Hamster Blastocysts Research Articles

Expression and function of interleukin‐1β is required for hamster blastocyst hatching: Involvement of hatching‐associated cathepsin proteases

In mammals, the phenomenon of blastocyst hatching is an essential prerequisite for successful implantation. Blastocyst hatching is regulated by various molecules. Of them, cytokines, expressed by preimplantation embryos, are thought to be functionally important in blastocyst development and hatching, but their mechanistic roles are not clearly understood. Here, we examined the involvement of two cytokines,namely,interleukin-1β(IL-1β) and its natural antagonist, IL-1ra, in blastocyst hatching in the golden hamster. Blastocysts expressed both cytokines and their receptor, IL-1rt1. Supplementation of IL-1β to cultured eight-cell embryos improved blastocyst hatching (84.1% ± 4.2% vs. 66.6% ± 6.8%; treated vs. control). This improvement was diminished by IL-1ra treatment (23.6% ± 12.9% vs. 76.4% ± 12.9%; treated vs. control). Interestingly, IL-1β-treated embryos showed increased messenger RNA expression of zonalytic proteases, that is, cathepsin-L and -B by 1.9 ± 0.5- and 3.5 ± 0.1-folds, respectively. This was accompanied by their increased enzyme activities; cathepsin-L by 2.8 ± 0.7 fold and -B by 2.3 ± 0.7-fold. Strikingly, proteases and IL-1β were intensely colocalized to trophectodermal projections of hatching blastocysts. This is the first report to show the involvement of embryonic IL-1β in regulating hatching-associated proteases required for blastocyst hatching.

Expression and function of cyclooxygenase-2 is necessary for hamster blastocyst hatching

Blastocyst hatching is critical for successful implantation leading to pregnancy. Its failure causes infertility. The phenomenon of blastocyst hatching in humans is poorly understood and the available information on this stems from studies of rodents such as mice and hamsters. We and others showed that hamster blastocyst hatching is characterized by firstly blastocyst deflation followed by a dissolution of the zona pellucida (zona) and accompanied by trophectodermal projections (TEPs). We also showed that embryo-derived cathepsins (Cat) proteases, specifically Cat-L, -B and -P act as zonalysins and are responsible for hatching. In this study, we show the expression and function of one of the potential regulators of embryogenesis, cyclooxygenase (COX)-2 during blastocyst development and hatching. The expression of COX-2 mRNA and protein was observed in 8-cell through hatched blastocyst stages and it was also localized to blastocyst's TEPs. Specific COX-2 inhibitors, NS-398 and CAY-10404, inhibited blastocyst hatching; percentages achieved were only 28.4 ± 5.3 and 32.3 ± 5.4%, respectively, compared with >90% with untreated embryos. Interestingly, inhibitor-treated blastocysts failed to deflate, normally observed during hatching. Supplementation of prostaglandins (PGs)-E2 or -I2 to cultured embryos reversed the inhibitors' effect on hatching and also the deflation behavior. Importantly, the levels of mRNA and protein of Cat-L, -B and -P showed a significant reduction in the inhibitor-treated embryos compared with untreated embryos, although its mechanism remains to be examined. These data provide the first evidence that COX-2 is critical for blastocyst hatching in the golden hamster.

Cellular and molecular regulation of mammalian blastocyst hatching

In mammals including humans, failure in blastocyst hatching and implantation leads to early embryonic loss and infertility. Prior to implantation, the blastocyst must hatch out of its acellular glycoprotein coat, the zona pellucida (ZP). The phenomenon of blastocyst hatching is believed to be regulated by (i) dynamic cellular components such as actin-based trophectodermal projections (TEPs), and (ii) a variety of autocrine and paracrine molecules such as growth factors, cytokines and proteases. The spatio-temporal regulation of zona lysis by blastocyst-derived cellular and molecular signaling factors is being keenly investigated. Our studies show that hamster blastocyst hatching is accelerated by growth factors such as heparin binding-epidermal growth factor and leukemia inhibitory factor and that embryo-derived, cysteine proteases including cathepsins are responsible for blastocyst hatching. Additionally, we believe that cyclooxygenase-generated prostaglandins, estradiol-17beta mediated estrogen receptor-alpha signaling and possibly NFkappaB could be involved in peri-hatching development. Moreover, we show that TEPs are intimately involved with lysing ZP and that the TEPs potentially enrich and harbor hatching-enabling factors. These observations provide new insights into our understanding of the key cellular and molecular regulators involved in the phenomenon of mammalian blastocyst hatching, which is essential for the establishment of early pregnancy.

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel and embryoblast. GLUT3 was exclusively localised in the apical membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.

Interfamily pregnancy and expression of CD57, CD68 in deciduas between golden hamster and mouse

Pregnancy between different species is one of the key steps to interspecific somatic cell cloning. Although interspecific clone embryos have been constructed, they could not develop to birth after being transferred to recipients. In order to clarify the mechanism of this phenomenon, interfamily pregnancy between golden hamste (Mesocricetus auratus) and mouse (Mus musculus) was studied. Co-culture results indicated that the adhesion ratios of golden hamster blastocysts on mouse uterine epithelia monolayer 12, 24, 48 and 72 h after co-culture were all significantly lower than those of mouse blastocysts. The outgrowth ratios of golden hamster blastocysts on mouse uterine epithelia monolayer 48, 72 h after co-culture were both significantly lower than those of mouse blastocysts (P < 0.01). Golden hamster’s blastula could be implanted and develop to D 11 of pregnancy after being transferred to mouse uterus (the 7th day after embryo transfer). Compared to the transfer of mouse embryo to mouse uterus, the successful ratio of interfamily embryo transfer was lower and the bulk of fetus was smaller than that of intraspecific fetus. Compared to intraspecific pregnancy of mouse, the remote decidual tissue of interfamily pregnancy on D8 is looser. At the same time, expressions of CD57 and CD 68 in remote deciduas were both higher than those in the secondary deciduas in both intraspecific and interfamily pregnancy. However, expressions of the two molecules in interfamily pregnancy were lower than those in intraspecific pregnancy. These results showed that interfamily pregnancy could be established between golden hamster and mouse. But the development of fetus in interfamily pregnancy was slower than that in intraspecific pregnancy. The expression difference of CD57 and CD68 indicates the difference of immunoreaction between interfamily and intraspecific pregnancy, which may be one of the reasons leading to interfamily pregnancy termination.

Regulation of peri-attachment embryo development in the golden hamster: role of growth factors

The molecular regulation of mammalian peri-implantation development is complex and difficult to study in vivo. We successfully cultured hamster blastocysts through hatching and peri-attachment stages, using a chemically defined medium, HECM-2h. Using this system, we showed that a species-specific, embryonic cysteine-like protease is involved in blastocyst hatching and that the process is modulated by growth factors. In particular, heparin binding-epidermal growth factor (HB-EGF) or leukemia inhibitory factor (LIF) enhance blastocyst hatching, and the former also improves attachment and trophoblast outgrowth. We observed interesting changing patterns of expression of mRNA and/or immunoreactive protein for EGF, HB-EGF, LIF and transforming growth factor-β (TGF-β) in the embryo and/or endometrial tissue, during peri-implantation development. Together, it appears that hamster blastocyst hatching, attachment and trophoblast outgrowth are regulated by autocrine and/or paracrine growth factors, produced by the embryo-endometrial tissues.

Evidence for the involvement of a species-specific embryonic protease in zona escape of hamster blastocysts.

The source and nature of zona lytic factors during zona escape of hamster blastocyts were investigated. When cultured in hamster embryo culture medium (HECM)-2h, all 8-cell embryos (n = 135) developed to zona escaped-blastocysts with complete zona lysis. In addition, 2-cell embryos, when co-cultured with zona escaping-blastocysts (at a ratio of 1:10), exhibited zona lysis. Various other embryos at the 1-8-cell stages also showed zona lysis when cultured with zona-escaping blastocysts. However, zonae from mice, rats, sheep and humans were resistant to lysis under these conditions. Pronase treatment resulted in rapid zona lysis in hamsters (7 +/- 1 s), whereas in other species zona lysis was much slower: mouse (662 +/- 27 s), rat (532 +/- 16 s), sheep (120 +/- 12 s) and human (104 +/- 8 s). When cysteine protease inhibitors (antipain, leupeptin, E-64 and p-hydromercuricbenzoate) were tested, they completely inhibited zona escape, while trypsin inhibitors (TLCK and SBTI) did not. Uterine zona lysin contribution in zona escape was discounted since: (i) uterine luminal flushing and endometrial extract from day 4 (the time of zona escape in vivo) pregnant females failed to lyse zonae and (ii) endogenous oocytes and transferred 2-cell embryos (to day 3 pseudopregnant recipients) were all zona-intact, while 71% of transferred blastocysts exhibited zona escape, following their recovery after 24 h. These observations suggest that a species-specific, embryonic proteolytic factor, with a cysteine protease-like activity, is involved in the zona escape of blastocysts in hamsters.

Kinematics of trophectoderm projections and locomotion in the peri-implantation hamster blastocyst.

The behavior of golden hamster blastocysts was studied in vitro by continuous time-lapse videomicrography and computer imaging, during and immediately following escape from the zona pellucida. This study revealed numerous small cytoplasmic trophectoderm projections (TEPs) approximately 18 microns long that penetrated the zona pellucida both radically and tangentially and appeared to be actively involved in zona escape in vitro. After escape from their zonae, some blastocysts moved across the culture dish by an endogenous means of locomotion, most likely involving activity of the small TEPs. Several hours after zona escape, embryos expressed large TEPs up to 46 microns long that moved in an undulating manner and showed rapid cycles of extension and retraction; the timing of their appearance suggested that these TEPs are normally involved in attachment to the uterine epithelium. Embryos fixed in utero, during the developmental interval between zona loss and embryo attachment, exhibited large TEPs similar in morphology to those expressed by cultured blastocysts. These observations document for the first time that mammalian blastocysts are capable of endogenous locomotion, confirm TEPs as components of normal blastocyst activity, reveal that there are two kinds of TEPs that differ temporally and morphologically, and extend earlier reports of TEP activity in guinea-pig embryos to the hamster.

Kinematics of trophectoderm projections and locomotion in the peri‐implantation hamster blastocyst

Kinematics of trophectoderm projections and locomotion in the peri‐implantation hamster blastocyst

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

Perivitelline space of mammalian oocytes: extracellular matrix of unfertilized oocytes and formation of a cortical granule envelope following fertilization.

Extracellular matrices (ECM) present around unfertilized and fertilized mammalian oocytes were studied ultrastructurally in samples prepared in the presence of ruthenium red to facilitate stabilization of extracellular materials. Unfertilized mouse, hamster, and human oocytes have an ECM comprising granules and filaments in their perivitelline spaces (PVS). This matrix is more abundant in the human than in hamsters and mice. The granule/filament matrix appears identical to the matrix seen between cumulus and corona radiata cells following ruthenium red processing and previously shown to comprise protein and hyaluronic acid. By including ruthenium red during fixation, it is possible to demonstrate the existence of cortical granule exudate in the PVS of fertilized oocytes from hamsters, mice, and humans. Much of the cortical granule exudate is trapped in the PVS and forms a new coat around the fertilized oocyte. This material is particulate when stained with ruthenium red and appears to be uniformly dispersed around the entire oocyte surface. We refer to this new coat as the cortical granule envelope. This envelope is observed in the PVS of all developmental stages up to and including blastocysts in all three species. Following hatching of mouse and hamster blastocysts, the cortical granule envelope is no longer present. Possible functions of this envelope are discussed.

Assessment of hamster blastocysts derived from eight-cell embryos cultured in hamster embryo culture medium-2 (HECM-2): Cell numbers and viability following embryo transfer

The viability of hamster blastocysts, cultured from the eight-cell stage using hamster embryo culture medium-2, was examined by embryo transfer. Approximately 15-20 cultured hamster blastocysts were surgically transferred unilaterally to uterine horns of pseudopregnant recipient hamsters that had been mated to vasectomized males 3 days previously. Control recipients received in vivo developed, freshly recovered eight-cell embryos or blastocysts on day 2 or 3 of pseudopregnancy, respectively. Of the successful embryo transfers, the experimental group (receiving cultured blastocyts; n = 10) gave 51.8% implantations and 28.2% live pups. These values were closely similar to those of the controls; the percentages of control implantations and offspring were 51.1 and 34.0%, respectively, for eight-cell embryo transfer (n = 7, P greater than 0.69) and 48.5 and 28.9% for blastocyst transfer (n = 6, P greater than 0.52). To evaluate the quality of cultured hamster blastocysts, the following two parameters were examined. (1) The mean number of cells per blastocyst was 24.4 +/- 0.7 for cultured blastocysts. This value was similar to that (range, 14-24) obtained in this laboratory for in vivo developed freshly recovered blastocysts. (2) Oxygen consumption analysis revealed that cultured blastocysts actively respired at a level close to that observed with freshly recovered eight-cell embryos (slopes of oxygrams: 0.25 and 0.26, respectively). From these results, it is concluded that hamster blastocysts, cultured from the eight-cell stage, are (a) qualitatively similar to freshly recovered in vivo developed blastocysts and (b) biologically viable as revealed by the production of live offspring upon embryo transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of estrogens by rabbit blastocysts: Formation of estrogen glucosides and preferential conversion of estrone to estradiol-17β

When day 6 rabbit blastocysts were cultured (3 embryos/mL) in medium 199 containing 3.68 μM estradiol-17β (E 2), 40% of E 2 was metabolized in 24 h, at a rate of 18 pmol/embryo(b)h, yielding 4 major metabolite fractions. Two of them were identified to be estrogen glucosides: 17β-hydroxyestra-1,3,5(lO)-trien-3-yl β-D-glucopyranoside (E 23G) (12 pmol/b/h) and 17-oxoestra-1,3,5(10)-trien-3-yl β-D-glucopyranoside (E 13G) (0.5 pmol/b/h). If the blastocysts were cultured in 3.68 μM E 1 medium, 75% of E 1 was metabolized in 24 h (34.1 pmol/b/h); most of it appears as E 2 (8 pmol/b/h), E 13G (16 pmol/b/h), and E 23G (6 pmol/b/h). Thus, the 17β-hydroxysteroid dehydrogenase activity in the rabbit blastocysts catalyzes mainly in the direction of the E 1→E 2 conversion, with little or no E 2→E 1. This may be responsible in part for the faster metabolism of E 1 than E 2 by the rabbit blastocyst. In comparison with the rat, mouse, and hamster blastocyst, the rabbit embryo shows an additional capability to conjugate large amounts of estrogens into glucosides by steroid glucosyltransferase.

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.

Vitamins and amino acids stimulate hamster blastocysts to hatch in vitro.

Vitamins and amino acids stimulate hamster blastocysts to hatch in vitro.

Mammalian Embryo Culture from the Pre‐Implantation Stage, and Analysis of Developmental Failures due to Chromosomal Abnormalities in Vitro*

Abstract In vitro culture methods make it possible to manipulate mammalian embryos experimentally, and continuous observation of the developmental processes furnishes significant information for mammalian embryology. The advancement in the culture methods of the mouse embryos from the pre‐implantation through the limb bud stage is reported here, along with a description of one of the applied experiments of embryo culture from the pre‐implantation stage in the Chinese hamster.A few mouse embryos at the limb bud stage cultured for 10 or 11 days from the blastocyst stage were obtained, but the frequencies were very low. There is a barrier to assure further development in the stationary culture of mouse blastocysts till the formation of early egg cylinder. It was suggested that trophectoderm inhibited the development of inner cell masses by enveloping them, but embryos could not develop without trophectoderm afterwards. After the egg cylinder or primitive streak stage, the medium and gas around the embryos needed to be exchanged with rotation or shaking.Chinese hamster blastocysts could be cultured as late as the early egg cylinder stage with methods similar those employed with mouse blastocysts. Sonta (1982) has established several lines of Chinese hamster bearing various reciprocal translocations. Thus, one can obtain embryos with abnormal karyotypes from these parents. By cultivation of these embryos from the pre‐implantation stage, the present authors investigated the relationship between the developmental failures and the chromosomal abnormalities.

Metabolism of progesterone by hamster blastocysts and the ontogeny of progesterone metabolic capability.

Progesterone (P) is required for the differentiation of reproductive tracts and maintenance of pregnancy. This study investigates whether the hamster blastocyst is capable of metabolizing P and, if so, at what stage of preimplantation development such capability becomes detectable. When the blastocysts collected from superovulated hamsters on Day 4 of pregnancy were cultured in 0.4 microM P medium, P metabolism was easily detectable at 1.25 h of culture and over half was metabolized by 7.5 h. Two major metabolites were generated: 5 alpha-pregnane-3,20-dione (or 5 alpha-dihydroprogesterone; 5 alpha-DHP) and 5 alpha-pregnane-3 beta-ol-20-one (or allopregnanolone; AP), about 90-95% and 5-10%, respectively. This indicates the activity of two enzymes: delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The rate of P metabolism increased with P concentration (0.4-6.4 microM), indicating a high capacity of the enzymes. Studies of embryos collected on Days 1-3 showed that P metabolism was not detectable up to 0100 h of Day 3 (2-4-cell), but was detectable with two metabolites, 5 alpha-DHP and AP, at 1515 h of Day 3 (morula) and thereafter. This indicates that, by the morula stage, the hamster embryo has already acquired the enzymatic capability (5 alpha-reductase and 3 beta-HSD) to metabolize P. These results, together with our earlier finding of 17 beta-hydroxysteroid dehydrogenase activity in Days 1-4 embryos, suggest that hamster preimplantation embryos can metabolize both P and estrogens, thus possibly modulating local actions of these hormones and causing local effects in the reproductive tract.

Estrogen synthesis and metabolism in the hamster blastocyst, uterus and liver near the time of implantation

The steroidogenic potential of hamster tissues, just prior to implantation of the blastocyst in the uterus, was characterized by incubating blastocysts (14) and pieces of endometrium with [1,2- 3H]-androstenedione for 24 h. [ 3H]-2-Methoxyestradiol was synthesized, but intermediate estrogens were not found. To obtain a more quantitative assessment and comparison of steroidogenic activity, especially aromatase activity, in these tissues as well as in the uterine myometrium and liver and to increase the possibility of recovering estradiol, microsomes were isolated from 244 blastocysts and portions of the other tissues. Microsomes were incubated with [1α,2α- 3H]-testosterone plus [1β- 3H]-testosterone for 6 h. During this time [ 3H]-metabolites were synthesized by all tissues as indicated by HPLC. [ 3H]-Androstenedione was noted and values were higher than control levels (medium alone or microsomes from uterine flush fluid) in all samples but liver. [ 3H]-Estradiol was detected at an elevated level only in the blastocyst sample; however, addition of unlabeled estradiol during the subsequent incubation of endometrial, myometrial and liver microsomes increased the recovery of [ 3H]-estradiol. Identities of [ 3H]-2-methoxyestradiol from the first experiment and [ 3H]-androstenedione and [ 3H]-estradiol from the second experiment were confirmed by recrystallization. The formation of 3H 2O from [β- 3H]-testosterone was used as an index of aromatase activity. After subtracting control medium values, blastocysts were 24-fold more active (dpm/μg protein) than the endometrium and myometrium in synthesizing 3H 2O. While there was no difference in synthetic potential between endometrium and myometrium, aromatase activity in these tissues was greater than that of the liver. Microsomes from uterine flush fluid displayed no capacity for synthesizing 3H 2O indicating that the elevated blastocyst levels were not caused by contaminating endometrial cells. These results indicate that all of the tissues examined have the capacity to metabolize C 19-steroids to a variety of hormones, including estrogens, and further, that estrogen metabolism occurs rapidly in these tissues. This capacity may be important for providing a suitable hormonal milieu at the time of implantation.

Viruslike Particles in Hamster Embryos, Fetuses, and Tumors&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Intracisternal viruslike particles were present in outbred Syrian golden and inbred ALB/Mey Pfd hamster blastocysts, embryos, and fetuses. Their morphology was identical to that of the "spoked" viruslike particles found in hamster tumors. They were released in great numbers in the embryonal tissues until day 9 of pregnancy and were found until day 13 in the fetal tissues. Thereafter, these viruslike particles were no longer observed in the fetus. They were never recorded in normal adult hamster tissues.

The Ultrastructure of Preimplantation Hamster Blastocysts Developed In Vivo and In Vitro

Scanning electron microscopy of zona-free blastocysts showed bulging trophoblast cells possessing slender microvilli and many small projections of various lengths. The trophoblast cells contained mitochondria in two shapes. One type consisted of spheroids with a few mitochondrial inner membranes. The other type had a more conventional appearance. Fibrous material varied in amount. The trophoblast cells from blastocysts cultured for 1 day had an appearance similar to that of trophoblast tissue fixed in vivo. However, the large, dense mitochondria with few cristae had diminished in number, and globular mitochondria occurred more frequently. Furthermore, the fibrous material was more sparsely distributed after culture. The experiments demonstrated that cultured hamster blastocysts have ultrastructural characteristics similar to those of in vivo blastocysts. Experiments are in progress to test whether the in vitro technique can determine the growth requirements of the hamster blastocyst.