Exam 3 - Repro

follicular phase

• proestrus

• estrus

follicular phase goals

• allow a follicle to reach mature stage (ovulation)

• display behavioral estrus

_____ is the shortest part of the follicular phase

estrus

tonic center

• like a dripping faucet of GnRH

  • more drips during puberty onset + puberty

  • less drips during luteal phase

• specific nuclei

  • arcuate nucleus

  • ventromedial nucleus

• following luteolysis - progesterone decreases

• decrease in P4 =

  • decreases negative feedback on tonic GnRH

  • increased the pulse frequency

• approx. 5 pg/mL pulse of GnRH every 1-2 hrs

• stimulates basal LH

  • 5 pg/mL

  • needed by the theca cells for androgen synthesis

• increased release in FSH

  • needed by granulosa cells for aromatization of androgen to estrogen

• both lead to increased estrogen production and follicle growth and maturation

surge center

• akin is a gushing faucet

• differential effect of estrogen

• specific nuclei (3)

  • preoptic nucleus

  • suprachiasmatic nucleus

  • anterior hypothalamic

• during luteal phase

  • high P4

  • low E2

    • has a negative feedback on the surge center (hypothalamus)

    • no GnRH surge released

    • FSH/LH stores up the gonadotrope cells

• during follicular phase

  • low P4

  • high E2

    • has a positive feedback on surge center (hypothalamus)

    • threshold E2 indues synchronized action potential firing of GnRH neurons

• GnRH neurons dumps GnRH into the portal plexus

• 20-100+ pg/mL

• travels to gonadotropic cells in the anterior pituitary gland

• stored up LH is dumped into the blood

• 100-150 ng/mL LH

summary

• CL regresses — decreases P4

• increase FSH and LH — increases follicle growth with increase E2

• as follicles grow inhibin is increased leading to decreased FSH slowing things down

• however, E2 continues to rising hitting a threshold

• this causes GnRH release causing LH surge

• ovulation

• when threshold E2 is reached

• LH surge leading to ovulation

• decreased E2

• CL forms leading to increased P4

follicle growth and atresia

• follicle growth occurs throughout the estrous cycle

• growth depends on FSH and LH in the blood

• final growth stage termed the “follicular phase”

antral follicles

• categorize follicles based on size and dependent on species

recruitment

• a small subset of primary follicles are selected and initiate growth

• the process by which the follicle are selected is not well defined

• we do know certain factors that appear to play a role

• believed to be follicles with the right amount of growth factor receptors start and then inhibit those around them

• therefore, they continue to grow while inhibiting those around them

• others will often regress via atresia

2 recruitment take aways

1.) follicles entering the recruited pool secrete AMH inhibiting those around them

2.) the oocyte orchestrates follicle progression

dominance

• surviving follicles will continue to grow

• this increases the level of E2

• start to synthesize and secrete inhibin

• leading to decreased FSH

atresia

• hormone controlled apoptosis

• fate of the vast majority of follicles (including most antral)

• monotocous species - all but one follicle will regress

• polyocous species - system uses E2/inhibin ratio to determine how many will survive

which is the one (dominance/atresia)?

• selective advantage is not well understood

• could be timing

  • gonadotropic support

  • growth factors

  • resist inhibition factors

  • receptors

  • blood supply

  • combination

• ends with the selected follicle expressing more LH receptors

follicle waves

• in most species a group or “wave” of pre-antral follicles are selected

• this group is selected during the pre-ovulatory FSH surge

• continue during periods of anuetrus

  • prior to puberty

  • during pregnancy

  • during other periods of anestrus

  • stops only with reproductive senescnece

follicle waves in cattle

• first wave

  • identifiable during metestrus

• second wave

  • during mid-diestrus (10-11mm)

    • but CL progesterone

    • limits follicle growth

    • follicles regress and become atretic

• third wave

  • CL is regressing therefore P4 is decreasing

  • FSH and LH increase

  • follicle continues maturing

  • dominant follicle producing more E2

    • estrus behavior

    • LH surge

  • ovulation and mating

wave number

• in cattle the number of waves per cycle varies

• either 2,3,4

• varies amoung cows

hormone function review

• FSH

  • stimulates follicles to start growing

  • stimulates estrogen production by granulosa cells

• LH

  • stimulates steroid production by theca internal cells

  • initiates ovulation and lutenization

    • signal of ovulation

    • GnRH surge = LH surge

steroid production

• 2-cell theory

• theca and granulosa

  • produces estradiol

estradiol

• diffuses through the basement membrane to capillaries

• distributed throughout the body

• binds to receptors:

  • hypothalamus (GnRH)

    • surge center specifically

  • reproductive tract

    • endometrial layer mainly

  • brain (behavior)

    • females level of receptivity

• causing immune cells totcome into the uterus with the increase of E2 to protect the uterus

ovulation and lutenization

• oocyte must exit the follicle (go through these layers first)

  • granulosa

  • basement membrane

  • theca internal and external

  • intracellular connective tissue

  • tunica albuginea

  • peritoneum/surface epithelium

LH surge

• induces cascade of events

• hypermia (increased blood flow) to the ovary and follicle(s)

• breadown of tissues forming the follicle wall

• contraction of the ovarian smooth muscle

hypermia

increased blood flow to the ovary/follicle

• increase in PGE2 a histamine (vasodilation)

• increase vasodilation, angiogenesis

• increase blood flow to ovary and pre-ovulatory follicle

  • resulting in antrum size increasing

  • increased pressure

wall breakdown

• blocks enzyme (17 alpha-hydroxylase) in theca internal that converts progesterone towards testosterone

• steroid synthesis stops at progesterone

  • estrogen decreasing

• local progesterone promotes the synthesis of collagenase

• collagenase breaks down the collagen in the follicle wall

• apex of follicle weakens and pushes out to form “stigma”

wall contraction

• increased local PGF2 alpha production

• PGF2 alpha increases contraction of the “myoid” (smooth muscle) components of the ovary

* if you block smooth muscle contraction via inhibitors ovulation is blocked

lysosomes

• cleaning crew + recycle debreis when the egg ovulates and ruptures the walls

• lysosomes within granulosa rupture

• enzymes further disrupt connective tissue

• collagenase

  • due to local P4 production

spontaneous ovualation

• cyclic changes based on predictable hormonal event

  • ex.) cow, ewe, mare, pig

induced ovulation

• termed “reflex” ovulatory

• requires the tactile stimulation of the vagina/cervix

• LH amplitude increases with increased mating

  • 50% of cats ovulate with only 1 mating

  • increased mating increases % ovulating

• ex.) cat, rabbit, ferret, llama

oocyte maturation

oocytes must develop from immature primordial germ cells to mature ovulated oocytes in order to be functional

oocyte maturation phases

• prenatal mitotic divisions

  • PGC formations

• nuclear arrest in dictyotene (prolonged diplotene) of prophase I

• cytoplasmic growth (cytoplasmic reticulum)

• resumption of meiosis at ovulation

mitotic divisions

• occur prior to birth

• generally held that born with all ova (no more divisions)

• 100,000’s to millions depending on species

• last mitotic division is oogonia to primary oocyte

meiosis

• primary oocyte develops and enters into meiosis

• meiosis does not complete but stops at dictyotene stage (prophase I)

  • where the eggs are

• resulting primary oocyte can remain dormant (years)

• unless stimulated in which it enters the growing pool

meiosis I

first division of meiosis

• prophase I: each chromosome duplicates (sister chromatids) and remains closely associated — crossing over can occur during the latter part of this stage

• metaphase I: homologous chromosomes align at the equatorial plate

• anaphase I: homologous pairs separate with sister chromatids remaining together (different from mitosis and meiosis II)

• telophase I: Two daughter cells are formed with each daughter containing only one chromosome of the homologous pair

meiosis II (no replication)

second division of meiosis: gamete formation

• prophase II: DNA does not replicate

• metaphase II: chromosomes align at the equatorial plate

• anaphase II: centromeres divide and sister chromatids migrate separately to each pole

• telophase II: cell division is complete - 4 haploid daughter cells are obtained

meiosis - genetic variation

• during normal cell growth, mitosis produces daughter cells identical to parent cell (2n to 2n)

• meiosis results in genetic variation by shuffling of maternal and paternal chromosomes and crossing over

• no daughter cells formed during meiosis are genetically identical to either mother or father

• during sexual reproduction, fusion of the unique haploid gametes produces truly unique offspring

independent assortment

• number of combinations = 2 to the power of n

  • where n = the number of chromosomes

• humans: 23 chromosomes = >8 million

• cattle: 60 chromosomes = >1 quintillionth

2 options (random)

• maternal copy left/paternal copy right

• paternal copy left/maternal copy right

crossing over - prophase I

• chiasmata - occur in synapsis

• exchange of genetic material between non-sister chromatids

• crossing over produces recombinant chromosomes

chiasmata

site of crossing over

end result of meiosis = ________ gametes + _______ variation

haploid, increased

oocyte growth

• primary oocyte accumulates cytoplasm

• secondary oocytes develop zona pellucida

  • the substrate provided by granulosa cells (some species)

  • oocyte synthesizes and secretes

  • aids in single sperm fertilization

polar bodies

• nucleus divides and half chromosomes extruded into small membrane-bound polar body

• polar body has little cytoplasm and remains dormant

• now have a mature oocyte

concluding the follicular phase

• the process of an immature primordial oocyte progressing to a mature ovulated oocyte

• the process of follicular progression from primordial to ovulated mature follicle

summary of oocyte maturation

• progesterone break removed

• high estrogen environment

• allows increased frequency of GnRH (hypothalamus) increased LH/FSH (anterior pituitary)

• follicles grow and fight for dominance (inhibin leads to decreased FSH)

• estrogen reaches threshold - behavioral estrus, LH surge

• ovulation - released inhibition of meiosis

luteal phase

interval from ovulation to luteolysis

• metestrus and diestrus

• dominant hormone

• progesterone

major events in the luteal phase

• formation of the CL

• synthesis and release of P4

• luteolysis (if pregnancy does not occur)

ovulation and CL formation

• LH surge

  • angiogenesis occurs (localized edema)

  • increased follicle pressure

  • stimulates synthesis and release of enzymes

  • initiates breakdown of basement membrane (outer)

  • stimulates collagenase production (tunica albuginea breakdown)

  • smooth muscle contraction

  • follicle wall collapses

  • folds into itself allowing cell types to mix

CL cell types

• large luteal cells

• small luteal cells

large luteal cells

• used to be granulosa cells

• increases in size as luteniation occurs (hypertrophy)

• relatively large (20-40 microns)

• produce the bulk of P4

• produces oxytocin and relaxin (late pregnancy)

small luteal cells

• former theca cells

• increase in number (hyperplasia) - proliferative

• relatively small (<20 microns)

• irregular shape

• mostly lipid droplets

• less P4 ad LH receptors fine tune P4 secretions

CL formation/regression

during ovulation, blood vessels rupture in theca externa

CL formation/regression - day 1-3

• blood appearance

• termed corpus hemorrhagium (CH)

• temporary structure immediately after ovulation

• similar to a blood clot

CL formation/regression - day 3-5

• CH grows (hypertrophy and plasia)

• loses hemorrhagic color (blood is absorbed)

• some retain central blood clot

• outer appearance is that of a CL

CL formation/regression - day 5 (mid-cycle)

• increase in functional tissue mass

• can palpate or ultrasound

• P4 is produced per unit of tissue

• so growth increases P4 concentration

CL size

mature size depends on:

• number of cells

• size of cells

• degrees of vascularization

• to maintain pregnancy requires a minimum amount of P4

• if P4 calls to low pregnancy will end in abortion

following ovulation the high progesterone environment has a ____ effect on the hypothalamus

negative

FSH and LH play different roles during folliculogenesis. It can be said that _____ is important early during follicular development and _____ at the late stages

FSH, LH

ovulation is signaled by the LH surge. What events is/are important steps for ovulation?

• increased blood flow

• increased collagenase production

• contraction of smooth muscle

ovulation can be induced by injecting ________

GnRH

meiosis resumption occurs largely due to _______

the breakdown of gap junctions

progesterone synthesis requires ______ and _______

LH, cholesterol

the CL is caused to luteolyse following what signal from the endometrium

prostaglandin F2 alpha

inhibin directly causes down-regulation of FSH? (T/F)

true

the follicular phase is stronger than the luteal phase? (T/F)

true

follicular growth occurs even during anestrus? (T/F)

true

induced ovulation is an example of a neural reflex? (T/F)

false

the end result of oocyte maturation (completion of meiosis) following ovulation is the oocyte having a ________ genotype

haploid

progesterone synthesis requirements

• cholesterol substrate

• tonic (basal) LH simulation

  • negative feedback on the hypothalamus

luteolysis

• if not pregnant — demise/destruction of the CL

• signals the end of the luteal phase

• occurs over 1-3 days

• irreversible degeneration of steroidogenic tissue

luteolysis results

rapid P4 decline

luteolysis is controlled by…

• oxytocin from the CL itself

• PGF2 alpha from the uterine endometrium

• positive (+) cascade of interactions between oxytocin and PGF

local control of luteolysis

long known if uterus is removed (hyterectomy) CL is retained similar to pregnancy

partial hysterctomy

• removal of contralateral horn (opposite CL) and CL regresses normally

• remove ipsilateral horn (same side as CL) and CL retained as pregnant

control of luteolysis - what is happening

• local same side control of uterus over CL regression

• product from uterus getting to ovary on the side but not the other

counter current exchange

• scientists discovered a counter-current exchange mechanism

• hormone in area of high concentration diffusing to low

• ovarian artery winds around the utero-ovarian vein carrying blood away from the uterus

• PGF is in high concentration leaving the uterus

• transfers to ovary

• PGF enters the ovarian artery without going through the lungs

  NOT PRESENT IN MARES

counter current exchange - sow

• PGF metabolized slower

• only 40% cleared in one pass through the lungs

counter current exchange - mares

• PGF metabolized even slower

• and mare CL is much more sensitive to PGF

• therefore no need for counter current exchange system

response of CL to PGF

• early metestrus

  • CL has no PGF receptors yet

  • no response to exogenous pGF

• PGF receptors synthesized and appear

  • in cow and ewe: days 3-5

  • in sow: days 12-14

• receptors must be in place for commercial PGF products to work

role of oxytocin

• endometrium synthesis oxytocin receptors

• each release of oxytocin is followed by the release of PGF from the uterus

• large luteal cells also synthesize oxytocin and store in granules

• as CL regresses oxytocin granules dumped into circulation

• increase pulse frequency of PGF surges

PGF/oxytocin cascade

• luteal cells

  • synthesize PGF receptors

  • the greatest number on large luteal cells (majority of P4)

• endometrial cells

  • synthesize oxytocin receptors (~day 14)

early luteal phase

• minimal P4 exposure

  • P4 required to synthesize and time release of PGF

• BUT P4 blocks the synthesis of oxytocin receptors

• few oxytocin receptors

• few PGF pulses from the uterus

PGF/oxytocin cascade - late luteal phase (if not pregnant)

• PGF pulse frequency increases as P4 increases

• P4 loses its ability to block the synthesis of oxytocin receptors

• increased oxytocin receptors

• small PGF pulses cause the release of oxytocin from CL

whole cascade

• oxytocin goes from CL to the uterus

  • causes more PGF to be released

• positive feedback mechanism

• more PGF released = more oxytocin

• leads to CL regression

effect of PGF on CL

• disruption of CL’s local circulatory system

• vasoconstriction of arterioles

• capillary beds in CL begin to degenerate

• this leads to relative ischema (reduced blood flow) to cells

• apoptosis

apoptosis

• PGF binds to membrane receptors of large luteal cells

• activates protein kinase-C which inhibits P4 synthesis

• PGF-receptor complex opens Ca++ channels

• Ca++ influx initiates cell death because there is no way to remove from the cell

3 effects of PGF2 alpha

1.) vasoconstriction causes relatie ischemia

2.) activates protein kinase-C (PK-C), inhibiting progesterone synthesis

3.) PGF causes calcium to enter the cells leading to apoptosis

structural regression

• macrophages and lymphocytes

  • produce cytokines

  • initiate immune response

  • decreased cell function

  • decreased structural integrity

• macrophages phagocytize dying luteal cells

final stages of luteal phase

• the luteal cells disappear

• the reminaing connective tissue forms small solid fibrous scar tissue

  • termed corpus albicans (CA)

primate CL

• secrete E2 too:

  • further suppress follicle growth

  • new follicles must start smaller

  • longer follicular phase (~14 days)

menstrual cycle of primates

• no defined period of sexual receptivity (no estrus)

• period of endometrial sloughing (menstruation)

• cycle starts with observable menses (day 0)

• follicular phase (~14 days)

lack of cycling

• amenorrhea

• menopause

amenorrhea (recall no estrus)

• lack of cyclicity in otherwise reproductively normal females

• generally stress induced

menopause

• lack of cyclicity in aged females

• ovaries are depleated

removal of uterus - primates

• shows lack of uterine signaling during luteolysis

• ex.) if uterus is removed the cycling still occurs

controlling the cycling

• our understanding of the reproductive cycle is a powerful tool

• allows us technologies such as AI

protocol downsides

• longer time

• more expensive

  • CIDER is cheaper and does the samne

exogenous options

• CIDR (progesterone) - mimicking CL

• PGF2 alphaa - luteolysis (removes CL)

• GnRH - leads to LH surge (ovualation)

stages of spermatogenesis

• spermatocytogenesis

• spermiogenesis

spermatocytogenesis

spermatogonia to secondary spermatocyte

• cell differentiation

• mitosis

• meiosis

spermiogenesis

spermatid to sperm cell

• structural and conformational changes from spermatid to spermatozoa

major difference in males and females with gonadal development/germ cells

• during gonadal development female germ cells enter meiosis (pause at prophase I)

• male germ cells remain mitotic until puberty

• this is because females can metabolize retinoic acid better than males

summary of gonadal development/retinoic acid

• retinoic acid (RA) induces meiosis by up-regulating meiosis factor Stra8

• Stra8 expression leads to SYCP3 expression and meiosis

• in male Cyp26b1 causes the metabolism of RA presenting meiotic entry until puberty

• therefore, male germ cells remain mitotic while females enter meiosis