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Ri Chang 1
- 1. Human Embryology and Assisted Reproductive Technology (ART) Introduction of Human Embryology; Assisted Reproductive Technology (ART); Chromosome in eggs and embryos; Screening of embryos for genetic disease; Experimental Embryology Course: ANAT-381
- 2. Introduction of Human Embryology
- 3. Life cycle
- 7. Hypothalamus and pituitary glands
- 9. At puberty the female begins to undergo regular monthly cycle; These ovarian cycles are controlled by the hypothalamus; Gonadotropin-releasing hormone (GnRH) produced by the hypothalamus acts on cell of the anterior pituitary gland, which in turn secretes gonadotropins; Ovarian cycle
- 10. Gonadotropins Gonadotropins are hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), stimulate and control cyclic changes in the ovary; FSH stimulates follicular development, and LH surge triggers follicular rupture and ovulation.
- 12. Follicular development The adult ovary can be subdivided into three regions: the cortex, medulla, and hilus regions. The cortex consists of the surface epithelium (se), tunica albuginea (ta), ovarian follicles (primordial, primary (pf), secondary (sf), small, medium, large Graafian follicle (gf)) and corpora lutea (cl). The medulla consists of large blood vessels and nerves. The hilus contains large spiral arteries and the hilus or ovary Leydig cells. (Bloom W, Fawcett DW: A Textbook of Histology. Philadelphia, WB Saunders Company, 1975)
- 13. In human females, all primordial follicles are formed in the fetus between 6 and 9 months' gestation. During this period, there occurs a marked loss of oocytes due to apoptosis. The number of primordial follicles decreases progressively as a consequence of recruitment, until very few if any are present after the menopause at ~50 years of age. (Baker TG: Radiosensitivity of mammalian oocytes with particular reference to the human female. Am J Obstet Gynecol 110:746, 1971. Reproduced with permission from Mosby, Inc.)
- 15. Diagram illustrating the major histological changes that accompany the gonadotropin-independent period of preantral folliculogenesis (Erickson, Gregory F. Normal ovarian function. Clin Obstet Gynecol 21:31, 1978. Reproduced with permission from Lippincott-Raven Publishers.)
- 16. Photomicrographs of the early stages of human preantral folliculogenesis. A) Primordial follicle; arrow, squamous granulosa cell. B) Recruitment showing the primordial-to-primary transition; arrow, cuboidal granulosa cell. C) Primary follicle with multiple cuboidal granulosa cells. D) fully grown primary follicle at the primary-to-secondary transition stage; arrow, formation of a secondary layer of granulosa cells. All photos are 40x.
- 17. A typical healthy secondary follicle. It contains a fully grown oocyte surrounded by the zona pellucida, 5 to 8 layers of granulosa cells, a basal lamina, a theca interna and externa with numerous blood vessels. (Bloom W, Fawcett DW In A Textbook of Histology. WB Saunders Company, Philadelphia 1975. With permission from Arnold.)
- 18. Photomicrograph of an early tertiary follicle 0.4 mm in diameter at the cavitation or early antrum stage. zona pellucida (ZP); granulosa cells (GC); basal lamina (BL); theca interna (TI); theca externa (TE); granulosa mitosis (arrowheads). (Bloom W, Fawcett DW In A Textbook of Histology. Philadelphia, WB Saunders Company, Philadelphia 1975. With permission from Arnold.)
- 19. Diagram of the architecture of a typical Graafian follicle. (Erickson GF: Primary cultures of ovarian cells in serum-free medium as models of hormone-dependent differentiation. Mol Cell Endocrinol 29:21, 1983. Reprinted with permission from Elsevier)
- 20. Photomicrograph of ovulation of a mature egg-cumulus complex through the stigma. (Hartman CG, Leathem JH In Conference on Physiological Mechanisms Concerned with Conception. Pergamon Press, New York 1959)
- 21. Diagram of the cellular mechanisms by which the preovulatory surge of FSH and LH causes ovulation. (Erickson GF: The Ovary: Basic Principles and Concepts: In Felig P, Baxter JD, Broadus AE, Froman LA, (eds): Endocrinology and Metabolism, 3rd edition. New York, McGraw Hill, 1995)
- 22. Photomicrograph of a human corpus luteum. (Bloom W, Fawcett DW (eds) in A Textbook of Histology. WB Saunders Co., Philadelphia 1975 with permission from Arnold)
- 25. First week of development: from ovulation to implantation
- 26. Human embryo development (5 days) 1 2 3 4 5 6 7 8 9
- 27. Spermatogenesis
- 28. Spermatogenesis
- 30. Sperm morphology
- 31. Sperm Under light microscope
- 32. Insemination ( In vivo =1:300-500 / in vitro =1:1x10 6 ) In vitro In vitro
- 33. Scanning electron micrograph (SEM) of sperm binding to the zona pellucida In vitro
- 34. Fertilization The process by which male and female gamete fuse; It occurs in the ampullary region of uterine tube; Sperm may remain viable in the female reproductive tract for several days; The isthmus of the uterine tube serves as a sperm reservoir;
- 35. Capacitation It is a period of conditioning in the female reproductive tract that in the human lasts approximately 7 hours; During this time a glycoprotein coat and seminal plasma proteins are removed from the plasma membrane that overlies the acrosomal region of the sperm; Only capacitated sperm can pass through the corona and undergo the acrosome reaction;
- 36. Acrosome reaction Which occurs after binding to the zona pellucida ; It is induced by zona proteins; This reaction culminates in the release of enzymes needed to penetrate the zona pellucida, including acrosin;
- 38. The three phases of sperm penetration
- 39. Penetration reactions Cortical and zona reactions; Resumption of the second meiotic division; Activation of the oocyte;
- 40. Cortical and zona reactions: As a result of the release of cortical oocyte granules, which contain lysosomal enzymes The oocyte membrane becomes impenetrable to other sperm; The zona pellucida alters its structure and composition to prevent sperm binding and penetration; These reactions prevent polyspermy (penetration of more than one sperm into the oocyte)
- 41. Resumption of the second meiotic division: The oocyte finishes its second meiotic division immediately after entry of the sperm; The second polar body forms;
- 42. Activation of the oocytes The activating factor is probably carried by the sperm; Formation of female and male pronuclei; Each pronucleus is haploid;
- 43. Main results of fertilization Restoration of the diploid number of chromosomes, half from the father and half from the mother; Determination of the sex of new individual;
- 44. Early embryonic development After fertilization, the zygote undergoes a series of mitotic divisions, increasing the numbers of cells; These cells become smaller with each cleavage division; The cleaved cells are known as blastomeres;
- 45. Compact morula After 8-cell stage, blastomeres maximize their contact with each other, forming a compact ball of cells held together by tight junctions; This process calls compaction, it occurs in 16 or 32-cell stage embryo called morula;
- 46. Blastocyst formation Approximately 5 days after fertilization, the compacted morula divide to form blastocyst (>32-cells); The cleaved cells constitute the inner cell mass (or embryoblast), and surrounding cells compose trophoblast;
- 47. Blastocyst hatching Inner cell mass (ICM) develops to fetus, and trophoblast contributes to the placenta.
- 48. Implantation
- 51. Changes of endometrium during menstrual cycle
- 52. Events during the first week of human development
- 53. Clinical correlations Assisted Reproductive Technology (ART): First week of embryonic development; Contraception methods: 1. Barrier techniques; 2. Contraceptive pill; 3. Vasectomy and tubal ligation;
- 54. What kind of hormones produced from hypothalamus? What kind of hormones secreted from pituitary gland? Where fertilization occurred in vivo? What kind of hormones secreted from corpus luteum? In the human, after fertilization, how long the embryo takes to migrate into uterine for implantation? Questions for Introduction of Human Embryology:
- 55. 1. Langman’s Medical Embryology (9 th Edition); ISBN: 0-7817-4310-9; Author: T. W. Sadler, Ph.D. Publisher: Lippincott Williams & Wilkins. 2. Human Reproductive Biology (3 rd Edition); ISBN 13:978-0-12-088465-0 Authors: Richard E. Jones & Kristin H. Lopez Suggested reading
Editor's Notes
- #3: He is giving us a repeat of what we learned before.
- #4: This is the human life cycle. For the female the meiosis produces egg and for the male mitosis produces a sperm. The sperm and the egg fuses and it makes a zygote which further develops into an embryo. Then this implants. This is the human reproduction. Today we discuss how the meiosis for male and females occur and for the fertilization how is the embryo developing for one week before implantation?
- #5: There is a difference for mitosis and meiosis. Mitosis division is seen here – it is diploid . For meioses from diploid you can produce haploid . This is the difference between both.
- #6: For the female it is oogenesis . For one oocyte, it produces one egg . For the spermatogenesis , they form a germ cell from diploid and they produce many sperm cells . The sperm cell is haploid. This is the difference between females and males.
- #7: Meiosis is controlled by endocrine systems . You can see here that it relates to reproduction is the hypothalamus, pituitary, and gonad. We can see that for females it is the ovaries and for the male it is testis. This means meiosis is controlled by the endocrine system.
- #8: This is a system for hypothalamus and pituitary glands . You can see the hypothalamus and the pituitary. It is a control for meiosis . It is in the testis for males and in the ovary for female.
- #9: This is the ovarian cycle where the egg is produced. Primordial follicles are seen and germ cells for females and then the primordial follicle is further developed to become primary follicles which becomes the secondary which becomes pre-ovulatory follicles. Then there is the mature egg and this is fertilized when it meets sperm, and it becomes an embryo and it is implanted into the uterus, which gives rise to children.
- #10: This the ovarian cycle – at puberty the female begins to undergo their regular menstrual cycle. The ovarian cycle is controlled by the endocrine system as he just mentioned what this was – it was an endocrine system . The hypothalamus produces GnRH . This hormone is further controlled to the pituitary gland which produces gonadotropins (which includes 2 kinds of hormones).
- #11: Gonadotropns include FSH and LH. Normally we say that FSH promotes follicle development and LH is the trigger for ovulation .
- #12: It is controlled by the endocrine system. We can see the human case here. It is different for mice. We see the pituitary and gonadotrpan cycle is controlled by the ovarian cycle. We can see here that FSH is produced by the pituitary gland and promotes follicle development . LH triggers ovulation . The ovary produces some hormones as well. Some are estrogen and progesterone . Based on this endocrine system, you can see the ovary and the follicle as it develops controlled by FSH and during ovulation , the sights come to the luteal phase. You see the uterus with its endometrium must be synchronized with endocrine in the system . If the egg is fertilized and it becomes to embryo the uterus and the endometrium must prepare for implantation. This is why you can see here that it goes from very thin to very thick. If this egg implantation continues, progesterone will be secreted to maintain the pregnancy and if not then the pregnancy will not continue.
- #13: No
- #14: Another concept for the human case is that we can see here the primordial follicle during the fetus development in the ovary - the primordial follicle is a germ cell marker and is increased a lot but it is during the fetus life and the 6-9 months that it reaches the peak and starts to reduce the number of the primordial follicle in the ovary . At birth, the number is reduced quite a lot. This means that at the birth, the number of the primordial follicles in the ovary is already decided. This is based on after puberty how the primordial follicle is produced by each for each cycle. Women of 50 years old are at menopause. For the whole life, women ovulate around 400 eggs.
- #15: No
- #16: This is the follicle development in ovaries. It is a primordial follicle – the side is quite small. This black part is the zona pellucida . The white part is the oocytes . The outer layer is the granulosa cell layer. Once the Primordial follicle goes from 40 microns to 400 microns, it means the antrum is formed .
- #17: Once the antrum is formed, in the human case, it looks like this – the granulosa cells are around and the size is increased .
- #18: Here we see how many granulosa cells there are in one follicle.
- #19: So the antrum is formed . Once the follicles develop and the antrum forms, the oocytes really become to a full sized growing oocyte. Regardless, the follicle antrum becomes big . The oocyte’s size become a full growing oocyte.
- #20: This antrum is bigger – more than 400 microns. This middle sight isn’t increased anymore but the outer part is increasing. The size of the oocyte is the same but the antrum is increasing .
- #21: This follicle is supposed to ovulate. Ovulation is triggered by LH - hormone triggered during the menstrual cycle. Once LH is surged, this egg will ovulate. It is at the surface of the ovary . This ovulated egg meets sperm in the oviduct, goes to the fallopian tube and becomes a zygote and then an embryo and then migrates to the uterus for implantation. This is a human case.
- #22: We have a lot of mechanisms for ovulation. You can see here that FSH is produced by pituitary gland . This promotes follicle development . LH surge is triggered by ovulation.
- #23: After ovulation, the sight of ovulation in the ovary is becoming the corpus luteum . This corpus luteum secretes progesterone . This means that the egg is fertilized and became a zygote which further developed to an embryo to implant. If this occurs, the corpus luteum secretes progesterone to maintain the pregnancy . This is the function of corpus luteum.
- #24: You can see the ovary and the follicle development during one menstrual cycle . You can also see the primordial follicle promoted by FSH which makes it a big follicle. It is follicle growing. The mature follicle comes to a secondary and the antrum is forming. Then it comes to a the pre-ovulatory follicle. It is controlled by the endocrine system. The LH surge triggers the ovulation. The zygote comes to the embryo and if the embryo implants, we can see the ovulatory sight . This comes to the corpus luteum which is fully developed and secretes progesterone. This implants and this leads to a pregnancy. You can see a uterus and the match with this cycle is controlled by endocrine. It is ready for implantation. If it doesn’t implant then the corpus luteum degenerates because you don’t need progesterone anymore .
- #25: This is a uterus. You can see primordial follicles generating and it becomes pre-ovulatory. After ovulation it migrates to the fallopian tube. The fimbriae catch the egg that just ovulated. The sperm and the egg meet in the oviduct (fallopian tube) at the region of the ampulla. The eggs migrate down to the isthmus and then to the uterus to be implanted. For the human case, the sperm comes from the cervic and migrates up to the fallopian tube and isthmus . Where the sperm and egg meets each other is in the ampulla area . Each migration reduces the sperm number. Where the sperm and the egg meets in vivo- only one egg meets 300-500 sperm. It is different in the in vitro case.
- #26: The sperm and egg meet each other in the ampulla region. The fertilized egg becomes a zygote . On day 1 it has become 2 cells. Day 4 you see the morula and day 5 is the blastocyst . The blastocyst almost goes into the uterus and to implant into the uterus. This is early human embryology. It relates to reproductive technology. Usually we do fertility treatment – we do it in the first week .
- #27: How do we know the egg is really fertilized? It is called the mature egg . We see that it is ready to fertilize . If we see the first polar body , we know the egg is diploid -it is not ready to fertilize . After fertilization, we see one pronuclei is from sperm and the other is from the egg. We see the egg fertilized normally because we see it on the microscope that there is 2 pronuclei . If you further culture this egg it is 30 hours after insemination – we can see 2 cells. It is further cultured and in day 2 after insemination we can see 4 cells and further on day 3 we can see 8 cells. On day 4 we can see the compacting morula and in day 5 we can see a human blastocyst. We detect a zona pellucida. This one migrates into the uterus. For the fertility treatment, we can transfer the embryo at any stage into the uterus . They can develop in the uterus and this one is hatched zona pellucida and we can implant this into the uterus .
- #28: The male has spermatogenesis which is sperm regeneration. This is a testis. You can see here the tube where they generate sperm. After they generated the sperm it migrates to the epididymus where sperm from immature stages come to mature stages . The mature sperm comes out and is ejaculated. If we bring this tube we can do a section and we see on the right. The spermatocyte further matures and it gets longer and this becomes sperm, it comes to the lumen. They stall there in the mature epididymus. Normally the spermatogonia to come to sperm takes 3 months .
- #29: From spermatogonia it becomes spermatocyte which further matures. It is gets longer and this leads to the lumen.
- #30: It forms one spermatogonia comes to spermatozoa. 1 spermatognia cam become many . For oocytes it is different. One ogonia comes only to one oocyte.
- #31: We see the sperm morphology. You see the head. The black part is the DNA part – nuclear and for any kind of mamallary sperm of any species, they have an acrosome . They have an outer and inner part and it helps penetrate the egg (zona pelllucida) . This is the device for penetration. Sperm is always moving so they must have an engine – this metal piece produces energy. They have a long tail for the sperm. This shows us the structure of the human sperm.
- #32: Under a light microscope we can see the head, the middle piece, and the tail. It is a bright field. For human cases such as the eggs, the size is 120 microns and for the sperm the size is smaller than the egg – its total length depends but around 60 microns .
- #33: This is insemination that they do in their lab for in vitro fertilization. How we do insemination in vitro is seen here. This is a human egg. The ratio of the insemination in vitro because we know in which sperm is good. We won’t put one egg and one sperm in the Petri dish. Normally we put the one egg vs. million sperm together. In vivo, when the sperm and egg met each other the region is in the fallopian tube and normally it is 1 egg vs. 300-500 sperm in vivo cases.
- #34: This is on the scanning EM and we can see how in vitro fertilization there is so many sperm attached to the zona pellucida and only one sperm can penetrate into the egg.
- #35: The fusion is called the process of fertilization . This is occurring in the fallopian tubes in the ampulla region. We know that in vivo cases, the sperm may remain variable in the female reproductive tract for several days so we can do fertility treatments. In vivo cases, the patients are given advice – we do an ovulation induction to make sure the patient ovulates. Other wise they have intercourse and then 2 days later they help - sperm needs times to migrate to the ampulla region. This is why you have the sperm survival several days in the female reproductive tract . The isthmus of the uterine tube serves as a sperm reservoir.
- #36: Sperm and egg meet each other. The sperm is undergoing capacitation - if not the sperm cannot penetrate the egg. We call this capacitation. This procedure is needed for human cases – 7 hours. If you see the sperm and the egg mixed together in the Petri dish, the sperm cannot penetrate- it needs 7 hours. They have a protein code on the surface because for the human, male semen is ejaculated . They have seminal fluid in there as well . This processes covers the sperm head. It has to be removed before it penetrates the egg . This process is called capacitation . Only capacitated sperm can pass through the corona . Normally we have these cumulus or ganulosa cells. This is a protector for the egg. The acrosome reaction helps it penetrate into the zona pellucida of the egg .
- #37: This is when the sperm binds to the zona pellucida . Some enzymes are released.
- #38: This is the acrosome reaction . You have sperm and you can see that the PM is out and the seminal proteins is covered by this sperm head . The capacitation has removed its coat . Of this capacitated sperm, this one meets the egg. To do this, there has to have an acrosome reaction. They have an outer and inner membrane, and they release their contents which is acrosin . This one can penetrate the zona pellucida and into the egg.
- #39: For fertilization, we have 3 phases for sperm penetration to this egg. The egg have the granulosa and cumulus cells. The sperm penetrates this. The sperm can approach the egg . They enter to the phase which has to be when the sperm comes to capacitate. They have to have the acrosome reaction which can let this sperm penetrate the zona pellucida. After it penetrates the zona pellucida, the egg membrane and the sperm membrane fuses together which is fertilization . There are 3 phases for penetration in the human case.
- #40: Sperm penetrated the egg. The egg also has reactions – otherwise many sperm can penetrate the egg. The reaction is the cortical granulosa and zona reaction. One sperm is penetrating the zona pellucida and so the egg has a reaction to refuse extra sperm penetrating the egg . This is called the cortical granulosa action – the cortical release is a reaction. The sperm stops and is continuing meiotic division. Also this egg is activated.
- #41: The enzymes released let the zona pellucida be as such so that no extra sperm can penetrate . This is the zona reaction. However we do in vitro fertilization and infertility treatments. We can see 3 or 4 sperm penetrate one egg and this is not normal fertilization .
- #42: Read slide. By this morphology 2 pronuclei are formed which is normal fertilization for the human zygote.
- #43: The activation factor is coming from sperm. There are 2 pro nuclei . One is a male and one is a female pronuclei. They migrate together. This process is called a singamy . We see the 2 pronuclei approach each other and form a diploid . It becomes a 2 cells stage .
- #44: It is the restoration of diploidy . Half of the set of the chromosomes is from sperm and half is from the egg. At fertilization the sex is decided . If the X sperm penetrates, the embryo is a female. If a Y sperm penetrates, it is a male. This means at fertilization the embryo sex is decided.
- #45: After fertilization it becomes diploid . It underwent mitotic division to increase the cell number . This embryo is cleaved as a blastomere. It is inside the zona pellucida and they just divide. From bigger blastomeres, it becomes 4 - they divide, become the smaller and smaller.
- #46: Once you come from 16-32 cell stage, it is compacting. The zona pellucida is there. They cannot become too big. This is why the blastomere is compacting and compacting and of the 32 cell stage this embryo is the morula stage .
- #47: Normally it is more than a 32 cell stages so they form the blastocyst stage . They have an ICM. This is a human case. This normally has become to the fetus . We call the ICM or embryoblast becomes the fetus and we call the other layer the trophoblast which becomes the placenta after implantation.
- #48: The zona pellucida has become a thing. This blastocyst is further inlies. It must have hatched from the zona pellucida . This is one hatched blastocyst. It implants into the uterus . We can see the ICM which becomes the fetus and this trophoblast develops into the placenta.
- #49: This is implantation -This is a hatched blastocyst in the uterus . How does it implant? We can see the ICM. It is not like human embryology. There is a lot of mechanisms involved.
- #52: We know the ovarian cycle so we must know how the endometrium and uterine cycle can accept this embryo for development . This is controlled by the hypothalamus which produces GnRH. The pituitary gland secretes gonadotropins – FSH and LH. The mean time they control the endometrium too . We can see there and synchronize with the ovarian cycle. Once they ovulate, they come to day 5 and the blastocyst implants. They continue for developments for the fetus and placenta. If this ovulated egg is not fertilized, the corpus luteum is degenerated and there so no more progesterone secreted. The endometrium cycle corresponds with the endocrine system too .
- #53: We must know for human cases how is after 1 week the embryo develops. When is the sperm and egg meeting each other? Normally we said after ovulation. It means that this zygote further develops – haploid comes to diploid. The embryo cleavage is mitosis , not meiosis. After ovulation, 30 hours it is 2 cells and day 3 is 8 cells. You can see that day 4 and 5 have hatched a zona pellucida . We must know that these procedures so that we can show fertility treatments for an infertile couple.
- #54: We do infertility treatment and try and help the couple. The first week of embryonic development is important with ART. We can do contraception methods. We prevent the sperm and egg from meeting each other . The reproduction system is controlled by the endocrine system so we can disrupt this too . Lastly we can do surgery . We can block or cut the oviduct or the fallopian tube so that the sperm and the egg cannot meet in the female body . Or prevent the sperm from ejaculating.
- #55: This was for this lecture.