Academic Portfolio

The Great Egg-scape

An educational tool based on the journey of the oocyte from oogenesis through ovulation, fertilisation and blastocyst formation until it reaches implantation

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-ABOUTHelp me Learn
Help me Learn is a student created revision aid which allows students to produce and share collections of electronic revision notes. These would not only help other students learn and revise, but also consolidate their own understanding of the material. The homepage would have a contents section outlining various university disciplines, for example Help me learn Physics, Help me learn Computer Science or Help me learn History etc, as seen in figure 1. Within these pages there would be a whole range of subtopics which address the major subject areas of the course, relative to the year of study. Students would be encouraged to play to their strengths and explore different ways to present work in a user-friendly manner. Furthermore, revision questions and a comprehensive reading list could be included so students using the site can further enhance their learning. The initial idea for this tool was to create an easy to use system where students could become interactive and cooperative with their exam revision. The tool is heavily based on the theme of peer support, as first-hand experience has shown that scientific information is more easily understood after group discussion and debate. Hopefully Help me LEARN would become a platform where students can learn and share methods of revision and enrich their learning experience.

Figure 1: Schematic plan of the 'Help me Learn...' website

The Great Egg-scape

This portfolio falls into the discipline of Reproductive Biology, in particular addressing topics covered in the third year of study. It was inspired by an essay I wrote, titled: The action of Leukaemia Inhibitory Factor on endometrial receptivity during implantation. I wanted to explore the basic concepts of pre-implantation biology, including current concepts being researched in the field at the moment. I specifically aimed to enhance my skills at creating informative and easy to understand diagrams of each of these, as I have always been able to make sense of even the most complicated scientific ideas through diagrammatic representation. Furthermore, images are much easier to retain in memory and so hopefully these would serve well for revision purposes, and be clear enough to be easily reproduced in the exam environment.

-OOGENESISOverview
Oogenesis encompasses the formation, development and maturation of an oocyte through various stages, which begins during fetal development and is completed at the time of fertilization1. The process of oogenesis occurs simultaneously with the development of the human follicular unit2, folliculogenesis, which starts as soon as the oocyte is surrounded by somatic cells creating the primary developmental unit, the primordial follicle. These follicles are believed to be the complete reserve of oocytes available throughout a womans reproductive life span1,2. Primordial follicles develop in the ovary around 10-30 weeks after conception and these give rise to primary follicles. At puberty, when the first menstrual cycle begins, around 12-20 of these follicles start to develop under the influence of elevated follicle stimulating hormone (FSH) leading to the formation of secondary follicles2. On day 9 of the cycle, most of the follicles have undergone atresia leaving one dominant to ovulate, which ovulates on day 14 in response to a surge of leutinizing hormome (LH) from the pituitary gland3. The empty follicle then forms the corpus luteum (CL) which produces progesterone necessary for later stages of the cycle. The duration of human folliculogenesis is estimated to span >175 days from primordial through to the preovulatory phases1,3.

Meiosis
Before an oocyte can be fertilised it must gain meiotic competence, which occurs at defined times of oogenesis, by accumulating specific factors at each stage of development3. Meiosis I begins in primordial oocytes during embryonic development and is halted in diplotene stage of prophase I, arrested in G2 of the cell cycle by high levels of cAMP produced by surrounding somatic cells2. Appropriate signals are given at the time of ovulation which stimulates the resumption of meiosis I generating a secondary oocyte and the first polar body, figure 2. Immediately after this, meiosis II occurs which is also halted at metaphase II until fertilisation. Upon penetration, the spermatozoa activates the oocyte to resume meiosis II and the second polar body is extruded, figure 2, which then degenerates along with the first one, leaving the oocyte to mature into an ovum. Polar bodies are there to discard haploid sets of chromosomes resulting from meiosis3.

Primordial to primary
All oocytes derive from primordial germ cells (PGCs), which migrate to and colonize the gonadal ridge in early fetal development1. As PGCs migrate they undergo mitotic divisions, and on reaching the gonadal ridge they undergo the first meiotic division

becoming primary oocytes in meiotic arrest3. PGCs make up a quiescent resting pool of oocytes estimated to hold around 7 million follicles at week 20 of gestation, which progressively depletes during fetal and throughout adult life3. Although it is believed that a finite number of oocytes is established around the time of birth, recent publications of work carried out in murine models suggest that ovarian follicles are renewed from germline stem cells. Further work is required however, to determine such dynamics of small follicle formation2. Oocytes originally develop in the ovary as clusters called germ cell cysts. These cysts break down and become surrounded by somatic cells, generating primordial follicles which are in turn activated to become primary follicles1. The mechanisms of primordial follicle activation are not fully understood, however it is currently described as a delicate balance between the actions of factors which promote proliferation, growth and differentiation and those promoting apoptosis. Some primordial follicles are activated and some remain quiescent for months and even years, maintained by the complex control system of factors secreted locally by surrounding somatic cells3.

Primary to secondary to preovulatory

The transition from primary to secondary follicles brings about the continued growth of the oocyte, granulosa (GCs) and theca cells (TCs) simultaneously which are all gonadotrophin independent processes3. The volume of the oocyte increases during the growth phase due to an accumulation of mRNA and rRNA which support protein synthesis during meiotic maturation, fertilisation and early embryogenesis1. As a woman reaches puberty, the HPG axis matures resulting in a pulsatile release of FSH and LH from the anterior pituitary gland. This leads to the cyclic development of antral follicles in a gonadotrophin dependent manner, the onset of ovulation and the first menstrual cycle3. Secondary follicles develop an antrum and are then termed antral or pre-ovulatory follicles. An increase in FSH prevents follicular atresia, allowing a cohort of follicles to mature and then increased inhibin A and estradiol inhibit follicular growth, creating a specific window in which antral follicle development occurs3. All stages of oogenesis are outlined in figure 2.

The dominant follicle

During ovarian development many follicles are lost and during each cycle, a single follicle is selected to ovulate2. This dominant follicle is chosen through follicle selection to continue the last phase of growth in the early to mid-follicular phase of the menstrual cycle3. It has more GCs with FSH receptors making it more sensitive to the gonadotrophin than subordinate follicles. These cannot survive in the decreasing FSH concentrations and so undergo atresia while the dominant one remains3. At a diameter of 16-29mm the dominant follicle has reached the preovulatory stage and continues to grow everyday leading up to ovulation, under the influence of both intraovarian and endocrine factors including AMH, inhibin A, estradiol, GDF9 and BMP153.

The dominant follicle is also responsible for producing large amounts of estradiol during the late follicular phase, through cooperation between somatic cells: GCs have aromatase activity on days 5-8 of the menstrual cycle; and TCs produce androgens to provide substrate for GC estradiol production. Estradiol negatively feeds back to the pituitary which decreases its FSH secretion leading to the death of subordinate follicles. As estradiol is secreted from the dominant follicle, LH receptors increase on GCs making the follicle less dependent on FSH and more responsive to LH during the selection process3. As estradiol production peaks a surge of LH is released and ovulation is induced.

Figure 2: Oogenesis and folliculogenesis

Oocyte-somatic cell interactions

The coordination of oogenesis and folliculogenesis is heavily dependent on somatic cell-oocyte interactions and the bidirectional communication of essential factors1 through transzonal projections2. Oocytes secrete factors to influence GCs such as GDF9, which stimulates the kit ligand (KL) pathway in GCs to promote follicle activation. This shows a complex relationship where oocytes secrete GDF9 to induce KL secretion in GCs which in turn stimulates oocyte growth through activation of c-kit2. Through transgenic mouse studies and genomic methodologies many genes, both germ cell and somatic cell-derived, have been identified to be crucial in regulating various stages of oogenesis, folliculogenesis and early embryonic development1. Figla and Nobox are transcription factors limited to the oocyte which regulate genes responsible for establishing the oocyte gene expression patterns whilst supressing the

testis pattern. They have both been implicated in the transition of germ cell cysts to primordial follicle stages1. Sohlh1 and Sohlh2 (spermatogenesis and oogenesis specific basic helix 1 and 2) are transcription factors with roles in early folliculogenesis and spermatogenesis to mediate the transition from primordial to primary follicles1. Yy1 is a transcription factor of gene expression essential for development, expressed in oocytes and GCs throughout folliculogenesis and stimulates the transition from primary to secondary stages2. These are just a few genes which represent transcriptional networks within the oocyte critical for directing oocyte development2.

-OVULATIONOverview
Ovulation is a unique event whereby a preovulatory follicle matures to a specific point and ruptures through the ovarian surface epithelium (OSE), to release a competent oocyte destined for fertilization4. It occurs once midway through every menstrual cycle of the mammalian female, the timing of which is verified and stimulated by a surge of leutinizing hormone (LH) from the pituitary gland5. This stimulates both GCs and TCs to stimulate cAMP and protein kinase signalling cascades which induce the transcription of4 multiple genes involved in the complex transition of a preovulatory follicle into one that can ovulate, and the process of ovulation itself6. The ovary has to go through a series of sequential events in preparation for ovulation: small follicles mature to preovulatory follicles; the oocyte gains meiotic competence; GCs produce oestrogens and acquire the ability to respond to LH; TCs increase output of androgens4. The LH surge stimulates germinal vesicle breakdown and the alignment of chromosomes on the 1st meiotic spindle. Unequal division of the cytoplasm and equal division of the chromosomes results in the extrusion of the 1st polar body. Meiosis II occurs after ovulation but arrests at metaphase II making it capable of fertilisation. If fertilization does not occur then the oocyte degrades between 12 and 24 hours after ovulation5.

The LH surge
The LH surge, displayed in figure 3, is the physiological trigger that kickstarts the events of ovulation7 through many biochemical and molecular changes in follicular cell functions8. In this way, ovulation is controlled by the hypothalamus in the brain and the release of FSH and LH from the anterior pituitary gland. Kisspeptin neurons have an essential role in the preovulatory LH surge as they interact with and stimulate GnRH secretion to mediate a positive feedback effect of estradiol to generate the surge. Kisspeptin neurons project to GnRH fibres so the kisspeptin protein can act upon them and give the pituitary a heightened sensitivity to the gonadotrophin9. LH initiates the complex process of ovulation4 by binding to its receptor on GCs and activing adenylate cyclase and the A-kinase pathway, leading to specialised and transient expression of genes. These include the progesterone receptor, cyclooxygenase-2 and prostaglandin endoperoxide synthase-2 (PGS-2)6. The resulting actions are implicated in: increasing steroidogenesis; increasing prostaglandin and leukotriene production; and increased plasminogen activator to convert plasminogen to plasmin7. Such products enhance ovulation by stimulating follicular collagenase, essential for digestion of the ovarian surface and follicular wall5. LH also increases blood flow to the preovulatory follicle due to local mediators such as VEGF,

cytokines, nitric oxide and oxygen radicals5. Therefore there is a tight coordination of the proteolytic and vascular cascade, which together culminate in rupture of the follicle wall8.

Figure 3: Ovulation and associated hormone fluctuations

Proteolytic cascade
The LH surge stimulates a cascade of proteolytic events in the preovulatory follicle and cells of the adjacent OSE, to initiate and control ovulation5. Follicle rupture requires the ECM associated with thecal layers and tunica albuginea to be digested. Following this, the cumulus-oocyte complex (COC) is released and moves to the peritoneal cavity, shown in figure 3, where it is caught by fimbriae and enters the oviduct beginning the journey to the uterus6. The OSE secretes urokinase plasminogen activator (uPA) to convert plasminogen to the active plasmin to breaks down the thecal- blood vessel epithelium. Sex steroids and PGs weaken the follicle epithelium causing lytic enzymes such as TNF- to leak out which induces the expression of MMPs, apoptosis and inflammation. MMPs break down collagen in the follicular wall, forming a stigma at the apex5,6 and stimulate collagenase to degrade the collagen fibre meshwork of the follicular wall7. Studies

with pharmacological blockage of these enzymes show inhibition of follicle rupture and a great reduction in ovulation5. The cascade is controlled by local tissue inhibitors in the theca of growing follicles which protects them from enzymes diffusing from ovulatory follicles5.

Inflammation
After the LH surge the connective tissue around the follicular wall thins and, there is an accompanying inflammatory reaction where inflammatory cells such as leukocytes and macrophages invade to have actions in remodelling and resolve. This process shares features with normal inflammation such as: increased vascular permeability, oedema, vasodiulation and tissue remodelling4,6. Inflammatory cells secrete cytokines and prostaglandins (PGs) which induce expression of plasminogen activators, MMPs and kallikreins, which all activate or inhibit collagenases and other protease cascades6, for enzymatic digestion of the follicle wall and OSE. This form of inflammation is controlled by an increasing cortisol concentration in follicle fluid which provides an anti-inflammatory environment to counteract the process hence limiting damage8.

Leutinization
Once the COC is released, the somatic cells undergo leutinization, the tissue remodelling process where GCs and TCs lose their concentric structure and differentiate into the irreversible luteal cell phenotype. The follicle wall collapses and the antrum is filled by a clot which is invaded by granulosa and theca lutein cells (GLCs, TLCs) to form a solid structure called the corpus luteum (CL), figure 3. The bloodfollicle barrier is broken down and LDL-cholesterol enters where it is metabolised to progesterone by steroidogenic enzymes in GLCs6. This progesterone is required to thicken and mature the uterine endometrium in preparation for blastocyst implantation9.

-FERTILISATIONOverview
Fertilisation encompasses the fusion of the nuclei of male and female gametes generating a new individual organism known as a zygote, which subsequently develops into a diploid embryo10. The spermatozoa swim through the female reproductive tract and reach the oocyte in the fallopian tube where they interact with the cumulus-oocyte complex (COC). The sperm produces hyaluronidase generating hyaluronan fragments to induce chemokine secretion in the cumulus cells to aid sperm migration through the corona radiata10. Gene knock out studies have allowed for the identification of various factors in both males and females essential for successful fertilisation. These include the zona pellucida binding protein B4galt1, acrosomal protease acrosin (Acr) and fertilin, an egg fusion protein10. Fertilization-related molecules in the plasma membranes of sperm and egg such as ADAM-1 in sperm heads and CD9 on the egg membrane, facilitate initial binding of cellular membranes11. Microvilli on the oocyte plasma membrane engulf the sperm and the sperm head passes into the oocyte cytoplasm. The two gametes have now fused to form the zygote where the two pronuclei form and migrate to the centre. DNA synthesis occurs and chromosomes merge on the spindly at the first mitotic division of the zygote12.

Sperm changes
As sperm cells migrate through the female reproductive tract they undergo a series of biochemical and morphological changes before they make contact with the oocyte10. These changes are essential for the sperm to achieve fertilization capability, the most important being capacitation. Capacitation comprises the removal of cholesterol and other sterols from the sperm surface, creating a destabilised and more fluid membrane environment which has increased permeability to Ca2+ and fertilization capacity10. Both tyrosine phosphorylation and calcium ion influx are essential for capacitation12. The acrosome reaction occurs on contact with the oocyte. The acrosome covers the tip of the sperm head and is a golgi-derived exocytic organelle. On binding to the ZP, progesterone secreted from cumulus cells induces the contents of the acrosome to be released. This includes surface antigens which are necessary for fusion with the oocyte plasma membrane, and enzymes necessary in aiding the sperm digest a path through the ZP and into the oocyte cytoplasm, as shown in figure 4.

Figure 4: Fertilisation of an oocyte by a spermatozoan

Zona Pellucida
The ZP is the final hurdle to the sperm before it meets the egg. It is comprised of 3 glycosylated proteins ZP1,2 and 3: ZP3 is the primary sperm receptor and induces the acrosome reaction; ZP2 is a secondary receptor which binds acrosome-reacted sperm; and Zp1 crosslinks 2 and 3 creating the filamentous structure of the ZP. The ZP maintains interactions between GCs and oocytes during maturation10,13. Proteolytic cleavage of ZP proteins by sperm cell surface proteases such as acrosomal enzymes like Acr, clears a path for the incoming sperm. In studies where Acr is knocked out however fertilization still occurs, and so 5 new testis- specific serine proteases have been identified termed Tesp1-511. After ZP penetration the sperm and egg plasma membrane fuse, a fusion mediated by factors such as Mn9, Cd46 and Izumo110.

Blocks to Polyspermy
It is vital that only one sperm fertilises the oocyte otherwise an abnormal polyploidy embryo which would normally die, persists. There are two blocks to polyspermy. The fast block occurs within a few seconds of sperm binding to the oocyte plasma membrane and involves an influx of sodium ions which changes the resting

membrane potential from -70mV to +20mV12. Other sperm cannot bind at this resting potential, an effect that only lasts a few minutes. The slow block occurs within a few minutes of sperm binding to the oocyte plasma membrane and involves the cortical granules fusing with the oocyte plasma membrane and subsequent release of contents into the perivitelline space. Enzymes cleave ZP2 and remove sugars form ZP3 which alters the sperm binding sites, preventing another sperm from binding and fertilising the egg. This is called zona hardening or the zona reaction13.

Egg activation
Before fertilization, the oocyte is quiescent and arrested at meiotic metaphase II. Sperm-oocyte fusion causes metabolic and physical changes that together are called egg activation and bring about the resumption of meiosis II and early embryonic development11. Fertilization causes intracellular calcium (Ca2+) oscillations in the oocyte cytoplasm, released from internal stores, modulated by upstream pathways and soluble sperm factors12. The sperm binds to the oocyte ZP and a sperm specific phospholipase C (PLC) is released which catalyses the conversion of phosphatidyl-inositol bisphosphate (PIP2) into diaglycerol (DAG) and inositoltriphosphate (Ip3) in the egg13. PIP2 is located in the plasma membrane and DAG stays in the membrane, acting as a second messenger by activating the enzyme protein kinase C (PKC) to stimulate downstream signalling pathways critical for embryonic development. Ip3 releases Ca2+ from intracellular stores in the ER, which diffuses to the plasma membrane to activate more Ca2+ channels and leading to a secondary influx of Ca2+ 10,13. Furthermore, increased Ca2+ causes cortical granule exocytosis, establishing the block to polyspermy11.

-BLASTOCYST FORMATIONOverview
Upon fusion of the male and female gametes at fertilization, a single celled zygote is formed which subsequently splits into 2 cells then 4 cells then 8 cells etc, outlined in figure 5. When it reaches 32 cells approximately it becomes known as the morula. The period where a zygote develops into a blastocyst corresponds with days 14 to 21 of the menstrual cycle, during which time the uterus is being prepared for implantation by the actions of progesterone secreted by the CL14. This marks the end of the germinal stage and the beginning of the embryonic stage of development15. From the morula, cell divisions and cavitation result in the formation of the blastocyst with a fluid filled blastocoel cavity, inner cell mass (ICM) and outer trophoblast layer. Blastocyst formation is around 4-5 days post fertilization16.

Zygote to Blastocyst
Early embryonic development is independent of direct cell-cell contacts with reproductive tissues until implantation17. As the zygote traverses the oviduct, the cells undergo cleavage divisions, differentiation and apoptosis followed by compaction at the 32-cell stage to create the morula. During compaction cells become epithelial like and polarised with microvilli on the outer apical surface, organelles in the apical region and the nucleus on the inner basal region. The outer cells of the morula have an ATP-pump system to pump NA+ out of cells and K+ into cells, found no their basal surface. NA+ is pumped into the blastocyst cavity during a process termed cavitation, and water is drawn in by osmosis, creating the internal fluid filled cavity called the blastocoel15. Key paracrine growth factors secreted from tract cells, including insulin like growth factor (IGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF), enhance the development of zygotes into morphologically effective blastocysts14, comprising an outer layer called the trophoblast and an ICM which will later form the fetus, displayed in figure 5. Three primary lineages are established at the late blastocyst stage: the epiblast or inner cell mass which later develops into the entire fetus, primitive endoderm which will become the yolk sac and trophectoderm which will give rise to the placental trophoblast cells17. As the blastocyst is forming, the uterine surface is changing and differentiating to become receptive to allow for apposition and attachment of the blastocyst16.

Figure 5: Development of the zygote to the blastocyst stage

Zona Hatching
Before the blastocyst can implant within the uterine wall, it has to hatch from the ZP16, a process mediated through the actions of urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9). Two novel implantation serine proteinases have also been identified as being expressed in morulae and blastocysts during hatching and outgrowth16: the ISP1 gene encodes the embryo-derived enzyme strypsin which initiates hatching; and the ISP2 gene encodes a related tryptase expressed in the endometrial glands, which is a zona lysis proteinase17. Both of these are mediated by the actions of progesterone. Hatching is localised to the abembryonic pole of the blastocyst and occurs around the 4th day after fertilization. It involves the global rupture of the ZP and release of the blastocyst mediated by the co-expression of many factors which have pivotal roles in ensuring successful hatching and implantation16.

Genome activation
Early development is partly controlled by the maternal genome which makes the oocyte rich in maternally encoded RNA and protein17. However, there is a transition from maternal to embryonic control through three phases: the minor zygote genome activation (ZGA) which occurs mostly in the male pronucleus with only a few genes activated during the 2 to 4 cell stage; the major ZGA which involves extensive reprogramming of the gene expression pattern from the 4 to 8 cell stage; and the midimplantation genome activation (MGA) which involves major gene expression changes before the 8 cell stage, preceding morula compaction14.

During the minor and major ZGA phases, there is an increased expression of growth factors like IGF-1 and GM-CSF which increase the likelihood of successful blastocyst formation. The MGA genes include ZO1 and Cx43 which are adhesion molecules needed during and after compaction15, and Oct4, an octamer-binding transcription factor required for the formation of the ICM which becomes persistently expressed in the trophectoderm of developed blastocysts. Cdx2, another transcription factor, is needed for trophectoderm development and is detectable in the blastocyst approximately 5 days after fertilization in the morula before cavitation occurs17. By analysis of ligand-receptor pairs in early embryos, it has been found that ligands in the cell cytoplasm bind to receptors on the plasma membrane demonstrating autocrine and paracrine signalling between cells of the developing blastocyst15.

-IMPLANTATIONOverview
Implantation is the biological process where a newly developed blastocyst embeds in the maternal endometrium, coordinated through a series of cellular and genetic interactions18. Whilst the blastocyst is developing the cells of the uterus are proliferating and differentiating under the influence of estrogen and progesterone, to become receptive to the incoming blastocyst18. The trophectoderm synchronously differentiates during blastocyst formation turning into invasive trophoblasts to mediate invasion into the uterine wall13. Human embryos implant around day 6-8 post fertilization. Implantation occurs in three complex stages: apposition, adhesion and invasion, which depend on paracrine signalling pathways between the embryo and endometrium to ensure success19. The outer layer of the blastocyst comprises trophoblast cells which proliferate and secrete proteolytic enzymes on initial contact with the luminal epithelium (LE) following this, the trophoblasts interdigitate between endometrial cells and eventually the blastocyst implants and makes contact with the maternal spiral arteries to establish a blood supply to acquire essential maternal nutrients19. Placentation and pregnancy follows, however if the egg is not fertilised, the endometrial lining is shed during menstruation13.

Uterine receptivity
For implantation to be successful, the blastocyst must come into contact with the endometrium during the period of receptivity called the implantation window19. There is a complex interaction between blastocyst and endometrium that ensures timely development and maturation of both19, mediated by a whole array of factors18. Ovarian estrogen and progesterone are the dominant hormones which ready the endometrium for successful embryo implantation19. Other determinants of uterine receptivity include: cytokines which prepare the uterus and mediate embryonic attachment; Hox 10 and 11 which induce stromal cell differentiation and uterus formation; and integrins and selectins which mediate the initial adhesion between the embryo and endometrial wall16. As the endometrium changes from the neutral state to the receptive state, several morphological changes occur. The luminal epithelium of the pre-receptive endometrium is covered in mucin-1 (Muc1) which prevents infection, long microvilli and a high surface charge, all of which resist blastocyst attachment20,21. During the receptive phase, the mucin coat is shed, microvilli shorten and the surface charge decreases to allow integrin-extracellular interactions21. Once receptive, these cells cannot go back to the neutral stage however, if there is no pregnancy, the

endometrium goes into the refractory period, which is once again resistant to implantation20.

Figure 6: Implantation of a developing blastocyst

Apposition, Attachment and Invasion

Apposition is where the blastocyst gains polarity and assumes the correct orientation for attachment19, bringing about the close contact of the trophoblast and the LE. The heparin binding EGF-like growth factor (HB-EGF) gene is the initial attachment molecule expressed in the epithelium in response to soluble materials secreted from the incoming blastocyst20. It mediates a tethering by binding to ErbB and heparin sulphate proteoglycans expressed by the trophoblasts18,21. Attachment ensures that the association between the blastocyst and epithelial layer is greater so the blastocyst will not get dislodged or flushed through the uterine lumen21. Numerous glycoproteins and carbohydrates with associated receptors are expressed locally in the LE and trophoblast cells around the time of attachment, with integrin v3, L-selectin and cadherins being the most important in the initial stages of embryo attachment18,20. Invasion is where the blastocyst penetrates through the LE and burrows into the underlying uterine stroma establishing a strong connection with the mothers spiral

arteries to later set up a placental bed19. An increased vascular permeability is set up at the implantation site, mediated by the action of prostaglandins under influence by COX1 and 2 enzymes18. During invasion, the stroma undergoes decidualisation, differentiation into the decidual cell phenotype, and the LE at the site of implantation is lost18. Cell-cell interactions are critical to ensure the appropriate amount of invasion occurs for the correct type of placentation21. All three stages can be seen in figure 6.

Decidualization
During decidualization, underlying stromal cells differentiate into a secretory phenotype, displayed in figure 6. These produce MMPs to promote angiogenesis and tissue remodelling21 which allow for the blastocyst to embed and establish a blood supply to the developing embryo18. Stromal cell decidualization causes the overlying LE cells to undergo apoptosis which can be loosened by trophoblast cells as they penetrate through LE and the basement membrane20. The trophoblast and decidua interact with each other to adjust the degree of invasion: trophoblast secrete MMPs to degrade maternal tissue which expresses high levels of MMP inhibitors; decidua expresses IGF-binding protein-1 which binds 15 intergrin on the trophoblast to limit invasion20.

Successful implantation
The implantation of a healthy blastocyst requires a multitude if essential factors, those that work both locally and systemically20. Leukaemia-inhibitory factor (LIF) is an interleukin-6 cytokine expressed transiently in epithelial cells of the uterine glands in the endometrium, with receptors found on pre-implantation embryos21. It is thought that LIF plays a role in human implantation and blastocyst growth, as LIF expression peaks at implantation, LIF receptors are found in the endometrium and infertile women have lower LIF expression18,19. It also has a role in shortening the microvilli on the uterine surface by stimulating the loss of polarity in luminal epithelial cells20. Homeobox transcription factors have been found to be important. Hoxa10 and Hoxa11 are expressed during the receptive phase in the uterine stromal cells which remains high during uterine preparation and decidualisation18. For the implanting conceptus to survive, it must signal to the mother to maintain the progesterone support from the CL through one of two systems: the luteotrophic mechanism is where chorionic gonadotrophin (hCG) is secreted by the trophoblast to maintain the CL 19: and the anti-luteolytic mechanism, where trophoblast cells secrete interferon tau to inhibit endometrial PGF2 secretion and prevent CL breakdown. Once the placenta is established the luteo-placental shift occurs and the placenta takes over in the secretion of progesterone19

-APPLICATIONSAssisted Reproductive Technologies

Assisted reproductive technologies (ARTs) have advanced considerably over the past decades and are continuously evolving with the aim of increasing the success rates of in vitro fertilisation and pregnancy19. It is of the utmost importance to research into the processes of pre-implantation biology including: oocyte and follicle development and maturation; fertilisation and the subsequent formation of the blastocyst; and the complex process of implantation with the interactions between endometrium and blastocyst15. Insight into these aspects of reproductive biology holds many opportunities for enhancing ARTs, with research focussing on: Optimizing ovary stimulation protocols to release plentiful follicles Improving embryonic culture conditions to ensure successful maturation Investigating biomarkers of uterine receptivity, both morphological and molecular, to establish the correct timing of embryo transfer, and Enhancing embryo selection, to choose the embryos with the greatest chance of survival19,20.

Such knowledge and research therefore is vital to progress in ARTs and the understanding of reproductive biology.

-Practise QuestionsOogenesis
Outline how an oocyte grows from a PGC Describe meiosis in female germ cells How are oocyte-somatic cell interactions crucial to oogenesis?

Ovulation
Outline the LH surge Explain what is meant by the dominant follicle Describe the proteolytic cascade stimulated at ovulation Is ovulation is an inflammatory event?

Fertilisation
Describe the changes in sperm prior to fertilization Outline how polyspermy is avoided Explain how the egg is activated following fertilization

Blastocyst formation
Describe the development of a zygote into a blastocyst Briefly outline embryo genome activation

Implantation
Describe what is meant by uterine receptivity Outline the three stages of implantation

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