Getting to the right site:

1. Getting to the right site: • Helping the sperm: • At ovulation, the cervical mucus increases in amount and becomes less thick, allowing easier sperm transport. • Passage of the sperm through the uterus and oviduct occurs mainly due to muscular contractions of these organs and flagellar movement of sperm. • Oocyte: • The ends of the oviducts come into close contact with the ovary during ovulation. • Fimbriae of oviduct ends “sweep” the ovulated ovum into the oviduct. • Peristaltic waves of oviduct musculature bring the ovum into the ampulla of the oviduct. • Ciliary movements of epithelial cells of oviduct.

2. The OviductH & E × 10M = muscleBL = broad ligamentS = serosa with vascular supporting tissue From Wheater’s Functional Histology, 4th ed., 2000.

3. Morphology of the Oviduct: Fallopian tube S = smooth muscle H & E × 150 E = ciliated epithelium Fallopian tube Azan × 320 From Wheater’s Functional Histology, 4th ed., 2000.

4. Capacitation: readying the sperm • Sperms cannot fertilize oocytes when they are newly ejaculated. • The process of capacitation takes 5-7 hours. • Capacitated sperms are more active. • Location: capacitation occurs in the uterus and oviducts and is facilitated by substances of the female genital tract. • The acrosomal reaction cannot occur until capacitation has occurred. • Glycoprotein coat and seminal plasma proteins are removed from plasma membrane of acrosomal region

5. Stage 1 of fertilization: • The acrosome reaction must be completed before the sperm can fuse with the secondary oocyte • Occurs when sperms come into contact with the corona radiata of the oocyte • Perforations develop in the acrosome • Point fusions of the sperm plasma membrane and the external acrosomal membrane occur • The acrosome reaction is associated with the release of acrosome enzymes that facilitate fertilization • Passage of sperm through the corona radiata depends on enzyme action: • hyaluronidase released from sperm acrosome • Tubal mucosal enzymes • Flagella action also aids corona radiata penetration • It is thought that the other sperms aid the fertilizing sperm in penetrating the barriers protecting the female gamete. Capacitated sperm pass freely through corona cells

6. Ovum and sperms: (In vitro) From this photograph, it should be clear that the heads of human sperm are less than 1/20 the diameter of human eggs.  Arrows point to sperm heads The surfaces of unfertilized eggs are usually smooth in appearance. The mottled look of this egg is not normally seen, but apparently all the ova from this woman had this appearance.

7. Stage 2 of fertilization: • Penetration of the zona pellucida around the oocyte: • Acrosomal enzymes: esterases, acrosin, and neuraminidase cause lysis of the zona pellucida • Once sperm penetrates zona pellucida, the zona reaction occurs: • This contact results in release of lysosomal enzymes from cortical granules lining the plasma membrane of the oocyte.In turn, these enzymes alter properties of the zona pellucida (zona reaction) to prevent sperm attachment and penetration • This reaction makes the zona pellucida impermeable to other sperms. • Inactivates species specific receptors for spermatozoa on zona pellucida • When more than one sperm manages to enter the ovum (dispermy = 2; triploidy = 3), the fetus nearly always aborts. • 1

8. Stages 3 & 4 of fertilization: • Fusion of plasma membranes of oocyte and sperm • Head and tail of a sperm enter the cytoplasm of the oocyte, but the sperm plasma membrane remains behind. • 2nd meiotic division of oocyte is completed • The secondary oocyte was previously arrested in metaphase of the 2nd meiotic division, and now forms the mature ovum and another polar body.

9. Fusion of egg and sperm As soon as the spermatozoon has entered the oocyte, the egg responds in three ways: • Cortical and zona reactions. • Resumption of the second meiotic division. • Metabolic activation of the egg.

10. Stage 5 of fertilization: • Formation of male and female pronuclei: • Chromosomal material of the sperm decondensates and enlarges • Chromosomal material of the ovum decondensates following the completion of meiosis • At this stage, the male and female pronuclei are indistinguishable. • As they grow, the pronuclei replicate their DNA  still 1N (haploid)- 23 chromosomes, each in chromatid pairs

11. Fusion of the pronuclei: (in vitro) • The male and female pronuclei are indistinguishable from one another. • The second polar body can be seen (arrow). • The plasma membranes of the two pronuclei are dissolving and one diploid nucleus will remain.

12. Stage 6 of fertilization: • Membranes of the pronuclei break down, chromosomes condense and arrange themselves for mitotic cell division • On membrane dissolution, there is 1 cell with 46 chromosomes = diploid (2N) • The first cleavage follows shortly, leaving 2 cells, each with 46 chromosomes. • Mitosis in the new zygote uses centrioles derived from the sperm. The oocyte has no centrioles.

13. Fertilization facts: • Completed within 24 hours of ovulation • Approximately 400 to 600 MILLION sperms are deposited at cervical opening during ejaculation. • Some sperm are held up by the folds of the cervix and are gradually released into the cervical canal; this gradual release increases the chances of fertilization. • Most human sperms do not survive longer than 48 hours in the female genital tract. • Only about 200 sperms reach the fertilization site; most degenerate and are absorbed by the female genital tract.

14. The results of fertilization: • Stimulates the secondary oocyte to complete meiosis. • Restores the normal diploid number of chromosomes (46). • Cleavage starts • Results in variation of human species as maternal and paternal chromosomes intermingle. • The embryo contains only maternal mitochondria because the sperm mitochondria are dispersed into the egg cytoplasm and discarded. • Determines the sex of the embryo. • The sex chromosome (Y or X) carried by the successful sperm determines embryonic sex.

15. Twins: still 1 sperm per egg • Monozygotic (monoovular): • A fertilized, single egg splits into two developing zygotes at a very early stage. • Identical twins; same sex. • Dizygotic (polyovular): • Result from the fertilization by two sperm of two separate ova that have reached maturation at the same time. • Not identical twins; can be different sexes • Incidence increases with age of the mother

16. How can fertilization go awry? • Too many sperm = dispermy or triploidy • Leads to spontaneous abortion in most cases. • Infertility • Bad timing: • The sperm can only survive 48 hours within the female genital tract. • In vitro studies show the ovulated egg cannot be fertilized after 24 hours.

17. Triploidy (in vitro) There are 3 pronuclei within this one zygote. In the laboratory, such embryos are discarded. In vivo, such embryos almost always abort spontaneously.

18. A little more on sex determination: • Not so clear-cut as X and Y: • The presence of other genes, located on the X, Y and other chromosomes, influence the determination of embryonic sex. • The SRY gene: • Normally present on the Y chromosome • Induces the sexless embryonic gonad to develop into a testis • Can sometimes be present on the X chromosome, leading a genetic female (XX) to develop testis • 1990: a fertilized mouse embryo with the XX configuration was injected with SRY and developed as a male both anatomically and behaviorally.

19. Infertility • Infertility is a problem for 15% to 30% of couples. • In vitro fertilization (IVF) • Follicle growth in the ovary is stimulated by administration of gonadotropins. Oocytes are recovered by laparoscopy from ovarian follicles with an aspirator just before ovulation when the oocyte is in the late stages of the first meiotic division. The egg is placed in a simple culture medium and sperm are added immediately. Fertilized eggs are monitored to the eight-cell stage and then placed in the uterus • to develop to term.

21. Another technique, gamete intrafallopian transfer (GIFT), introduces oocytes and sperm into the ampulla of the fallopian (uterine) tube, where fertilization takes place. Development then proceeds in a normal fashion. In a similar approach, zygote intrafallopian transfer (ZIFT), fertilized oocytes are placed in the ampullary region. Both of these methods require patent uterine tubes.

22. intracytoplasmic sperm injection (ICSI) • Severe male infertility, in which the ejaculate contains very few live sperm (oligozoospermia) or even no live sperm (azoospermia), can be overcome using intracytoplasmic sperm injection (ICSI). With this technique, a single sperm, which may be obtained from any point in the male reproductive tract, is injected into the cytoplasm of the egg to cause fertilization. This approach offers couples an alternative to using donor sperm for IVF.

23. Therapeutic cloning or somatic nuclear transfer • Nuclei are taken from adult cells e.g., skin and introduced them into enucleate oocytes • Oocytes are stimulated to differentiate into blastocyst

24. Preselection of the Embryo's Sex • The differential swimming abilities of the X and Y sperms • Different speeds of migration of sperms in an electric field • Differences in the appearance of X and Y sperms • DNA difference between X (2.8% more DNA) and Y sperms • The use of a selected sperm sample in artificial insemination may produce the desired sex.

25. Cryopreservation of Embryos • Early embryos resulting from in vitro fertilization can be preserved for long periods by freezing them with a cryoprotectant (e.g., glycerol). Successful transfer of four- to eight-cell embryos and blastocysts to the uterus after thawing is now a common practice.

26. Surrogate Mothers • Some women produce mature oocytes but are unable to become pregnant, for example, a woman who has had her uterus excised (hysterectomy). In these cases, in vitro fertilization may be performed and the embryos transferred to another woman's uterus for development and delivery.

27. Cleavage • Blastomeres • Until the eight-cell stage, they form a loosely arranged clump • compaction • This process segregates inner cells, which communicate extensively by gap junctions, from outer cells after 3 division.

28. Development of the zygote from the two-cell stage to the late morulastage. The two-cell stage is reached approximately 30 hours after fertilization; thefour-cell stage, at approximately 40 hours; the 12- to 16-cell stage, at approximately3 days; and the late morula stage, at approximately 4 days. During this period, blastomeresare surrounded by the zona pellucida, which disappears at the end of the fourth day.

29. 16-cell morula (mulberry)(Approximately 3 days after fertilization) • inner cell mass • outer cell mass

30. Blastocyst Formation • Blastocele • Cells of the inner cell mass, now called the embryoblast, are at one pole, and those of the outer cell mass, or trophoblast, flatten and form the epithelial wall of the blastocyst • In the human, trophoblastic cells over the embryoblast pole begin to penetrate between the epithelial cells of the uterine mucosa about the sixth day

31. uterus at the time of implantation • The uterus at the time of implantation is in the secretory phase, and the blastocyst implants in the endometrium along the anterior or posterior wall. If fertilization does not occur, then the menstrual phase begins and the spongy and compact endometrial layers are shed. The basal layer remains to regenerate the other layers during the next cycle.

32. Starting of implantation • L-selectin on trophoblast cells and its carbohydrate receptors on the uterine epithelium mediate initial attachment to the uterus • Further attachment and invasion by trophoblast involve integrins and laminin and fibronectin by extracellular matrix • Integrin and laminin results in attachment while integrin and fibronectin stimulates migration