| dc.description.abstract | 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) | en_US |
