1.) GAMETOGENESIS AND FERTILIZATION Primordial germ cells (PGCs): ~ Originate from epiblasts (pluripotential embryonic cells which can become either germ/somatic) ation based on: ~ Location (posterior primitive streak region of embryo that's fated to become hindgut) ~ Signaling factors e.g. BMP (bone morphogenetic proteins) from extraembryonic ectoderm ~ Suppression of somatic cell genes e.g. Blimp-1 (transcriptional repressor) from extraembryonic tissue - Migration from yolk sac endoderm to gonadal ridge (3-5 wks gestation) ~ Controlled by various molecular mechanisms e.g, distant soluble factors, local signals, interactions with ECM molecules - Any PGCs that are left along migratory route (i.e. don’t reach ridge) are killed by apoptosis, + ifnot eliminated, pediatric midline germ cell tumors e.g. dysgerminoma ~ PGC at gonadal ridge don’t die b/c steel factor on somatic cells in ridge binds cKit receptor on PGCs Sdownregulates pro-apoptotic genes ~ Type of somatic cell in gonad determines whether PGC become oogonia vs. spermatogonia ‘Spermatogenesis (64 days/cycle, occurs in seminiferous tubules of testes) - At puberty, PGCs will differentiate into spermatogonia, seminiferous tubules mature Spermatagonia divide mitotically Spermatagonia > Primary spermatocytes Primary spermatocytes enter Meiosis | > Secondary spermatocytes Secondary spermatocytes enter Meisosi Il > Spermatids Spermatids undergo spermiogenesis (loss of cytoplasm, development of sperm tail and acrosome) > Mature sperm (aka spermatozoa) 6) Spermatozoa break connection with Sertoli cells, released into lumen of seminiferous tubules (spermiation) - Spermatocytes joined by cytoplasmic bridges as they divide ~ Division takes place btwn Sertoli cells (large cells transversing entire seminiferous tubule; tight junctions btwn them form blood testis barrier; nurture developing sperm) - _ Acrosome derived from Golgi of spermatid, contains enzymes needed to penetrate secondary oocyte - Tail has three segments, middle part has mitochondria/ATP - Spermatogonia can self-renew (<<1%) or differentiate into Primary spermatocytes ~ SSC (Spermatogonial Stem Cells) allows for continual sperm production through lifetime - Sertoli cells secrete GDNF (glial cell line-derived growth factor) > binds GRA1 or RET receptors on spermatogonia, induces SSC formation - “In vivo” sperm culturing - implant male germline stem cells into another male carrier Ysune ogenesis - PGCs divide mitoticaly and differentiate into oogonia and enter meiosis by 5 months gestation 1) Oogonia > Primary oocytes (9 wks gestation) ‘a. No primary oocytes formed after 20 wks gestation b. High oocyte attrition: 7 million > 2 million during fetal period; then > 400,000 by puberty 2), Primary oocytes enter Meiosis I, arrest in prophase (13 wks gestation) 3) Stromal cells form single layer flat epithelial cells (granulosa cells) around oocyte = primordial follicle 4) Primordial follicle > Primary follicle as primary oocyte grows, granulosa cells become columnar5) After puberty, FSH (follicle stimulating hormone, from pituitary) promotes growth of several Primary follicles (granulose cells divide to form lots layers around oocyte, closest layer called cumulus cells) -> Secondary follicle Only one follicle is ovulated per month, the rest degenerate Zona pellucida (an ECM made from glycoproteins secreted by oocytes) forms around oocyte Secondary follicle forms antrum (fluid filled cavity) surrounding oocyte Follicle enlarges until forms swelling on ovary surface > mature or Graafian follicle LH (luteinizing hormone, from pituitary) promotes Primary oocyte to resume and finish Meiosis | > Secondary oocyte and first polar body (asymmetric division) a Secondary oocyte gets all the cytoplasm, but chromosomes split evenly 10) Secondary oocyte enters Meiosis l, arrests at metaphase until fertilization - Complex factors governing oocyte development e.g, TGFB subfamilies, Steel/c-Kit - Oocyte arrest is maintained by complex processes: = Cross talk btwn oocyte and pregranulosa cells - _ Steel/c-kit protect preantral follicles from apoptosis - Anti-mullerian hormone secreted by follicle suppresses growth of surrounding primordial follicles ‘Ovulation 1) At mid-cycle (14 days), mature follicle growth spurt, high estrogen conc causes increase in LH 2), LH induces ovulation = oocyte w/cumulus cells expelled from ovary into Fallopian tube (coaxed by fingerlike fimbriae) 3) Remaining follicle tissue becomes corpus luteum ‘a. Corpus luteum secretes progesterone to prepare endometrium for implantation b. if oocyte fertilized, CL enlarges and increases hormone production, embryo's extraembryonic tissues secrete hCG (human chorionic gonadotropin) to prevent CL degeneration c_fnot fertilized, CL degenerates after 10-12 days, estrogen and progesterone decrease ~ Oocyte to zygote transition requires: synthesis of new proteins, removal of mRNA that maintain prophase arrest, protein degradation, reshuffling organelles Fertilization (occurs in ampulla of Fallopian tube) 1) 200-600 million sperm deposited, but only 200 survive trip to ampulla, takes min-1hr 2), Sperm undergo capacitation before they can fertilize ‘a. Takes 7hrs, increased sperm motility, loss of glycoprotein coat and seminal proteins in acrosome 3) Acrosomerxn a sperm contacts the cumulus cells and zona pellucida around oocyte b, acrosome membrane perforates and fuses w/sperm plasma membrane proteolytic enzymes e.g, hyaluronidase and acrosin released, help sperm penetrate cumulus cells and zona pellucida d. glycoprotein ZP3 found in ZP can initiate acrosome rxn 4) Sperm fuses with egg plasma membrane, mediated through CD9 (egg) and Izumo (sperm) 5) Sperm head decondenses, releases phospholipase C-zeta -> Ca2+ increase in egg > cortical granule exocytosis (granules release enzymes to modify ZP to prevent polyspermy) 6) Ca2+ increase induces finish of Meiosis ll > Secondary oocyte emits second polar body 7) Male and female pronucleiform, replicate DNA, chromosomes condense, first mitosis~ _ Infertility = inability to conceive after 1 yr of trying; 20% and rising in US - “functionally sterile” = sperm counts below 20 million/mL semen - Invitro fertilization more successful if woman younger ~ ICSI (intracytoplasmic sperm injection) ~ sperm injected directly into egg cytoplasm; circumvents sperm motility defects, low sperm count (CLEAVAGE AND IMPLANTATION Early Cleavage - _ Embryo surrounded by zona pellucida, first cleavages as travel thru Fallopian, no overall size increase after divisions; these cells are truly totipotent - Compaction at & cell stage - Ca2+-dep event, mediated by E-cadherin (aka uvomorulin) ~ First event in embryo that causes cells to differ from one another — cells no longer totipotent ~ Cells on inside become inner cell mass (ICM) (forms embryo) ~ Cells on outside become trophectoderm (forms extraembryonic membranes, implantation) - Cell differentiation based on LOCATION - Embryo now a morula (borders of individual cells not distinguishable now) - Regulation — before compaction, all cells same, so can remove one and still be normal; take ZP from three 8-cell embryos and aggregate them, they'll compact into one morula and form a normal mouse Blastocyst Formation ~ Outer cells of morula pump fluid towards embryo center to form blastocoele (fluid-filled cavity); embryo now called blastocyst - Two distinct cell types: outer = trophectoderm; inner = ICM - ICM cells are pluripotent (can make germ line, endoderm, ectoderm, mesoderm but NOT placental or extraembryonic cells), but can’t self-renew; ICM is what’s used to make ES cells in vitro - Hatching -ZP degenerates, blastocyst free floats in uterus Implantation - _ Blastocyst attaches to endometrial epithelium of uterine wall - Implantation occurs in superior (upper) part, posterior wall of uterus (6 days after fertilization) - _ HB-EGF (heparin-binding EGF-like growth factor) induces attachment of embryo - Regulated by growth factors that are selectively expressed in luminal epithelium - _ Embryonic pole of blastocyst attached to uterine wall now defines dorsal-ventral axis of embryo - Trophoblast cells differentiate into: syncytiotrophoblast (directly above ICM) and cytotrophoblast (around blastocoele} - _ Syncytiotrophoblast secrete enzymes to let embryo embed further into endometrium - _ Syncytiotrophoblast secrete HCG that enters maternal bloodstream to maintain corpus luteum, but placenta will ake over this role and secrete progesterone = Decidual cells (maternal stromal cells) loaded w/elycogen and lipids, provide nutrients for embryo - Implantation regulated by uterine expression of LIF (leukemia inhibitory factor) and L-selectin on trophoblasts - May failed pregnancies spontaneously terminate around time of implantation - Ectopic pregnancy = implantation outside uterus, usually in uterine tube, due to inflammation/scarring of tube