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For many cattle operations, profitability depends on the success of reproductive management programs. Opportunities for improving fertility exist within the numerous challenges related to reproductive management. Non-conventional, creative tools for reproductive management could help producers overcome these challenges. In an effort to produce information that could be used to improve reproductive performance of cattle, the following studies were undertaken. The objectives of these studies were threefold: to determine whether GnRH administered as an epidural injection causes ovulation in healthy cows and heifers, to evaluate whether the follicular environment (specifically, follicle fluid) surrounding the oocyte during the maturation phase affects the ability of the cumulus-oocyte complex to progress through early embryonic development, and to investigate the relative effects of estradiol and progesterone on oocyte maturation and early embryo development. Ability of GnRH to elicit an ovulatory response when administered as an epidural was evaluated in crossbred angus cows and heifers. The preliminary study evaluated this route of administration in crossbred angus cows. Animals were assigned randomly to either intramuscular or epidural administration, and ovaries were visualized via transrectal ultrasound every 6 h until ovulation of the dominant follicle. Results indicated that epidural administration of GnRH was able to trigger an ovulatory response, but timing of ovulation was not measured. The main experiment evaluated incidence of ovulation, time to ovulation, and ovulatory follicle size in crossbred angus heifers administered GnRH either epidurally or intramuscularly. Heifers were randomly assigned to treatment and ovaries were visualized every 4 h via transrectal ultrasound until ovulation of the dominant follicle. Results indicated that epidural administration of GnRH was able to elicit an ovulatory response in heifers, and the timing of ovulation and ovulatory follicle size was not different between administration route. Further investigation is needed to determine if characteristics of the ovulatory response (such as the luteinizing hormone surge) and circulating concentrations of GnRH are altered by epidural administration, which may impact fertility. GnRH administration is standard practice in many estrous synchronization programs. For fixed-time artificial insemination programs, the detection of estrus prior to insemination has been shown to improve conception and decrease early embryonic loss. The impact of behavioral estrus expression on the oocyte and early embryo were evaluated. Oocytes were matured in vitro in follicle fluid collected from synchronized cows who were classified as having expressed behavioral estrus or not expressing estrus. Embryo cleavage was not affected by estrus expression, but there was a tendency for improved blastocyst development in embryos matured in follicle fluid from animals who had expressed estrus. Cell number was not affected by estrus expression, but future research is needed as to the effect on oocyte acquisition of competence and early embryonic development. Despite the progress that has been made in culture conditions for in vitro produced embryos, developmental capacity following fertilization is limited at best, with only around one-third of oocytes placed into maturation resulting in viable embryos. During in vivo maturation, the oocyte undergoes final maturation within the follicle, surrounded by a changing microenvironment of estradiol and progesterone. Although the effects of steroids on oocyte development in vitro have been studied on an individual basis, a direct comparison between the ratio of estrogen and progesterone relative to follicle size has not been investigated Effects of steroid hormones estradiol and progesterone on oocyte maturation and early embryonic development were evaluated. Oocytes were matured in vitro in media supplemented with either estradiol, progesterone, or a combination of estradiol and progesterone. Oocytes were fertilized after maturation and cultured for 7 d until development to blastocyst stage. Addition of estradiol alone did not support oocyte maturation or early embryonic development in vitro, and a combination of estradiol and progesterone exhibited an inhibitory effect on oocyte maturation and early embryonic development. Addition of progesterone alone resulted in improved development when compared with estradiol alone or a combination of estradiol and progesterone. These results indicate that efficiency of reproductive management programs is controlled by multi-faceted factors and opportunities for improvement of reproductive outcomes exist in all of these factors. Although ovulation can be elicited via epidural administration, the impact of this ovulatory trigger on fertility requires further investigation. Display of estrus after synchronization for fixed-time artificial insemination improves conception and decreases early embryonic loss and has a may improve blastocyst development. This effect on early embryo development could be the focus of future research, further improving fertility and possibly the efficacy of in vitro embryo production. Steroid hormones play crucial roles in oocyte competency and the addition of progesterone during in vitro maturation improves development compared with estradiol alone or a combination of estradiol and progesterone. / Ph. D. / Reproductive success is critical for economic sustainability for many cattle operations. Creative tools for fertility management could help cattle producers overcome many challenges to fertility. In an effort to produce information that could be used to improve reproductive performance of cattle, the following studies were undertaken. The objective of these studies was to determine whether hormone administration as an epidural injection causes ovulation in healthy cattle (young and mature cattle assessed). Additionally, the second study evaluated whether the follicle (fluid-filled compartment surrounding the egg on the ovary) environment affects the female egg prior to ovulation, and the early embryo after fertilization. Finally, the third study looked at the impact of follicle fluid and specific hormones on embryo growth. An experiment was conducted in cows and heifers to determine if administering a hormone as an epidural injection, as opposed to conventional methods, could cause ovulation of the follicle. Animals received either an intramuscular or epidural hormone injection, and the ovaries of the animals were observed on an ultrasound until the follicle ruptured, releasing the egg. Epidural administration of the hormone was indeed able to trigger the rupture of the follicle. Hormone administration is standard practice in many cattle fertility programs. To maximize fertility, animals need to come into “heat” or estrus (period of sexual receptivity). Coming into heat is important for fertility in the female as it is indicative of impending ovulation and preparation of the egg for fertilization. In some reproductive management systems, reproductive cycles can be controlled in ways that deemphasize the need for behavioral estrus. Recent reports have suggested that animals in these systems that exhibit behavioral estrus are more fertile, as it makes it more likely for the female to conceive and stay pregnant compared to females who do not come into heat. The impact of heat on the female egg and early embryo of the cow has not been investigated. To evaluate the impact of heat on embryos, eggs were taken from the ovary of the cow and matured in a cell culture lab overnight in media containing fluid taken from the follicles of animals who came into heat, and animals that did not come into heat. The eggs were then fertilized, and embryos developed. There was only a tendency for improvement in embryo development for those matured in fluid from animals in heat compared with animals not in heat. When growing embryos in a culture lab, success rates are lower than embryos developing in the animal. When the egg is being prepared for release, it goes through important maturation steps to enable fertilization and eventual growth into a calf. Hormones in the follicle fluid facilitate maturation, and the conditions in the follicle are not easily replicated in the lab. The addition of these critical hormones to the lab conditions may help facilitate improved development in lab-produced embryos. Two hormones (estrogen and progesterone) were added to follicle fluid that was used in the lab culture environment to determine their effect on embryo growth. When progesterone was added, embryos grew well, matching the development rate of the control medium. When estrogen was added, embryos experienced poor development. Neither resulted in embryo development that exceeded the control medium. These results indicate that control of reproduction in cattle is complex, and multiple opportunities exist to improve fertility. Future research on how the oocyte and embryo react to their environment is needed and will facilitate further improvement of reproductive management systems in cattle. Improved reproductive management will enhance efficiency, sustainability and profitability of cattle production systems.

<p>Ovarian stimulation in assisted reproduction often leads to the production of a high number of oocytes. After fertilization of these oocytes, the resulting embryos can be cryopreserved for later use. Vitrification is a recently introduced method for cryostoring embryos, showing high survival rates for both cleavage stage embryos and blastocysts. Characteristic of vitrification are high concentrations of cryoprotectants and ultra fast freezing which makes the material glassily. A major concern with vitrification has been the direct contact of the cryo-solutions with liquid nitrogen. Therefore, sealed containers have been developed and one of these is the Rapid-i™ made by Vitrolife Sweden AB.</p><p>We evaluated this new device using embryos not suitable for embryo transfer or cryopreservation for clinical purposes. Embryos at cleavage stages were first vitrified and then warmed. Outcome parameters were cryosurvival and development to the blastocyst stage. Blastocysts were randomised between the established VitroLOOP™ and the Rapid-i™ as carriers. Outcome parameters were cryosurvival and further development. Our results show that Rapid-i™ gives good survival rates in vitrification for cleavage stage embryos and blastocysts.</p>

Ovarian stimulation in assisted reproduction often leads to the production of a high number of oocytes. After fertilization of these oocytes, the resulting embryos can be cryopreserved for later use. Vitrification is a recently introduced method for cryostoring embryos, showing high survival rates for both cleavage stage embryos and blastocysts. Characteristic of vitrification are high concentrations of cryoprotectants and ultra fast freezing which makes the material glassily. A major concern with vitrification has been the direct contact of the cryo-solutions with liquid nitrogen. Therefore, sealed containers have been developed and one of these is the Rapid-i™ made by Vitrolife Sweden AB. We evaluated this new device using embryos not suitable for embryo transfer or cryopreservation for clinical purposes. Embryos at cleavage stages were first vitrified and then warmed. Outcome parameters were cryosurvival and development to the blastocyst stage. Blastocysts were randomised between the established VitroLOOP™ and the Rapid-i™ as carriers. Outcome parameters were cryosurvival and further development. Our results show that Rapid-i™ gives good survival rates in vitrification for cleavage stage embryos and blastocysts.

ABSTRACT Infertility is a medical condition that affects approximately 16 % of Swedish couples in childbearing age and is a condition found in both men and women. When a couple has been trying to conceive for more than 12 months without success they need to see a health care provider. Infertility can be treated with medicine, insemination or assisted reproductive technology such as in-vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). The aim of this study was to validate a morphokinetic selection model, KIDScore™ D3 Basic, against morphological selection model that is currently in use at the Fertility unit at University hospital in Orebro. A total of 782 embryos with known implantation data were assessed both y use of KIDScore™ D basic and the morphological selection model. The results from the study showed that KIDScore™ D3 Basic better discriminates between implanting and non-implanting embryos. Another strong finding in this study was the notion that presence of multinucleation at the two cell stage decreased chance of implantation significantly. The majority of implanting embryos belonged to the best morphokinetic class, whereas for morphology the majority of implanting embryos belonged to the second best class. The combination it of morphology and morphokinetic is to recommend as clinical use as it provided the best selection tool.

Made available in DSpace on 2014-06-11T19:25:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-28Bitstream added on 2014-06-13T18:26:27Z : No. of bitstreams: 1 000736004.pdf: 627827 bytes, checksum: 411ff86dc11bb8a1a240b7427a561cdf (MD5) / A maturação in vitro (MIV) é considerada uma das etapas mais desafiadoras da produção in vitro (PIV) de embriões bovinos. O meio de MIV deve fornecer condições necessárias para que a maturação citoplasmática e nuclear ocorra de modo próximo ao fisiológico. As gonadotrofinas hipofisárias são essenciais para que o oócito adquira (in vivo) a competência para ser fertilizado; entretanto, se as gonadotrofinas (hipofisárias ou coriônicas) são essenciais, na MIV, as concentrações a serem utilizadas ainda são controversas. Os objetivos, com este estudo, foram estabelecer concentrações otimizadas de gonadotrofinas coriônicas (eCG e hCG) para a substituição daquelas comumente utilizadas de FSH e LH; comparar o efeito de baixas concentrações de eCG e/ou de hCG, isoladamente ou em conjunto com gonadotrofinas hipofisárias; e analisar a necessidade da utilização do FSH e do LH, individualmente, na PIV de embriões livre de suplementação proteica. Complexos cumulus-oócito (CCOs) obtidos de ovários de vacas provenientes de abatedouro, foram selecionados e utilizados em três experimentos: 1) CCOs maturados em meio MIV com seis concentrações distintas de eCG (0; 0,015; 0,15; 1,3; 1,5 e 4 UI mL-1 ) dando origem aos grupos experimentais (C+; E1; E2; E3; E4 e E5, respectivamente), e seis concentrações distintas de hCG (0; 0,05; 0,5; 0,67; 2 e 5 UI mL-1), originando também seis grupos experimentais (C+; H1; H2; H3; H4 e H5, respectivamente); 2) Meio MIV com concentrações mínimas funcionais de eCG e hCG obtidas no experimento anterior, resultando em sete grupos experimentais: sem quaisquer gonadotrofinas; apenas eCG (0,015 UI mL-1); apenas hCG (0,05 UI mL-1); eCG mais hCG; eCG mais LH; hCG mais FSH; e SFB com FSH (0,5 μg mL-1) e LH (5 μg mL-1); e 3) MIV na presença de FSH (0,5 μg mL-1) ou LH (5 μg mL-1) sem nenhuma suplementação proteica. Os CCOs maturados foram fertilizados e... / The in vitro maturation (IVM) is considered one of the most challenging steps of in vitro production (IVP) of bovine embryos. The IVM medium must provide the conditions required for nuclear and cytoplasmic maturation to occur similarly to physiological. The pituitary gonadotropins are essential components that enable oocytes for fertilization (in vivo), but as the gonadotropins (pituitary or chorionic) are essential for IVM, their employed concentrations are still indefinite. We aimed to establish optimal concentrations of chorionic gonadotropin (eCG and hCG) to replace those of FSH and LH commonly used; to compare the effect of low concentrations of eCG and/or hCG alone or in combination with pituitary gonadotropins; and to analyze how necessary is the use of FSH and LH, individually, on the IVP of embryos free from any protein source. Cumulus-oocyte complexes (COCs), obtained from ovaries of slaughterhouse were selected and used in three experiments: 1) COCs matured on the IVM medium with six different concentrations of eCG (0, 0.015, 0.15, 1.3; 1.5 and 4 IU ml-1) yielding the experimental groups (C +, E1, E2, E3, E4 and E5, respectively) and six different concentrations of hCG (0, 0.05, 0.5, 0, 67, 2 and 5 IU ml-1), also yielding six experimental groups (C +, H1, H2, H3, H4 and H5, respectively); 2) IVM medium with minimal concentrations functional of eCG and hCG obtained in the previous experiment, resulting in seven groups: without any gonadotropin; only eCG (0.015 IU mL-1); only hCG (0.05 IU mL-1); eCG plus hCG, eCG plus LH; hCG plus FSH; FCS with FSH (0.5 mg ml-1) and LH (5 mg ml-1); and 3) IVM in the presence of FSH (0.5 mg mL-1) or LH (5 mg ml-1), without protein supplementation. The matured COCs were fertilized and cultured in incubator (38.3 °C, 5% CO2 and maximum humidity). Embryo development was evaluated in terms of cleavage, blastocyst and hatching rates (at 72, 168 and 240 hours post-insemination, ...

Orientador: Marcelo Fábio Gouveia Nogueira / Banca: Anthony César de Souza Castilho / Banca: Gisele Zoccal Mingoti / Resumo: A maturação in vitro (MIV) é considerada uma das etapas mais desafiadoras da produção in vitro (PIV) de embriões bovinos. O meio de MIV deve fornecer condições necessárias para que a maturação citoplasmática e nuclear ocorra de modo próximo ao fisiológico. As gonadotrofinas hipofisárias são essenciais para que o oócito adquira (in vivo) a competência para ser fertilizado; entretanto, se as gonadotrofinas (hipofisárias ou coriônicas) são essenciais, na MIV, as concentrações a serem utilizadas ainda são controversas. Os objetivos, com este estudo, foram estabelecer concentrações otimizadas de gonadotrofinas coriônicas (eCG e hCG) para a substituição daquelas comumente utilizadas de FSH e LH; comparar o efeito de baixas concentrações de eCG e/ou de hCG, isoladamente ou em conjunto com gonadotrofinas hipofisárias; e analisar a necessidade da utilização do FSH e do LH, individualmente, na PIV de embriões livre de suplementação proteica. Complexos cumulus-oócito (CCOs) obtidos de ovários de vacas provenientes de abatedouro, foram selecionados e utilizados em três experimentos: 1) CCOs maturados em meio MIV com seis concentrações distintas de eCG (0; 0,015; 0,15; 1,3; 1,5 e 4 UI mL-1 ) dando origem aos grupos experimentais (C+; E1; E2; E3; E4 e E5, respectivamente), e seis concentrações distintas de hCG (0; 0,05; 0,5; 0,67; 2 e 5 UI mL-1), originando também seis grupos experimentais (C+; H1; H2; H3; H4 e H5, respectivamente); 2) Meio MIV com concentrações mínimas funcionais de eCG e hCG obtidas no experimento anterior, resultando em sete grupos experimentais: sem quaisquer gonadotrofinas; apenas eCG (0,015 UI mL-1); apenas hCG (0,05 UI mL-1); eCG mais hCG; eCG mais LH; hCG mais FSH; e SFB com FSH (0,5 μg mL-1) e LH (5 μg mL-1); e 3) MIV na presença de FSH (0,5 μg mL-1) ou LH (5 μg mL-1) sem nenhuma suplementação proteica. Os CCOs maturados foram fertilizados e ... / Abstract: The in vitro maturation (IVM) is considered one of the most challenging steps of in vitro production (IVP) of bovine embryos. The IVM medium must provide the conditions required for nuclear and cytoplasmic maturation to occur similarly to physiological. The pituitary gonadotropins are essential components that enable oocytes for fertilization (in vivo), but as the gonadotropins (pituitary or chorionic) are essential for IVM, their employed concentrations are still indefinite. We aimed to establish optimal concentrations of chorionic gonadotropin (eCG and hCG) to replace those of FSH and LH commonly used; to compare the effect of low concentrations of eCG and/or hCG alone or in combination with pituitary gonadotropins; and to analyze how necessary is the use of FSH and LH, individually, on the IVP of embryos free from any protein source. Cumulus-oocyte complexes (COCs), obtained from ovaries of slaughterhouse were selected and used in three experiments: 1) COCs matured on the IVM medium with six different concentrations of eCG (0, 0.015, 0.15, 1.3; 1.5 and 4 IU ml-1) yielding the experimental groups (C +, E1, E2, E3, E4 and E5, respectively) and six different concentrations of hCG (0, 0.05, 0.5, 0, 67, 2 and 5 IU ml-1), also yielding six experimental groups (C +, H1, H2, H3, H4 and H5, respectively); 2) IVM medium with minimal concentrations functional of eCG and hCG obtained in the previous experiment, resulting in seven groups: without any gonadotropin; only eCG (0.015 IU mL-1); only hCG (0.05 IU mL-1); eCG plus hCG, eCG plus LH; hCG plus FSH; FCS with FSH (0.5 mg ml-1) and LH (5 mg ml-1); and 3) IVM in the presence of FSH (0.5 mg mL-1) or LH (5 mg ml-1), without protein supplementation. The matured COCs were fertilized and cultured in incubator (38.3 °C, 5% CO2 and maximum humidity). Embryo development was evaluated in terms of cleavage, blastocyst and hatching rates (at 72, 168 and 240 hours post-insemination, ... / Mestre

Bovine embryos produced in vitro consistently display decreased quality in terms of their potential to reach the blastocyst stage as well as post-transfer survival. Media formulations, oocyte quality, and inferior expression of needed transcripts are all causes of this reduced developmental potential commonly present in in vitro-produced (IVP) bovine embryos. Recently our lab has confirmed interleukin-6 as an embryokine whose capabilities include increasing inner cell mass (ICM) numbers and promoting bovine blastocyst development. LIF is another family member of the IL6 cytokine family and has been shown to produce several positive effects when supplemented during oocyte in vitro maturation. IL6 has predominantly been studied as being supplemented post-fertilization. However, published transcriptomic work described receptors for IL6, IL11, and LIF as present in cumulus cells at the time COCs are removed from their follicles. Consequently, we wanted to investigate if supplementing 25 ng/ml of IL6, IL11, or LIF would improve IVM bovine oocyte competency. Several experiments were completed (4replicates/experiment; 30-60 cumulus-oocyte complexes (COCs)/replicate). In Experiment 1, COCs were in vitro matured for 16 or 22 hours, then meiotic stage was assessed after denuding, fixation, and DNA staining. No cytokine treatment influenced the percentage of oocytes that achieved metaphase II at either time point. In Experiment 2, COCs were in vitro matured for 4 hours before snap freezing. and processing to examine changes in five cumulus-expressing transcripts associated with oocyte competency (CX43, CX37, AREG, TNFAIP6, HAS2). Our chosen housekeeping gene, HPRT1, served as the internal control. An increased abundance of AREG occurred following exposure to LIF but not with the other treatments. Supplementation with IL6 and IL11 but not LIF tended to increase TNFAIP6 abundance (P<0.10). No other transcript differences were detected. In Experiment 3, we examined whether supplementing these cytokines during IVM affects subsequent fertilization and blastocyst rates. No effects were detected on cleavage rates but at day 8 increases in blastocyst yield were detected for LIF and IL11, but not IL6. LIF showed a tendency to increase hatching rates. In Experiment 4, we aimed to assess how the cytokine treatments affected cryosurvivability. Blastocysts (5-10/replicate, 9 replicate studies) were frozen at a rate of -0.6 degrees C/min until reaching -32 degrees C, then were stored in liquid nitrogen for 4-8 weeks before being thawed and incubated in conventional embryo culture medium (SOF-BE1) for 3 days. No treatment effects were noted for re-expansion, hatching, and overall survivability. In summary, these results implicate IL11 and LIF as potential mediators of oocyte competency. However, the evidence presented here suggest that IL6 and IL11 may function differently than LIF when provided during COC maturation. / Master of Science / The numerous similarities in the regulation of early embryonic development between human and cow has made bovine embryos an excellent model for exploring how to optimize assisted reproductive techniques (ARTs) and other methods for improving and preserving fertility in humans. Pregnancy loss is also very similar in both cattle and humans. In beef cattle, more than 50 percent of reproductive failures occur before day 16 of gestation. In women, approximately 15 percent of all clinically recognized pregnancies result in spontaneous loss, however, several more pregnancies fail prior to ever being clinically recognized. Various ARTs are used to treat sub-fertile conditions in cattle, and these technologies are generally deemed as a viable way to improve fertility. However, IVP embryos are inferior in their ability to properly fertilize and develop to the blastocyst stage, the stage when embryos are normally transferred. Furthermore, IVP-generated embryos are inferior at maintaining pregnancies. There are two primary restraints to the IVP process: a low percentage of oocytes that become fertilized and produce transferable embryos and transferred IVP embryos have decreased chances of maintaining a viable embryo than embryos produced in vivo. Very little is known about the various hormone and molecular factors that promote oocyte and embryo development. Therefore, a primary objective for bovine oocyte and embryo research is to classify these factors and implement them into their maturation and culture media to improve overall IVP efficiency. My lab studies members of the IL6 cytokine family as potential factors that might play a role in the development of oocytes and embryos. The aim of this work is to assess the capacity of three molecules within this family, IL6, IL11, and Leukemia inhibitory factor (LIF) to improve oocyte development, fertilization rate, and blastocyst yield when supplemented during in vitro maturation (IVM). This work revealed that both IL11 and LIF improved IVP bovine blastocyst development at day 8. Unfortunately, none of the treatments had any effect on fertilization rates. LIF increased the expression of a cumulus-specific transcript known to aid in cumulus expansion. Cumulus cells are the somatic cells immediately surrounding the oocyte. Cumulus expansion is a key indicator of proper oocyte maturation. We did not observe any treatment effect on post-thaw survival of cryopreserved bovine embryos. This indicates that our treatments did not help the embryos maintain viability after undergoing a slow-freeze cryopreservation protocol followed by thawing and culture. In summary, this work showed that IL11 and LIF have potential benefits to the in vitro production of bovine embryos when supplemented at IVM. However, future work is needed to assess how these molecules are causing these improvements. Our results indicate that IL11 and LIF may function differently from IL6 when supplemented during IVM.

During folliculogenesis, oocytes and cumulus cells undergo many morphological and physiological changes. Transcriptome data were produced from single oocytes and corresponding cumulus cells (CC) to infer the differences in the transcript abundance from fully grown versus growing phase oocytes and surrounding CC. Using cow ovaries from an abattoir, COC were collected from follicles ranging from 3 to 8 mm in diameter. Cumulus-oocyte complexes were incubated in the supravital stain brilliant cresyl blue (BCB) as a means of separating oocytes based on the growth phase. Fully developed oocytes remained stained and were categorized as BCB+, whereas oocytes in the growing phase were colorless and were categorized as BCB-. Following the classification, COC were used for in vitro embryo production. Blastocyst yield from COC classified as BCB+, BCB- and unstained controls were 20%, 14% and 16.5%, respectively (P=0.18). Transcriptome data were also produced from oocytes and cumulus cells from BCB+ and BCB- COC. Transcripts from one long non-coding gene were differentially abundant in fully grown oocytes compared to oocytes in the growing phase. Eleven protein-coding genes were differentially expressed in cumulus cells collected from COC containing growing and fully grown oocytes. The results indicate no significant variation of transcript abundance of protein-coding genes in oocytes and limited regulation of transcript abundance in cumulus cells relative to the oocyte's growth phase in mid to large antral follicles. / Master of Science / The implementation of assisted techniques for achieving pregnancy is becoming increasingly adopted in the production of agriculturally important animals. However, most artificial reproductive methods have limited success, including in vitro embryo production. While many factors can contribute to reduced pregnancy rates relative to natural breeding, the developmental competence of the female egg is one of the many limiting factors. During its residence within the ovary, until it is fertilized by sperm, the egg is surrounded by layers of supporting cells. The egg and somatic cells interact by exchanging micro and macromolecules, but there is limited knowledge of the dynamics involving this interaction. It also remains unclear how these connections aid the egg in proceeding through development. Using a blue stain, the eggs were separated based on their stage of maturation. Then I investigated the interaction of the egg and the surrounding cells by measuring gene transcripts, which are a proxy of cellular function. Changes in the transcript abundance in the egg's surrounding cells were identified, which may be related to the egg's ability to be fertilized and proceed through embryonic development.

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).