Molecular Human Reproduction, Vol. 6, No. 11, 1005-1012,
November 2000
© 2000 European Society of Human Reproduction and Embryology
Embryo development |
Evidence for the involvement of a species-specific embryonic protease in zona escape of hamster blastocysts
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore-560012, India
Abstract
The source and nature of zona lytic factors during zona escape of hamster blastocyts were investigated. When cultured in hamster embryo culture medium (HECM)-2h, all 8-cell embryos (n = 135) developed to zona escaped-blastocysts with complete zona lysis. In addition, 2-cell embryos, when co-cultured with zona escaping-blastocysts (at a ratio of 1:10), exhibited zona lysis. Various other embryos at the 1–8-cell stages also showed zona lysis when cultured with zona-escaping blastocysts. However, zonae from mice, rats, sheep and humans were resistant to lysis under these conditions. Pronase treatment resulted in rapid zona lysis in hamsters (7 ± 1 s), whereas in other species zona lysis was much slower: mouse (662 ± 27 s), rat (532 ± 16 s), sheep (120 ± 12 s) and human (104 ± 8 s). When cysteine protease inhibitors (antipain, leupeptin, E-64 and p-hydromercuricbenzoate) were tested, they completely inhibited zona escape, while trypsin inhibitors (TLCK and SBTI) did not. Uterine zona lysin contribution in zona escape was discounted since: (i) uterine luminal flushing and endometrial extract from day 4 (the time of zona escape in vivo) pregnant females failed to lyse zonae and (ii) endogenous oocytes and transferred 2-cell embryos (to day 3 pseudopregnant recipients) were all zona-intact, while 71% of transferred blastocysts exhibited zona escape, following their recovery after 24 h. These observations suggest that a species-specific, embryonic proteolytic factor, with a cysteine protease-like activity, is involved in the zona escape of blastocysts in hamsters.
blastocyst/hamster/hatching/protease/zona escape
Introduction
In mammals, the escape from the zona pellucida (ZP) or hatching enables the blastocyst to attach and invade the receptive uterine endometrium. This process is the rate-limiting step for implantation and establishment of early pregnancy. However, little is known about this critical developmental event occurring during the peri-implantation period because of the difficulties in studying this in vivo. The low success rate of implantation of IVF-produced embryos in assisted reproductive technology is believed to be partly due to the impairment of blastocyst hatching (Cohen et al., 1990
; Magli et al., 1998). There is a need to study and understand the cellular and molecular mechanism of mammalian zona escape.
In vitro, zona escape is known to occur in most species, including rodents (Bergstrom, 1972; Surani, 1975; Seshagiri et al., 1999), cow (Massip and Mulnard, 1980), pig (Niemann and Elsaesser, 1987), rhesus monkey (Seshagiri and Hearn, 1993), and human (Lopata and Hay, 1989), by shedding. In all these cases, the fully expanded blastocyst exerts mechanical pressure creating a nick in the zona and then it is actively extruded through this hole, leaving the empty zona intact. Nonetheless, there are reports proposing the involvement of protease-like hatching factor(s) of embryonic origin assisting in the rupture of zona in vitro (Denker and Fritz, 1979; Perona and Wassarman, 1986; Menino and Williams, 1987; Sawada et al., 1990; Vu et al., 1997). In addition, zona escape in vivo by uterine zona lysin has been proposed for mouse (Mintz, 1971), rat (Joshi and Murray, 1974), rabbit (Denker, 1977) and hamster (Orsini, 1965; Gonzales and Bavister, 1995; Thrasher et al., 1999).
In the golden hamster (Mesocricetus auratus), several interesting developmental features have been observed with regard to the zona escape of blastocysts in vivo (Parkening, 1976; Gonzales and Bavister, 1995). Firstly, zonae undergo gradual thinning characterized by global dissolution leading to their complete disappearance. Secondly, in contrast to other species, there is no blastocoelic expansion and by 3.5 days of pregnancy, most blastocysts (and even preblastocyst-stage embryos) are azonal. Thirdly, trophoectodermal projections of deflated blastocysts facilitate the process of zona escape (Gonzales et al., 1996). Complete disappearance of the zona, therefore, indicates the existence of a potent lytic factor of embryonic and/or endometrial origin to aid the removal of the zona barrier between trophectodermal cells and uterine epithelium. However, the source and the nature of such factor(s) associated with the phenomenon of zona escape, in vivo as well as in vitro, are virtually unknown in the hamster.
A high percentage of successful hamster blastocyst development (Ain and Seshagiri, 1997) and zona escape (Kane and Bavister, 1988) has been achieved in chemically defined culture media. We recently showed that blastocysts, cultured from the 8-cell stage in hamster embryo culture medium, (HECM)-2h, underwent 100% zona escape, accompanied by peri-attachment development (Mishra and Seshagiri, 1998). The developmental pattern observed in HECM-2h closely mimicked that seen by others in vivo (Gonzales and Bavister, 1995). This is characterized by: (i) blastocysts undergoing deflation followed by focal zona thinning preceding focal zona rupture; (ii) embryos emerging from their hemizona cases with zonal dissolution; (iii) non-starters, i.e. preblastocyst-stage embryos also exhibiting zona lysis in the presence of zona escaping-blastocysts; and (iv) azonal blastocysts having the ability to attach and exhibit trophoblast outgrowth (Mishra and Seshagiri, 1998; Seshagiri et al., 1999). These observations strongly indicate the possibility of de-novo production of zona lytic factor(s) by embryos undergoing zona escape in vitro.
As HECM-2h successfully supports the development of hamster embryos through the peri-attachment stages, it would be quite useful to exploit this culture system to study the cellular and molecular nature of the phenomenon of zona escape in vitro since this would be difficult to investigate in vivo. Therefore, the present study aimed to determine: (i) the production of zona lytic factors by hamster blastocysts; (ii) the biochemical nature and species specificity of such factor(s); and (iii) whether or not a uterine zona lysin contributes to the process of zona escape of blastocysts.
Materials and methods
Animals
Sexually mature golden hamsters, Swiss mice and Wistar rats maintained on a 14 h light:10 h dark lighting schedule (lights on at 06:00) and normal temperature (24–26°C), from our Institute colony were used for the present study. Procedures for handling and experimentation followed the guidelines on the Use of Laboratory Animals for Research (INSA, New Delhi, India).
Recovery and culture of embryos
Ovulation was stimulated in female hamsters with an i.p. injection of 25 IU pregnant mare's serum gonadotrophin on the day of post-oestrous discharge and mated 3 days later. Embryos developed in vivo were collected in the evening of day 2 (2-cell) and day 3 (8-cell) of pregnancy by flushing the excised reproductive tracts with 0.5–1.0 ml of equilibrated hamster embryo culture medium (HECM)-2h (Ain and Seshagiri, 1997; Mishra and Seshagiri, 1998). Unless indicated otherwise, reagents were purchased from Sigma Chemical Company (St Louis, MO, USA). The HECM-2h medium contains 116.5 mmol/l NaCl, 3.2 mmol/l KCl, 2 mmol/l CaCl2, 0.5 mmol/l MgCl2.6H2O, 25 mmol/l NaHCO3, 10 mmol/l sodium lactate, 0.25 mmol/l sodium pyruvate, 1 mmol/l glutamine, 0.5 mmol/l succinate, 0.1 mg/ml polyvinyl alcohol, 100 IU/ml penicillin G, 3 mg/ml bovine serum albumin (BSA, fraction V), 3 µmol/l pantothenate, 5 µmol/l choline chloride, 3 µmol/l inositol and 0.1 mmol/l MEM non-essential amino acids (No 11140–019; Gibco BRL Life Technologies Inc, Grand Island, NY, USA).
Flushings were collected into organ-tissue culture dishes (Falcon Plastics, Oxnard, CA, USA) containing equilibrated silicone oil. The collection procedure was performed in a room equipped with orange-red filters over all light sources to protect embryos from light damage. Embryos (2- or 8-cell) were selected for culture in 100 µl drops of the desired culture medium (without or with test compounds), overlaid with 2.5 ml of silicone oil (Sigma) in 35 mm plastic culture dishes (Greiner Labortechnik, Frickenhausen, Germany). Dishes were placed in an incubator maintained at 37°C with a humidified atmosphere of 5% CO2 in air. Embryo development was monitored every 12 h until 72 h using a Olympus IMT-2 inverted microscope equipped with Nomarski DIC optics (Mishra and Seshagiri, 1998).
Co-culture of preblastocysts/oocytes with zona escaping-blastocysts
Eight-cell embryos were cultured in HECM-2h for a period of 48 h (when blastocysts initiate zona escape in vitro; Mishra and Seshagiri, 1998). At this time, freshly recovered 2-cell embryos were added to the culture drop in a ratio of 10:1 (blastocyst:2-cell) and were co-cultured for an additional 48 h. As controls, 2-cell embryos from the same donor were cultured independently in adjacent drops. Embryo development and changes in the integrity of zona were observed microscopically. In addition, 1-, 3-, 4-, 5- and 8-cell embryos were also co-cultured with zona escaping-blastocysts and monitored for signs of zona lysis. Similarly, in-vivo developed 2-cell embryos from rat/mouse and oocytes from human/sheep were co-cultured with zona escaping-hamster blastocysts. Changes in the integrity of zonae were recorded. Rejected human oocytes from an IVF programme (Hope Infertility Clinic, Bangalore, India) and follicular oocytes from sheep ovaries (local abattoir) were used both in this and the following experiments as a source of zonae from these species (see below).
Lysis of zonae by proteases
Zonae from 2–8-cell hamster, mouse and rat embryos, as well as sheep and human oocytes were treated with 0.4% pronase (Sigma; 5.6 IU/mg) at pH 7.4, 37°C. The time taken for complete dissolution of zonae was recorded and expressed as zona dissolution time. It provides a measure for comparative susceptibility of zonae to protease. To test the lysis of hamster zonae by different classes of proteases, equivalent amounts (5 IU) of chymotrypsin, trypsin (serine proteases), papain (cysteine protease), collagenase (metalloprotease) and pepsin (acid protease) were added to zona-intact (2–8-cell) embryos at pH 7.4, 37°C, and the zona dissolution time for individual zonae was recorded. For experiments involving the use of uterine luminal flushing to induce hamster zona lysis, 50 µl drops were used. This was prepared by flushing each uterine horn (from day 4 pregnant animals; 5 AM) with 100 µl of the culture medium. Zonae (n = 25) were incubated for 72 h and their status evaluated.
Influence of protease inhibitors on zona escape
Inhibitors specific to different classes of proteases were tested using three sets of 8-cell embryo cultures. Inhibitors tested in the first set of experiments were antipain and leupeptin (both trypsin and cysteine protease inhibitor; each 50 µg/ml). In the second set, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64; cysteine protease inhibitor; 25 µg/ml), parahydromercuric benzoate (PHMB; cysteine protease inhibitor; 100 µmol/l), N-tosyl-L-phenylalanine chloro- methyl ketone (TPCK; chymotrypsin inhibitor; 25 µg/ml), pepstatin (aspartate protease inhibitor; 50 µg/ml) were tested. In the third set, soybean trypsin inhibitor (SBTI; 100 µg/ml) and N
-p-tosyl-L-lysine chloromethyl ketone (TLCK; trypsin inhibitor; 100 µmol/l) were tested. The concentrations of inhibitors used had been optimized and were not deleterious to the development of embryos. Percentages of blastocyst development (24 h) and zona escape (72 h) were monitored in these treatments. To ascertain the quality of blastocysts in the presence of antipain or leupeptin (where maximum inhibition of zona escape was observed), mean cell number per blastocyst was determined for 15 blastocysts per treatment by Hoechst dye staining (Ain and Seshagiri, 1997).
Influence of superoxide dismutase (SOD) and catalase on embryo development
SOD (1000 IU/ml) or catalase (250 IU/ml) was tested in 8-cell embryo cultures with an appropriate control, as described above. About 40 embryos per treatment were used and six replicate experiments were performed.
Zymography
Endometrial protein extracts were prepared by homogenizing endometrial scrapings from day 1–4 pregnant hamsters in 50 mmol/l Tris–HCl buffer containing 0.25% Triton X-100. Samples were centrifuged and supernatants were analysed by gelatin zymography (Heussen and Dowdle, 1980). Briefly, samples (100 µg total protein per lane) were electrophoresed on 10% polyacrylamide gels co-polymerized with substrate (1 mg/ml of gelatin). Gels were washed twice (30 min) in 2.5% Triton X-100, incubated for 18 h at 37°C in a substrate buffer (50 mmol/l Tris–HCl, 10 mmol/l CaCl2, 1 mmol/l ZnCl2, pH 7.5), stained in Coomassie Blue R250, and destained in water. Negative staining(s) indicated the presence of active protease(s) in the analysed samples.
Uterine transfer of 2-cell embryos/blastocysts to pseudopregnant females
Freshly recovered 2-cell embryos or cultured blastocysts were transferred unilaterally to uterus of day 3 pseudopregnant recipients (12:00 h). About 24 h later, i.e. on day 4 (when zona escape is complete in normal pregnancy), both uterine horns (transferred and contralateral) were flushed to recover transferred embryos as well as endogenous oocytes of the recipients. Numbers of recovered embryos/oocytes with or without zonae were counted and their developmental status was assessed.
Statistical analysis
Mean values were calculated from the total number of embryos cultured in a particular treatment. To control between animal variations, a block design was used in which 8-cell embryos from each female were distributed randomly and equally into all treatments and was considered as one replicate experiment (block). A minimum of three to six replicate experiments were carried out. Percentage values were transformed by arcsin transformation to normalize the variance and the transformed data were subjected to a paired Student's t-test.
Results
Zona lysis of 2-cell/preblastocyst stage embryos co-cultured with zona escaping-blastocysts
All cultured 8-cell hamster embryos developed to blastocysts and underwent zona lysis (100%; Table I). Out of 13 co-cultured 2-cell embryos, 10 (77%) exhibited either partial (n = 6) or complete (n = 4) lysis of their zonae (Figure 1A), whereas all independently cultured 2-cells remained with intact zonae (Table I). It was further observed that lysis of zonae occurred irrespective of the developmental stage of the preblastocysts, as 1-, 2-, 3-, 4-, 5- and 8-cell embryos also showed lysis of their zonae when co-cultured with hatching blastocysts. Representative photomicrographs of these embryos are shown in Figure 2.
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Species-specificity of hamster zona lysin and susceptibility of zonae to proteases
Two-cell embryos from mouse and rat and oocytes from sheep and human (n = 6, each), when co-cultured with hamster blastocysts, failed to show any signs of zona lysis (Figure 1B
). Under these conditions, all cultured hamster blastocysts (n = 60) underwent complete zona dissolution. The zona dissolution time was found to be drastically different when zonae from hamster, mouse, rat, sheep and human were treated with pronase (Table II). Hamster zonae were most susceptible to the action of pronase and they disappeared within 7 ± 1 s, while mouse zonae took 663 ± 27 s to dissolve. The zona dissolution times for rat, sheep and human zonae were 532 ± 16 s, 120 ± 12 s and 104 ± 8 s respectively (Table II). Treatment with different classes of proteases showed that chymotrypsin and papain were most efficient in dissolving hamster embryonic zonae (25 ± 3 s and 27 ± 1 s respectively; Table III). Trypsin and collagenase took much longer time to bring about zona dissolution (249 ± 14 s and 822 ± 21 s respectively) whereas in the presence of pepsin, zonae remained intact for more than 40 min (Table III).
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Influence of protease inhibitors on blastocyst development and zona escape
Embryos (8-cell), cultured in the presence of specific inhibitors to different classes of proteases, developed to blastocysts but showed varying extents of zona escape (Figure 3
). Blastocyst development in the presence of antipain (91 ± 4%) and leupeptin (89 ± 8%) was comparable with the control (94 ± 4%). However, zona escape in the presence of these inhibitors was completely blocked (Figure 3A). The mean cell number per blastocyst in the presence of antipain (28 ± 1) and leupeptin (30 ± 2) were similar and comparable with those of control blastocysts (32 ± 2). Other cysteine protease-specific inhibitors, E-64 and PHMB also completely inhibited zona escape, whereas in the control 96 ± 3% embryos underwent zona escape (Figure 3B). Chymotrypsin inhibitor, TPCK, and an acid protease inhibitor, pepstatin, curtailed zona escape by 41 and 30% respectively (Figure 3B). Blastocyst development, in comparison with the control (94 ± 2%) was also inhibited by E-64 (63 ± 9%, P < 0.05), and to a lesser extent by PHMB, TPCK and pepstatin (80 ± 7%, 75 ± 9% and 79 ± 6% respectively). Specific inhibitors to trypsin, TLCK and SBTI, however, did not have any inhibitory effect either on blastocyst development (88 ± 7 and 90 ± 6% respectively) or zona escape (100%) when compared with control in which blastocyst development and zona escape were 95 ± 5 and 100% respectively (Figure 3C).
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) and zona escape (after 72 h, 
). Inhibitors were tested in three sets of experiments (A–C) at appropriate concentrations, as detailed in the text. Each set had a separate control treatment. Values represent mean ± SEM. a,b,c,d,e,f,gValues with same superscripts were significantly different (P < 0.05).Influence of SOD and catalase on blastocyst development and zona escape
The possibility of a chemical means of zona dissolution involving reactive oxygen species (ROS) was tested. Addition of SOD to the culture medium did not inhibit blastocyst development (96 ± 4% versus control, 98 ± 2%) and zona escape (91 ± 5% versus control, 96 ± 2%). In the presence of catalase, although an apparent decrease in blastocyst development was observed (60 ± 7% versus control, 78 ± 6%), there was no significant difference in the zona escape (80 ± 10% versus control, 94 ± 6%).
Lack of zonalytic activity of maternal origin
When zonae or embryos (n = 25 for each treatment) were incubated with either luminal flushings or endometrial extracts from day 4 pregnant females, no alteration in zona thickness was found and the zonae remained intact. Moreover, protease profiles of endometrial samples were found to be similar on days 1–4 of pregnancy with the predominant band of molecular weight ~60 000. Two minor bands with molecular weights of 82 000 and 97 000 were also detected in day 1 sample but were absent in the other samples (Figure 4).
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The data on the state of the oocytes and the outcome of 2-cell/blastocyst transfer to day 3 pseudopregnant recipients is shown in Table IV
. Endogenous oocytes of pseudopregnant recipients, recovered either from the transferred horn (n = 17 and n = 12) or from the contralateral horn (n = 25 and 14) were all zona-encased (Figure 5A). Similarly, all the recovered embryos (44%) out of the 2-cell transfers (n = 68; 3 recipients) cleaved further to the 6–8-cell stage and they remained zona-encased (Figure 5B). When a total of 103 blastocysts were transferred to five recipients, there was a recovery rate of 44%. However, 71% (n = 32) of the recovered blastocysts were azonal (Figure 5D), while 29% (n = 13) were zona intact. The latter blastocysts showed variable zona thickness, indicating signs of lysis (Figure 5C). Such blastocysts eventually underwent complete zona escape when cultured in HECM-2h for an additional 12–24 h.
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Discussion
The present study shows that hamster blastocysts are self-sufficient to undergo zona escape with concomitant and complete lysis of zonae, by their intrinsic ability to produce zona lytic factor with a cysteine protease-like activity. This activity, secreted by and/or associated exclusively with zona escaping-blastocysts, induces zona dissolution of preblastocyst-stage embryos only from hamsters but not from other species, thereby indicating that the embryo-derived zona lytic activity probably is species-specific. Interestingly, a uterine zona lysin appears unlikely to be involved in the phenomenon of zona escape of blastocysts in hamsters.
The observation on the preblastocyst-stage embryos exhibiting zona lysis is interesting and it is similar to that occurring with in-vivo developing embryos (Orsini, 1965; Mintz, 1971; Gonzales and Bavister, 1995). As expected, preblastocyst stage-embryos cannot produce zona lytic factor and fail to undergo zona escape when cultured independently. The observed zona lysis in the preblastocyst stage-embryos, in the co-culture setup (Table I), must be due to a high zona lytic protease activity, produced transiently into the medium by the zona escaping blastocysts and, surprisingly, this activity appears to be hamster zona-specific.
It is possible that enzymatic digestion of zonae differs between species. This is reflected by an extreme susceptibility of hamster zonae to the action of pronase, in contrast with the other species studied, of which mouse zonae were the most resistant (Table II). This is also consistent with the observation made on boar and bull spermatozoal acrosins, which could lyse the zonae of hamsters but not those of mice (Mansouri et al., 1985). Despite their high susceptibility to pronase, it is remarkable that hamster zonae are not equally prone to lysis by other proteases (Table III), signifying their selectivity to the type of protease for undergoing zona lysis.
One of the most striking observations made in the present study is that all cysteine protease inhibitors completely restricted the developed blastocysts from undergoing zona escape, without affecting their quality (MCN). In contrast, specific inhibitors of trypsin (SBTI/TLCK) had absolutely no effect on either blastocyst development or zona escape (Figure 3). This strongly implies that the hamster `zona lysin' could be a cysteine protease. These results are in sharp contrast to that observed in the mouse. In this species, serine protease, in particular, trypsin-like enzyme (strypsin/hepsin) has been reported to be involved in the process of hatching (Perona and Wassarman, 1986; Sawada et al., 1990; Vu et al., 1997). Similar observations have been made in other mammalian species, e.g. rat (Ichikawa et al., 1985), rabbit (Denker and Fritz, 1979) and cow (Menino and Williams, 1987). The lack of involvement of trypsin-like activity in hamster zona lysis is of particular interest here. Our report is the first to implicate cysteine protease-like activity in the phenomenon of zona escape of mammalian blastocysts. It is rather puzzling why hamsters could have evolved this enzymatic mechanism for zona escape. However, it should be mentioned that cysteine proteases have important roles throughout early development, from fertilization (Ichikawa et al., 1984), through peri-implantation (Ichikawa et al., 1985; Afonso et al., 1999) to proper fetal development (Grubb et al., 1991). It is conceivable that this category of protease could play an important role during the critical period of zona escape/loss, at least in the hamster. Identification and characterization of this putative enzyme remains to be investigated. However, it could be speculated that cysteine proteases, e.g. cathepsin which have been shown to be present in developing embryos (Denker and Tyndale-Biscoe, 1986), may have a role in the zona escape of hamster blastocysts.
We also considered the possibility of a chemical means of zona dissolution involving ROS, in addition to proteases. Failure of SOD and catalase to inhibit zona escape eliminates the involvement in this process of superoxide radical and hydrogen peroxide, in case they are generated by embryos. This is in contrast to that reported for the mouse (Thomas et al., 1997). This difference coupled with the type of zona lytic protease requirement between the two species may be attributable to the different modes of zona escape observed in them and/or to the physicochemical nature of their zona matrix. The difference in protease-induced zona lytic activity observed may suggest that the hamster ZP matrix architecture (Keefe, et al., 1997) is different from the mouse (Green, 1997). In this context, it should be emphasized that, in comparative terms, the hamster zona shows extraordinary sensitivity to exogenous proteases (Tables II and III) as well as to the embryonically produced cysteine-like protease (Table I, Figures 1–3). It would be interesting to study the molecular characteristics of the latter enzyme produced by hamster embryos.
In addition to embryonic proteases, the involvement of uterine zona lysin(s) in the process of zona escape in vivo has been the subject of debate (Orsini, 1965; Dickmann, 1969; Mintz, 1971; Joshi and Murray, 1974; Gonzales and Bavister, 1995; Thrasher et al., 1999). Our observations on the luminal flushings and endometrial extracts failing to induce zona lysis in vitro and the uterine milieu unable to facilitate pre-blastocyst embryos to undergo zona escape in vivo (Table IV; Figure 5), eliminates the possibility of maternal tissue contributing to zona escape. This is supported also by the finding that the zymogram of endometrial extracts did not show appearance of any specific (new) protease (with gelatinase activity) on day 4 of pregnancy (Figure 4), coinciding with the timing of in-vivo zona escape (Gonzales and Bavister, 1995). Other reasons supporting the lack of contribution of uterine lysin for zona escape are: (i) in rats, blastocysts, but not morulae, undergo hatching when transferred to pseudopregnant recipients (Dickmann, 1969; (ii) trophectoderm-derived zona lysin is involved in blastocyst hatching (Schiewe et al., 1995); (iii) zona thinning seems to be induced by blastocysts (Confino et al., 1997); (iv) hatching of mouse blastocysts can occur at ectopic sites devoid of uterine influence (Dickmann, 1969); and (v) embryos from non-mammalian, lower organisms, e.g. echinoderms (Edwards et al., 1977), amphibia (Katagiri, 1975) and teleosts (Hagenmaier, 1974) are known to secrete hatching enzymes, which are required to digest their oocyte coverings. These findings, taken together, eliminate the role of `uterine zona lysin' in the process of zona escape and strongly support our hypothesis that blastocysts are self-sufficient to produce lysins, both in vivo and in vitro, thereby undergoing zona escape in hamsters.
It is not known how zona lytic activity in embryos is regulated at the molecular level, either in vivo or in vitro. It is believed that a variety of regulatory molecules, e.g. epidermal growth factor (EGF), transforming growth factor ß (TGFß) and leukaemia inhibitory factor (LIF), possibly produced by both blastocysts and interacting endometrial tissues, could play important roles during peri-implantation development, including zona escape/hatching (Harvey et al., 1995; Kane et al., 1997; Seshagiri et al., 1999). Of relevance in this context, are our preliminary results which indicate that the addition of these factors or their antibodies profoundly influence the ability of cultured embryos to undergo zona escape (A.Mishra and P.B.Seshagiri, unpublished data). These encouraging observations prompt a detailed investigation of the molecular regulation of zona escape in hamsters.
In conclusion, this study sheds light on the possible nature and source of the zona lytic agent in hamsters and gives definitive answers to questions relating to the phenomenon of zona escape. The study also marks interesting species differences (especially with mouse) which could provide new leads to our understanding of differences in the mechanism of zona escape in mammals in general. Moreover, it is envisaged that additional knowledge on the molecular nature of agents mediating and regulating the crucial event of blastocyst hatching will be of great relevance to the improvement of human assisted reproductive technology.
Acknowledgments
Financial support from the Department of Science and Technology, New Delhi is gratefully acknowledged. The authors thank Dr T.C. Anandkumar, Hope Infertility Clinic, Bangalore, for providing rejected human oocytes from IVF programme, during the course of this work; Ms H.S.Lalitha for technical support and Ms M.S.Padmavathi for her help during the preparation of the manuscript.
Notes
1 To whom correspondence should be addressed at: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, India. E-mail: polani{at}serc.iisc.ernet.in 
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Submitted on March 7, 2000; accepted on July 24, 2000.
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