Embryology Colloquium I Summary (PDF)

Colloquium I Embryology 4th Semester Embryology 1. Teratology and embryology: main terms, historical aspects and actuality of investigations. Ontogenesis. Experimental embryology. Teratology - "Gr: miracle" is the scientific branch of embryology dealing with malformations occurring through embryonic environment. This science dates back to ancient times, where the term "monster" was used to describe certain birth defects. The view on malformations in pre-medieval times was a supernatural one, where a malformed child was a positive sign indicating happiness. This was however changed in the medieval era, when the view became an infranatural one indicating possession of the devil and death. Teratology became of great importance in modern medicine due to several reasons: - 1941 - virus infection of pregnant mother gave birth defects in newborn - 1945 - atomic bomb disaster of Hiroshima and Nagasaki caused a lot of birth defects in following generations - 1959-1962 - the thalidomide catastrophe in Germany (harmless sedative) caused defects Incidence of birth defects throughout the world greatly differs in literature, but amounts to 2-5,6 percent, but is doubled during first years of life when physiological functions of child develops. Incidence of malformations is extremely high; 2/3 of zygotes with chromosomal anomalies fail implantation. Abortion is a beneficial reaction of nature to prevent children with birth defects to be born. Light anomalies, s. stigma, have also been described. They are combined with the life and functioning of the body, and include all abnormalities of ear, mouth, increased size of thumbs in upper and lower limbs etc. Teratogen: inducer of congenital malformation. Embryology is the scientific branch dealing with the part of ontogenesis (development of an organism) from zygote until birth. The field of embryology includes: comparative, descriptive and experimental embryology. Founder of embryology is credited the German embryologist Reichert, which described the first two-week-old human blastocyst in 1873. The interest of the human society about the improper development of mammalians, including humans, is older than embryology, which means that embryology is also connected with teratology. Experimental embryology: Main role today is to provide: 1) Prevention of the developing body from the possible malformations, which includes detection of the sensitive and critical stages in the development of each structure - Based on knowledge of sensitive and critical stages, which differs in time for different systems and tissues. Sensitive stage for CNS development are during whole intrauterine development, while critical stage is between weeks 3-5. 1 Colloquium I Embryology 4th Semester - Structural damage of heart may develop under influence of teratogens between weeks 3-5.5, but functional heart malformations appear if teratogens persist until end of 8th week. Critical stage for limb development is between weeks 4-7, but for external genitalia: weeks 7-12. - Prevention also includes antenatal consultation, strictly no medications, drugs, alcohol or nicotine during first 90 days. However, all antibiotics pass through placenta and reach level of 20-25% from concentration in maternal blood in the blood of fetus. 2) Research in in vitro fertilization to improve outcome 3) Intrauterine surgery development - Started in 60s with successful blood transfusion on fetus with anemia due to Rh- factor incompatibility with mother, but fetus died following. In 1963 similar surgery performed, successful and fetus survived. - Intrauterine surgeries are very expensive, and the fetus is very fragile - organs and tissues are very soft and can easily be damaged. 4) Research in the field of stem cells, and the frames of ethical considerations and laws concerning their use - Stem cells are a special branch of experimental embryology and reproductive biology - First human stem cells obtained in 1998 from human embryos. - "Stem cells are unspecialized cells that renew themselves for long periods through cell divisions, and under certain physiological and/or experimental conditions can be induced to become cells with particular functions". - Stem cells exist in specific locations within a given tissue; comprise a small percentage of the total cell populations; ultra-structurally unspecialized; pluripotent; slow cycling (but may be induced to proliferate more rapidly); have proliferate potential that exceeds an individual’s lifetime; an intermediate group of more rapidly proliferating cells exists that can form clones; microenvironment of a stem cell plays and important role; many cancers arise from stem cells. - There are 5 types of stem cells: conceptus, embryonic, fetal, umbilical cord and adult / induced stem cells. The most totipotent are the conceptus stem cells. Totipotency decrease from conceptus to adult stem cells. Two main properties of all stem cell types have to be investigated to develop successful usage of these cells for diseases: 1) Precise determination of how stem cells remain unspecialized and self-renewing for many years 2) Precise identification of signals that cause stem cells to become specialized cells - Easiest to obtain adult stem cells, and are found in brain, bone marrow, peripheral blood, blood vessels, skeletal muscles, skin and liver. There is a very small number of cells in each tissue, where they remain non-dividing for many years until activation by disease or injury. - Mesenchymal stem cells have been investigated most; can transform into: smooth muscle cells, endothelial cells, epithelial cells equally well; their development expands over the 2 Colloquium I Embryology 4th Semester border of one germ layer - Umbilical cord stem cells are limited in number, and can also develop into many forms: adipocytes, pancreatic cells, neurons, glial cells, osteoblasts, endothelial cells, SMC and are suitable for long-term storage in stem cell banks - elaborated laws for collection and storage. - Numerous problems in working with stem cells: large number of cells needed, risk of contamination into the storage environments, embryonic stem cells from a donor could cause transplant rejection and formation of teratomas (embryonic tumors) or teratocarcinomas. Terms: Agenesia - total absence of organ or structure Aplasia - failure to develop a functioning organ Atresia - absence of opening Dyschromia - disorders in developmental time Ectopia - atypical localization of organs Heterotrophia - presence of an organ in atypical position Hypoplasia - underdeveloped organ or structure in size and mass Hypotrophia - underdeveloped fetal mass Macrosomia - increase in body length 2. Ethological factors for development malformations and their classification Each cell has a genetic program, and the groups of cells form blastema with reciprocal control for the differentiation of tissues and organs. Malformations will result if disturbing factors cross threshold of ontogenic adaption. There are numerous factors, classified into genetic and environmental factors 1. Genetic factors: - Mutagens that affect fetal germ cells give rise to congenital malformations in the following generations e.g. ionized radiation (X-rays) and many chemicals. - Intervals between mutation of germ cell and phenotypic manifestation can include several generations. Mutations of somatic cells may result in malformations or in tumors without transmitting to next generations. Chromosomal abnormalities - May appear in numerical or structural chromosomal aberrations, causing severe malformations in embryo, often resulting in spontaneous abortion or death after birth i. Numerical - According to the formula of human karyotype that is set to be: 46XX or 46XY ii. Gonosomal aberrations concern sex chromosome abnormalities - Monosomy - one sex chromosome, about 97% is aborted, vital forms are the females; Turner's syndrome. 3 Colloquium I Embryology 4th Semester - XXY-trisomy - Klinefelder syndrome - male with small testes, azoospermia, defective masculinity - XYY-trisomy - supermen, aggressive behavior, lesser intelligence etc. iii. Autosomal aberrations concern the number of autosomes Some forms of trisomy (2n+1) result in different syndromes - Trisomy 21 - Downs syndrome (1/1000 births) - Trisomy 18 - Edwards syndrome (abortion; mean survival 2 months) 2. Environmental factors - All spaces outside the chromosomes; intercellular and extracellular spaces, placenta, intra maternal space, external environment - Human teratogens producing congenital malformations are numerous, divided into biological, chemical and physical factors i. Biological teratogen groups: - All microorganisms causing congenital defects, including viral infections (especially in last trimester of pregnancy may cause postnatal epilepsy) - Additional infectious agents: toxoplasmosis, syphilis, HIV ii. Chemical teratogens - include medicaments, chemicals, heavy metal compounds, hormones, hypo- and hypervitaminosis during pregnancy, alcohol, drug and nicotine abuse. iii. Physical factors: noise and vibrations, irradiation, cosmic radiation, radioactivity. Additional factors such as hypoxia and stress are responsible for malformations. 3. Sensitive phases of intrauterine life. Periods of intrauterine life. Sensitive and critical phase of body development. It is extremely important to clarify the post sensitive and critical phase for each organ development. Teratogenic determination period is the phase of the development in which a teratogen becomes effective; the time of excessive mitotic activities in the blastema during their differentiation. Each organ has its critical period depending on time and duration of differentiation. Generally, there are 3 phases during intrauterine life. However, one phase, gametogenesis (related to parent's reproductive cells) is realized before fertilization, and has impact on the further development of zygote, because chromosomal abnormalities occur in this stage. 1. Blastogenesis - from fertilization till day 16 - development of blastula and blastocyst. Development of bilaminar embryo (hypoblast, epiblast). Impact of teratogens on conceptus causes blastopathies, mainly concern embryoblast and trophoblast, and result in 4 Colloquium I Embryology 4th Semester abortions. In case of conceptus survival, heavy malformations develop. 2. Embryogenesis - day 16-60 - further development of trilaminar embry (all three germ layers), and further devleopment of primordia in main organs, fetal membranes and placentation. Teratogens cause embryopathies, concerns embryo, amnion and chorion. 3. Foetogenesis - day 61 till birth - differentiation and maturation of organs. Foetopathies concern the fetus and placenta, and cause metabolic disorders, physical and mental retardations and also some kinds of malformations. 4. Gametogenesis. Embryonal gonads. Gametogenesis - the development of a specific structure in germ cells capable of fertilization. The main event of a gametogenesis is the reduction of chromosomal number in half by meiosis in female and reproductive cells The first primordial germ cells, or gonocytes, become visible on 24th day after fertilization in the endoderm of yolk sac. They are large, rich with alkaline phosphatase, glycogen and are able to carry out amoeboid-like movement. They migrate through gut endoderm to posterior body well, at level of Th10 gonads development. During migration, 100 cells increase by mitosis to 400. Gonocytes and gonads (organ developing gonads, primitive gonads at Th10) interact, results in incorporation of gonocytes into the primitive gonads. In migration disorders, gonocytes can stop somewhere on their way, or migrate to other regions, following transformation into teratomas, which show aggressive growth. Primordial germ cells undergo rapid mitotic division into gonads, where number increase to millions. However, pattern of mitotic division differs between male and female. Primordial cell for females are named oogonia, and undergo oogenesis later on. Counterparts are spermatogonia, undergo spermatogenesis. There are differences between both types of gametogenesis already during embryonic life. Number of oogonia increase via mitotic division from 2 million at 2nd embryonic month, to 7 million at 5th month, and then undergo atresia, reduce number to 1-2 million at moment of birth. Mitosis of oogonia does not continue after birth of a girl, but atresia continues in postnatal life, and affects all types of oocytes in the ovaries, ends at menopause. Number of primordial oocytes in the ovaries capable of oogenesis therefore varies between 10-40 000 around puberty, but during female reproductive life (20 years) approximately 400 undergo ovulation. Mitosis of spermatogonia starts during embryogenesis, but continues throughout life. In puberty, spermatogonia in tubuli seminiferi contortii of testis undergo periodic mitotic division, and enter meiosis as synchronous groups. Spermatogenesis continues until old age, and diminishes with age due to decrease of testosterone. 5 Colloquium I Embryology 4th Semester 6 Colloquium I Embryology 4th Semester 5. Ovogenesis. Characterization of ovocyte I. Oogenesis consists of 3 periods: division, growth and maturation. Division period relates to embryogenesis, and ends with development of 1-2 million oogonia (44+XX) at moment of birth. Growth period starts at fetal period, with entrance of oogonia into first meiotic division, and appearance of primary oocytes (responds to primordial follicle). After birth, primary oocytes enter diplotene stage of meiosis I during the first months of life (responds to primary follicles). Later, meiotic process is blocked in primary oocytes (44+XX, 4n) until puberty. During diplotene stage, mammalian oocyte produces less rRNA and mRNA, as well as synthesizes more than 5000 cortical granules to prevent invasion of excess spermatozoa to the egg during fertilization. Maturation period starts with puberty, when 10-30 primary oocytes complete the first meiotic division with each menstrual cycle, and continue their development. Other primary oocytes remain arrested in diplotene stage. Primary oocyte, most sensitive to FSH, develops a columnar cell ring around itself, called corona radiata, and the following surrounding cell layers are called granular folliculocytes. A thick zona pellucida separates oocyte from the surrounding cells, and is called secondary follicle then. After completing first meiotic division, two cells unequal in size appear, one is called secondary follicle, other is small and called polar body I. Both cells display 22 chromosomes + X, and are of 4n (responds to 3rd follicle of ovum). Multilayered granular folliculocytes develop stratum granulosum, surrounded by special C.T. - theca layer. Theca interna contain epithelium-like cells producing estrogens, but theca externa is rich in C.T., fibers, and blood vessels. Liquor folliculi is produced in granular cells of secondary follicle, but is more pronounced in tertiary follicle, where antrum is developed. Liquour folliculi is similar to plasma, and contain plasma proteins, 20 different enzymes, hormones like FSH, LH and steroids, and proteoglycans that bind water. Tertiary follicle is also called Graafian follicle. Second oocyte begins the second meiotic division, but again meiosis is blocked in metaphase stage, which can only be eliminated by fertilization, while unfertilized oocytes fail to complete the second meiotic division. Second meiotic division after fertilization is also unequal, and results into a fertilized ovum and 7 Colloquium I Embryology 4th Semester polar body II. At fertilization, cells are 22+X, n, and as polar body I also undergoes division, the result is the development of 1 fertilized secondary oocyte, and 3 polar bodies, that remain beneath the zona pellucida until entering uterus. Dies within 12-14 hours if not fertilized. Hormonal control of female reproductive cycle by release of inhibiting factors from hypothalamus that is acting on adenohypophysis, causing release of FSH and LH. FSH stimulates growth of follicle in the follicular stage. Meanwhile, estrogens are produced by granular folliculocytes of the ovaries. Tertiary follicle is most sensitive for LH, becomes a Graafian follicle, and a sudden soar in LH and FSH after peak of estradiol in the blood causes ovulation. Remnant of ovulated follicle becomes corpus luteum, which produces progesterone in case of pregnancy. The secondary oocyte will then be released from the secondary follicle into the fallopian tube to either take part in fertilization or get released with menstruation. (Remember that this secondary oocyte is still in metaphase II of secondary meiosis until the sperm will fertilize.) 6. Spermatogenesis. Consists of 4 periods: division, growth, maturation and differentiation. Division starts in early embryogenesis, presents the increase of cell number via mitotic division already described above. The result is the development of two types of spermatogonia: A and B. Type A give primordial stem cells and B spermatogonia that will participate into further events, while the other parts of A spermatogonia are reserve cells. Spermatogonia cells have chromosome number 44+XY, 2n. Growth period: spermatogonia transform into the primary spermatocytes (stimulated by testosterone from leydig cells) doubled DNA (44+XY, 4n). This stage is sometimes called preleptotene. Maturation includes two divisions, meiotic and mitotic. First meiotic division (reduction division, meiosis I) has a prolonged prophase with 5 stages: - Leptotene - thin, prolonged chromosomes with indistinct spiralization. - Zygotene - pairing of homologous chromosomes with indistinct spiralization. - Pachytene - spiralization resulting in thick, short chromosomes - Diplotene - formation of tetrads consisting of 4 chromatids - Diakinesis - continues spiralization of chromosomes 8 Colloquium I Embryology 4th Semester Metaphase I demonstrate lining of tetrads into equatorial plane of division spindle. Anaphase I start movement of 2 chromatids to the opposite poles of division spindle. Meiosis I end with development of 2 secondary spermatocytes, with haploid chromosome complex (22+X, or Y, 2n). Prophase I take around 22 days. Second meiotic division, equational division, is similar to ordinary mitotic division, with exception that in this process, the division cells are haploid. Division starts after meiosis I, and carries out in 6-8 hours. Period of synthesis is absent. In metaphase II, chromosomes line up along equatorial plane and centromeres between sister-chromatids divide. In anaphase II, sister chromatids migrate to opposite poles of the spindle. Telophase II ends with development of two haploid spermatids (22+X, or Y, n) from one secondary spermatocyte. Cytokinesis results into the development of 4 spermatids. Spermatids enter the last differentiation stage connected by cytoplasmic bridges, which provide synchronic differentiation. Differentiation is called spermiogenesis. The spermatids will become mature sperm cells with help from sertoli cells which give nutrients and phagocytose the cytoplasm from our spermatid. 7. Differences between oogenesis and spermatogenesis Female Male Meiosis initiated once in a finite population Meiosis initiated continuously in a of cells. mitotically dividing stem cell population. One gamete produced per meiosis (+2 polar, Four gametes produced per meiosis unequal bodies) Completion of meiosis delayed for months or Meiosis completed in days or weeks years Differentiation of gamete occurs while Differentiation of gamete occurs while diploid, in first meiotic prophase haploid, after meiosis ends All chromosomes exhibit equivalent Sex chromosomes excluded from transcription and recombination during recombination and transcription during first meiotic prophase meiotic prophase Oogenesis occurs at normal body Spermatogenesis occur at 33,6 degrees temperature (36.6) The meiotic division in oogenesis give 2 cells In spermatogenesis, divisions are equal of unequal size; unequal division Oocytes are only maturing after fertilization Spermatozoa are mature cells (arrested in metaphase after 1st meiotic division) Oogenesis begins before birth Spermatogenesis begins at puberty All ova are present at birth Spermatozoa develop at puberty and onwards 9 Colloquium I Embryology 4th Semester Oocytes are immotile Spermatogenesis involves the development of flagella 8. Spermiohistogenesis and characterization of spermatozoa. Spermatids undergo four following stages: Golgi stage, head development, acrosome development and maturation of spermatozoa. Golgi stage includes condensation of the Golgi apparatus at apical end of nucleus, and rising of the acrosome. Head development starts with the reduction of size and form of the nucleus, and condensation of the chromosomal material. Condensation of nucleus material continues in acrosome stage. The acrosome is an enzyme-filled structure containing digestion enzymes, covering 2/3 of nucleus. Both centrioles move behind nucleus and form axoneme (flagellum), which starts from neck region. Mitochondria are arranged around proximal part of the flagellum, and represent the main motoric center of spermatozoa. In maturation stage, the cytoplasm streams away from the nucleus, form residual bodies that are later phagocytized by Sertoli cells. Length of spermatozoa: 0.06 mm, head is 4-5um x 4-5 um, and tail reaches 50 micrometers. During spermiogenesis, plasma membrane of head is partitioned into antigenetically distinct molecular domains, removed only in the female reproductive tract during capacitation. Spermatozoa in testis are mature, but are non-motile and incapable of fertilizing an oocyte. Cells form testis are carried to epididymis, undergo biochemical maturation (covered with a glycoprotein coating), which is continued when ejaculated sperm is mixed with secretions of prostatic gland and seminal vesicles during ejaculation. Spermatogenesis varies from 64-72 days, depends on influence of testosterone. In males, LH hormone stimulates the Leydig cells to produce testosterone, and follicle-stimulating hormone (FSH) acts on the Sertori cells. Feedback inhibition decrease production of pituitary hormones. The level of testosterone decreases under stress and after 60 years of age. 9. Fertilization. Stages and biological sense. Abnormal fertilization. Preconditions: 1. Number of reproductive cells - it is considered normal to have 100-400 million spermatozoa in 1 mL of sperm, while one oocyte is usually enough for fertilization. Border of infertility is 30 million spermatozoa in 1 mL of sperm. 2. Alive reproductive cells - oocytes survives 1-2 days after ovulation, while spermatozoa may survive for 1-2 days in female reproductive tract, and for one week in the mucous of the female reproductive tract. 30 % of all spermatozoa may be defective. 10 Colloquium I Embryology 4th Semester 3. Motility of spermatozoa - 70 % of reproductive cells in 1 mL of sperm has to be motile. Normal amount reaches 3-4 mL, and lesser can be sign of infertility. Motility of cells varies from 2-3 mm per minute, and 5-6 hours after sexual act, cells reach abdominal serous cavity where fertilization may take place. Sperm passage through different parts of female reproductive tract is not fully understood. Two main modes. Rapid transport - spermatozoa reach uterine tubes within 20 minutes from ejaculation, depending on muscular movements of female reproductive tract. Slow movement involves passage of spermatozoa through cervical canal from 2-4 days 4. Presence of mucous in female reproductive tract - varies from 20-200 mL. Normally, due to cervical mucin and soluble components, the mucous is not penetrable. Between days 9-16 of menstrual cycle, water content of mucous increase, stimulates passage of sperm via female reproductive tract. Mucous is called E-mucous. Fertilization: The contact of a sperm with an ovum, ends with fusion of haploid pronuclei, result in development of zygote(diploid) - the product of fertilization. Fertilization usually realized in the ampulla part of uterine tube, in oviduct between ampulla and isthmus, or in the abdominal serosa cavity. 100-400 million sperm pass from vagina via cervical canal into uterine cavity, then into uterine tube and reach ampulla of the uterine tube. Before fertilization, the spermatozoa have to undergo capacitation, and acrosome reaction. Capacitation is the elimination of the inhibiting factors (glycoproteins) from the spermatozoa head; takes part in female genital tract. Plasma membrane of spermatozoa alters, and cells become hyperactive; detach from tubal epithelium. Acrosome reaction is the effect of enzymes, which enable the spermatozoa to penetrate corona radiata, and the zona pellucida. Before penetration, the outer acrosome membrane fuses with the plasma lemma of spermatozoa, so enzymes are released. Fertilization can be divided into 6 steps: 1. Capacitation - is the process of eliminating inhibiting factors (glycoprotein) from the spermatozoa head. (Najeeb: The glycoproteins are on the head and we have to wash the head with help of cilia.) 2. Penetration of the corona radiata - affected by hyaluronidase and movements of the sperm (Hylaronic acid in the CT of the corona radiata that holds the cells together will be cleaved by 11 Colloquium I Embryology 4th Semester hyaluronidase.) 3. Penetration of zona pellucida - affected by acrosin. Zona pellucida is 13 micro meters thick, and consists of 3 binding proteins. (When the acrosome touches the zona pellucida --> fusion --> dissolvation of ZP by acrosine enzyme.) 4. Cytoplasm fusion - between head of sperm and the ovum, realized in previtelline space. Sperm protein fertilin binds to the integrin molecules on the oocyte surface. Active fusion between cells brings their cytoplasm into the continuity, which is not possible if acrosomal reaction has not taken place. Sperm, including tail, enters oocyte. 5. Cortical reaction - response of the ovum, and divides in the two blocks of polyspermy. Fast block presents rapid electrical depolarization of the plasma membrane, exist for 5 minutes, and avoids new sperm fusion to the oocyte. Slow block of polyspermy begins with Ca2+ elevation in the zone of sperm-egg contact. This event releases the content of cortical granules into the previtelline space. (Ca+ binds to cortical granules granules will be exocytosed to zona pelluzida and release lysosomal granules impermeable for other sperm cells) (Najeeb: When the oocyte membrane and the posteriod plasma membrane of the sperm will fuse --> the oocyte will then realease the lyzozomal enzymes to make zona pellucida impermeable to other sperms.) 6. Second meiotic division - forms mature ovum, and a second polar body is completed after the incorporation of sperm. Nucleus of mature ovum becomes female pronucleus, and sperm nucleus becomes male pronucleus. Both replicate DNA, and chromosomes become organized for a regular mitotic division. Maternal and paternal chromosomes develop a new diploid cell, the zygote. (After ovulation the mother will still have the double DNA chromosome. It will start its second division after ovulation but it wont finish until the sperm enters the oovum. So it has to finish second meiotic division and form one polar body and one definate oocyte. The pronucleus of female will go together with the pronucleus of male to form a diploid zygote.) Results of fertilization: 1. Restoration of diploid number of chromosomes (46) 2. Determination of chromosomal sex of the embryo 3. Variation of human species 4. Initiation of cleavage (mitotic division of zygote into blastomeres) Abnormal fertilization: 1. Dispermia - when two sperms enter oocyte; diploid embryo contain 69 chromosomes; extremely seldom, leads to death of embryo 2. Superfetatio - fertilization during pregnancy 3. Superfecundatio - two or more oocytes are fertilized by different men at the same time 4. Partenogenesis - specific process when a new embryo develops only from oocyte, under influence of some physical or chemical factors (without sperm) 12 Colloquium I Embryology 4th Semester 10. Cleavage. Blastocyst formation. Embryoblast. Trophoblast. During the passage along the uterine tube, the zygote (zygote is when there is only one cell, as soon as there are 2 cells it is called embryo!) undergoes cleavage, asynchronic mitotic division. The new cells, known as blastomeres, become smaller with each mitotic division. Usually one blastomere (darker one) undergoes mitotic division after the bright blastomere division. Thus, the number of cells might be unequal in each next division. After several divisions, the conceptus (embryonic and extraembryonic structures developing from zygote; term is used in any time of gravidity) starts compaction during which blastomeres adhere via gap and tight junctions. Now the conceptus is called morula (compacting embryo) and is still surrounded by zona pellucida. Entering the uterus (about 3-4 days after fertilization), fluid passes into the intercellular spaces of the blastomeres, this process is called cavitation. New structure formed is the blastocyst (or blastula) (there are several forms: early-, mid-, late-). As the blastocyst cavity (blastocoel) increases, cell becomes separated into two parts: trophoblast (consisting of external cell mass) and the embryoblast (consisting of inner cell mass). The trophoblasts consist of flattened cell ring, thus called cytotrophoblast, and gives rise to the placenta. The embryoblast gives rise for the embryo itself. (Remember that all of this still happens before implantation) The second polar body, usually persist until late blastocyst stage, marks anterior end of anteroposterior axis of conceptus. The blastomeres are all totipotent up to the 8-cell stage, but some blastomeres retain the ability to form any cell type of the body even in the 16-cell stage. Cells lose their totipotency, and become pluripotent later on. Gene and growth factor expression starts already in the 4-cell stage. 11. Implantation. Conceptus, different decidua. Ectopic implantation. Corona radiata is stored around the zygote for the first 2 days from the beginning of cleavage. Blastocyst leaves zona pellucida by digesting a hole with a trypsin-like enzyme on the 5th day after fertilization, into the uterus cavity. (So zona pellucida has to be gone for the ovum to attach) Implantation takes place in the anterior or posterior wall of uterus. In case of implantation close to cervix uteri, the so-called "placenta praevia" develops and it has to be controlled by regular ultrasound, and normal labor has to be excluded. The trophoblast attaches to the endometrium, and implantation starts about 5-6 days after fertilization. The next implantation stage starts with digestion enzymes, forming a niche into the endometrium, which is usually well-closed and filled with digested maternal tissue 13 Colloquium I Embryology 4th Semester and blood. (So, the digestion enzymes also break down the mothers blood vessels located in the functional layer of the endometrium! This gives nutrients to embryo and will eventually form the placenta.) This is used by the conceptus, to "feed the embryo". Implantation ends around 13,5th day, when conceptus is completely covered by endometrium. Functional layer of endometrium during pregnancy is called decidua, and there are 3 types: 1. Decidua capsularis - endometrial tissue between conceptus and the uterine cavity 2. Decidua basalis - endometrial tissue between conceptus and basal part of the functional endometrium (placental site) 3. Decidua parietalis - all other layers of functional endometrium For a short time period, functional layer is occupied by decidua cells, rich in glycogen and serving for the trophic function of the embryo. They degenerate afterwards, and hormonally active cells of the peripheral trophoblasts invade the basal decidua. Implantation of embryo in site other than the uterus is named ectopic or extrauterine pregnancy. Most often the sites are the ampullar, isthmic of fimbral part of oviduct, abdominal and rectouterine pouch part, ovaries and cervical part of uterus. 13. Bilaminar embryo. Extraembryonic mesoderm. After 7,5 days, during the 2nd week, the embryoblasts form a layer of the hypoblast (ACCORDING TO PILMANES BOOK Will form Endoderm: Digestive system+ Lungs + Pancreas + Bladder BUT IN REALITY ALL GERM LAYERS ARE FORMED BY EPIBLASTS) and the epiblast (WILL MIGRATE TO THE PRIMARY STREAK TO FORM Mesoderm, endoderm AND WILL ALSO FORM ECTODERM FROM THE SURFACE WHERE IT IS ORIGINALLY PLACED). The hypoblast is formed by cuboidal cells along the ventral surface of the embryoblast, and the epiblast consists of columnar cells. The remaining cells MOST LATERALY OF EPIBLAST LAYER become amnioblasts and amniotic cavity appears on the epiblast side as a small space (day 8). 14 Colloquium I Embryology 4th Semester The primitive yolk sac is enclosed by cells, which possibly derive from the hypoblast and cytotrophoblast, forming a thin "Heuser" membrane. Soon the primitive yolk sac is exchanged by a secondary yolk sac. Two main functions provided by yolk sac: hosting of gonoblasts until the 3rd week, until their migration and participation in the intravascular hemopoiesis (megaloblastic period). The bilaminar embryo, or germ disc, is composed of two cell layers: the hypoblast and the epiblast. Development of extraembryonic mesoderm (13,5 days): Delamination of the cytotrophoblast cells gives rise to the development of extra embryonic mesoderm (magma reticulare) that fills the space between the cytotrophoblast, the amnion and the primitive yolk sac. Isolated spaces soon appear in the extraembryonic mesoderm, they merge to form a cavity called the extraembryonic coelom (chorionic cavity) at the middle of week 2. This cavity splits the extraembryonic mesoderm into the extraembryonic somatopleuric mesoderm, or extraembryonic external lamina (covers internal part of the cytotrophoblast in blastocyst and developing amnion), and extraembryonic splanchnopleuric mesoderm or extraembryonic internal lamina (covers the yolk sac). The bilaminar germ disc is connected to the cytotrophoblasts by a connecting stalk that is the primordia of the umbilical cord. Cells of the hypoblast that replace the Heuser membrane and form its caudal part line the secondary, or definite, yolk sac. Primordia of allantois grow into the connecting stalk. The primary yolk sac disappears; the rest can survive as exocoelic cysts in the extraembryonic coelom. In the cranial pole of the hypoblast, proliferating cells form the prechordal plate, which participates in the development of the head region. The normal amount of amniotic fluid is between 800-1000 mL for full-term placenta. An excessive amount is named hydramnion (in cases of multiple pregnancies, esophageal atresia, and anencephaly), and an amount less than 500 mL is called oligohydramnion (in cases of bilateral agenesis of kidneys, premature rupture of amnion in 1 % of all pregnancy cases). 15 Colloquium I Embryology 4th Semester Normally, fluid is without smell and color (green color indicates intrauterine infection of fetus), and it expands the female reproductive tract shortly before labor, protects fetus from temperature changes and mechanical damage during pregnancy. Contains about 200 proteins from fetus and material body. Amniocentesis is the withdrawal of amniotic fluid by needling through the mother's abdominal wall and uterus into the amniotic cavity for prenatal diagnoses. Increased concentration of alfa-fetoprotein is the indicator of anomalies in the central nervous system. This has to be detected twice if the fetus is suspected for Downs’s syndrome. Urea levels in amniotic fluid increase in case of intrauterine hypoxia in fetus. Lecithin to sphingomyelin ratio predicts possible lung immaturity. Estradiol is the indicator of aliveness of the fetus. A constituent of amniotic fluid is fetal urine; swallowed by the fetus is an amount of approximately 20mL/h starting from the 2nd trimester of pregnancy. 13. Trilaminar embryo / germ disc. Gastrulation. Development of chord. The process of germ layer formation is called gastrulation; this is the beginning of embryogenesis, day 15 or 16. Trilaminar embryo is composed of three germ layers: ectoderm, mesoderm and endoderm. On dorsal surface of epiblast, cell proliferation and migration can be recognized as a primitive streak caudally in the median plane under the influence of 4 signaling molecules. As it elongates, the proliferating cells of its cranial part form an elevate primitive know, surrounding a small primitive pit. The primitive know / node expresses molecular markers: chordin (signaling molecule) and transcriptional factors, goosecoid, and hepatic nuclear factor 3β (HNF-3β). HNF-3β initiates notochord functions, goosecoid organizes other genes, but chordin and other molecules associated with the node regulate the left-shift asymmetry of the body. During development, cells from the epiblast invaginate to form mesoderm and migrate into the hypoblast, now called the embryonic endoderm - the remaining cells in the epiblast form the ectoderm. Additionally, epiblast cells secrete hyaluronic acid that avoids aggregation of out-migrating cells, and the presence of fibronectin association with basal membrane beneath the epiblast plays an important role in cell spreading. 16 Colloquium I Embryology 4th Semester Notochordal process - formed by migrating cells from the primitive knot between the ecto- and mesoderm, ending at prechordal plate, where ectoderm and endoderm are fused, forming the oropharyngeal membrane. At caudal end of primitive streak is the cloacal membrane where the embryonic disc remains bilaminar; indicates future site of the anus. Notochord defines primitive axis of embryo, around which vertebral column is formed. During fetal period it degenerates and disappears, small remnants persist as nucleus pulposos in the intervertebral disc. Notochordal process cells spread out and fuse with embryonic endoderm. In such way, canalis neurentericus appears as a small temporarily communication between amnion cavity and yolk sac. Later, the notochord cells separate from the endodermal root of yolk sac and form the definite notochord. 14. Neurulation. Neural plate, neural crest and neural tube. At day 18, the notochord (formed from the mesoderm) induces the ectoderm to develop the neuroectoderm; a process called neurulation. (The nervous system is the first type of system to develop.) The neural plate is the primordia of the central nerve system (brain and spinal cord); neural plate is transformed into a neural groove flanked by neural folds(see pic), which fuse to a neural tube. The open ends of the tube are called neuroporus anterior (closes around day 24) and neuroporus posterior (closes 2 days later). The neural crest develops when the neural folds fuse, and some neuroectoderm cells migrate along each side. Neural crest cells migrate widely in the embryo, and give rise (PNS) to spinal and autonomic ganglia, meninges, pigment cells, and skeletal and muscular components of the head. Nasal placode, lens placode and otic placode become visible in the heard region. 15. Development of paraxial intermediates and lateral mesoderm. Mesoderm - gives the material for the construction of body walls and extremities. 17 Colloquium I Embryology 4th Semester (Muscle, Bone, Kidney and Gonads.) The mesoderm cells form: - Paraxial mesoderm along each side of notochord (day 17), segments into somites - Two embryonic lamina of lateral mesoderm, visceral and parietal, which continue and fuse with the extraembryonic mesoderm around the amnion, known as parietal mesoderm or somatopleura, and around the yolk sac, known as visceral mesoderm of splanchnopleura - The intermediate mesoderm, which connects the paraxial and lateral mesoderm; material provides basis for kidney tissue, and is also called nephrotome - Lateral mesoderm give rise to body wall structures and is continuous with the extraembryonic mesoderm. 16. Tissue induction, differentiation and determination. Induction - the initiation of the development under environmental factors (epigenesis). Chemical substances produced by distinct cell groups cause induction. Cell group providing induction is called inductor. There are multistep inductions on the level of differentiated tissues. Primary mesodermal induction induces transformation of eye vesicles from diencephalon. Secondary induction by eye vesicles induces formation of lens from surface ectoderm. Tertiary induction includes development of cornea from ectoderm under stimuli excreted by lens. Induction depends on sensitivity of a cell group to react to inductors. There are three main inductor groups: 1. Paracrine factors - induce material from distance (hormones, growth factors) - Fibroblast growth factors - development of angiogenesis, mesoderm and growth of neuronal processes - Transforming growth factor beta - many subgroups that impact development of mesoderm - Hedgehog genes - development of cell junctions, symmetry of body and regional GI organs - WNT genes - development of derma myotomes - Ephrins - unknown, but blood vessel typing 2. Extracellular matrix proteins - require special receptors 3. Cell surface proteins capable of juxtacrine induction (via receptors) Determination - process for development of a distinct tissue and/or organs from the blastema. Cells lose omnipotence, ability to differentiate in different direction, and further development depends on epigenesis. Usually realized during gastrulation, by blocking distinct 18 Colloquium I Embryology 4th Semester genes. Determination indicates perspective significance of the cells. Differentiation - development of specific structures with distinct functions from the cells that are together realized into a phenotype. It limits developmental potency of cells, and develop in accordance to their functions. Without this, all cells would express the same phenotype, and there would be no multicellular organisms. 17. Tissue growth and factors influencing the growth process Growth is the increase of cell number, size and extracellular matrix. Cell size does not depend on the size mass, and/or length of the individual. Growth process is stimulated by growth factors, and also hormones and substances with growth properties. Some of the main growth factors during embryogenesis are insulin, EGF, TGF alfa and beta, and PTHRP (parathyroid hormone related protein) and oncogenes. Growth is inhibited by specific inhibitory substances, chalones, expressed by differentiated and aged cells in S phase of cell cycle. Other substances, antichalones, interrupt the influence of chalones. 18. Derivate of the germ layers "Germ layer" introduced by Karl von Baer. Most organs develop under the influence of all three germ layers, so attention is paid to the origin of the epithelium to state the germ-layer origin. Ectoderm: two types: 1. Surface ectoderm: epidermis(dermis and subcutaneous CT by paraxial mesoderm), hair, hair follicles, nails, skin glands, adenohypophysis, enamelum (outer layer of teeth), and sensory organs (lens, cornea, inner ear). Epithelium of mouth and anal canal (Rest of the GI from endoderm). Pituetary and pineal gland. Adrenal medulla (adrenal cortex from splanchopleura of lateral mesoderm) 2. Neuroectoderm: - Neural tube – (CNS) brain hemispheres, spinal cord, retina, pigmented epithelium of retina, ciliary muscle, epiphysis and neurohypophysis - Neural crest – (PNS) autonomic ganglia, adrenal medulla, pigmented cells, schwann cells A special part of ectoderm is head mesectoderm providing material for head mesenchyme; neurocranium; meninges; head muscles; dentinum/cementum. Endoderm: Inner stuff (GI tract + glands, Resp, other glands and epithelium of reproductive) - Epithelium of GI tract, pharynx, larynx, trachea, cavum tympani, tonsils, thyroid gland, parathyroid gland, thymis, bronchi, lungs, liver, pancreas, urinary bladder 19 Colloquium I Embryology 4th Semester Mesoderm: (MUSCULOSKELETAL SYSTEM) three types 1. Paraxial mesoderm – skeleton(Bone) and trunk muscles, dermis and subcutaneous C.T. (since BV is formed by mesoderm it makes sence that dermis which is mostly BV will also be formed by mesoderm) 2. Intermediate mesoderm - kidney and reproductive organs with their duct system 3. Lateral mesoderm - Splanchnopleura – SM(since SM it means all the blood vessels also! And if blood vessels then also lymphatic system and heart!), heart, hemapoietic cells (blood since it is also CT), mesothelium of inner organs, BV, adrenal cortex, spleen - Somatopleura - pericardial mesothelium, omentum 19. Cephalocaudal flexion and lateral folding. Development of intraembryonic coelom. Embryo folding is the change of embryonic disc from a flat form into a 3D structure characteristic for vertebrates. It starts in the 4th week, because of the rapid differential growth of various embryonic structures. Since embryo grows faster in length than width, reflections are deeper at the caudal and cranial end of the embryo. Developing notochord, neural tube and somites stiffen the dorsal axis of the embryo, so most of the folding is concentrated in the thin, outer rim of the disc. Folding starts in the cephalic and lateral region on day 22, but in the caudal region on day 23. Midline fusion transforms flat embryonic endoderm into a gut tube with foregut, midgut open to the yolk sac, and hindgut open to the allantois As a result of midgut folding, vitelline duct forms. When edges of ectoderm fuse along midline, space formed within the lateral plate mesoderm is enclosed in the embryo and becomes the intraembryonic cocelom. This is a horseshoe shaped cavity, which at 2nd month is divided into 3 body cavities; the percardial cavity around heart, the pleural cavities around lungs and the peritoneal cavity around abdominal and pelvic organs. The abdominal part of the gut becomes suspended in the coelom by a thin, bilayered reflection of serosal membrane; the dorsal mesentery (developed from mesenchyme, caused the broad attachment of the gut to the dorsal body wall). Some of the visceral organs develop in the body wall, and are separated by the coelom by a covering of serious membrane. These organs are said to be retroperitoneal. Some parts of the gut tube adhere to the body wall during their development 20 Colloquium I Embryology 4th Semester and become secondarily retroperitoneal: ascending and descending colon, duodenum and pancreas. The thoracic cavity and abdominal cavity is later separated by the diaphragm, which develops from 4 anlagen at the end of the 4th week: the septum transversum, the dorsal mesentery of esophagus, the pleuroperitoneal membranes and the somatopleura. The separation between the pleural and pericardial cavity is realized by the pleuropericardial membranes. 20. Chorion leave and chorion frondosum. Primary, secondary and tertiary villi. Anchor villi. Chorion (fetal membrane) (choreon from the synchiothrophoblasts formed from trophoblasts during implantation) with decidua encircles the chorion cavity, which contains the bilaminar embryo, the amnion and the yolk sac. Chorio-amniotic membrane is the fused amnion with the chorion around the 6th week. Placentation starts at day 13, while development of villi is observed from start of 2nd week. 3 types of chorionic villi: 1. Primary chorionic villi - consisting of epithelium from syncytiotrophoblast and cytotrophoblast, migrate into the endometrium. Maternal blood from eroded vessels flows in the lacunae. 2. Secondary chorionic villi - contain additional extraembryonic mesoderm of the chorion 3. Tertiary chorionic villi - develops when mesenchymal cells differentiate into blood capillaries. These capillaries connect with the blood vessels in the allantois, which join the intraembryonic blood vessels. Until week 8, chorionic villi cover the whole surface of the chorion, and later they become atrophic on the side towards the lumen of the uterus, which is called chorion leave (smooth chorion). All chorionic villi at the embryonic pole increase rapidly in size, and develop the chorion frondosum (villous chorion). Both chorions are connected by a chorionic plate, which consists of amniotic columnar epithelium, extraembryonic mesoderm, blood vessels, cyto- and syncytiotrophoblast (or fibrinoid after 4th embryonic month). The terminal portion of villi remains trophoblastic, called 21 Colloquium I Embryology 4th Semester cytotrophoblastic cell column. Mixtures of the cytotrophoblast develop proliferation buds, providing trophoblast cell spread on all parts of the placenta. Sometimes these proliferations buds may detach from the placenta and enter maternal blood, reaching the lungs and expiring afterward. The cytotrophoblast is called peripheral trophoblast after the spread. Additionally, an attachment of tertiary villi by growing cytotrophoblasts at the decidua basalis develops the anchor villi. 21. Allantochorion. Development of umbilical cord. The allantochorion is a compound membrane formed by fusion of the allantois (that is used for waste products) and the chorion (forms villi/placenta). The umbilical cord develops from the connecting stalk, which is removed to the ventral side of the embryo during the folding. The connecting stalk (with allantois and its vessels) and the yolk sac (aka umbilical vesicle since in humans it does not contain yolk, instead it has function to produce red blood cells.) (with vessels) are getting closer and become enveloped by the expanding amnion. The extraembryonic mesenchyme of the stalk and yolk sac changes into tissue of Wharton's jelly, which acts as a protective layer for the blood vessels in the mature umbilical cord. The allantois and yolk sac with vessels degenerate, and the extraembryonic umbilical coelom that is temporary used as a space for the extruding intestinal loop disappears. The allantois vessels persist as umbilical vessels, the right vein degenerates, and then the mature umbilical cord contains one vein and two arteries. During perinatal period, the umbilical vessels might be used for blood transfusions. The length of umbilical cord is about 50 cm and could sling around the fetus' neck, giving rise to anoxia or death of fetus. In cases when cord encircles limbs, strangulation grooves may develop, leading to hypoplasia of the encircled structure. Umbilical cord is not innervated by nerves. In 0,5 percent of cases, umbilical cord displays only one artery instead of two, leading to cardiovascular anomalies in 15-20 percent of the cases. 22. Amnion. Amnion liquor, it's content. Fetal liqour. Amnion (fetal membrane) encloses the amniotic cavity (where the embryo lies), consists of columnar amniocytes (from ectoderm layer) and a thin layer of extraembryonic mesoderm (from mesoderm layer). Amniotic fluid, which derives from maternal capillaries and amniotic cells increase to 1000 mL till 35 weeks, but reduces to 800 mL at birth. It allows fetal movements, and serves as a protective cushion (mechanical insults, dryness, temperature balance, adhesions etc). Amniotic fluid consists of 99 percent water (prevent from drying out!), desquamated fetal epithelial cells, proteins, fats, carbohydrates, hormones, pigments and fetal urine. It is 22 Colloquium I Embryology 4th Semester replaced every 3 hours, and by maternal circulation, the fluid passes through the amnion and the fetal gut into the maternal blood. 23. Paraplacental pathway. The paraplacental pathway is an alternative nutrition pathway between chorion leave and decidua capsularis. This pathway is of no significance unless the mother dies, and the developing fetus can be held alive for about 2 hours, during which time we may perform surgery in order to save the developing fetus. 24. Cotyledonis. Significance of different trophoblasts. With development the decidua basalis is eroded by syncytotrophoblasts forming intervillous spaces. Some of the decidua remains and it forms compartments, which contain a villous tree with many branches called a cotyledon. At the start of placentation (13,5 days), there are about 200 cotyledons, which decrease to about 50-60 before birth. These can be recognized in the placenta as lobules. The trophoblasts have two main functions: 1. Developing into h ormonal active cells of p eripheral trophoblasts which secrete: - HCG(human chorionic gonadotropin) - used as a pregnancy detector, as it appears in maternal blood 9 days after fertilization. Its function is to prevent disintegration of corpus luteum -> corpus luteum graviditionis, which maintain progesterone production. - Placental lactogen - stimulate general growth - Chorionic corticotrophin - similar effect of ACTH by stimulating metabolism and cardiovascular function in the mother - Later it produces progesterone (So in beginning it has HCG to keep getting progesterone and estrogen from corpus luteum, but after a while it can supply itself!) - Estrogens in cooperation with fetal liver and adrenal gland - Prostaglandins and interleukins 2. Replacing the endothelium of the maternal spiral arteries in decidua basalis - in order to stabilize the maternal blood supply to the placenta. If this does not occur, pre-eclampsia may develop (maternal hypertension). Cytotrophoblasts of the villi may conglomerate, and form proliferation buds, which aid growth of the villi. Some of these proliferation buds may detach and enter the maternal blood flow where they may reach the lungs, possibly causing tumors. 25. Fetal part of placenta. Fetal part of placenta consists of the chorionic plate, and tertiary villi. The chorionic plate consists of amnioblast (columnar epithelium from the epiblasts), extraembryonic mesoderm, vessels and after the 4th month also fibrinoid. The anchor villi build a bridge between the fetal and the maternal part of the placenta. 23 Colloquium I Embryology 4th Semester 26. Maternal part of placenta. The maternal portion is formed by decidua basalis and tertiary villi. The decidua contains decidual cells, which disappear in the 1st trimester, and hormonal active cells of peripheral trophoblasts will secrete several substances already mentioned earlier. 27. Placental circulation. Intervillous space. During placentation (13,5 day), the syncytiotrophoblasts (remember, the throphoblast layer of the blastocyct develops into the outer syncytiotrophoblasts and inner cytothrophoblast layers during implantation) secrete hydrolytic enzymes such as trypsinase, which will degrade the endometrium/decidua basalis, and form spaces called lacunae, where the branching villi grow into. Some parts of the decidua remain forming the intervillous septs. (The enzymes released from the chorion will also break down the BV of the mother in order to fill the decidua basalis with blood so that the chorionic villi can absorb the nutrients from this blood) The placenta is a fetomaternal organ consisting of fetal and maternal material. The portions are bridged by anchor villi. And during erosion of decidua basalis by syncytiotrophoblast, intervillous spaces are excavated. Parts of the decidua remain as placenta septa; they divide the placenta into a number of compartments. Each compartment contains a cotyledon (a villous tree with many branches). In the beginning of placentation, there are around 200 cotyledons, but eventually there is only 50-60. Mature placenta is disc-like in shape, 3 cm thick and 20 cm in diameter. Weight about 500 grams. Fetal part is recognizable by the umbilical cord, by maternal part divided into 35 lobes. Grooves between lobes present placental septa arising from decidua basalis, and each lobe contains several cotyledons, where each one is actually the once branched villous tree. Maternal placental circulation is called uteroplacental circulation. Oxygenated maternal blood is pressed into the intervillous spaces from spiral arteries in the decidua basalis; deoxygenated blood leaves the intervillous spaces via endometrial veins. All lacunae are connected, and amount of blood in common intervillous space is around 150 mL, renewed every 3 hours. Blood flows into lacuna under pressure, but in the intervillous space, the circulation slows down. Invasive cytotrophoblast cells from 24 Colloquium I Embryology 4th Semester the anchoring villi migrate into the wall of spiral arteries, and exchange endothelial cell layer. These cells secrete ECM that results into widening of common arteries lumen, except the open end, and blood in these parts leaves under much higher pressure than normal arterial pressure. In cases when this endothelial ell exchange is disturbed, eclampsy of pregnancy might occur, causing cramps in placental blood vessels due to differences of arterial pressure, and danger for survival of fetus The first maternal blood that bathes the trophoblast of villi is not reach with cells, the oxygen tension is low. Fetal RBC in this period contains embryonic hemoglobin, which is adapted to bind oxygen under low tension. This changes after 12th week, when maternal blood in intervillous space contains large number of erythrocytes and is more oxygenated. Fetus also switches on the production of fetal hemoglobin, which requires higher oxygen tension for its binding. Fetal placental circulation: oxygenated blood reaches the fetus through (left) umbilical vein (right one degenerates). Deoxygenated fetal blood leaves the fetus by 2 umbilical arteries via umbilical cord. 28. Functions of the placenta. Adaption (aging) of placenta. Fibrinoid. - Acts as endocrine gland. See Q24 - Gas exchange (no lungs in fetus) and nutrient exchange (glucose,aa etc.) btw fetal blood and mothers blood (takes place in chorionic villi) - The semipermeable membrane of the chorionic villi that takes place in gas exchange will also protect against bacteria of the mothers blood and the small sized IgG can pass through to give protection of the fetus. - Waste product removal (urea, uric acid, creatinine) Common placental functions are: respiration, nutrition, excretion, protection, storage and hormonal production. Placenta brings from mother to the fetus: oxygen, water, electrolytes, nutrients, hormones, antibodies, vitamins, iron, trace elements, but also drugs and toxic substances, alcohol and some viruses. Carbonic acid, water, electrolytes, creatinine, bilirubin, hormones and erythrocyte AG are all transferred from the fetus to the mother. Maturation or aging of placenta includes: - Development of fibrinoid from fibrinogen of maternal blood and degenerative cytotrophoblast and its deposition into the placenta from 4th embryonic month. This adaption takes place generally already during placental development, but increases from the 2nd trimester. - Prominent fibrinoid depositions into the placenta are observed in cases of immune conflicts where Rh- mother conflicts with Rh+ fetus, or due to unknown reasons where mother possess blood group I, while fetus has blood group II. Commonly, abundance of fibrinoid depositions in placenta suggests some problematic pregnancy conditions. 25 Colloquium I Embryology 4th Semester - Total surface of placenta expands up to 15m2 due to growth of microvilli on surface of syncytiotrophoblasts - Hofbauer cells develop from mesenchyme into tertiary villi, provide role of m/ph - Deposition of calcium into extraembryonic mesoderm of tertiary villi - Development of syncytiotrophoblast "bridges" present many thinned areas and thickened "nodal" places. Thickened regions provide greater hormone production, while thinner allows for easier diffusion via placental barrier. - Decrease of extraembryonic mesoderm in villi, appearance of numerous sinusoids instead of blood capillaries, abundant branching of villous trees in cotyledons; all these factors decrease diffusion time via placental barrier. 29. The formation and the role of the fibrinoid in full-term placenta. Fibrinoid is a deposition of maternal fibrinogen and derivate of trophoblast cells from tertiary villi. This is a normal process and it starts already after placentation, but production spikes at 4th month. Excessive amounts of fibrinoid can cause infarction of fetal blood supply. This can lead to cardiovascular anomalies. A placenta after birth should be elastic, but will appear rough if excessive fibrinoid is accumulated. Fibrinoid accumulation increases with immune conflicts: erythroblastosis fetalis, blood group conflict and HIV positive mothers, or mothers with hepatitis. 30. Umbilical cord. See question 21. 31. Morphofunctional basis of delivery. Labor is a complicated and often prolonged process (especially when it happens for the first time). It starts with hormonal changes in placenta. Progesterone level decreases, and estrogen increase causing the first uteral contractions. Fetal sac breaks and amniotic fluid flows out. (“My water broke”) The cervix also dialates and flattens out to allow the head of the fetus moves downward and irritates the pelvic mechanoreceptors. Irritation stimuli reach hypothalamic nucleus paraventricularis, with the following expression of oxytocin. The last one additionally evaluates the contractions of the uteral smooth muscles. Finally, a child is born and the placenta also leaves the uterus within 30 minutes. Not a risk-free process. Even in developed countries such as Sweden, the rate of maternal mortality is 10 deaths in every 100 000 births. Complications could be: placenta previa, poor uterine strength, large babies and cesarean sections. Factors such as coitus, spicy food, hot baths etc have been said to induce child birth, but in the clinic, oxytocin is given per injection to induce labor. 32. Prenatal diagnostic methods. In modern times, fetus has become a patient. We screen and perform diagnostic 26 Colloquium I Embryology 4th Semester exercises on the fetus on a daily basis. Methods are: - Amniocentesis (trans abdominal needling into amniotic cavity) for analysis of amniotic fluid and fetal cells for chromosomal sex analyses and abnormalities. - Ultrasound screening used for detecting morphological details of the embryo; the most important and successful method in early stages (w 18-22) - Chorionic villus sampling (CVS) during weeks of gravidity 8-12 is used for cyto- and chromosomal diagnostic - Amnio- and fetoscopia (insertion of endoscope in the uterus) enables direct observation of fetus - Amnio-fetography (AFG) - an x-ray method used in addition to ultrasound. - Cordocentesis is the obtaining of fetal blood from the umbilical vein (also in case of blood transfusion in the case of fetal hemolytic disease) - Preimplantation diagnostic complex - obtaining of one blastomere from embryo after IVF for diagnostic reasons - Detection of cotinine, folic acid and choline in the blood serum of the woman. 33. Development of body external shape. Morphofunctional principles and inductors for common body development. The human body follows certain principles of body development: - Division of the body into caudal and cranial poles - Metametric division of certain organs. E.g.: two lungs, two eyes, two kidneys etc - Regional development is usually provided by dermatomes and myotomes developing together In the head however, these principles are controlled by induction centers which are specific for this region: - Proencephalon (will become cerebrum and thalamus)- induction to eyes, nose and anterior base of skull - Rhombencephalon (will become the midbrain)- inducts ears, occipital skull and brachial apparatus - Hindbrain (will become pons cerebellum and medulla)- inducts medulla oblongata, spinal cord, vertebral column and GI tract. The existence of these centers is provided by developmental conditions such as cyclopia. Molecular control of skeleton formation: Under control of Hox genes. Transcriptional factor Sox-9 activates collagen IIA gene, essential for transfer of mesenchymal cells into precartilage. Core binding factors alfa 1 controls differentiation of osteoblasts from mesenchymal cells. Bone morphogenetic proteins (BMPs) responsible for embryonic bone development. 27 Colloquium I Embryology 4th Semester 34. Development of vertebral column, ribs and sternum. Vertebral column derives from somites, and its development undergoes 3 stages: mesenchymal, cartilaginous and bone. Mesenchymal: during 4th week sclerotome cells surround notochord; each vertebra is made from caudal half of one sclerotome that fuses with loosely arranged mesenchymal cells from cranial half of sclerotome below. Notochord persists as nucleus pulposus in the intervertebral discs. Pax-1 gene expression regulates separation of vertebral bodies Spinous, transverse and costal processes arise. The two processes that form the vertebral arch fuse in median plane to enclose spinal cord. By growth, myotome cells move at level of intervertebral discs and during the development, each developing muscle connects with cranially and caudally located bodies as well as the furthest ones. In such way, the vertebral column "hangs" in the muscle, which gives possibility for free movements horizontally into the foramina intervertebralia located spinal nerves. Exception is atlas and axis, where the dense axis develops protruding upwards. Cartilaginous: vertebrae undergo endochondral ossification that starts from 3 primary centers (1 ventral and 2 dorsal). The dorsal ossification centers fuse and melt together with vertebral body (3-5 years after birth). Failure of fusion result in spina bifida. Bone: after birth, secondary ossification centers appear at the upper and lower side of the vertebral body and on its processes, they act as growing centers. The ossification of vertebrae ends first in the lumbar region around 14-16 years of life. Immobile os sacrum develops because of all ossification centers fuse. But, os coccygeus develops only one ossification center, thus undergoes incomplete development. Fusion of all ossification centers ends the growth of vertebral column (21-25 years). The curvatures (lordosis and kyphosis) are formed during the first year after birth. The expression of Hox (a-d) genes influence the development of the vertebral column, and increase from the cervical vertebra towards the coccygeal one. Ribs and sternum: Develop from condensed mesenchymal cells lateral to the centrum: proximal part (head, neck and tubercles) develops from ventromedial sclerotome, but distal part derives from the ventrolateral part of adjacent cranial somite. Until the beginning of ossification, ribs separate from the vertebra. Ossification ends around 20 postnatal years. Sternum initiates as two lateral mesodermal condensations in the ventral body wall takes place, and one interclavicular blastema. Sternum undergoes ossification only between years 21-25. 35. Development of cranium: chondrocranium and desmocranium. The skull develops from unsegmented head-mesectoderm, prechordal mesoderm, 4 cranial somites and mesoderm of 2 first branchial arches. 28 Colloquium I Embryology 4th Semester All bones which are prepared by intramembranous ossification form the desmocranium (vault of the skull, viscerocranium), and all the bones resulting from endochondral ossification forms chondrocranium (base of skull, surrounding oral cavity, pharynx, and upper respiratory ways - neurocranium). Chondrocranium starts with an independent development of cartilaginous lamina at the base of the skull and in nasal and ear region. Later, these primordia fuse and endochondral ossification affects os occipitale, os sphenoidale, os ethmoidale, os temporale with pars petrosa nad pars mastoidea. However, some bones of the chondrocranium incorporate also membranous elements and thus membranous neurocranium develops: os parietale, os rontale, and pars squamosa of os temporalis, and pars interparietalis of os occipitalis. Chondrocranium primordia rise as result of epithelial induction on surrounding mesenchyme. Desmally ossified bones of cartilaginous viscerocranium: primordia of branchial arch I: malleus, incus (Meckel's cartilage); primordia of branchial arch II (Reichert's cartilage): stapes, proc. styloideus and bones of viscerocranium: maxilla, mandibula, os zygomaticum, os nasale, os lacrimale, os palatinum, vomer, lamina pterygoidea, tympanic ring. At birth, the bones are separated from each other by sutures (desmocranium) and synchondroses (chondrocranium), which act as growing centers. Unossified membranes are the fontatelles: anterior fontanelle located between the two frontals and two parietal bones, closes at 1,5 years after birth; posterior fontanelle located between the two parietal bones and single occipital bone, closes at end of 1st postnatal years; two lateral fontanelles, located between the parietal, frontal and occipital bone in each lateral side; close around 3rd postnatal month. 36. Ossification of cranium and limb bones. Chondral ossification is characteristic for os ethmoidale, concha nasalis inferior, os sphenoidale, os temporale (pars petrosa), os occipitale (pars basilaris) and ear ossicles. Desmossification is characteristic for maxilla, os zygomaticum, os palatinum, vomer, os nasale, os lacrimale, os frontale, os parietale, os temporale (pars squamosa et tympanica), os sphenoidale (pars lamina medialis), processes pterygoideus, ala major, os occipitale (pars squamosa and mandibula). Ossification of limb bones: limb skeleton develops via endochondral ossification under influence of BMP. The first ossification centers appear and ossification starts at week 6-7 and by the 3rd month, the diaphysis will be ossified. The epiphysis will ossify after birth with the growth zone marking the growth of the limbs. 37. Development of limb skeleton. Main skeleton malformations. Limbs develop as outgrowth of ventro-lateral body wall. Beginning of it is unclear, but generally starts by expression of fibroblast growth factor 10 in prelimbar lateral mesoderm, and influence of retinoic acid and Hoxb-8 gene is also important, initiating appearance of signaling centers in limb bud. Limb buds are formed by local proliferation of somatic mesoderm in response to signals form adjacent somites. Early limb mesoderm express T-box family molecules - Tbx-4 29 Colloquium I Embryology 4th Semester indicates lower limb development, and Tbx-5 for upper limb. Upper limb buds appear on day 24, as lateral body wall at about C5-C8 level. Lower limb buds appear on level of L3-L5 on 28th day. Early limb buds are self-regulating system, able to neutralize surgical damages. Limb development goes along three axes: 1. Proximodistal - (extends rom base of limbs to tips of digits) - FGF-2, FGF-4, FGF-8, Msx-b1, Hoxa and Hoxd influences growth. 2. Anteroposterior - (runs from first (anterior) to fifth (posterior) digit) - SHH molecules, Hoxb-8, Gli family influences growth. 3. Dorsoventral - (back of the hand or palm is dorsal, while palm or sole are ventral) - Wnt-7a, Lmx-1b, En-1 influences growth. In rapid growth, the buds prolongate to flipper-like limbs and the distal ends flatten to form paddle-shaped hand/foot plates. Mesenchymal tissue in plates condense to form digital rays, soon after Notch signaling between rays appear, tissue breakdown separates digits (fingers and toes). Differentiation of limb buds occurs between week 5 and 8: Day 33: upper limb with hand plate, forearm, arm and shoulder appears, and lower limb with rounded caudal part in distal tip: the next foot, appears Day 37: hand plate derives carpal region, digital plate; thigh, leg, foot become distinct Day 38: finger rays are visible; apoptosis rakes place in radial necrotic zones between digital rays, and foot plate is clearly defined Day 44: grooves between fingers are deeper, elbows become clearly seen, and toe rays are visible Day 47: horizontal flexion is seen in upper limb, and lower limb toe rays separate; flexion begins towards the parasagittal plate Day 52: bends appear at the elbow and tactile pads; lower limbs become longer, digital plates are visible Day 56: all regions are well developed, the fingers of two hands overlap at the midline Day 60: lower limbs are also fully developed Main abnormalities of skeletal development: Achondroplasia - hereditary condition, where long bones do not develop fully - dwarf Osteogenesis imperfecta - congenital conditions, bones break easily Spina bifida Craniostenosis - premature ossification of bones in skull Acrania - defect of vault Craniosynostosis - premature closure of sutures between membranous bones of chondrocranium Brevicollis - short neck, reduced number of cervical vertebra Amelia - congenital absence of limbs 30 Colloquium I Embryology 4th Semester Micromelia - short limbs Phocomelia - absence or shortening of proximal limb segments Brachydactyly - shortened digits Split hand or foot - absence of central components in hand or leg Brachypodism - shortening of limbs Syndactylia - fusion of fingers or toes Club foot - disloaction of lower limb 38. Skeletal striated and smooth muscle development. The skeletal muscles develop from somites -> myotome cell that differentiate into myogenic cells, and start mitotic division, and become post-mitotic myoblasts. These post-mitotic myoblasts will differentiate into multinucleated muscle fibers with cross striations. General smooth muscle growth is under influence of FGF and TGF beta. The formation of actin and myosin is regulated by insulin related growth factor. First developing myotubes are primary, but secondary myotubes soon differentiate around them from the fetal myoblasts. Satellite cells are though to develop from separate cell line. Phenotypes of muscle fibers depend on specific proteins, light and heavy myosin chains. However, phenotype of muscle fibers is not fixed and can change in accordance to plasticity (hypertrophy, atrophy and denervation). Muscles of the trunk develop from myotomes that divide into the epimers, that produces the epaxial muscles or extensor of vertebral column, and the hypomer that gives three layers from which hypaxial or flexor muscles of trunk develop. Dorsal branches of spinal nerves innervate all muscles of epimer, and ventral branches innervate the hypomer derivate. Head muscles develop from cranial somites (muscles of the tongue), prechordal mesoderm with influence of migrating neural crest cells (external eyeball muscles) and branchial arches 1-4 (masticatory, facial and pharyngeal muscles) Smooth muscles derives from lateral mesoderm (except the sphincter and dilatator pupillae muscle). Mesenchymal cells of the splanchnopleura form the smooth musculature of the intestine. Local mesoderm differentiates into the smooth musculature of blood vessels. Heart musculature derives from splanchnopleura. 39. Development of muscles in inner organs, heart, body, viscera, limbs, diaphragm. Muscle development anomalies. See previous questions. Cardiac muscle develops from splanchnic mesoderm that envelops the endothelial heart tube. Myoblasts adhere to each other and intercalated discs develop at their junctions. The conducting system is formed by special muscle cells with irregular distribution of myofibrils. Diaphragm derives from four sources: 1. Septum transversum (central tendon) 2. Mesentery of the esophagus (crura of diaphragm) 3. Pleuroperitoneal membranes (lateral parts of the diaphragm) 31 Colloquium I Embryology 4th Semester 4. Somatopleura, the myoblasts migrate from the body wall, the innervation of the diaphragm by the phrenic nerve (C3-C5) indicates its origin Abnormalities of musculature development: - Aplasia of muscles - no significance if main muscles aren't affected - Muscle defects of anterior body wall with herniation - Diaphragmal defects with herniation - Muscle variations - Muscular dystrophy shows degeneration and regeneration of different muscle groups - Tortiocollis - pathological shortening of m. sternocleidomastoideus 40. Periods of embryonic haemopoiesis. Blood develops in 3 periods: 1. Day 13-14-18 - megaloblastic period: groups of cells form blood islets in the extraembryonic mesenchyme of the yolk sac, connecting stalk and chorion. First cells are called hemangioblasts with bipotential capacity to transform into endothelial cells or hematopoietic cells. Additionally, this first hematopoiesis is intravascular, which means that blood cells in blood vessels develop simultaneously with the development of the latter. 2. Intraembryonic hematopoiesis - from 7th week - although the first nucleated erythrocyte enter bloodstream shortly before day 22, when first heart pulsation takes place. Starting from day 28, paraaortic hematopoietic islets of cell clusters provide intraembryonic hematopoiesis. Weeks 5-6 initiate the beginning of liver hematopoiesis. Erythrocytes of liver are different from yolk sac cells as they are enucleated and possess different chains of hemoglobin. A small amount of blood cells originates also in spleen, omentum and skin. In the beginning, hematopoietic islets appear regionally over all the surface of the skin, but around 5th month they are present only in the skin above main joints. Liver hematopoiesis decline after 6th month. 3. After 6th month - medullar period - in red bone marrow, when red and white blood cells are produced. This shift is controlled by cortisol and in its absence; hematopoiesis remains dominant in the liver. Hox gene family plays a role in hematopoiesis, by regulating cell proliferation. Shift from fetal to adult hemoglobin appears around the 30th week of development. 41. First blood vessels and circulatory system. During somite formation, many independent primordia of blood vessels appear in embryo. Part of blood vessel is formed by angioblasts in the extraembryonic mesenchyme (week 3). Separate blood vessels in the embryo join those in the extraembryonic mesenchyme. 32 Colloquium I Embryology 4th Semester In embryo, vascular precursors or angioblasts organize the primary capillary plexus via vasculogenesis. Later on, the growth of new blood vessels is realized via angiogenesis, a process that might continue in postnatal life. Both vasculogenesis and angiogenesis depend on growth factors and receptors. Development of angioblasts from mesoderm depend on vascular endothelial growth factor 2 (VEGF-2), under influence of VEGF-A. Angiogenesis depends on angiopoietin 1. Endothelial cells release platelet-derived growth factors (PDGF), which stimulates the migration of mesenchymal cells close to the vascular walls. Transforming growth factor beta stimulates mesenchymal cells into smooth muscle cells or pericytes. First main blood vessels of embryo are precardinal and postcardinal veins that return blood to the embryo's heart, vitelline veins that transport blood from the yolk sac, and umbilical veins from the placenta. Two dorsal arteries appear at first, but fuse in the caudal half of the embryo to form a single dorsal aorta. The intraembryonic blood circulation starts with the first heart pulsation, at day 22. 42. Heart development: endocardial tube, septation of atrium and primitive ventricle, conductive system, coronary arteries. On day 17-18-19, mesenchyme cells form cardiogenic area in front of prechordal plate. Cords of cells spread out from the splanchnomesoderm, and become canalized, producing two endocardial tubes (endocardial tubes are produced by progenitor cells that migrate from the epiblast through the streak, into the splanchic layer of lateral plate – forming cluster called primary heart field.). With transversal folding of embryonic disc, the tubes fuse to a single endothelial tube. The cephalocaudal flexion causes the incorporation of endothelial tube into the pericardial cavity. But endothelial tube remains attached to posterior wall of pericardial cavity by dorsal mesocardium. The endothelial tube possesses the outflow and the inflow part with a slightly different cell origin. Major components of the outflow tract derive from neural crest, but endothelial components arise from paraxial and lateral mesoderm. Splanchnic mesenchyme forms myoepicardial mantle around endothelial tube, consists of three layers: outer layer producing epicard, middle layer that differentiate into myocardium, and internal layer, the endocardium (jelly-like). The cardiac jelly contains adherons with proteoglycans, fibronectin and matrix proteins that induce atrioventricular cells to transform into mesenchyme, along with TGF beta cells. The transformed cells secrete proteases, destroying the adherons, and form the endocardial cushions responsible for development of main part of heart valves. Disturbance of events lead to heart anomalies. After third week, the heart tube undergoes characteristic dextral looping under influence of cardiac transcriptional factors HAND molecules, influencing heart asymmetry. Result of cardiac looping, is the formation of heart tube - a series of constrictions and dilatations of the endothelial tube - S-shaped heart with distinct regions. (SEE PIC DAY 23) 1. Sinus venosus - caudal end receiving blood returning from cardinal, vitelline and 33 Colloquium I Embryology 4th Semester umbilical veins (opens into right atrium) 2. Primitive atrium 3. Atrioventricular channel 4. Primitive ventricle 5. Bulbus cordis 6. Truncus arteriosus - dilates to form aortic sac, where aortic arches derive from Cardiac valves: Atrium and ventricle of endothelial tube communicate by atrioventricular canal. Proliferations of mesenchyme called endocardial cushions develop and divide the canal into right and left. Primitive valves are of endocardial cushion origin, while the definite ones develop from superficial epicardially growing tissue of atrioventricular groove. Together with the lateral endocardial cushions, they form the left, bicuspidal (mitral) and the right, tricuspidal valve. Septation occurs when a primitive septa called septum primum grows from the superior wall (5th week= down towards the endocardial cushions, dividing the atrium into right and left. The septa does not reach the cushion, but stops to form the primary opening. 34 Colloquium I Embryology 4th Semester Second septa, septum secundum, forms to the right of septum primum, overlaps foramen ovale, that develops as space between right and left atrium. In second trimester of pregnancy, 30 % of blood is shunted from the right atrium to the left, but in 3rd trimester, decreases to 20 %. In such way, the blood flows from the right atrium to the right ventricle, then to left ventricle, then leaves chamber through pulmonary outflow. As lungs are still closed, blood flows via ductus arteriosus

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