The mammalian embryo is encased in a glycoproteinaceous covering, the zona pellucida (ZP/zona) during preimplantation development. Prior to implantation into the recipient maternal endometrium, the blastocyst has to hatch out of this zona. This is a critical and an important event for the successful establishment of pregnancy. Hatching in mammals is characterised by the expansion of the blastocyst, followed by the nicking of the zona and extrusion of the blastocyst by repeated contraction-expansion cycles, thereby leaving the empty zona behind. In species such as the mouse, cow and primates, the empty zona is left behind in the uterine lumen. However, in the hamsters, the features associated with hatching are characteristic for this species. Firstly, the blastocyst remains predominantly in a deflated state. Secondly, the zona undergoes focal rupture which is followed by the complete dissolution of the zona. Third, trophectodermal projections (TEPs) present in the blastocysts, aid the hatching of the blastocyst. Hence, this study was aimed to identify the molecular players involved in hamster blastocyst hatching and to study their embryo-endometrial expression. Earlier work in the laboratory has demonstrated the involvement of cysteine protease-like factors in hamster blastocyst hatching (Mishra and Seshagiri, 2000a). Broad spectrum cysteine protease inhibitors, E-64 and PHMB, completely blocked the hatching of blastocysts. To identify the class of cysteine proteases involved in this phenomenon, class-specific inhibitors were used in this study. Calpain and caspase inhibitors, calpastatin and Z-VAD- FMK, respectively, did not block hamster blastocyst hatching (Fig 2.2). Cathepsin (cts)-specific inhibitors, cystatin-C and peptidyl diazomethane (PPDM) blocked hatching of embryos in a dose-, time- and embryo-stage dependant manner (Figs 2.5, 2.6, 2.10 and 2.11). Continuous exposure of 1.0 µM cystatin to expanded or deflated blastocysts completely blocked hatching (Fig 2.3Aii, iii), without effecting their viability (Fig 2.3Bii and iii). Deflated blastocysts exposed transiently to 1.0 µM cystatin, for 12 or 6 h failed to hatch, but could overcome the inhibition and exhibited hatching, when transferred to fresh, inhibitor-free medium (Fig 2.6). Effect of the inhibitor was less pronounced in the deflated blastocysts when compared to expanded blastocysts (Figs 2.5 and 2.6). The viability of the cystatin-treated embryos was not affected as assessed by vital-dye staining and also their ability to attach and exhibit trophoblast (TB) proliferation on serum-coated dishes was not compromised. The area of the TB outgrowth of cystatin-treated embryos was similar to that of the untreated embryos (Fig 2.9). The inhibitory effect of PPDM, an irreversible inhibitor of cts, on blastocyst hatching was demonstrated. Expanded and deflated blastocysts exhibited a dose-dependent inhibition in hatching, following the exposure to 0.5 and 1.0 µM of PPDM (Figs 2.10A and 2.11A). When treated with the inhibitor for 6 or 12 h, there was a transient inhibition in hatching, as blastocysts could overcome the inhibition and exhibit hatching following transfer to inhibitor-free, fresh medium. Inhibitor-treated hatched blastocysts, when transferred to serum-coated dishes, attached and exhibited TB outgrowth, similar to untreated embryos (Figs 2.13 and 2.14). A PPDM-interacting protease was localised to the cytoplasm of the embryonic cells in the hamster blastocyst, suggesting that the embryo is the source of the zona lysin. Two forms of the enzyme, a probable variant zymogen of molecular mass 65 k and an active form of molecular mass 32 k were detected in the blastocysts (Fig 2.15). In vitro susceptibility of hamster zona to cathepsins is significantly different from that of other species zonae such as the mouse, rat, monkey and human zonae (Table 2.2). All these lines of evidence unequivocally demonstrate the involvement of cathepsins in hamster blastocyst hatching, which is in sharp contrast to what is observed in the mouse, where serine proteases such as strypsin/hepsin, ISP-1 and -2 are reported to play an important role in blastocyst hatching. However, since extensive inhibitor studies were not performed using embryos from other species, it is possible that cysteine proteases maybe involved in the hatching of blastocysts from other species. Having shown the role of cathepsins in hamster zona dissolution, expression of the cathepsins in preimplantation embryos was investigated. Hamster specific cts–L, -B and –P were amplified from day 14 placenta using mouse primers and the amplicons were found to be highly homologous to the cts of other species (Fig 3.2). Hamster and mouse preimplantation embryos i.e., 8-cell, morula and blastocyst were found to express cts–L, -B and –P transcripts (Figs 3.6 and 3.10). Cts-P, present only in the TBs of the placenta (Fig 3.4), for the first time, was also shown to be present in the preimplantation embryos. The immunoreactive cts-L and -P proteins were detected in blastomeres of 8-cell embryo, in the inner cell mass (ICM) and trophectoderm (TE) of the blastocyst (Figs 3.7 and 3.8). These cathepsins could probably correspond to the PPDM- interacting enzymes of molecular mass 32 and 65 kDa, described above. (fig) Fig 5.1. Overview of the expression and the role of embryo-endometrial cathepsins in blastocyst hatching in the golden hamster. Cathepsins ( ) produced by the inner cell mass ( ) or the trophectoderm ( ) of the blastocyst or the endometrial cells ( ) act on the zona matrix ( ), bringing about its lysis. The cathepsins are secreted into the peri-vitelline space or are carried by trophectodermal projections (TEPs, yellow projections) to the zona. Also shown are endogenous inhibitors and growth factors that can regulate these cathepsins. A striking observation made in this study was the detection of the immunoreactive signals for cathepsins in the zona matrix of blastocysts. Since hamster blastocysts possess extracellular projections (TEPs), it is possible for these projections to participate in the transport of cathepsins from TE cells to the zona; as the localisation of the proteases to these projections was demonstrated (Fig 3.9). Also, since the actin-based projections are highly undulating structures, they might potentiate the mechanical rupture of the zona during hatching, apart from acting as carriers for the proteases. Hence, during hatching of the hamster blastocyst, cathepsins, expressed in the ICM and the TE, might be secreted transiently into the peri-vitelline space, whereby they can act on the ZP. Alternatively, in the absence of any apoptotic cells in the embryo that can release the cell contents (Fig 3.13), the cathepsins may be deposited by TEPs in specific pockets of the zona matrix, thereby causing focal zona lysis. In vivo, the hatching of the blastocysts is brought about by both embryonic and maternal proteases. Cts–L and -B transcripts were detected in the maternal endometrium during different stages of the reproductive cycle and early pregnancy (Fig 4.1 and 4.3). Immunoreactive cts-L protein was detected in the uterine luminal epithelium and the stromal cells (Fig 4.5). In the uterus, the PPDM-interacting 32 kDa form was in abundance compared to the 65 kDa form (Fig 4.6). Hence, uterine cathepsins might play a major role in the remodelling of the extracellular matrix during estrous cycle and pregnancy. However, the role of these cathepsins in causing zona dissolution during blastocyst hatching, along with embryonic proteases cannot be ruled out. Reports of recurrent miscarriages in women with low serum cystatin levels imply a role for cysteine proteases in early pregnancy events like blastocyst development, hatching and implantation. Hence, these studies, described in the thesis, could form a basis to investigate the role of cathepsins in early human development. Taken together, the results demonstrate the involvement of embryo-derived cathepsins in hamster blastocyst hatching. These cathepsins may be secreted into the peri-vitelline space or transported to the zona matrix by TEPs (Fig 5.1). Additionally, in vivo, endometrial cathepsins might aid the embryonic zona lysins in the complete zona dissolution. The regulation of these proteases by growth factors, cytokines and their specific inhibitors needs to be explored. (For figure pl see the original document)

The zona-pellucida (zona, ZP) is an adhesion-refractory, acellular coat enclosing the rapidly growing, free-living mammalian preimplantation embryo which undergoes successive cleavage divisions to form the blastocyst, composed of ICM-cells surrounded by outer TE-cells. For further development, the blastocyst must ‘hatch’ or ‘escape’ out of the zona before it can implant into the endometrium for further development (Fig. 5.1A). Hence, the event of hatching or ‘zona escape’ assumes critical importance for the establishment of a successful pregnancy. The golden-hamster blastocyst offers a very unique paradigm to understand hatching, whereby upon attainment of a fully-expanded state, the blastocyst undergoes a dramatic (and molecularly unexplained) deflation event, followed by appearance of TE-derived dynamic cellular projections called TE-projections, whose appearance in an embryonic-stage and -time dependant manner suggest an intimate association with the hatching phenomenon (Fig. 5.1B). Thirdly, embryo-derived zonalytic proteases have been shown to bring about a focal-lysis of the ZP followed by global zona dissolution. Earlier work in the laboratory had demonstrated the intimate involvement of signaling molecules like LIF, HB-EGF, TGF-β and (ER)-α with hatching (Seshagiri et al., 2002, 2009). Investigations also revealed the involvement of cysteine-proteases of the cathepsin (cts) family, especially cts-L, -B and-P to be involved in zona lysis (Sireesha et al., 2008). In order to achieve a better understanding of mammalian preimplantation development, especially hatching, it was important to investigate the role and impact of other critical regulators of developmental and reproductive physiology. COX-2 is one such key signaling moiety and it was decided to investigate the role, if any, of COX-2 and its derived PGs in hamster peri-implantation events. COX-2 transcripts and immunoreactive COX-2 protein were detected in the different preimplantation stages, from 8-cell onwards. COX-2 protein was abundant in both the ICM and TE, but was especially enriched in the TE-cells of the late blastocyst. In order to investigate the function of this enzyme in preimplantation development and hatching, two very-specific inhibitors of COX-2 catalytic action, NS-398 and CAY-10404, were tested in identical concentrations of 25, 50 and 75 μM on in vitro cultured hamster blastocysts. In order to assess the impact of COX-2 inhibition on an embryo-stage and time-dependant manner, inhibitors were tested on freshly recovered 8-cell embryos or early blastocysts, continuously for 72 h or 48 h, respectively. COX-2-selective inhibitors inhibited hamster blastocyst hatching in a dose-dependant manner with maximum inhibition observed in the 75 μM dose. Surprisingly, there was a profound dose-dependent failure of deflation of late-blastocysts upon inhibitor treatment and embryos which hatched, did so in an inflated state and retained intact zonae in cultures. Moreover, embryos subjected to NS-398 treatment phenocopied those subjected to CAY-10404 treatment. Results demonstrate that the effect of inhibitors, and hence the need for COX-2 mediated signaling events is more pronounced in 8¬cell embryos than with early-blastocysts, indicating that COX-2 dependant molecular and cellular processes required for blastocyst morphogenesis and ZP-lysis may have been initiated prior to compaction and cavitation. The reversal of effects of COX-2 inhibition on hatching with exogenous addition of PGE2 and Iloprost (a stable PGI2 analogue) to inhibitor cultures, show that COX-2-derived eicosanoids could, in effect bring about hamster hatching, which is in agreement with previous reports (Davis et al., 1999) and augment peri-implantation development including hatching (Huang et al., 2003). Additionally, it has been successfully demonstrated that PGE2 was superior to PGI2 in augmenting blastocyst hatching in inhibitor-cultures. In this study, the modulation of critical cts-L, -B, -P proteases in COX-2 mediated hamster zona hatching has been verified by quantifying cts in transcripts in control and inhibitor-subjected embryonic samples which was further substantiated by the decreased intra-embryonal protein levels of cts-L and -P. These results demonstrate that COX-2 mediated signaling components directly and effectively modulate hamster preimplantation development, especially zona-hatching phenomenon by transcriptional-regulation of the critical zonalytic proteases. Another potential hatching-associated molecule i.e., NF-κB which is known to exert a great deal of influence on overall reproductive and developmental biology, was investigated in this study. Its specific effects on mammalian preimplantation development, especially hatching, remain totally uninvestigated. This formed the rationale to investigate the reach and impact of NF-κB signaling network in the modulation of peri-hatching events. Transcripts and immunoreactive NF-κB protein of crucial pathway-components like IKK, IκB-β and RelA were detected from 8-cell embryo to the zona-free blastocyst. In order to ascertain the impact of NF-κB signaling on peri-hatching events, two very-specific inhibitors of the NF-κB pathway, BAY-11-7082 and JSH-23 were employed which acted at two strategic signaling points. In order to assess the impact of NF-κB inhibition on an embryo-stage and time-dependant manner, inhibitors were tested on freshly recovered 8-cell embryos or early blastocysts, continuously for 72 h or 48 h, respectively. NF-κB-selective inhibitors inhibited blastocyst hatching in a dose-dependent manner. Interestingly, a profound dose-dependent failure of deflation of late-blastocysts upon inhibitor treatment was observed and embryos which hatched did so in an inflated state and also retained intact zonae in cultures. Moreover, embryos subjected to BAY-11-7082 treatment phenocopied those subjected to JSH-23 treatment, indicating specificity of inhibitor action. Time-course experiments demonstrated that the need for efficient NF-κB mediated signaling is distinctively more for 8-cell embryos than early-blastocysts, indicating that NF-κB dependant molecular and cellular processes required for blastocyst morphogenesis and ZP-lysis may have been initiated prior to compaction and cavitation. Moreover, modulation of zonalysins cts-L, -B and -P by NF-κB-signaling, during the event of zona lysis, both by real-time quantitation of its transcripts and intracellular protein levels has been demonstrated. These results demonstrate that NF¬κB mediated signaling components directly and effectively modulate hamster preimplantation development, especially zona-hatching phenomenon by transcriptional-regulation of the critical zonalytic proteases. The profound inhibition of hatching and effects on blastocyst morphogenesis observed by inhibition of COX-2 and NF-κB signaling systems demonstrate a fundamental need of the growing embryo for these critical signaling moieties. Moreover, the underlying similarity of consequences obtained upon inhibition of both signaling networks i.e., NF-κB and COX-2, perhaps indicate a linear mode of signaling between these principles. It remains to be tested, though, if it really is the case. A striking observation made in this study was the detection of immunoreactive signals for critical signaling moieties like ER-α, COX-2 and RelA onto TEPs of the deflated hamster blastocyst, in addition to earlier TEP-localisation of cathepsins. A B C (Figure) ENDOMETRIUM ENDOMETRIUM ENDOMETRIUM Fig 5.1. Schematic representation of the role of molecular and cellular factors in the regulation of concordant phenomena of mammalian blastocyst hatching and endometrial implantation. (A) Depicts a zona-intact well-formed blastocyst. Preimplantation embryo development and blastocyst formation involves close cooperation between several molecular principles (discussed in sections 1.3.1 to 1.3.3), (B) as the embryo prepares to hatch, prior to implantation, it initiates egression from the non-adhesive ZP coat by cathepsin (cts) protease-mediated lysis of zona (pink circles); there is concomitant appearance of cellular principles such as TEPs (undulating projections shown in green). Of interest is the intimate association of hatching-promoting molecules such as COX-2, NF-κB, ER-α, Cts etc. with the TEPs. (C) depicts a zona-free, TEP-rich blastocyst initiating implantation into the maternal endometrium. It is possible, that the embryonic TEPs with the associated hatching-regulatory molecules are also critical for implantation phenomena during the embryo-maternal recognition and implantation during the establishment of early pregnancy. Preliminary results indicate that TEPs could be the site of membrane lipid-rafts, focal points of membrane-based signaling. The definitive role of TEPs in peri-hatching events is yet to be confirmed, but it is presumed that these actin-based undulating structures, harboring several key molecules involved in peri-implantation events in the embryo as well as the maternal uterus could be instrumental in successfully bringing about the concomitant processes of hatching and implantation. Interestingly, during rodent implantation (hamster, guinea-pig, mouse and rat), the blastocyst orients in such a way that the ICM is oriented away from the endometrium and, at least in the hamster, the TEP-carrying abembryonic (mural) pole remains closest to the luminal epithelium (LE) (Gonzales et al., 1996b; Seshagiri et al., 2009; Fig. 5.1C). In contrast, in humans and other primates, the embryonic pole is closest to LE before implantation (Kirby, 1971; Lee and DeMayo, 2004). Although direct evidence is lacking, but these observations gives rise to a possibility that both hatching and implantation could be intimately related to the polar appearance of TEPs in the embryo. Several key signaling molecules like ER-α, LIF, HB-EGF and TGF-β have been already demonstrated to play crucial roles in mammalian hatching. In this thesis, we have exemplified the need for COX-2 mediated prostanoid signaling and the pleotropic NF-κB signaling system in bringing about mammalian blastocyst hatching. How exactly do these molecular entities communicate among themselves and with cellular principles like TEPs thereby effectively enabling peri-implantation development, remain to be understood. Taken together, these results demonstrate, for the first time, the involvement of embryo-derived signaling molecules, like COX-2 and NF-κB in an embryo stage-and time-dependant manner in mammalian peri-implantation events, especially blastocyst hatching. The association of TEPs with key molecules common to embryonic and maternal preparation for hatching and implantation, respectively, indicates towards a molecular and cellular continuity between the concomitant events. These fundamental findings on hamster blastocyst biology have profound clinical implications in the management of human infertility. (For figures pl see the abstract file).