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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).

Conclusion. Taken together, paternal factors carried in ASG secretion affect genomic imprinting of developing embryos. The outcome of research work described here deepens our understanding of the role of ASG in maximizing reproductive performance mediated by regulating the epigenetic marks of the genome and in particular the imprinted genes. / Introduction. Our previous in vivo studies in golden hamster have shown the accessory sex glands (ASG) secretion facilitate the development of embryos to term but the underlying mechanism is still not clear. Since the deleterious effect caused by the lack of sperm exposure to ASG secretion is heritable to developing fetus and even after birth, we hypothesized that the paternal factor carried in ASG secretion may change the epigenetic regulation and in particular the imprinted genes of embryonic genome. / Materials and methods. Golden hamster and ICR mouse were used in this study. Hamster is a well-established animal model to study the effect of individual ASG but the genetic background of hamster is poorly known. To verify the specificity of our molecular probe and antibodies used in hamster, a mouse model was also established. Five groups of male hamsters and two groups of male mice were established by surgical treatment. In hamster, (SH) sham-operated, (VPX) ventral prostate-removed, (TX) all ASG-removed, (VPVX) castrated with ASG-removed except ventral prostate and (VX) castrated with intact ASG were established. In mouse, SH and VPX were established. In single-mating of hamster, male was copulated with female at estrus for 15 min. In double-mating of hamsters, female mated with each male for 10 min each. In single-mating of mouse, male was caged with female for 1 h. Epididymal sperm, uterine sperm, fertilized oocytes, pre-implantation embryos and fetuses at 13 days gestation (E13) were collected. Global DNA methylation of sperm, fertilized oocytes, early embryos and E13 fetuses were investigated by indirect immunofluorescence and DNA dot-blot using antibody against methylated DNA. Using the same technique, histone acetylation at lysine 5 residue was detected in male pronuclei of fertilized oocytes, protamine 1 and 2 content were detected in sperm, DNA methyltransferase 1, 3a and 3b activities were detected in early embryos. The crown-rump length and weight of fetuses were measured. Morphology was also examined under scanning electron microscope. Two sets of co-ordinately regulated but oppositely expressed imprinted genes Igf2/H19 and Dlk1/Gtl2 were investigated. H19 differentially methylated region (DMR) and Gtl2 promoter were examined by bisulfite sequencing in sperm and E13 fetuses. Expression of Igf2 and Dlk1 were examined by in situ hybridization and real-time PCR in pre-implantation embryos and E13 fetuses. / Results. Uterine sperm in VPX and TX groups showed no change of DNA methylation level and protamine 1 and 2 content. Fertilized oocytes in VPX and TX groups showed similar DNA methylation level as SH group in both hamster and mouse. Histone hypoacetylation was observed in male pronuclei of hamster but not in mouse. Early embryos in VPX and TX groups showed abnormal level of DNA methylation and Dnmt3b during embryo development in hamster. Replenishment of ASG secretion to sperm from VPX and TX group by double-mating restored the DNA methylation level to normal in early embryos. E13 fetuses of VPX and TX groups in hamster and VPX group in mouse showed DNA hypomethylation. E13 fetuses of VPX group in hamster showed increase in average crown-rump length and body weight with larger variations between individuals. One E13 fetus of VPX group in mouse showed polydactyly and malformation in the head. Real-time PCR showed abnormal expression of Igf2 and Dlk1 in both pre-implantation embryos and E13 fetuses of VPX and TX groups. Bisulfite sequencing showed hypermethylation of H19 DMR in VPX and TX groups of hamster and hypomethylation of Gtl2 promoter in VPX group of mouse. / "August 2007." / Adviser: Pak Ham Chow. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4739. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 194-224). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

The current study examined the impact of plant sterols, stanols, sterol esters, and stanol esters on (i) cholesterol-lowering efficiency, (ii) gene expression of ABCG5 and ABCG8 sterol transporters in the liver and small intestine, and (iii) colon mucosal cell proliferation in hamsters. After 5 weeks on experimental diets, plasma total cholesterol levels were reduced ( P<0.05) by stanols, sterol esters and stanol esters compared to cholesterol-control diet. Different PS analogs did not alter ABCG5 and ABCG8 mRNA levels in small intestine and liver as compared to cholesterol control. In addition, colon mucosal cell proliferation was 21.4% lower (P<0.01) in group fed 0.7% stanol esters relative to cholesterol control. Results suggest that hypocholesterolemic effects of PS analogs are not associated with changes of liver and small intestine ABCG5 and ABCG8 sterol transporters. Data also indicated that plant stanol ester may possess anticarcinogenic properties.

The cholesterol-lowering effect of corn fiber oil, obtained from the seed coats of corn kernels, has been reported previously. Corn fiber oil contains phytosteryl fatty acyl esters, ferulate phytostanyl esters, and free phytosterols. To date, however, no studies have examined the cholesterol-lowering efficacy of ferulate phytostanyl esters. Moreover, although plant stanols and sterols have been established as cholesterol-lowering agents over the past five decades, their exact mechanisms of action are not clearly understood. One of the possible mechanism is that plant sterols/stanols disrupts the normal sub-cellular cholesterol absorption by down-regulation of the influx sterol transporters such as the Niemann pick C1 like 1(NPC1L1) protein and/or up-regulation of efflux sterol transporters such as the ATP binding cassette (ABC) G5 and ABCG8 protein. Hence, the objectives of this thesis were to assess the efficacy of corn fiber oil, ferulate phytostanyl esters and their parent compounds including sitostanol and ferulic acid, on plasma cholesterol levels. Further, objectives were to investigate their impact on parameters of cholesterol kinetics and gene expression of sterol transporters to obtain insight into their role in genetic control of regulation of cholesterol flux. Results of this experiment demonstrate that the hypocholesterolemic effect of corn fiber oil is mostly due to sitostanol, while esterification of ferulic acid and sitostanol yields no apparent synergistic cholesterol lowering effect. Present data exhibited a cholesterol absorption lowering effect of corn fiber oil and sitostanol and suggest that this effect may be due to up-regulation of intestinal enterocyte efflux sterol transporters such as ABCG5 and ABCG8.

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Chan Oi Chi. / "May 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 259-310) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The objective of this study was to examine the effects of a medium chain triglyceride oil mixture (MCTo), designed to increase energy expenditure and improve lipid profiles containing medium chain triglycerides, phytosterols and n-3 fatty acids in the form of flaxseed oil, versus the antioxidant alpha-lipoic acid (ALA). Forty-eight hamsters were fed (i) hypercholesterol emic (HC) control, (ii) HC MCTo, (iii) HC ALA, (iv) HC MCTo/ALA diets for 4 weeks. No effects on food intake, body weight, total body water, lean body mass, fat mass, and tissue thiobarbituric acid-reactive substances (TBARS) were observed. ALA alone had no effect on total cholesterol (TC); however, MCTo feeding increased TC with (p < 0.03) and without (p < 0.003) ALA when compared to control. ALA increased HDL levels compared to control (p 0.04) and MCTo/ALA (p < 0.007) groups. MCTo, with (p < 0.0001) or without (p < 0.006) ALA, increased non-HDL cholesterol levels versus control. The non-HDL:HDL ratio was decreased by ALA compared to MCTo (45%) and MCTo/ALA (68%) (p < 0.0001), a similar trend was seen when compared to the HC control (22%) group (p < 0.14). Triglyceride levels were not altered by any of the dietary treatments. Liver and heart tissue reduced glutathione (GSH) was increased (p < 0.05) by all three treatments when compared to control. Both tissues showed an increase (p < 0.05) in oxidized glutathione (GSSG) when fed ALA compared to all other treatments. Hamsters fed ALA had a lower (p < 0.05) GSH/GSSG ratio compared to all treatment groups. In conclusion, MCTo feeding does not elicit beneficial effects on circulating plasma lipids and measures of body composition. In addition, our results do not clearly support an improvement in oxidative status through supplementation of ALA. However, our results do support the existence of beneficial effects of ALA on circulating lipoprotein content in the hamster.