the rat preimplantation embryo was studied by incubating rat blastocysts recovered on days 5 or 6 of pregnancy in the absence or presence of increasing levels of glucose and/or insulin for 24 or 48 h. A differential cell-staining method allowed the separate counting of inner cell mass (ICM) and trophectoderm (TE) cells at the end of the incubation period. In a high-glucose medium (17 mM), ICM and, to a lesser extent, TE developments were significantly and irreversibly inhibited. Low insulin concentrations (3 pM) stimulated ICM and TE development in the presence of 1.1 or 6 mM glucose. Higher insulin levels (30-600 pM) in a 6-mM glucose medium, resulted in a dose-dependent inhibition of ICM and, to a lesser extent, TE development after both 24 and 48 h. This insulin-induced inhibition was reversible if insulin was removed from the medium after 24 h. In the absence of glucose in the medium, insulin was neither stimulatory nor inhibitory on ICM growth. Dead-cell occurrence in ICM after a 48-h incubation increased with increasing glucose concentration in the medium. Insulin alone did not increase dead-cell number but enhanced the effect of glucose. These results show that, in the presence of glucose, insulin might be stimulatory (at low concentrations) or inhibitory (at higher concentrations) on ICM development. A high glucose level was also inhibitory and increased dead-cell occurrence. The data suggest that insulin and glucose might interact and modulate blastocyst development as a function of their respective concentrations.

Stimulatory and inhibitory effects of glucose and insulin on rat blastocyst development in vitro

Stimulatory and inhibitory effects of glucose and insulin on rat blastocyst development in vitro

Immunolocalization of fibronectin and laminin within rat blastocysts cultured under serum-free conditions

Blastocysts (approximately 50 per female) were collected on Day 5 of gestation from immature Sprague-Dawley rats superovulated using FSH/hCG-loaded mini-osmotic pumps and a single injection of the LHRH analogue, des-gly10 (D-ala6)-LHRH-ethylamide. The cytoplasmic distribution of fibronectin and laminin was determined by immunofluorescence within these blastocysts, either immediately following their isolation or after they had been cultured in serum-free medium for 48-96 h (to allow trophectodermal cell attachment and outgrowth). In addition, inner cell masses (ICMs; isolated by immunosurgery) were cultured under serum-free conditions and immunofluorescently stained for the presence of the two adhesive glycoproteins. Within the freshly isolated blastocysts, positive immunostaining was obtained only for fibronectin and this was associated with the trophectodermal layer. After 48-96 h of culture, the cytoplasm of all trophectodermal cells contained both fibronectin (organized as a slightly granular network) and laminin (the staining pattern was distinctly punctate and perinuclear concentrations of immunoreactivity were evident). ICM-cells stained intensely for the presence of laminin at 48, 72 and 96 h of culture, but appeared to contain little to no fibronectin. While further studies using serum-free culture are needed to define the hormonal regulation of this process, these findings support a role for early gestation rat trophectodermal cells, in addition to the established involvement of ICM-derived parietal endodermal cells, in the synthesis of extracellular matrix components found in Reichert's membrane. The appearance of trophectoderm-associated fibronectin in freshly isolated blastocysts before the establishment of the parietal endoderm layer may implicate this glycoprotein in the provision of a substrate for the migration of these cells as they form an endodermal lining to the blastocoele.

Mouse blastocyst surface expression of galactose-containing epitopes coinciding with trophoblast differentiation

A galactose-containing cell surface epitope of mouse blastocysts was identified and partially characterized by means of immuno- and lectincytochemistry, using a mouse IgM anti-blastocyst monoclonal antibody (mAb N63) and four different galactose-binding lectins (BSL-1, DBA, PNA and SBA) as molecular probes. The mAb was produced by syngeneic intrasplenic immunization with adhesive mouse blastocysts, obtained 18 h after estrogen reactivation from facultative delay of implantation. Labelling of different mouse embryonic stages collected by uterine flushings revealed that the labelling of the epitope by monoclonal antibodies was restricted to the blastocyst stage. A peak labelling intensity was observed on late blastocysts. When examining blastocyst outgrowths, both trophoblast and embryoblast were weakly stained by mAb N63. Direct antigen characterization performed on blastocysts indicated that the mAb N63 recognized a galactose-containing glycolipid antigen. Immunochemistry of cryosectioned, unfixed mouse tissues including ovary, testis, uterus in delay and at implantation, Day 12 and term placenta, liver, kidney, brain, intestine, heart, striated muscle, and skin was negative. In addition, labelling of rat and hamster blastocysts was negative. In vitro experiments demonstrated that the galactose-containing blastocyst surface epitope was not involved in blastocyst attachment to plastic culture dishes. The appearance of the epitope at the embryonic surface in vivo coincides with the time of trophoblast differentiation and implantation in the mouse.

Changes in electrical activity of myometrium during intrauterine distribution of rat blastocysts and after prazosin administration.

In the early pregnant rat, electrical activity of the myometrium consisted of regular bursts of spike potential, which appeared well propagated on Day 2 of pregnancy. During Day 3, there was a gradual disappearance of propagated activity. Concomitantly, there was a 7-fold increase (P less than 0.001) of uterine progesterone concentrations. At this stage, mean duration of bursts was 15.2 +/- 0.9 sec and intervals of complete quiescence between bursts were 84.2 +/- 7.0 sec. At 10:00 h on Day 4, there were peaks in the uterine concentrations of oestradiol and progesterone, +36% and +654%, respectively, compared with values on Day 2 (P less than 0.05). Between 10:00 and 20:00 h on Day 4, EMG activity exhibited a rapid and transient rise: bursts were of longer duration at the utero-tubal end of the horn (+60%, P less than 0.05) with an increased amplitude of spike potentials (+67% and +90% respectively at the tubal and cervical ends of the uterus, P less than 0.05). The administration of prazosin depressed EMG activity reversibly in a dose-dependent manner with maximal inhibition at about 2-3 h later. It is concluded that the changes observed during EMG recordings are relevant to the intrauterine distribution of blastocysts and related to changes in the steroidal environment and/or to catecholamine effects via alpha 1-adrenoceptors.

Induction of micronuclei in pre-implantation rat embryos in vivo

To test the capability of different chemicals to induce clastogenic effects in pre-implantation embryos in vivo, rat blastocysts were collected on the afternoon of the 4th day of gestation from the uterus of females treated on the morning of the 3rd day. Cyclophosphamide (40 mg/kg) and daunomycin (10 mg/kg) did induce micronuclei, but methotrexate (10 mg/kg) and CuSO 4 (8 mg/kg) did not. The micronucleus frequency was dose-related when 3, 9, or 18 mg/kg of mitomycin C were administered. These results confirm the sensitivity of the rat pre-implantation embryo to clastogenic chemicals also after in vivo exposure.

Effect of diclofenac on implantation and embryonic development in the rat

Recent evidence suggests that decidualization can be considered a modified inflammatory reaction. In this work we intended to determine whether the prostaglandin inhibitor diclofenac could affect implantation. Rat blastocysts were cultured in diclofenac in vitro, then implanted to host mothers on day 5 of pseudopregnancy. Large doses of diclofenac in culture were toxic. Smaller doses had a profound effect on implantation. Another group of host mothers received diclofenac i.p., one hour prior to transfer of untreated blastocysts. The results were compared to parallel controls without diclofenac treatment. Control animals had a 72% impantation rate, whereas there was only a 35–41% implantation rate after in vitro diclofenac treatment. In the treated host mothers only 7% of embryos were normal, while 34% were growth-retarded. More normal embryos were found when the blastocysts were cultured with diclofenac. The observations indicate that diclofenac administered to the mother inhibits the ongoing process of implantation and placentation, whereas following in vitro exposure the embryo can recover from the insult. The implications for a better understanding of the process of implantation are discussed.

Cryopreservation of rat blastocysts by vitrification

Rat blastocysts equilibrated with vitrification solution (VS1), consisting of dimethyl sulfoxide, acetamide, propylene glycol, and polyethylene glycol were plunged directly into liquid nitrogen. The embryo suspensions are solidified by an extreme elevation in viscosity of solution. The embryos are cryopreserved by vitrification without intra- and extracellular ice formation. The proportion of morphologically normal embryos after cooling and warming was 79% ( 117 149 ) and all ( 48 48 ) of the embryos cultured were developed to expanded or hatched blastocysts. Normal live young were obtained 41% of the time ( 28 69 ) after transfer of the cooled and warmed embryos to pseudopregnant recipients.

Trophectoderm surface expression of the cell adhesion molecule cell-CAM 105 on rat blastocysts.

A variety of cellular interactions is involved in the process of implantation of the mammalian embryo into the uterine tissue. Recent discoveries have demonstrated that intercellular recognition and adhesive events are governed by a class of cell surface molecules known as cell adhesion molecules (CAMs). In the present report, we have investigated the occurrence of the well-characterized cell adhesion molecule cell-CAM 105 on the surface of rat pre- and peri-implantation embryos of various stages. This was carried out by indirect immunofluorescence microscopy employing affinity-purified rabbit antibodies against cell-CAM 105. The embryonal stages investigated comprised morulae, normal day-4 blastocysts, and delayed and adhesive blastocysts obtained by using the method of experimentally delayed implantation. Cell-CAM 105 was absent in the early-morula stage, but in normal day-4 blastocysts and delayed blastocysts a specific staining for cell-CAM 105 was seen on the entire surface. However, adhesive-stage blastocysts exhibited a marked polarity with staining of the polar trophoblast cells. Scanning electron microscopy of adhesive-stage blastocysts revealed that the stronger staining of the polar region was not due to a greater number of microvilli on the polar trophoblast cells. Thus, it seems as if cell-CAM 105 is lost or masked from the surface of the mural trophoblast cells of adhesive-stage rat blastocysts. Since the mural trophoblast cells are the first to adhere to the uterine luminal epithelium during the onset of implantation and subsequently invade the uterine stroma, we suggest that the apparent downregulation of cell-CAM 105 in the mural trophoblast cells might be linked to the acquisition of trophoblast invasiveness.

Effects of relaxin on the intrauterine distribution and antimesometrial positioning and orientation of rat blastocysts before implantation.

An i.v. infusion of porcine relaxin was administered to rats from the afternoon of Day 4 of pregnancy to the morning of Day 6, a time by which implantation has normally occurred. Implantation sites were irregularly distributed and confined to the cranial half of each uterine horn. Histological sections of uteri fixed by vascular perfusion with osmium tetroxide revealed that the blastocyst was no longer invariably positioned antimesometrially within the lumen and that embryonic disc orientation was often abnormal. A reduced decidual cell reaction was observed around several of the implanting blastocysts.

In Vitro Growth of Rat Blastocyst under the Effect of Azathioprine: (rat/blastocyst/azathioprine/in vitro).

Rat blastocysts were isolated from the uterus on the 5th day after fertilization and set in culture. The effect of azathioprine (Imuran) on the blastocyst's development and on the early trophoblastic differentiation in vitro was investigated. Azathioprine, added to the medium of the blastocyst culture at various concentrations, dose dependently arrested development and had definite cytotoxic effect. In order to study the mechanism of action, a minimal dose of 5 μg/ml, which allowed the survival of about 60% of the blastocysts, was added to the medium after 48 hr of culturing. Under the effect of the substance the area of the spreading blastocyst cells was significantly restricted. It was found, by autoradiographic methods, that the azathioprine affects the development by restraining DNA synthesis in the throphoblastic cells. Concomitantly RNA synthesis was inhibited and protein synthesis was reduced. The observations indicate, that the impairment of the in vitro differentiation of the blastocysts can be a result of the intracellular inhibitory action of the substance.

Concanavalin A agglutinability and immunoadherent properties of rat blastocyst.

Preimplantation rat blastocysts were examined in vitro for concanavalin A agglutinability and antigenic properties on the 5th day of development after flushing and after 24 h in culture, following regional differentiation. Differences were found between the two trophoblastic cell populations with regard to cell membrane receptors to concanavalin A. Immunoadherence hemagglutination tests show that the early trophoblast possesses antigenic properties, which are similarly present in the mural and polar cells of the early preimplantation blastocyst. With later differentiation in vitro, differences can be found between the two trophoblastic cell populations with regard to detectable surface antigens.

The presence of 17 beta-hydroxysteroid dehydrogenase activity in preimplantation rat and mouse blastocysts.

When Day 5 rat blastocysts and Day 4 and 5 mouse blastocysts were cultured in 53 microliters of medium containing 1340 or 2680 pg [3H]estradiol (E2), large amounts of [3H]estrone (E1) were detected in the medium at daily intervals for up to 5 days. This indicates the presence of 17 beta-hydroxysteroid dehydrogenase in the embryos. The activity was higher at a higher concentration of E2 and was also higher in mouse than in rat blastocysts. In the mouse, the activity was higher in Day 5 than Day 4 blastocysts during the first day in culture; then it decreased in Day 5 but increased in Day 4 blastocysts. The importance of E2 in embryonic development and implantation as suggested by others may be related to the activity of 17 beta-hydroxysteroid dehydrogenase.

In vitro inhibition of trophoblast maturation and expansion of early rat blastocysts by an oestrogen antagonist

Rat blastocyst development and expansion were studied in an in vitro culture system. Early blastocysts collected from rats on the morning of day 5 were shown to develop normally and underwent expansion when placed in bovine serum albumin supplemented culture medium for 12 h at 37 degrees C. Nafoxidine, an oestrogen antagonist, prevented embryo expansion when added to the culture medium, and it was found that if oestradiol-17 beta was added to this medium this inhibitory effect was removed and the embryos again expanded. Ethanol, used as a solvent for steroids, and cholesterol were added to the medium containing nafoxidine but the blastocysts remained unexpanded after 12 h in culture. These results suggest a need for oestrogen action during blastocyst expansion in vitro. Cultured rat blastocysts were subjected to histochemical study for acid phosphatase (ACPase) and leucylnaphthylamidase (LNAse). In unexpanded blastocysts ACPase was found to be low while LNAse activity was higher when compared with enzyme distribution patterns in expanded blastocysts.

Absence of capillaries in the endometrium surrounding the implanting rat blastocyst

Absence of capillaries in the endometrium surrounding the implanting rat blastocyst

Immunosurgical studies on inner cell mass development in rat and mouse blastocysts before and during implantation in vitro

Eighty per cent of rat blastocysts (Wistar, SW72) cultured for 96 h in NCTC-109 supplemented with fetal calf serum (FCS) hatched from the zona pellucida and developed a trophoblast giant cell lyer. Thirty seven per cent from the rat blastocysts developed an inner cell mass (ICM) which, in about 7% consisted of two germ layers (ectoderm and endoderm) compared to 84% in NMRI mice. A significantly better ICM development was obtained with cultured rat blastocysts that had hatched in vivo. Similar to the in vivo situation LDH-5 was present in rat blastocysts after implantation in NCTC-109-FCS. Differentiation of C57BL mouse blastocysts in NCTC-109-FCS proceeded as poorly as in the rat. ICM development of rat and mouse blastocysts in NCTC-109-FCS was studied in detail. ICMs of the two species were isolated immunosurgically using complement from different species, e.g. human, rat and rabbit complement, since guinea-pig complement did not lyse trophectoderm cells of rat blastocysts. All immunosurgically isolated rat ICMs degenerated within 48 h, but mouse ICMs isolated with rat or rabbit complement developed significantly better than mouse ICMs isolated with guinea-pig complement. Determinations of the blastocyst total cell number (BTCN) and of the cell number of immunosurgically isolated ICMs were performed in rat and mouse blastocysts to investigate growth kinetics of the ICM before implantation in vitro. In the mouse an exponential increase in both BTCN and cell number of the ICM was observed during the 48 h before implantation in NCTC-109-FCS and also during the 16-24 h before implantation in vivo. In the rat, doubling of the BTCN was found only during the first 24 h in NCTC-109-FCS and there was hardly any increase in the cell number of the ICM during the first 48 h in culture. ICM growth of blastocysts in NCTC-109-FCS is therefore, stimulated in the mouse before and after implantation and in the rat it is inhibited already before implantation.

Concanavalin A binding capacity of preimplation rat embryos

AbstractThe number of concanavalin A (Con A) binding sites was determined in Days 2–5 rat embryos (Day 1 = sperm‐positive) by Scatchard plot analysis. There were 127 ± 13 million sites on Day 2 (2‐cell), which were decreased slightly to 88 ± 9 million sites on Day 3 (2 ∼ 4‐cell), then increased to 200 ± 24 million sites on Day 5 morning (early blastocyst) and 175 ± 21 million sites on Day 5 evening (late, implanting blastocyst). However, the apparent association constant decreased steadily with development, from about 3 × 106 M−1 on Days 2 and 3 to 1.3 × 106 M−1 on Day 5 evening.Thus, in comparison with the results of mouse embryos (Wu, '80), the number of Con A binding sites was not increased in the implanting rat blastocyst, as it was in the mouse, although there were comparable numbers of Con A binding sites up to the early blastocyst stage in both species. The data also suggest some changes in the sugar groups, and probably the surrounding groups, on the surface coat of the plasma membrane during the preimplantation stages of development.

Affinity of uterine luminal proteins for rat blastocysts.

The binding of 125I-labelled rat uterine luminal proteins from Day-5 pregnant rats showed higher binding affinity to blastocysts than did the binding of proteins in uterine fluid from pro-oestrous rats (Day 0), rat serum albumin (RSA) or bovine serum albumin (BSA). Apparently little uptake of proteins into cells by phagocytosis or entry into the blastocoelic cavity occurred since similar results were obtained in the presence of sodium azide or cytochalasin B. Autoradiographic studies showed that the proteins were localized on the outer surface of the blastocyst. The binding was Ca2+-dependent. Denaturation of Day-5 uterine proteins at 80 degrees C reduced the counts to the values obtained with undenatured RSA and Day-0 fluids; this residual binding was considered as non-specific. The binding of labelled Day-5 uterine proteins was substantially reduced in the presence of unlabelled Day-5 proteins but to a lesser extent in the presence of RSA or rat serum. The dissociation of the bound labelled Day-5 uterine proteins occurred most rapidly in the presence of unlabelled Day-5 proteins. However, dissociation occurred within 2 h in the presence of other macromolecules, suggesting that the binding was not strong.

Ultrastructural studies on maternal-embryonic cell interaction during experimentally induced implantation of rat blastocysts to the endometrium of the mouse

Rat blastocysts collected around noon on Day 5 of pregnancy were transferred to the uterus of the mouse on Day 3 of pseudopregnancy. Out of 430 rat blastocysts transferred, a total of 37 were recovered as xenogeneic implants from the recipient mice killed 36, 48, 52, 58, 72, 96, and 120 hr after transfer. None of the transferred blastocysts was found surviving in the host uterus beyond 96 hr after transfer. Electron microscopic examination of the recovered implants revealed that rat blastocysts can successfully undergo the stages of ovum implantation in the mouse uterus from the early attachment to the initial phase of the trophoblastic invasion of the endometrium. During the late attachment stage, desmosomes (maculae adhaerentes), intermediate junctions (zonulae adhaerentes), and gap junctions (nexuses) were formed xenogeneically between the foreign trophoblast and the uterine epithelial cells of the host. Trophoblast cells of xenogeneic implants were destroyed shortly after the penetration of the basement membrane of the luminal epithelium of the host endometrium, leading to the degeneration and sloughing off of the transferred embryos.