An Sci 434 Oral Exam

• CONTRAST: o The cow's cervix has 4-5 annular rings and a fornix vagina o Cows have a bicornuate uterus: one cervix, one uterine body, one vagina, and medium size uterine horns. The fetus will grow in the uterine horns. o Humans have a simplex uterus: there is one cervix, one vagina, and one large uterine body. Humans do not have uterine horns, so the fetus will grow in the uterine body. o Cows also have caruncles on the endometrium where the cotyledons of the placenta attach. These cotyledons surround the entire placenta. o Humans have a discoid placenta where the points of attachment are on one side for gas, nutrient, metabolic, and waste exchange occur. o The cow has a labia majora (external vulva) where the human has a labia majora and minora (external vulva) o In cows, the CL secretes progesterone to maintain pregnancy. If pregnancy does not occur, then the CL regresses. CL regression is caused by prostaglandin secretions from the uterine horn ipsilateral to the CL. The posterior pituitary and the CL release oxytocin which triggers the uterus to release prostaglandin which causes regression of the CL (Luteolysis). o In humans, the CL secretes progesterone and estrogen. The CL in the human lasts for 12-14 days and if the person is not pregnant, then the CL self-destructs. The CL needs h CG to maintain pregnancy. o Cows have an estrus cycle and humans have a menstrual cycle. o Estrus cycle: Follicular phase • Proestrus: follicle enlarges, E2 increases • Estrus: allows males to mount, ovulation 24-48 h after LH surge Luteal Phase • Metestrus: low E2, ovulation in cow, corpus hemorrhagicum, FSH increases • Diestrus: fxnal CL and secretes progesterone, FSH growth of ovulatory follicle, CL regresses if not pregnant and repeat o Menstrual Cycle: Menses: blood tissue secretions out of uterus and into vagina, recruitment of follicles Follicular Phase: about 9 days, the development of dominant follicle, E2 AND P4 increases, LH surge Luteal phase: CL, high P4 and E2, regression of CL and repeat • COMPARE: o Ovulation over entire surface of ovary o Same outcome: a baby

• Type of penis: o Bull Sigmoid flexure, fibroelastic penis, small glans penis o Human No sigmoid flexure, muscular vascular, large glans penis • Type of glands: o Bull Larger semial vesicals (than human), prostate body, prostate disseminate, cowper's gland Large accessory glands means that there is a large volume of the ejaculate with lower concentration of sperm/ml o Human Seminal vesicals, larger prostate body (than bull), cowper's gland (no disseminate prostate) • Testicle orientation: o Bull Verticle Testis weight (g) is 300+ o Human Vertical Testis weight (g) is 25 • Erection/ejaculation: o Bull Rapid erection, short ejaculate time, medium semen volume, medium to high sperm concentration, medium sperm number o Human Slow speed of erection, moderate ejaculate time, medium semen volume (same), low sperm concentration, low sperm number

• Spermiation is release of spermatozoa (mature sperm) from sertoli cells into the lumen of the seminiferous tubule. o Sertoli cells phagocytizes the remaining cytoplasm of the spermatozoa, and only a small droplet is left on the neck of the sperm. • It is transported to the epididymis o In epididymis o Becomes fertile o Develops motility o Nuclear condensation o Cytoplasmic droplet is absorbed • Mixes with seminal plasma and ejaculated. • The sperm is produced in the seminiferous tubule, then travels to the • Rete testis • Vas efferentia • Caput (head) of the epididymis • Corpus epididymis • Cauda (tail) of the epididymis • Vas deferens • Spermatic cord • Accesory glands (ampulla, seminal vesicles, prostate, cowpers gland) • Bulbossponegous muscle, iscocavernous muscle, retractor penial muscle, sigmoid flexure, glans penis

• The Y gene controls testicular differentiation in mammals o SRY (sex reversal Y) Codes for a DNA binding protein Acts as a transcription factor to alter expression of other genes on the Y Causes development of the primary sex chord (seminiferous tubule), sertoli cells, and the Anti-Mullerian Hormone is produced by Sertoli cells Development of the testes o Spermatogenesis, androgen production, lone bone growth o The SRY gene expression causes the mesoderm of the genital ridge to differentiate into sertoli cells. These divide mitotically until puberty. Divide meiotically when puberty begins. o The SRY gene causes the testes to develop along with the primary sex chords (seminiferous tubule). The Sertoli cells then secret anti-mullerian hormone (AMH) and this causes the leydig cells to develop and produce testosterone. The Wolffian duct system develops and degeneration of the Mullerian duct system (Female). Wolffian duct: • The testis secrete testosterone and this diffuses across the cell membrane of the Wolffian ducts. It binds to a receptor in the nucleus and causes the DNA to change the gene expression (or development of the Wolffian duct system) • Contains testis, epididymis, vas deferens, and seminal vesicles • Phenotypic Sex: Testosterone is converted into dihydrotestosterone (DHT) by 5alpha reductase (an enzyme). This binds to the same receptor as Testosterone in the nucleus with high affinity and triggers events in transcription to happen. This causes the development of the penis, scrotum and accessory sex glands. • Descent of the testis: the gubernaculum rapidly grows and pulls the testis down through the inguinal ring and into the scrotum. The gubernaculum then regresses • Brain development: o The testis secrete testosterone. This crosses the blood brain barrier and testosterone is converted into estrogen in the brain. Estrogen acts on the surge center and it suppress development.

• Mesoderm develops into granulosa cells, and migrate into the sex cords to form egg nests. Divide mitotically but soon enter meiosis • There is not testis determining factor (or no SRY gene), so the ovaries develop, there is no testosterone, so the Wolffian ducts regress. There is no AMH (anti-mullerian hormone) so the Mullerian ducts become the oviducts, uterus, cervix and part of the vagina. • The Mullerian ducts connect to the urogenital sinus and begin to fuse. Unfused is the uterine horns. • Since there is no testosterone development, it can't be converted into DHT, so the female phenotypic appearance is expressed. There is a clitoris, labia minora and majora. • Brain development: o The placenta and the follicles secrete estrogen. Estrogen binds to the protein alphaFP (which has a higher affinity for estrogen). Estrogen can't diffuse away from the protein so it can't cross the blood brain barrier. Since there are no steroids acting on the surge center, it can develop along with the tonic center.

• Luteal regression occurs when the body realizes it is not pregnant, so the CL starts secreting oxytocin. The posterior pituitary then also secretes oxytocin. The target cell of oxytocin is the uterus or uterine horn ipsilateral to the CL. The uterus then secretes prostaglandin and the CL regresses (luteolysis). • Oxytocin o Produced by the CL large luteal cells o Receptors on the uterus decline after ovulation as progesterone increases o After 10-12 days of progesterone, uterine oxytocin receptors begin to increase again o Oxytocin binds to its receptors on the uterine endometrium, PGF is released Oxytocin is a protein hormone, calcium second messenger system • The hormone binds to the cell receptor on the plasma membrane, G protein and PLP2 proteins within the membrane become associated with it and PLC from the cytoplasm becomes associated • The G protein links PLC activitating it which causes PIP2 head to break off and form IP3 into the cytoplasm, and DAG (lipid portion) remains in the membrane • The ER has a receptor for the IP3 and causes the ER to secrete calcium into the cytoplasm • Calcium activates protein kinase C (PKC) to have an influx of calcium move down its concentration gradient from the outside of the cell to the inside of the cell. PKC is associated with the plasma membrane. • The calcium activates secretory granules that contain PGF which are released through the membrane. • PGF is produced by the uterus in response to oxytocin o Progesterone from the CL stimulates production of PGF2alpha after day 15 in the cow o PGF synthesis by the uterine endometrium is release into the uterine vein and then PGF2alpha is picked up by the ovarian artery through counter current exchange and delivered back to the ovary where it causes lysis of the CL. o CL only sensitive to PGF after day 5-6 of cycle o Triggers release of oxytocin from CL to initiate positive feedback loop Ca2+ second messenger system o Protein kinase C inhibits P4 synthesis by blocking the mechanism of cholesterol to progesterone o Increase in intracellular calcium and kills luteal cells (apoptosis) • Immune response: o Macrophages and lymphocytes infiltrate the CL (luteal tissue) and increase at the time of luteolysis Phagocytose cells Cytokines are released and cause • apoptosis • Stop P4 production

• Puberty in the female: o At age of first estrus (heat), first ovulation, or age at which the female can support pregnancy o Puberty controlled by: hormonal, genetics, environment, and nutrition o Pre to puberty: Tonic center to surge and tonic center working o Pre: LH increases (and so does GnRH) and E2 increases: high levels of E2 so negative feedback and that inhibits LH and FSH: LH decreases, FSH decreases, and E2 decreases. So when E2 increases, LH decreases Minimal GnRH release FSH and LH low Minimal to no folliculargenesis (follicles are not growing) o Puberty: Increase in pulse freq and amp of GnRH release (GnRH surge) Increase in FSH and LH pulses Folliculargenesis occurs When E2 increases, LH increases too: negative feedback is decreased E2 reaches a level that stimulates positive feedback which stimulates a surge of GnRH release from the hypothalamus which causes a surge of LH and that triggers ovulation of the follicle. Progesterone is produced and the CL forms P4 takes over negative feedback and it keeps LH levels low, E2 levels low in general After some time, the CL regresses, P4 decreases and a decrease in negative feedback again and therefore GnRH and LH levels are going to rise, E2 levels increase until reach a level to trigger positive feedback and GnRH and LH surge and ovulation And the cycle repeats itself • Regulators of GnRH pulses at puberty o Development of surge center Present at very early age but become more responsive as puberty approaches o Change in the feedback sensitivity to E2 B4 puberty, the female has strong neg. feedback to e2 produced from small follicles At puberty neg feedback decreases and the hypothalamus changes by releasing more GnRH. This leads to folliculargenesis and therefore follicular E2 production. Once E2 levels are high enough the positive feedback loop occurs triggering a GnRH surge o Silent ovulation at puberty The first GnRH and LH surge is usually not associated with estrus or ovulation Cause by lack of P4 which is needed for E2 behavior and positive feedback. The brain is primed with P4

• Estrus cycle in a cow is Polyestrus and lasts for 21 days • Proestrus o Follicle enlarges o E2 increases o Vasculartity of the Female repo tract increases o Endometrial glands begin to grow o E2 levels peak • Estrus (18 hr) o Allows male to mount o E2 decreases o And LH surge and GnRH surge occurs o Ovulation 24-48 hr after LH surge, E2 decreases, follicle ovulates o Uterine motility high with contractions moving toward oviduct o Sperm transport o Cervical mucus volume increase and can be penetrated by sperm • Metestrus o E2 stays low, no follicles growing o Ovulation in cow (11 hr after end of estrus) o Corpus hemorrhagicum present, follicle tissue for CL o Uterus Contractions subsite because low E2 Endometrial glands continue to grow and become coiled. Getting ready for potential embryo from oviduct to uterus In cattle bleeding occurs o FSH increase which triggers the growth of follicles to produce E2 later. Follicle doesn't produce inhibin anymore so FSH increase • Diestrus o Majority of the time o Functional CL to secrete P4 o FSH: Increases for growth of ovulatory follicle (end of diestrus) o Uterus Secretes fluid but volume gradually decreases Contractions stop under influence of P4 CL regress at the end of this period if the female is not pregnant due to PGF release • PGF is produced by a non-pregnant uterus • Drop of P4, cycle returns to proestrus

• In cows it is a cycle manipulation: Co-Synch + CIDR • Day 0: GnRH + CIDR o GnRH induces LH and FSH for ovulation of a dominant follicle. This will eliminate the current follicular wave o CIDR: vaginal progestin, it will maintain the CL to prevent the animal from going into estrus • Day 7: pull CIDR + PGF injection o Taking the CIDR out, CL will regress o PGF also destroys the CL • Day 10: GnRH + AI o GnRH will induce LH and FSH to induce ovulation. This follicle will ovulate in the absence of a CL. The dominate follicle ovulating was induced by follicular wave that started by GnRH on day 0 or a little after.

• 28 days long • Menses, 5 days o Loss of uterine endometrium tissue: blood secretions o Recruitment of follicles and selection o FSH increases and initiates a follicular wave at 1-2 days and LH is low o FSH decreases • Follicular Phase, 9 days o As the follicle begins to grow during selection and dominance (select one large follicle), E2 is secreted by tertiary follicles. E2 has neg. feedback on the hypothalamus and anterior pituitary o Positive feedback of E2 causes the LH to rise and LH surge occurs on day 13 with ovulation on day 14 • Luteal Phase, 14 days o Dominated by CL o The CL produces E2 and P4 and causes the uterine endometrium to further proliferate. Growth and maintance in uterine endometrium o The CL is programed to last for 12-14 days and regresses if the female is not pregnant; P4 and E2 decrease and menses occur again o Luteolysis is not dependent on the uterus o Lack of h CG contributes to luteolysis because h CG is required to maintain pregnancy • Menstrual flow: endometrial PGF cause vasoconstriction and necrosis o As menses ends, the uterine endometrium begins to proliferate under the influence of E2 from growing follicles.

• =The production of fertile sperm o Hormonal regulation of the testis o Mitotic division of spermatogonia o Meiotic division of spermatocytes o Morphologic transformation of spermatids into spermatozoa • Occurs in the Sertoli cells in the seminiferous tubule • Spermatogenesis: o Every 13.5 days sperm are released o Release in stage 8 o The sperm goes though different stages Specific cellular association within a small segment of a seminiferous tubule Stages are not the same length in time 1 cycle=progression through sequence of all stages • The bull goes through about 4.5 cycle for a spermatogonia to spermatozoa • Spermatogenesis = 61 days in a bull o Spermatocytogenesis Spermatogonia (AIB) Mitotic divisions of spermatogonia • A divides and produces 2 daughter cells. One goes back into the proliferating pool to restart next generation • FSH regulates the mitotic divisions Cytoplasmic bridges form between daughter cells B divides into Primary spermatocyte (still mitosis) o Meiosis Primary spermatocyte Secondary spermatocyte Round spermatid o Most cells go through apoptosis Season, disease, trauma, heat, hormone levels (low FSH) o Spermiogenesis Morphological change: round spermatid spermatozoa 4 Phases: • 1)Golgi Phase o In a round spermatid, the golgi apparatus secrete granules to produce the acrosome o Acrosome migrates to one end of the nucleus o Centrioles migrate to the other end. Form implantation apparatus and axonene • 2)Cap Phase o Acrosome development like a cap over the nucleus o Golgi moves away from the nucleus o Flagellum elongates with microtubules • 3)Acrosomal Phase o Acrosome continues to spread around the nucleus o Manchette forms (fibers of microtubules in nuclear membrane) o Elongation of the axon of tail • 4) Maturation Phase o Machete disappears o Mitochondria migrate to the mid piece o Dense fibers form over the surface of the axon o The spermatid is now a spermatozoa o Spermiation The release of spermatozoa from sertoli cells into the lumen of the seminiferous tubule Contains a cytoplasmic droplet on its tail

• The changes within sperm that confer upon the sperm the ability to acrosome react in response to the appropriate stimulus: zona pellucida, cumulus cells, follicular fluid.--- add decapitation factors • Addition of sperm membrane components in epididymis allows fertility • Epididymal secretions and/or seminal plasma add decapicitation factors to stabilize the plasma membrane of the sperm • When the sperm enters the female tract, the decapitiation factors (proteins) are removed and undergoes the acrosome reaction (fused plasma membrane and outer acrosomal membrane). • Physical changes: o No morph changes o It's a motility change-hyperactivation (increase in c AMP and increase in power output of the flagellum) • Capacitation in the bovine: o Decapacitation factor (BSP in bovine) stabilizes plasma membrane and inhibits a premature acrosome reaction o Heparin displaces BSP proteins removes cholesterol from the plasma membrane o Removal of cholesterol into cholesterol-acceptors o Bicarbonate moves into the cell and H, protons move out of the cell. This causes an increase of pH inside the cell o Bicarbonate and the increase of pH inside the cell activates the sAC (soluble adenylate cyclase) which increases c AMP and stimulates PKA o PKA undergoes many mechanism and the result of these mechanism is Protein Tyrosine Phosphorylation. This causes a changes in the activity of protein. o Calcium flows into the cell due to inactivation of the plasma membrane. This fills the acrosomal calcium store in the cytoplasm. This leads to a change in activity of cytoplasmic enzymes o Variation in plasma membrane calcium ATPase begins to shut down.

• The sperm binds with ZP3 on the zona pellucida. The acrosome reaction occurs: o The plasma membrane of the sperm and outer acrosomal membrane fuse together and release proteins: acrosin and hyaluronidase. Acrosin digrests through the zona pellucia Hyaluronidase digests through the cumulus cells o The acrosomal matrix binds to ZP2 and the sperm digests through it. The sperm needs to be motile. o The sperm ends up in the perivitelline space o It binds to a site on the oocyte. This triggers a calcium release in the oocyte: Soluble Phospholipase C (PLC) is release from the sperm. This cleaves PIP2 in the plasma membrante which causes IP3 to bind to a receptor on the ER to release calcium. Calcium inactives MPF activity in the oocyte • Then metaphase spindle, anaphapse, telophase, and the 2nd polar body then chromatin (DNA + protein) remodels and condenses and then decondenses and swells and forms a pronuclei The sperm nucleus enters the cytoplasm of the oocyte and then disulfide bonds are broken down by glutathione. This alos the sperm nucleus to decondese o Cortical granules move to the plasma membrane and fuse and then release contents into the perivitelline space This modifies the zona pellucida to prevent another sperm from inducing the acrosome reaction and prevents polyspermy (many sperm from penetrating the oocyte) o The pronucli of the sperm and the oocyte fuse and fertilization occurs (syngamy)

• Imprinting o maternal and paternal genomes are not equivalent and need both to form a viable embryo maternal and paternal genomes are expressed differently in the embryo and fetus. Same genes, but different expression Androgenote: 2 sperm pronucleus-fails during embryo development Gynogenote: 2 egg pronucelus-fails midpregnancy o In the embryo, there is a 50% chance if a cell has maternal or paternal X inactivation o In the placenta, the paternal X is inactivated • Maternal gene expression o Proteins are made from mRNA during oocyte development and maturation o During these stages of embryo development, no new mRNA is made (no transcription) o Many cell regulatory functions are accomplished by phosphorylation of proteins (post translational modification) that were made during oocyte growth or after fertilization (translational of pre-existing mRNA) • Precompaction cleavage o cell size decreases as embryo divides blastomeres get smaller but zona pellucida remains the same size o inner cell mass and trophectoderm development asynchronous cleavage • some blastomeres divide faster and smaller blastomeres go into the insdie of the embro • the blastomeres on the outside of the mass (and slower dividing) get formed in the trophectoderm and part of the placenta • Embryonic gene expression o Transcription and translation activity resumes o Transition is initiated during a pause in G1 The embryo runs out of key factors needed to progress in the cell cycle So it triggers new transcription of the embryonic genome for a new protein • Movement into the uterus o Around day 4 after 8 cell stage o This is caused by a change in E2 to P4 o Open utrerotubual junction, the embryo goes down through the isthmus and out into the uterine horns • Early Pregnancy factor o 24-72 hr after fertilization o Sensitize the uterus for implantation o Basis for early pregnancy kit in cattle • Morula to Blastocyt o Polarization Initial microvilli surround most of the blastomere (polar) surrounding non-polar blastomeres that don't have microvilli Gap and tight junctions form between blastomeres where microvilli are not present • Tight junctions between outer cells o For blastocoel formation o Cells involved give rise to the trophectoderm • Gap junctions between inter cells and inter cells and inner cells and outer cells o Communicate and give rise to the inner cell mass Division of polarized blastomere • 2 polarized blastomere with vertical division • 1 polar and 1 non-polar blastomer if divison is horizontal or oblique Divison of a non-polar blastomere 2 non-polar blastomere o Compaction at fertilization membranes are close and begin to flatten differentiation event controlled by the embryonic genome microfilaments and tubules interact with plasma membrane to cause this to occur o Blastocyst formation Tight junctions among outside cells that give rise to trophectoderm • Tight junctions prevent ion and water movement between cells o Na/K pumps move Na into center of embryo Leads to an increase in osmotic pressure between cells in the center of the embryo Water diffuses through cells to dilute the ion concentration Leads to the blastocoel forming (cavity): as it gets larger, it pushes cells to one side (inner cell mass) and then there is a single layer of cells around the cavity (trophectoderm that forms the placenta) The blastocoel is not dependent on • Cell number, number of cell divisions • IT IS controlled by the embryonic genome o Blastocyst hatching Enzymatic digestion of zona pellucida from embryo and/or uterus Increase of size of blastocyte due to water pumping and the zona ruptures Occurs on day 9-10 in cattle

o Luteal Regression o Luteolytic: PGF: cow, sheep, mare o Lack of CL support: human o Luteal Maintenance o Antiluteolytic: prevent effects of PGF, PGF release o Luteotrophic: enhance survial of CL, support CL o Pig o Estradiol Antiluteolytic E2 produced causes PGF2a to be redirected into the circulatory system and into the uterine lumen. It prevents from reaching CL Requires 2 embryos per uterine horn o Critical days 11-12 o Human o HCG Luteotrophic Embryo produces it o Critical days 8-12 o Recognition of pregnancy is important because if the CL is not maintained the pregnancy will be lost and the animal will begin to cycle again.