Hatching Of Mouse Blastocysts Research Articles

An investigation of mechanisms underlying mouse blastocyst hatching: a ribonucleic acid sequencing study

To investigate the regulatory mechanisms and signaling molecules underlying hatching in mouse embryos. Experimental laboratory study using a mouse embryo model. University-based basic scientific research laboratory. A total of 40 B6C3F1 × B6D2F1 mouse embryos were used in this study. Frozen/thawed mouse embryos, at the 8-cell stage, were cultured invitro for 2 days. The resulting hatching and prehatching blastocysts were then used for complementary deoxyribonucleic acid (cDNA) library preparation and ribonucleic acid (RNA) sequencing analysis (n = 8 for each group). Differentially expressed genes were then used for downstream functional analysis. In addition, a list of genes related to developmental progression in humans was used to identify genes that were potentially related to the hatching of human embryos. Differentially expressed genes, enriched Gene Ontology terms and canonical pathways, clustered gene networks, activated upstream regulators, and common genes between a gene list of hatching-related genes in mice and a gene list associated with developmental progression in humans. A total 275 differentially expressed genes were identified between hatching and prehatching blastocysts: 230 up-regulated and 45 down-regulated genes. Functional enrichment analysis suggested that blastocyst hatching invitro is an adenosine triphosphate (ATP)-dependent process that involves protein biosynthesis and organization of the cytoskeleton. Furthermore, by regulating cell motility, the RhoA signaling pathway (including Arpc2, Cfl1, Gsn, Pfn1, Tpi1, Grb2, Tmsb10, Enah, and Rnd3 genes) may be a crucial signaling pathway during hatching. We also identified a cluster of genes (Krt8, Krt7, Cldn4, and Aqp3) that exerted functional roles in cell-cell junctions and water homeostasis during hatching. Moreover, some growth factors (angiotensinogen and fibroblast growth factor 2) and endocrine factors (estrogen receptor and prolactin) were predicted to be involved in the regulation of embryo hatching. In addition, we identified 81 potential genes that are potentially involved in the hatching process in human embryos. Our analysis identified potential genes and molecular regulatory pathways involved in the blastocyst hatching process in mice; we also identified genes that may potentially regulate hatching in human embryos. Our findings enhance our knowledge of embryo development and provide useful information for further exploring the mechanisms underlying embryo hatching.

Expression of IL-1β and implantation serine proteases is required for mouse blastocyst hatching.

Mammalian blastocyst hatching is a critically indispensable process for successful implantation. One of the major challenges in IVF clinics is to achieve superior embryonic development with intrinsically potent hatching-competent blastocyst. However, the molecular regulation of hatching phenomenon is poorly understood. In this study, we examined the expression and function of one of the cytokines, IL-1β during blastocyst hatching in the mouse. In particular, the expression of IL-1β (Interleukin-1β), IL-1ra (Interleukin-1 receptor antagonist) and their functional receptor IL-1rt1 (Interleukin 1 receptor type-1) in morulae, zona intact- and hatched-blastocysts was studied. Supplementation of IL-1β to cultured embryos accelerated blastocyst development with improved hatching (treated: 89.6 ± 3.6% vs untreated: 65.4 ± 4.1%). When embryos were treated with IL-1ra, blastocyst hatching was decreased (treated: 28.8 ± 3.1% vs untreated: 67.5 ± 3.8%). Moreover, IL-1β and IL-1ra influenced the expression of hatching enzymes viz., implantation serine proteases (ISP1 and ISP2). While IL-1β increased the embryonic mRNA expression of ISPs (Isp1: 2-4; Isp2: 9- to 11-fold), IL-1ra decreased expression. The protein localization studies revealed increased nuclear presence predominantly of ISP 2 in IL-1β-treated blastocysts. This is the first report to show the functional significance of embryonic IL-1β in regulating hatching-associated proteases, particularly ISP2. These findings have implications in our understanding of molecular regulation of blastocyst hatching and implantation failure in other species including humans.

Effect of laser-assisted three area zona thinning at the 8-cell embryo on the hatching of mouse blastocysts

Introduction: This study was aimed to preliminarily examine the effect of laser-assisted three area zona thinning (LAZT) during the cleavage stage of embryo development and the hatching process in mice based on female age. Materials and Methods: C57BL female mice of two different age groups (6-11 and 28-31 weeks) were superovulated and then mated. The 8-cell stage embryos were treated with LAZT in three areas of zona pellucida (ZP) at 120º intervals. The control group was embryos without LAZT. Results: In old mice, LAZT significantly increased blastocyst formation and hatching compared to the control group (82% vs. 38% in number of expanding blastocyst and 66% vs. 20% in number of hatching blastocyst). Conclusion: These results show that multi-area LAZT results in a significant improvement of blastocyst formation and hatching in mice compared to controls.

Mechanics of mouse blastocyst hatching revealed by a hydrogel-based microdeformation assay

Mammalian embryos are surrounded by an acellular shell, the zona pellucida. Hatching out of the zona is crucial for implantation and continued development of the embryo. Clinically, problems in hatching can contribute to failure in assisted reproductive intervention. Although hatching is fundamentally a mechanical process, due to limitations in methodology most studies focus on its biochemical properties. To understand the role of mechanical forces in hatching, we developed a hydrogel deformation-based method and analytical approach for measuring pressure in cyst-like tissues. Using this approach, we found that, in cultured blastocysts, pressure increased linearly, with intermittent falls. Inhibition of Na/K-ATPase led to a dosage-dependent reduction in blastocyst cavity pressure, consistent with its requirement for cavity formation. Reducing blastocyst pressure reduced the probability of hatching, highlighting the importance of mechanical forces in hatching. These measurements allowed us to infer details of microphysiology such as osmolarity, ion and water transport kinetics across the trophectoderm, and zona stiffness, allowing us to model the embryo as a thin-shell pressure vessel. We applied this technique to test whether cryopreservation, a process commonly used in assisted reproductive technology (ART), leads to alteration of the embryo and found that thawed embryos generated significantly lower pressure than fresh embryos, a previously unknown effect of cryopreservation. We show that reduced pressure is linked to delayed hatching. Our approach can be used to optimize in vitro fertilization (IVF) using precise measurement of embryo microphysiology. It is also applicable to other biological systems involving cavity formation, providing an approach for measuring forces in diverse contexts.

Methods to Study the Role of Progranulin in Preimplantation Mouse Embryo Development.

Progranulin is a 67-88kDa glycoprotein, also known as acrogranin, PC-cell-derived growth factor, granulin-epithelin precursor, and proepithelin. This protein is present in a variety of mouse, rat, and human tissues. Progranulin, which is a growth factor, mediates cell cycle progression and cell migration in normal and pathological conditions. In several types of cancers, progranulin expression is upregulated, whereas function-interfering mutations in the granulin gene in humans have been linked to a subset of heritable cases of frontotemporal lobar degeneration. Also, progranulin has important effects on mouse preimplantation embryo development in vitro, including regulation of the appearance of the epithelium in the developing mouse blastocyst and growth of trophectoderm. Furthermore, progranulin promotes mouse blastocyst hatching, adhesion, and outgrowth in vitro. In this chapter, we describe some of the techniques that may be useful in the study of progranulin in embryo development.

The effect of the site of laser zona opening on the complete hatching of mouse blastocysts and their cell numbers.

ObjectiveWe studied the effect of the site of laser zona opening on the complete hatching of mouse blastocysts and the cell numbers of the completely hatched blastocysts.MethodsMouse blastocysts were randomly allocated to the inner cell mass (ICM) group (zona opening performed at the site of the ICM, n=125), the trophectoderm (TE) group (zona opening performed opposite to the ICM, n=125) and the control group (no zona opening, n=125).ResultsThe rate of complete hatching of the blastocysts was not significantly different in the ICM and the TE group (84.8% vs 80.8%, respectively; p=0.402), but was significantly lower in the control group (51.2%, p<0.001). The cell numbers in the completely hatched blastocysts were comparable in the control group, the ICM group, and the TE group (69±19.3, 74±15.7, and 71±16.8, respectively; p=0.680).ConclusionThese findings indicate that the site of laser zona opening did not influence the rate of complete hatching of mouse blastocysts or their cell numbers.

Regulatory Role of Nitric Oxide in Development and Hatching of Mouse Blastocysts

To determine the regulatory role and mechanism of nitric oxide (NO) in the development and hatching of mouse blastocysts. The Kunming female mice were superovulated and then mated with mature male mice. On the day 2.5 of their pregnancy, morulae were flushed from their uterine horns with culture media. Morulae were cultured in different concentrations of N-nitro-L arginine methyl ester (L-NAME), sodium nitroprusside (SNP), or the combination of L-NAME and SNP in culture media for 48 hours. The development and hatching of blastocysts were examined on day 4 and day 5 and the total numbers of blastocyst cells and cysteinyl aspartate specific proteinase 3 (caspase 3) were observed under confocal laser scanning microscope. With the increase of the concentration of L-NAME or SNP, the hatching rate of blastocysts and the total number of blastocyst cells were significantly reduced. The addition of 10 nmol/L SNP in culture media with 5 mmol/L L-NAME significantly increased the development of blastocysts and promoted hatching of blastocysts. However, with increase of SNP concentration in culture media with 5 mmol/L L-NAME, the development and hatching rates of blastocysts were significantly decreased. L-NAME had no obvious effect on the expression of active caspase 3 in blastocyst cells. However,when being above 500 nmol/L,SNP significantly increased the expression of caspase 3 in blastocyst cells. NO plays an important role in development and hatching of mouse blastocysts. Excessively high or low NO can damage the division of blastomeres, resulting in the failure of the blastocyst development and hatching. Also, excessively high NO can lead to the apoptosis of the blastocyst cells.

Effects of partial or complete laser-assisted hatching on the hatching of mouse blastocysts and their cell numbers

BackgroundIt is still debatable whether a full-thickness assisted hatching (AH) is better than the partial zona thinning. In this research, we used a mouse model to study the effect of partial and complete laser-AH on the rate of completely hatched blastocyst and their cell numbers.MethodsIn experiment 1, mouse morulae had 0, 1, 2 or 3 full-thickness openings of 10 microns created in the zona pellucida with an infrared laser beam. In the second experiment, 0, 1 and 2 openings of 20 microns were studied. In the third experiment, a full-thickness opening of 20 microns or quarter-thinning of the zonal circumference to a depth of 90% was compared with non-AH controls.ResultsNo difference in blastocyst formation was found in laser-treated groups and in the controls. In experiment 1, the rate of completely hatched blastocysts was significantly lower than the controls. In experiment 2 when the size of the opening was increased, blastocysts completely hatched at a significantly higher rate than that in the controls. In experiment 3, the rate of completely hatched blastocysts was the highest in the full-thickness group. Cell numbers in completely hatched blastocysts from both AH groups were significantly fewer than those in the controls.ConclusionsFull-thickness opening resulted in a higher rate of completely hatched blastocysts than quarter zonal-thinning and controls, but the cell numbers were significantly decreased.

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH 2 to prostacyclin (PGI 2). It is reported that COX2 derived PGI2 2 plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARδ receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARδ expression starts at 2-cell stage of development. Our results suggest that PGI 2 may affect blastomeres number via the so called hypothesis of PPARδ nuclear receptor in autocrine manner.

An improved mechanical technique for assisted hatching

Varied clinical outcomes of assisted hatching (AH) have been reported. We attempt to investigate whether the size of the zona opening created by AH is adequate for blastocyst hatching, and, if not, set up a new method to improve it. A new AH technique, long zona dissection (LZD), was established, and experiments were performed to compare the effects of different sizes of zona opening on complete hatching of blastocysts in mouse and human embryos in vitro. The LZD technique can create a long zona slit on early embryos, even blastocysts, with the slit size beyond two-thirds of zona diameter. Compared with three-dimensional partial zona dissection, LZD can significantly enhance the hatching speed and the rate of complete hatching of mouse blastocysts (93.9%). All (100%) human blastocysts completely hatched following LZD; however, when the slit size after AH was about two-fifths of zona diameter, more of the larger inner cell masses (ICM) became trapped by the zona opening during hatching than the smaller ICM (53.3 versus 12.5%, P = 0.01). Zona opening of moderate size following AH is inadequate for the completion of blastocyst hatching in vitro; in some cases, however, it can be significantly improved by LZD.

Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo.

We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.

O-203. Hatching of mouse blastocysts grown in vitro depends on the total number of embryonic cells and differs from hatching in vivo

O-203. Hatching of mouse blastocysts grown in vitro depends on the total number of embryonic cells and differs from hatching in vivo

A programmed oxyradical burst causes hatching of mouse blastocysts.

The emergence of the mammalian blastocysts from their thick glycoprotein investment known as the zona pellucida is an important, but poorly understood, event in embryogenesis. In this paper, we demonstrate that peri-hatching blastocysts generate a considerably high quantum of an active oxyradical species for an extremely short period of time when compared to the pre-hatching (unhatched) and post-hatching (hatched) blastocysts. Hatching could be induced in pre-hatching blastocysts by exposing them to superoxide artificially generated to match the observed peri-implantation stage specific levels of superoxide, without impairing their viability. These observations suggest the operation of a superoxide-dependent hatching initiation in developing mammalian embryos.

Culture with Matrigel inhibits development of mouse zygotes.

It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F1 hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a "two-cell" block similar to the developmental blocks of other species. Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F1-derived zygotes. Matrigel may be a deleterious addition to embryo culture or coculture systems.

The influence of indomethacin on the hatching of mouse blastocysts

In order to investigate the effect of indomethacin for inhibition of the cyclooxygenase pathway on the hatching process of mouse blastocysts, 508 mouse blastocysts were cultured in modified HAM F10 medium containing 0, 8, 79 or 788 μM indomethacin, added after 24 h of incubation. Hatching was scored after 72 h of incubation. In another series of experiments, indomethacin was added only after blastulation had occurred. Indomethacin in doses of 8 μM and 79 μM, added after 24 h of incubation or after blastulation had occurred, did not influence successful hatching. A dose of 788 μM indomethacin interfered with hatching, but this dose appeared to be toxic for the embryo.

Micromanipulation of cleaved embryos cultured in protein-free medium: a mouse model for assisted hatching.

A mouse model for studying anomalies of human embryonic hatching following micromanipulation is proposed. Initiation and completion of mouse blastocyst hatching was severely impaired (34/292; 12% and 28/292; 10%, respectively) with protein deprivation, resembling the situation in human in vitro fertilization. Hatching ability was restored when an artificial gap was introduced in the zona pellucida by micromanipulation at the cleaved embryo stage. This enabled 77% (285/371) and 36% (134/371) of the embryos to initiate and complete hatching in protein-free medium. No differences were found in overall cell counts between the two groups of embryos. Transfer of micromanipulated blastocysts to pseudopregnant females resulted in development of healthy fetuses.

Inhibition of Mouse Blastocyst Hatching by Subsite-Specific Trypsin Inhibitors, Peptidyl Argininals1.

To explore the substrate or subsite specificity of a mouse hatching enzyme, effects of leupeptin [acetyl(P4 )-Leu(P3 )-Leu(P2 )-argininal(P1 )] and its analogs (peptidyl argininals) on mouse blastocyst hatching were investigated. The compounds containing benzyloxycarbonyl group (Z) in the P4 position inhibited the hatching more strongly than those containing acetyl group or unprotected N-terminal amino acid. Among five Z-Leu-P2 -argininals, a derivative containing a P2 Ser residue was the most potent inhibitor, and the derivatives containing Leu, Thr, Pro, and Gly in the P2 position followed in this order. Then, we synthesized four Z-P3 -Ser-argininals and tested their effects on hatching. The result indicated that the compound with Phe residue in the P3 position was the strongest inhibitor, and the Leu-, Pro-, and Ala-containing derivatives were ranked in this order. Thus, among Z-dipeptidyl-argininals tested, Z-Phe-Ser-argininal most potently inhibited the mouse embryonic hatching, suggesting the preference of the mouse hatching enzyme for Phe(P3 )-Ser(P2 )-Arg(P1 ) sequence as a substrate.

Lectin‐induced abnormalities of mouse blastocyst hatching and outgrowth in vitro

We have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachment, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL-1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin-1 (LTA-1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded blastocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their environment.

Mouse blastocysts hatch in vitro by using a trypsin-like proteinase associated with cells of mural trophectoderm

The mammalian blastocyst must hatch from its extracellular coat, or zona pellucida, to implant in the uterus and continue development normally. Results of experiments described here strongly suggest that a proteinase (74K M r), called “strypsin,” is directly involved in hatching of isolated mouse blastocysts in vitro. Strypsin is a trypsin-like proteinase, based on its substrate specificity and sensitivity to inhibitors, that is present in mouse blastocysts and exhibits certain properties characteristic of membrane-associated enzymes. Histochemical and autoradiographic evidence suggests that, prior to hatching of blastocysts, strypsin is found with cells of mural trophectoderm; not with polar trophectoderm or inner cell mass. Following hatching, strypsin is also found associated with empty zonae pellucidae, specifically at the opening through which the embryo emerged. These and other observations suggest that hatching of mouse blastocysts in vitro is initiated by limited proteolysis of the region of zona pellucida overlying mural trophectoderm.

Effects of indomethacin, prostaglandin E 2, prostaglandin F 2α and 6-keto-prostaglandin F 1α on hatching of mouse blastocysts

The present study has been performed to investigate how PGs would participate the hatching process. Effects of indomethacin, an antagonist to PGs biosynthesis, on the hatching of mouse blastocysts were examined in vitro. Furthermore, it was studied that prostaglandin E 2 (PGE 2), prostaglandin F 2α (PGF 2α) or 6-keto-prostaglandin F 1α (6-keto-PGF 1α) were added to the culture media with indomethacin. (1) The hatching was inhibited by indomethacin yet the inhibition was reversible. (2) In the groups with indomethacin and PGE 2, no improvement was seen in the inhibition of hatching and the inhibition was irreversible. (3) In the groups with indomethacin and PGF 2α, inhibition of hatching was improved in comparison with the group with indomethacin. (4) In the groups with indomethacin and 6-keto-PGF 1α, no improvement was seen. The above results indicated that PGF 2α possibly had an accelerating effect on hatching and a high concentration of PGE 2 would exert cytotoxic effect on blastocysts.