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Cellular and molecular regulation of mammalian blastocyst hatching
Journal of Reproductive Immunology 83 (2009) 79–84
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Cellular and molecular regulation of mammalian blastocyst hatching Polani B. Seshagiri ∗ , Shubhendu Sen Roy, Garimella Sireesha, Rajnish P. Rao Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India
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Article history: Received 11 March 2009 Received in revised form 7 May 2009 Accepted 21 June 2009 Keywords: Blastocyst Hamster Hatching Protease Trophectodermal projections
a b s t r a c t 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 actinbased 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-17 mediated estrogen receptor-␣ signaling and possibly NFB 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. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction During preimplantation development, the mammalian embryo remains enclosed in a glycoproteinaceous coat, the zona pellucida (ZP), from which it hatches prior to implantation into the receptive maternal uterine endometrium (Enders and Schlafke, 1967). Blastocyst hatching is a critical and tightly regulated phenomenon of fundamental importance for the subsequent viability and development of the embryo. Any dysregulation of the hatching process causes implantation failure leading to infertility (Petersen et al., 2005). The peri-hatching blastocyst development and implantation is governed by an extremely complicated but cooperative interplay of various cellular factors and biomolecules, which are regulated in a defined spatio-
∗ Corresponding author. Tel.: +91 80 309 2687; fax: +91 80 360 0999. E-mail address: [email protected] (P.B. Seshagiri). 0165-0378/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2009.06.264
temporal fashion (Simon et al., 1996; Hartshorne and Edwards, 1991; Seshagiri et al., 2002). Information on this developmentally critical phenomenon is scarce and hence it is necessary to investigate the cellular and molecular mechanisms of blastocyst hatching and zona lysis. In this article, we provide a comprehensive review on the current understanding of mammalian blastocyst hatching phenomenon with particular reference to early hamster development. 2. Biology of blastocyst hatching The mammalian blastocyst hatching phenomenon is intriguing despite the fact that ZP architecture is similar in most species (Green, 1997). This process is thought to be subject to both cellular and molecular control. Blastocyst hatching occurs by initial formation of a nick in the ZP, caused by a hydrostatic (mechanical) pressure exerted by the increasingly expanding blastocyst. Thus, the blas-
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Fig. 1. Confocal microscopic images of a hatched hamster blastocyst. (A) DIC image displaying finger-like trophectodermal projections (TEPs; arrow head), and (B) phalloidin-FITC (green) localization of actin-based TEPs and nuclear counterstaining with propidium iodide (PI, red). Note the predominant appearances of the TEPs at the abembryonic pole, at the opposite side of the embryonic pole (the embryonic half below the black line, with a greater number of PI-stained nuclei). Bar = 10 m, photographed with Zeiss confocal microscope.
tocyst gradually egresses from the zona and, in most in vitro cultured hatching blastocysts, the ZP remains largely intact. This hatching behavior is observed in the mouse/rat (Bergstrom, 1972; Seshagiri et al., 1999; Surani, 1975), cow (Massip and Mulnard, 1980), rhesus monkey (Seshagiri and Hearn, 1993) and human (Lopata and Hay, 1989). Moreover, it is demonstrated that both the hatching blastocyst and the endometrium produce potential zona lysins involved in hatching (Denker and Fritz, 1979; Ichikawa et al., 1985; Joshi and Murray, 1974; Menino and Williams, 1987; Mishra and Seshagiri, 2000a; Sawada et al., 1990; Vu et al., 1997). Interestingly, hatching in the golden hamster blastocyst is quite unique in that the blastocyst initially expands, followed by a very conspicuous deflation prior to hatching (Gonzales et al., 1996a; Kane and Bavister, 1988; Mishra and Seshagiri, 1998). Moreover, the peri-hatching blastocyst invariably exhibits extensive membranous extensions of the trophectoderm called trophectodermal projections (TEPs) and there is a complete dissolution of the ZP after post-hatching (Fig. 1). In this regard, the phenomenon of blastocyst hatching in hamsters is quite remarkable and a peculiar temporal regulation of cellular and molecular factors governs this event (Fig. 2). The exact mechanism is unknown but several hatching-associated factors coupled with TEPs, which may function as possible cargo carriers for hatching regulators, could be playing major roles in the hatching process.
pierce the ZP at the abembryonal pole and exhibit undulating movements during ZP penetration, following zona escape (Blandau, 1949; Blandau and Rumery, 1957; Spee, 1883). The biology of TEPs in the context of blastocyst hatching in hamsters was extensively pursued by Bavister and co-workers (Gonzales et al., 1996a,b) and also our group (Fig. 1; Mishra and Seshagiri, 1998; Seshagiri et al., 1999; Sireesha et al., 2008). The kinematics of TEPs during hatching has been examined and their criti-
3. Cellular factors and TEPs in blastocyst hatching During the period of blastocyst hatching, a cellular phenomenon involving TEPs has been demonstrated in a number of species (Gonzales et al., 1996b). This was first described in guinea pig blastocysts (Spee, 1883), and their existence was subsequently verified in other species (Blandau, 1949). Early findings showed that TEPs
Fig. 2. A hypothetical working model showing the roles of molecular regulators of embryo and endometrial origin in protease-mediated blastocyst hatching in the golden hamster. Abbreviations used are: cyclooxygenase-2 (COX-2), estrogen receptor-␣ (ER-␣), heparin binding-epidermal growth factor (HB-EGF), leukemia inhibitory factor (LIF), nuclear factor B (NFB) and transforming growth factor- (TGF-).
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Fig. 3. Effect of estrogen receptor antagonist (ICI 182, 780) on hamster blastocyst hatching. Panel A depicts percentages of blastocyst hatching in the absence or presence of the inhibitor. The antagonist (150 nM) inhibits hatching by about ∼80% when cultured for either 12 h ( ) or 24 h (). After culture in the presence of the inhibitor for 12 h ( ) or 24 h ( ) when embryos were transferred back to a fresh medium, they exhibited hatching. Panel B shows photomicrographs of embryos cultured in the absence (a and b) or presence (c and d) of the inhibitor. Control embryo (a) develop to blastocyst and hatch in about 24 h (b). The embryo (c) cultured in the presence of the inhibitor deflates but fail to hatch (d). Bar = 5 m.
cal role in normal hamster blastocyst hatching has been established. Also, their existence in other species such as bovine, equine, human, monkey and guinea pig was demonstrated (Gonzales et al., 1996a,b; Spee, 1883). Studies on the critical functional role of TEPs in the hatching phenomenon are extremely sparse in vitro and more importantly in vivo and these remain to be investigated. However, trophoblast-derived cellular protrusions extending into the endometrium during early implantation in rats (Enders and Schlafke, 1967), and similarly in mice (McRae and Church, 1990) were demonstrated. In addition TE-derived, actin-based cytoskeletal structures, named filopodia in blastocysts, traverse into the inner cell mass (ICM) and are involved in signal transduction as well as in the positioning of the eccentric ICM (Salas-Vidal and Lomeli, 2004). In view of the above, it is exciting to study the involvement of TEPs and filopodia in hatching phenomenon of developing peri-implantation blastocysts, in parallel with molecular regulators of blastocyst hatching. 4. Molecular regulators influencing blastocyst hatching One of the important aspects of blastocyst hatching is the involvement of regulatory molecules, primarily embryotrophic factors, which encompass second messengers, transcription factors, proteases, growth factors and cytokines (Kane et al., 1997; Sargent et al., 1998; Seshagiri et al., 2002; Simon et al., 1996). Our studies with cultured hamster embryos show that epidermal growth factor (EGF), heparin binding-EGF (HB-EGF), transforming growth factor- (TGF-) and leukemia inhibitory factor (LIF) accelerate hatching, associated with a complete dissolution of zona, predominantly brought about by embryo-derived cysteine protease-like activities (Mishra and Seshagiri, 2000b; Seshagiri et al., 2002). Moreover, our studies with
the estrogen receptor-␣ (ER-␣) antagonist (ICI 182, 780) demonstrate a profound reversible inhibition of hamster blastocyst hatching (Fig. 3), thereby ascribing a definitive regulatory role to ER-␣ in the process. Similarly, our ongoing studies indicate that inhibition of cyclooxygenase-2 (COX-2) or nuclear factor B (NFB) profoundly affect blastocyst hatching associated with a failure of deflation of hatching blastocysts (Sen Roy and Seshagiri, unpublished). Consistent with this is the observation made in the mouse that COX-2 derived prostaglandin (PG)-I2 , is crucial for peri-implantation development, including blastocyst hatching (Huang et al., 2004; Pakrasi and Jain, 2007). Unlike in the mouse (Thomas et al., 1997), zona dissolution in the hamster is predominantly protease-dependent and it is not brought about by any chemical means such as reactive oxygen species (Mishra and Seshagiri, 2000a). Various classes of proteases such as serine-, cysteine- or metallo-proteases are involved in hatching, depending on the species (Denker and Fritz, 1979; Ichikawa et al., 1985; Kimie et al., 1994; O’Sullivan et al., 2002; Perona and Wassarman, 1986; Sireesha et al., 2008). It is evident that key embryotrophic molecular regulators such as growth factors, cytokines, transcription factors and importantly cysteine proteases are critical for hatching (Fig. 2). 5. Cysteine proteases (cathepsins) in development and blastocyst hatching The role of cysteine proteases during embryogenesis is diverse. They are involved in follicular maturation and ovulation (Carnevali et al., 2006), fertilization (Ichikawa et al., 1985), preimplantation development (Afonso et al., 1997), including blastocyst hatching (Mishra and Seshagiri, 2000a; Sireesha et al., 2008) and trophoblast invasion and placentation (Sol-Church et al., 2002). Dysregulation of proteases during embryogenesis can lead to preg-
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nancy failure. Cysteine proteases, belonging to 6 clans and 43 families, are phylogenetically ubiquitous enzymes, broadly classified into legumains, caspases, calpains, and cathepsins (reviewed by Dickinson, 2002). Collectively, they are mainly involved in housekeeping functions (Chapman et al., 1997). At least in hamsters, a speciesspecific, blastocyst-derived cysteine protease-like activity is responsible for hatching of blastocysts and we earlier showed that several pan-cysteine protease inhibitors such as antipain, leupeptin, E-64 and P-hydromercuribenzoate suppressed cultured blastocyst hatching without affecting their development, while there was a negligible or no curtailment of hatching when inhibitors to other classes of proteases were used (Mishra and Seshagiri, 2000a; Sireesha et al., 2008). Our recent data show that blastocyst-derived cathepsins, presumably secretory or TEP-associated types, are involved in blastocyst hatching and zona lysis (Sireesha et al., 2008). Using cathepsin-class specific inhibitors such as cystatins and FYAD, we unequivocally showed the expression and critical involvement of cathepsins in hamster blastocyst hatching, unlike other cysteine-class enzymes such as calpains and caspases (Sireesha et al., 2008). It is significant to note that cathepsins are crucial for hatching, an event occurring immediately prior to the invasion events of blastocyst implantation, which are heavily dependent on cysteine (cathepsin) proteases as well as other proteases (Chapman et al., 1997). Interestingly, the ZP shows an extraordinary sensitivity to exogenous exposure of cathepsins (L, B and P) in hamsters, with effects mediated within seconds in this species compared with 10 min in other species. The hamster ZP dramatically exhibits complete dissolution, as observed with in vivo hatching (Sireesha et al., 2008). It appears that the mouse ZP is relatively less susceptible to proteolysis than hamster ZP (Mishra and Seshagiri, 2000a; Sireesha et al., 2008), indicating that there could be differences in ZP composition or architecture across species. For example, an additional ZP protein (ZP-4) in hamster ZP composition (Izquierdo-Rico et al., 2009) may make the hamster ZP differentially susceptible to protease digestion during hatching. 6. Functional association of TEPs and molecular regulators in blastocyst hatching The functional significance of actin-based TEPs associated with peri-hatching blastocyst developmental stages is not clearly established. The TEPs are thought to play a critical role in zona escape and in the initial attachment of the embryo to the uterine luminal epithelium (Blandau and Rumery, 1957; Sireesha et al., 2008; Spee, 1883). In addition, TEPs might be involved in the uniform spacing of implanting embryos and/or choosing functionally suitable endometrial sites for implantation. Also, TEPs could play an important role in the absorption of nutrients from the endometrium and in the embryo-maternal cellular and molecular dialogue during implantation (Hartshorne and Edwards, 1991; Seshagiri and Hearn, 1995). It is interesting to point out that the appearance of TEPs in hatching blastocysts at the abembryonic (mural) pole coincides with blastocyst orientation at implantation, as observed in
guinea pigs (Blandau and Rumery, 1957; Spee, 1883) and hamsters (Gonzales et al., 1996b; Fig. 1). Conversely, in primates where implantation occurs at the embryonic (polar) pole, the TEPs are also observed at the same pole (Boatman, 1987; Gonzales et al., 1996b). It is of great interest to examine the involvement of the highly dynamic and penetrative TEPs in blastocyst hatching. As described above, the hamster blastocysts hatch in a deflated state and therefore, blastocoelic pressure on zona during hatching is ruled out. Notwithstanding this, deflated blastocysts produce a nick in the zona, cause a focal lysis and completely lyse the zona. We have consistently observed the ‘focal’ lysis occurring at the TEP-associated ZP zone at the abembryonic pole. The highly extendible TEPs (up to 17 m in length; Gonzales et al., 1996b) are capable of reaching the zona which is approximately 5 m in thickness (Green, 1997) through the open peri-vitelline space to cause focal and then global lysis of the zona. One of our interesting observations is that TEPs predominantly harbor hatching-associated zona lysing molecules such as cathepsin-L, cathepsin-B and cathepsin-P (Sireesha et al., 2008) and other critical regulators such as ER␣, COX-2 and NFB (Sen Roy and Seshagiri, unpublished; see Fig. 2). Importantly, we believe that TEPs function as cargo carriers for the endogenous, embryo-derived zona lysins and act to deliver them to the ZP causing its lysis. TEPs could therefore potentially be playing a novel role as transporters of zona lysins onto the ZP during hatching (Fig. 2). TEP biology in the context of development, hatching and implantation of the blastocyst remains to be thoroughly investigated and a number of unanswered questions remain. These include (i) the basis of their spatiotemporal localization in the context both of embryonic polarity and of embryonic orientation at implantation into the endometrium, (ii) the molecular and cytoskeletal regulation of TEP biogenesis, and (iii) the in vivo functional significance of the TEP during the critical period of hatching across different species. Unfortunately, TEPs are not intensely investigated despite their profound biological significance. The reasons for this could be that most studies, with a few exceptions (Gonzales et al., 1996b), could have overlooked these structures. Their occurrence is limited to a narrow time window (for example about 4 h in hamsters) and they exhibit highly transient and microscopic appearances, with cycles of protrusions and retractions, occurring about every 4 min during embryonic development (Gonzales et al., 1996b). 7. Summary and future directions The phenomenon of blastocyst hatching still remains as one of the least studied aspects of early mammalian development, despite the fact that it is of paramount importance in the series of implantation events during the early establishment of pregnancy. Molecular signaling pathways regulate peri-hatching blastocyst development and a number of players are involved in hatching (Fig. 2). These include cathepsin proteases in cooperation with other potential zona lysins, a panel of growth factors (HBEGF, LIF), transcription factors (ER-␣, NFB), or ubiquitous second messengers (prostanoids) and actin-remodeling
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proteins. Together, these factors are responsible for the timely genesis of transient but functionally important TEPs, which appear to act as carriers for the deposition of key molecules into the ZP to induce its lysis during hatching (Fig. 2). Moreover, it is necessary to examine hitherto unstudied signaling mechanisms regulating proteases, particularly the role of cryptic players such as tumor suppressor p53 that regulates LIF expression (Wenwei et al., 2007). In the future, a high-throughput transcriptome (or proteome) assessment of the peri-hatching embryonic stages and its functional correlations in the context of hatching and implantation would be exciting possibilities in the cell and developmental biology of the phenomenon of blastocyst hatching in mammals. Acknowledgements Financial support from the Department of Science and Technology, New Delhi is gratefully acknowledged. The authors thank Mr. B. Sukesh for technical support and Ms. M.S. Padmavathi for her help during the preparation of the manuscript. References Afonso, S., Romagnano, L., Babiarz, B., 1997. The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation. Development 124, 3415–3425. Bergstrom, S., 1972. Shedding of the zona pellucida of the mouse blastocyst in normal pregnancy. J. Reprod. Fertil. 31, 275–277. Blandau, R.J., 1949. Observations on implantation of the guinea pig ovum. Anas. Rec. 103, 19–47. Blandau, R.J., Rumery, R.E., 1957. The attachment cone of the guinea pig blastocyst as observed under time-lapse cinematography. Fertil. Steril. 8, 570–585. Boatman, D.E., 1987. In vitro growth of non-human primate pre- and peri-implantation embryos. In: Bavister, B.D. (Ed.), The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation. Plenum Press, New York, pp. 273–308. Carnevali, O., Cionna, C., Tosti, L., Lubzens, E., Maradonna, F., 2006. Role of cathepsins in ovarian follicle growth and maturation. Gen. Comp. Endocrinol. 146, 195–203. Chapman, H.A., Riese, R.J., Shi, G.P., 1997. Emerging roles for cysteine proteases in human biology. Annu. Rev. Physiol. 59, 63–88. Denker, H.W., Fritz, H., 1979. Enzymatic characterization of rabbit blastocyst proteinase with synthetic substrates of trypsin-like enzymes. Hoppe Seylers Z. Physiol. Chem. 360, 107–113. Dickinson, D.P., 2002. Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease. Crit. Rev. Oral. Biol. Med. 13, 238–275. Enders, A.C., Schlafke, S., 1967. A morphological analysis of the early implantation stages in the rat. Am. J. Anat. 120, 185–226. Green, P.L.D., 1997. Three-dimensional structure of the zona pellucida. Rev. Reprod. 2, 147–156. Gonzales, D.S., Boatman, D.E., Bavister, B.D., 1996a. Kinematics of trophectoderm projections and locomotion in the peri-implantation hamster blastocyst. Dev. Dyn. 205, 435–444. Gonzales, D.S., Jones, J.-M., Pinyopummintr, T., Carnevale, E.M., Ginther, J., Shapiro, S.S., Bavister, B.D., 1996b. Trophectoderm projections: a potential means for locomotion, attachment and implantation of bovine, equine and human blastocysts. Hum. Reprod. 11, 2739–2745. Hartshorne, G.M., Edwards, R.G., 1991. Role of embryonic factors in implantation: recent developments. Baillieres Clin. Obstet. Gynaecol. 5, 133–158. Huang, J.C., Wun, W.S., Goldsby, J.S., Matijevic-Aleksic, N., Wu, K.K., 2004. Cyclooxygenase-2-derived endogenous prostacyclin enhances mouse embryo hatching. Hum. Reprod. 19, 2900–2906. Ichikawa, S., Shibata, T., Takehara, Y., Tamada, H., Oda, K., Murao, S., 1985. Effects of proteinase inhibitors on preimplantation embryos in the rat. J. Reprod. Fertil. 73, 385–390. Izquierdo-Rico, M.J., Jiménez-Movilla, M., Llop, E., Pérez-Oliva, A.B., Ballesta, J., Gutiérrez-Gallego, R., Jiménez-Cervantes, C., Avilés, M.,
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Cellular and molecular regulation of mammalian blastocyst hatching Polani B. Seshagiri ∗ , Shubhendu Sen Roy, Garimella Sireesha, Rajnish P. Rao Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India
a r t i c l e
i n f o
Article history: Received 11 March 2009 Received in revised form 7 May 2009 Accepted 21 June 2009 Keywords: Blastocyst Hamster Hatching Protease Trophectodermal projections
a b s t r a c t 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 actinbased 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-17 mediated estrogen receptor-␣ signaling and possibly NFB 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. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction During preimplantation development, the mammalian embryo remains enclosed in a glycoproteinaceous coat, the zona pellucida (ZP), from which it hatches prior to implantation into the receptive maternal uterine endometrium (Enders and Schlafke, 1967). Blastocyst hatching is a critical and tightly regulated phenomenon of fundamental importance for the subsequent viability and development of the embryo. Any dysregulation of the hatching process causes implantation failure leading to infertility (Petersen et al., 2005). The peri-hatching blastocyst development and implantation is governed by an extremely complicated but cooperative interplay of various cellular factors and biomolecules, which are regulated in a defined spatio-
∗ Corresponding author. Tel.: +91 80 309 2687; fax: +91 80 360 0999. E-mail address: [email protected] (P.B. Seshagiri). 0165-0378/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2009.06.264
temporal fashion (Simon et al., 1996; Hartshorne and Edwards, 1991; Seshagiri et al., 2002). Information on this developmentally critical phenomenon is scarce and hence it is necessary to investigate the cellular and molecular mechanisms of blastocyst hatching and zona lysis. In this article, we provide a comprehensive review on the current understanding of mammalian blastocyst hatching phenomenon with particular reference to early hamster development. 2. Biology of blastocyst hatching The mammalian blastocyst hatching phenomenon is intriguing despite the fact that ZP architecture is similar in most species (Green, 1997). This process is thought to be subject to both cellular and molecular control. Blastocyst hatching occurs by initial formation of a nick in the ZP, caused by a hydrostatic (mechanical) pressure exerted by the increasingly expanding blastocyst. Thus, the blas-
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Fig. 1. Confocal microscopic images of a hatched hamster blastocyst. (A) DIC image displaying finger-like trophectodermal projections (TEPs; arrow head), and (B) phalloidin-FITC (green) localization of actin-based TEPs and nuclear counterstaining with propidium iodide (PI, red). Note the predominant appearances of the TEPs at the abembryonic pole, at the opposite side of the embryonic pole (the embryonic half below the black line, with a greater number of PI-stained nuclei). Bar = 10 m, photographed with Zeiss confocal microscope.
tocyst gradually egresses from the zona and, in most in vitro cultured hatching blastocysts, the ZP remains largely intact. This hatching behavior is observed in the mouse/rat (Bergstrom, 1972; Seshagiri et al., 1999; Surani, 1975), cow (Massip and Mulnard, 1980), rhesus monkey (Seshagiri and Hearn, 1993) and human (Lopata and Hay, 1989). Moreover, it is demonstrated that both the hatching blastocyst and the endometrium produce potential zona lysins involved in hatching (Denker and Fritz, 1979; Ichikawa et al., 1985; Joshi and Murray, 1974; Menino and Williams, 1987; Mishra and Seshagiri, 2000a; Sawada et al., 1990; Vu et al., 1997). Interestingly, hatching in the golden hamster blastocyst is quite unique in that the blastocyst initially expands, followed by a very conspicuous deflation prior to hatching (Gonzales et al., 1996a; Kane and Bavister, 1988; Mishra and Seshagiri, 1998). Moreover, the peri-hatching blastocyst invariably exhibits extensive membranous extensions of the trophectoderm called trophectodermal projections (TEPs) and there is a complete dissolution of the ZP after post-hatching (Fig. 1). In this regard, the phenomenon of blastocyst hatching in hamsters is quite remarkable and a peculiar temporal regulation of cellular and molecular factors governs this event (Fig. 2). The exact mechanism is unknown but several hatching-associated factors coupled with TEPs, which may function as possible cargo carriers for hatching regulators, could be playing major roles in the hatching process.
pierce the ZP at the abembryonal pole and exhibit undulating movements during ZP penetration, following zona escape (Blandau, 1949; Blandau and Rumery, 1957; Spee, 1883). The biology of TEPs in the context of blastocyst hatching in hamsters was extensively pursued by Bavister and co-workers (Gonzales et al., 1996a,b) and also our group (Fig. 1; Mishra and Seshagiri, 1998; Seshagiri et al., 1999; Sireesha et al., 2008). The kinematics of TEPs during hatching has been examined and their criti-
3. Cellular factors and TEPs in blastocyst hatching During the period of blastocyst hatching, a cellular phenomenon involving TEPs has been demonstrated in a number of species (Gonzales et al., 1996b). This was first described in guinea pig blastocysts (Spee, 1883), and their existence was subsequently verified in other species (Blandau, 1949). Early findings showed that TEPs
Fig. 2. A hypothetical working model showing the roles of molecular regulators of embryo and endometrial origin in protease-mediated blastocyst hatching in the golden hamster. Abbreviations used are: cyclooxygenase-2 (COX-2), estrogen receptor-␣ (ER-␣), heparin binding-epidermal growth factor (HB-EGF), leukemia inhibitory factor (LIF), nuclear factor B (NFB) and transforming growth factor- (TGF-).
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Fig. 3. Effect of estrogen receptor antagonist (ICI 182, 780) on hamster blastocyst hatching. Panel A depicts percentages of blastocyst hatching in the absence or presence of the inhibitor. The antagonist (150 nM) inhibits hatching by about ∼80% when cultured for either 12 h ( ) or 24 h (). After culture in the presence of the inhibitor for 12 h ( ) or 24 h ( ) when embryos were transferred back to a fresh medium, they exhibited hatching. Panel B shows photomicrographs of embryos cultured in the absence (a and b) or presence (c and d) of the inhibitor. Control embryo (a) develop to blastocyst and hatch in about 24 h (b). The embryo (c) cultured in the presence of the inhibitor deflates but fail to hatch (d). Bar = 5 m.
cal role in normal hamster blastocyst hatching has been established. Also, their existence in other species such as bovine, equine, human, monkey and guinea pig was demonstrated (Gonzales et al., 1996a,b; Spee, 1883). Studies on the critical functional role of TEPs in the hatching phenomenon are extremely sparse in vitro and more importantly in vivo and these remain to be investigated. However, trophoblast-derived cellular protrusions extending into the endometrium during early implantation in rats (Enders and Schlafke, 1967), and similarly in mice (McRae and Church, 1990) were demonstrated. In addition TE-derived, actin-based cytoskeletal structures, named filopodia in blastocysts, traverse into the inner cell mass (ICM) and are involved in signal transduction as well as in the positioning of the eccentric ICM (Salas-Vidal and Lomeli, 2004). In view of the above, it is exciting to study the involvement of TEPs and filopodia in hatching phenomenon of developing peri-implantation blastocysts, in parallel with molecular regulators of blastocyst hatching. 4. Molecular regulators influencing blastocyst hatching One of the important aspects of blastocyst hatching is the involvement of regulatory molecules, primarily embryotrophic factors, which encompass second messengers, transcription factors, proteases, growth factors and cytokines (Kane et al., 1997; Sargent et al., 1998; Seshagiri et al., 2002; Simon et al., 1996). Our studies with cultured hamster embryos show that epidermal growth factor (EGF), heparin binding-EGF (HB-EGF), transforming growth factor- (TGF-) and leukemia inhibitory factor (LIF) accelerate hatching, associated with a complete dissolution of zona, predominantly brought about by embryo-derived cysteine protease-like activities (Mishra and Seshagiri, 2000b; Seshagiri et al., 2002). Moreover, our studies with
the estrogen receptor-␣ (ER-␣) antagonist (ICI 182, 780) demonstrate a profound reversible inhibition of hamster blastocyst hatching (Fig. 3), thereby ascribing a definitive regulatory role to ER-␣ in the process. Similarly, our ongoing studies indicate that inhibition of cyclooxygenase-2 (COX-2) or nuclear factor B (NFB) profoundly affect blastocyst hatching associated with a failure of deflation of hatching blastocysts (Sen Roy and Seshagiri, unpublished). Consistent with this is the observation made in the mouse that COX-2 derived prostaglandin (PG)-I2 , is crucial for peri-implantation development, including blastocyst hatching (Huang et al., 2004; Pakrasi and Jain, 2007). Unlike in the mouse (Thomas et al., 1997), zona dissolution in the hamster is predominantly protease-dependent and it is not brought about by any chemical means such as reactive oxygen species (Mishra and Seshagiri, 2000a). Various classes of proteases such as serine-, cysteine- or metallo-proteases are involved in hatching, depending on the species (Denker and Fritz, 1979; Ichikawa et al., 1985; Kimie et al., 1994; O’Sullivan et al., 2002; Perona and Wassarman, 1986; Sireesha et al., 2008). It is evident that key embryotrophic molecular regulators such as growth factors, cytokines, transcription factors and importantly cysteine proteases are critical for hatching (Fig. 2). 5. Cysteine proteases (cathepsins) in development and blastocyst hatching The role of cysteine proteases during embryogenesis is diverse. They are involved in follicular maturation and ovulation (Carnevali et al., 2006), fertilization (Ichikawa et al., 1985), preimplantation development (Afonso et al., 1997), including blastocyst hatching (Mishra and Seshagiri, 2000a; Sireesha et al., 2008) and trophoblast invasion and placentation (Sol-Church et al., 2002). Dysregulation of proteases during embryogenesis can lead to preg-
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nancy failure. Cysteine proteases, belonging to 6 clans and 43 families, are phylogenetically ubiquitous enzymes, broadly classified into legumains, caspases, calpains, and cathepsins (reviewed by Dickinson, 2002). Collectively, they are mainly involved in housekeeping functions (Chapman et al., 1997). At least in hamsters, a speciesspecific, blastocyst-derived cysteine protease-like activity is responsible for hatching of blastocysts and we earlier showed that several pan-cysteine protease inhibitors such as antipain, leupeptin, E-64 and P-hydromercuribenzoate suppressed cultured blastocyst hatching without affecting their development, while there was a negligible or no curtailment of hatching when inhibitors to other classes of proteases were used (Mishra and Seshagiri, 2000a; Sireesha et al., 2008). Our recent data show that blastocyst-derived cathepsins, presumably secretory or TEP-associated types, are involved in blastocyst hatching and zona lysis (Sireesha et al., 2008). Using cathepsin-class specific inhibitors such as cystatins and FYAD, we unequivocally showed the expression and critical involvement of cathepsins in hamster blastocyst hatching, unlike other cysteine-class enzymes such as calpains and caspases (Sireesha et al., 2008). It is significant to note that cathepsins are crucial for hatching, an event occurring immediately prior to the invasion events of blastocyst implantation, which are heavily dependent on cysteine (cathepsin) proteases as well as other proteases (Chapman et al., 1997). Interestingly, the ZP shows an extraordinary sensitivity to exogenous exposure of cathepsins (L, B and P) in hamsters, with effects mediated within seconds in this species compared with 10 min in other species. The hamster ZP dramatically exhibits complete dissolution, as observed with in vivo hatching (Sireesha et al., 2008). It appears that the mouse ZP is relatively less susceptible to proteolysis than hamster ZP (Mishra and Seshagiri, 2000a; Sireesha et al., 2008), indicating that there could be differences in ZP composition or architecture across species. For example, an additional ZP protein (ZP-4) in hamster ZP composition (Izquierdo-Rico et al., 2009) may make the hamster ZP differentially susceptible to protease digestion during hatching. 6. Functional association of TEPs and molecular regulators in blastocyst hatching The functional significance of actin-based TEPs associated with peri-hatching blastocyst developmental stages is not clearly established. The TEPs are thought to play a critical role in zona escape and in the initial attachment of the embryo to the uterine luminal epithelium (Blandau and Rumery, 1957; Sireesha et al., 2008; Spee, 1883). In addition, TEPs might be involved in the uniform spacing of implanting embryos and/or choosing functionally suitable endometrial sites for implantation. Also, TEPs could play an important role in the absorption of nutrients from the endometrium and in the embryo-maternal cellular and molecular dialogue during implantation (Hartshorne and Edwards, 1991; Seshagiri and Hearn, 1995). It is interesting to point out that the appearance of TEPs in hatching blastocysts at the abembryonic (mural) pole coincides with blastocyst orientation at implantation, as observed in
guinea pigs (Blandau and Rumery, 1957; Spee, 1883) and hamsters (Gonzales et al., 1996b; Fig. 1). Conversely, in primates where implantation occurs at the embryonic (polar) pole, the TEPs are also observed at the same pole (Boatman, 1987; Gonzales et al., 1996b). It is of great interest to examine the involvement of the highly dynamic and penetrative TEPs in blastocyst hatching. As described above, the hamster blastocysts hatch in a deflated state and therefore, blastocoelic pressure on zona during hatching is ruled out. Notwithstanding this, deflated blastocysts produce a nick in the zona, cause a focal lysis and completely lyse the zona. We have consistently observed the ‘focal’ lysis occurring at the TEP-associated ZP zone at the abembryonic pole. The highly extendible TEPs (up to 17 m in length; Gonzales et al., 1996b) are capable of reaching the zona which is approximately 5 m in thickness (Green, 1997) through the open peri-vitelline space to cause focal and then global lysis of the zona. One of our interesting observations is that TEPs predominantly harbor hatching-associated zona lysing molecules such as cathepsin-L, cathepsin-B and cathepsin-P (Sireesha et al., 2008) and other critical regulators such as ER␣, COX-2 and NFB (Sen Roy and Seshagiri, unpublished; see Fig. 2). Importantly, we believe that TEPs function as cargo carriers for the endogenous, embryo-derived zona lysins and act to deliver them to the ZP causing its lysis. TEPs could therefore potentially be playing a novel role as transporters of zona lysins onto the ZP during hatching (Fig. 2). TEP biology in the context of development, hatching and implantation of the blastocyst remains to be thoroughly investigated and a number of unanswered questions remain. These include (i) the basis of their spatiotemporal localization in the context both of embryonic polarity and of embryonic orientation at implantation into the endometrium, (ii) the molecular and cytoskeletal regulation of TEP biogenesis, and (iii) the in vivo functional significance of the TEP during the critical period of hatching across different species. Unfortunately, TEPs are not intensely investigated despite their profound biological significance. The reasons for this could be that most studies, with a few exceptions (Gonzales et al., 1996b), could have overlooked these structures. Their occurrence is limited to a narrow time window (for example about 4 h in hamsters) and they exhibit highly transient and microscopic appearances, with cycles of protrusions and retractions, occurring about every 4 min during embryonic development (Gonzales et al., 1996b). 7. Summary and future directions The phenomenon of blastocyst hatching still remains as one of the least studied aspects of early mammalian development, despite the fact that it is of paramount importance in the series of implantation events during the early establishment of pregnancy. Molecular signaling pathways regulate peri-hatching blastocyst development and a number of players are involved in hatching (Fig. 2). These include cathepsin proteases in cooperation with other potential zona lysins, a panel of growth factors (HBEGF, LIF), transcription factors (ER-␣, NFB), or ubiquitous second messengers (prostanoids) and actin-remodeling
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