ANS 220 Exam 3

How is the estrous cycle divided into 2? And sections within them?

Based on ovarian structures:

1) Follicular Phase

2) Luteal Phase

Based on hormone profiles:

1) Proestrus

2) Estrus

3) Metestrus

4) Diestrus

1 and 2 are in follicular phase with estrogen from follicle

3 and 4 are in luteal phase with progesterone from CL

Dominant structure and hormone during follicular phase?

Dom structure: preovulatory follicle

Dom hormone: estrogen

What starts follicular phase? What ends it?

Regression of corpora luteum (CL)

Ovulation

What percent of cycle is follicular phase?

25-33%

Draw graph of cycle with follicular area boxed in

Dominant structure and hormone during luteal phase?

Dom structure: corpus luteum

Dom hormone: progesterone

What starts luteal phase? What ends it?

Starts with ovulation

Ends with luteolysis

What percent of cycle is luteal phase?

66-75%

Draw graph of cycle with luteal area boxed in

Main goals of follicular vs luteal phase

Growing follicle - preparing to ovulate

Growing CL - preparing for pregnancy

Which phase is target for understanding?

Luteal b/c longest

4 estrous phases graph with E2 and P4 drawn on

What happens to hormones during proestrus? Draw graph. What is proestrus called?

Increasing E2 - follicles growing

Decreasing P4 - CL regressing

Transition stage

What happens to hormones during estrus? Draw graph. What is estrus called? What is it the period for? LH and FSH? Ovulation?

Peak E2 - max follicle size

Lowest P4

Called heat b/c body temp increases

Period of sexual receptivity

LH and FSH surges due to increased feedback from E2

Ovulation during estrus

-Mare and sow

-Long estrus time

What happens to hormones during metestrus? Draw graph. What is metestrus called? Ovulation?

Increasing P4 - formation of CL following ovulation

Low E2 and LH

Transition stage

Ovulation in metestrus

-Cow and ewe

-Short estrus time (hours); while LH surge has been initiated it takes time to activate system

What happens to hormones during diestrus? Draw graph. LH and FSH? How long is diestrus?

Max P4 production - CL is dominant structure

Lowest E2 though follicles continue to grow in waves and undergo atresia in domestic livestock so small increases

Tonic secretions of LH and FSH but not surge secretions b/c P4 blocks it

Longest period of cycle

Species Comparison Estrus Length vs Ovulation

Cattle, Sheep, Goats: short estrus, ovulate in metestrus

Swine, Horses: long estrus; ovulate in estrus

Review types of cycles and ovulators

Cycles:

Polyestrus: cats, swine

Monoestrus: cows, dogs

Seasonally polyestrus: horses, sheep, goats

Ovulators:

Spontaneous: cows, pigs, sheep, horses

Induced: cat, rabbit

Estrous and Menstrual Cycles Difference. Time difference. Ovulation time. What animals is menstrual?

Cycle based on what we see

-Menses (sloughing of endometrial lining) vs estrus (sexual receptivity)

Menstrual: 50 follicular, 50 luteal

Estrous: 25 follicular, 75 luteal

Ovulation on day 14 of 28

Primates

Does menses have clear sexual receptivity period?

No

Menstrual cycle sequence events (6)

1) Menstruation

2) Follicular Growth

3) Ovulation

4) Lutenization - formation of CL

5) Endometrial growth - thickening for embryo

6) Luteolysis - decreases P4 so support for endometrial lining leaves so menses

1-3 are follicular phase and 4-6 are luteal phase

Estrous vs menstrual cycle timeline

Estrous vs menstrual cycle graphs

Different estrogen graph curves?

Ruminant: estrogen peak prior to ov

Mare or sow: estrogen higher at ov

What does FSH curve look like? What does it cause?

Tonic release but blip at ov

Increases E2

What does PGF2a do? Where does it come from?

Increase amplitude and frequency before ov to kill CL

Uterine endometrium

Full hormone graph

Graph description of order

↑ FSH → ↑ E2 → + feedback loop → LH surge → ovulation → CL forms → ↑ P4 → not pregnant→ ↑ PGF2a → ↓ P4 → beginning

Parts of follicle and process

Oocyte - all follicles have one

-Oogenesis - development of oocyte

Theca cells: produce T that goes into granulosa

Granulosa cells: makes E2 from T

-Cumulus surrounds oocyte

-Mural surrounds other

Follicular fluid

-Nutrients

-Cell communication

Parts of oocyte

Zona pellucida: thick hardened shell for protection and assists fertilization as sperm cells bind to it

Perivitelline space: air sac buffer btwn two layers

Vitelline/plasma membrane: surface layer of oocyte

Cytoplasm (yolk): contains proteins, enzymes, nutrients, needed for survival of zygote

-Has all needed for mitosis and meiosis

Germinal vesicle: nucleus containing genetic info

What is final stage of embryo development

Zygote

Describe oogenesis and some stages

Oogenesis: formation and development of ovum (egg)/gamete

Primordial germ cells are start

-Fixed # encapsulated in tunica albuginea + loss from medulla region

Oogonium: primordial cell which develops into oocyte

-After mitosis

-Earliest stage

Oocyte: early, not yet fully developed ovum

-Primary oocyte: most prevalent; frozen in meiosis 1

-Secondary oocyte: if activated

Ovum: egg; capable of developing into new individual

What are primary oocytes formed from?

Mitotic divisions of oogonia

What does primary oocytes enter and why?

Enter meiosis 1

Become dormant/frozen to protect them

-Done by surrounding follicular cells

When are max # of primary oocytes formed? What happens once max number is attained?

Mid to late fetal life

Atresia (programmed cell death) or natural degeneration begins and continues for life

Flow map of follicle and oocyte

How many primary oocytes stimulated to develop? And when?

Only a select few and after puberty

Oocyte number in humans at birth then puberty

1-2 mil

300,000

Human oocyte wastage - fetal vs adult

Loss:

99.98% - fetal

97.92% - adult

Primordial follicle: cell type, state, cues to grow, when seen

1° oocyte

Single layer of squamous epithelial

Resting state

Growth factors in ovary: transforming growth factor beta - AMH after puberty

Seen in fetal life and into puberty

Primary follicle: cell type, changes

1° oocyte

Single layer but expand into cuboidal/columnar

Zona pellucida forms

Secondary follicle: cell type: changes

1° oocyte

Multiple granulosa cell layers and theca layer forms (division of cells)

Pre-antral (no follicular fluid)

Describe folliculogenesis

Classification and regulation of follicle growth

Growth and development of 3° follicle with 1° oocyte

What cells provide E2 in follicle?

Mural granulosa

What cells provide meiotic inhibition to oocyte?

Cumulus granulosa

Granulosa and theca: where are they from; responsive to what; convert what to what

Granulosa (mural)

-From cortex

-Responds to FSH

-Convert T to E2

Theca

-From stroma

-Responds to LH

-Convert cholesterol to T

Explain 2-cell-2-gonadotropin model

In Theca/Leydig:

AP → LH → LH receptor → activates g-proteins → adenylate cyclase converts ATP to cAMP → increase in cAMP → activates protein kinases → cholesterol moved into mitochondria → steroidogenesis → testosterone

In Granulosa/Sertoli:

AP → FSH → FSH receptor → activates g-proteins → adenylate cyclase converts ATP to cAMP → increase in cAMP → activates protein kinases → protein kinases increase aromatase (CYP19A1) → test converted to estrogen → estradiol

*Note this is paracrine b/c some of the testosterone that will be converted to estrogen came from theca/leydig cell + test can pass freely through cell membrane b/c lipophilic to become substrate

What transition occurs as follicles grow in regards to FSH and LH

More dependent on FSH to LH as follicle grows

Follicular fluid (antral follicle) components

Hormones

-Steroids (E2, T, P4)

-Gonadotropins (LH, FSH)

-Prostaglandins

Proteins

Enzymes

Carbs

Follicular fluid (antral follicle) function

Supports follicle growth

Aids in oocyte development and health

Mediates granulosa cell functions

Cells live in hypoxic environ b/c no blood supply so follicular fluid keeps them alive

What are the follicular growth stages and what happens in each?

Primordial → 1° → 2° → ovarian follicular pool → 3° →

1) Recruitment: increase number of follicles and low E2 → atresia of many →

2) Selection: decrease number of follicles, increase follicle size, increase E2 b/c granulosa cells increase with follicle size →

3) Dominance: decrease number of follicles until 1 dom prev-ovulatory follicle that is at max size (monovulatory species), high E2

What is follicular growth driven by

LH and FSH support

How long does follicular growth take

45-60 days

Early astral (tertiary)

1° oocyte

Oocyte reaches max size

Granulosa cells expand so more E2

Antrum forms

Theca layer more prominent so more T

-Interna

-Externa

Graafian follicle (preovulatory follicle)

1° oocyte

Last stage of tertiary follicle

Largest domestic follicle

Max # of granulosa cells + follicular fluid

How do follicles grow?

In waves and numbers

What is follicular development regulated by

Gonadotropins (FSH + LH)

Growth factors

Activin: activates FSH from AP from small follicles granulosa

Inhibin: inhibits FSH from AP from large follicles granulosa

Follicle fates

Ovulation: needs low P4

Atresia: follicular death; fate of most

Cysts: diseases causes them not to ovulate or die; produce low E2 +P4

->25 mm in cattle

-Often in high-producing dairy cattle where hormonal regulation from hyp-pit doesn’t always get to follicle

What is PCOS? Affects how many women? What is it? Strong association with? Stuck at what?

Polycystic ovarian syndrome

5-10%

Continuous exposure to E2 blocks LH surge and increases thickness of uterine endometrium

Strong association with obesity

Doesn’t get past small recruitment stage

What happens at dominant stage in litter bearing species

Increased number recruited and selected so co-dom follicles

What does P4 need to be at for dom follicle ovulate

P4 low → ovulate

P4 high → atresia

Different waves of follicular growth. And signals

Ovulatory vs non-ovulatory

Dominant (dom, pre-ov follicle) vs subordinant (recruited + selected follicles)

Signals: LH, FSH, Inhibin (from dom follicle)

Waves description

Non-ov wave when P4 is high so atresia of dom follicle

Ov wave when P4 is lowered so removes inhibition so surge center can be activated by E2 from large dom follicle

Signals for follicle growth

FSH

-Granulosa cell mitosis

-Increases LH receptors

-Steroidogeneis: increase E2

-Effects preantral and antral follicles

LH

-Steroidogeneis: increase T

Effect antral follicles

Surge starts ov

Resumption of meiosis in oocyte

FSH increases LH receptors on granulosa cells

How many follicular waves? Cows? Which is more fertile

1-6

2-3 average in cows

-3 more fertile b/c less P4 exposure in same time frame

What inhibits follicle growth

Presence of dom follicle: large so takes up space + prevents others from growing

Inadequate production of gonadotropins b/c consumed by dom b/c so big

Steroids from dom: E2 and androgens

Inhibin from dom: inhibits follicle growth by decreasing FSH

How to increase number of ova in cow

FSH to keep # recruited follicles high

Two ovulation stimuli

LH surge whether induced or spontaneous

FSH: don’t know why but it surges at ov

Changes in follicle from LH surge and changes in oocyte

What phase do female reproductive scientists focus on

Luteal phase b/c long

What is characteristic of luteal phase? How long is luteal? How does it end?

CL formation

-Lutenization (in metestrus)

P4 production: (-) feedback

66-75%

Ends with luteolysis if not preg

What is lutenization? What is it called? Triggered by what?

Process that turns granulosa and theca cells into luteal cells

Terminal differentiation so can’t switch back

Triggered by LH surge

Describe what happens to follicle after ov

Ovary tissue damaged after ov with ruptured blood vessels to corpus hemorrahgicum forms (blood clot early in metestrus that lasts hours to days)

Then corpus luteum forms and has new blood vessels for more nutrients to come in and P4 to go out

-Heterogenous population of cells meaning small and large luteal cells

Functional capability of CL (P4 produced). 2 components. And match granulosa and theca to slc and llc

Number of luteal cells
-Large cells undergo hypertrophy (3 fold increase) - granulosa

-Small cells undergo hyperplasia (5 fold increase) - theca

Vascularization of CL

-Initiated by angiogenic factors from follicle (luteotropins)

-Vascularity effects CL steroid synthesis and delivery of hormones (more nutrients)

What is insufficient CL function

Failure to maintain luteal phase and/or preg

Changes in steroid production before LH surge and after

Before LH surge: E2 is primarily produced - from granulosa cell

-Lacks aromatase to convert T to E2 in theca cell

After LH surge: P4 is primarily produced - from granulosa and theca cells

-Upregulation of P450 and 3BHSD so SLC and LLC can produce tons of P4

Sows and primates and mares produce E2 and P4 from luteal cells

Action of progesterone order

1) Reduces GnRH pulse amplitude and frequency

2) Stops pre-ov LH surge - basis of many hormonal contraceptions

3) Prevents behavioral estrus

4) Reduces myometrial tone to relax uterus

5) Stimulates endometrial gland development and secretion

6) Stimulates alveolar development in mammary gland

-If long term P4 exposure from gestation

What are the luteotropins for each species

What would LH antibodies do?

Antibodies bind to LH + prevent LH from binding to receptor so P4 decreases to prevent ov and pregnancy

Vaccine for LH in wild mares

-Initial + booster lasts 5 years

Difference in P4 profiles

Gradual increase in sow

How is progesterone used as diagnostic tool

ELISA assay: recommend sampling 3 times over 21 days

Radioimmunoassay or standard assay: serum concentration of 1ng/ml or higher =cycling

Give different milk assay examples and meanings. And graph

What is needed for her to be cycling

P4 change

What initiates luteolysis/is main luteolytic agent

Prostaglandin F2a secreted by endometrium

Describe PF2a blood flow

Cross current exchange with high to low concentration

Uterine vein (from uterus to heart and body)

Uterine-ovarian artery (heart to ovary+uterus)

Artery wraps around vein

PGF2a in vein moves into artery so it isn’t deactivated in lungs

-In ruminants and swine one pass through lungs can deactivate 90% of PGF2a

Effect of hysterectomy on CL length and its regression. What was found out?

Found that uterus produced something that regressed CL (except in primate/woman where it regressed even without uterus)

Effect of 4 uterectomy types on CL regression. Found what?

Found that PGF2a blood flow is important in regressing CL

How is luteolysis controlled in primate? CL lifespan?

Doesn’t require uterus

CL lifespan in human is 12-14 days unless preg

In absence of preg, CL produces its own PGF2a

Does PGF2a always induce luteolysis? What days?

Works:

Cattle and sheep: Days 7-17 (10 d effectiveness window)

Swine: Days 12-17 (5 d effectiveness window)

Doesn’t work:

Cattle and sheep: Days 1-6/7 (7 used as safety net)

Swine: Days 1-12

*Because acquisition of luteolytic capacity

-CL has to become sensitive to PGF2a

Doesn’t work:

Days 17-21 b/c already undergoing luteolysis so PGF2a has no effect

2 luteolysis stages

1) Functional regression

-CL can’t make and secrete P4

2) Structural regression

Luteolysis in depth steps

Decreased blood flow due to vasoconstriction from PGF2a

Cellular response

-P4 synthesis decreases

-Apoptosis

Immune response

-Increase in lymphocytes and macrophages that attack tissues

Why is a lot of estrous synchronization work done in cattle first

B/c 9 month gestation so not much room for error with timing

-Only 60 day window to get preg

Non-synchronized percent preg vs synchronized

Non-synch: after 3 cycles 88% are preg

Synch: add extra cycle to breed so after 4 cycles 94% preg

-9 day age advantage so calves from beginning of breeding season weigh more more money

What hormone isn’t used in US?

Estrogen due to carcinogenic effect

Estrous synch advantages

Can shorten breeding season

Shortened time to Breeding

Utilizes labor more efficiently

No heat checking needed

Reduced days to first service

Groups timing of parturition

-Uniform crop of offspring=more valuable

Estrous synch disadvantages

Increased labor costs

Requires skilled labor and periods of intensive management

-Multiple injections

-Timing is everything

Requires adequate facilities

-Less time spent observing animals so can miss detecting non-cyclers in herd

Higher input costs

-Is it worth it

What controls the cycle? Brand names?

Progesterone

Blocks dom follicles from ovulating so they die

Progestins mimic progesterone

-MGA (melengesterol acetate)

-Norgestomet

-PRID (progesterone releasing intravaginal device)

-CIDR (controlled internal drug release)

What does PGF2a do? Brand names

Kills CL to decrease P4 and cause estrus in 72 hrs (cattle)

-Decreases P4 and increases E2

Lutalyse, estimate, prosolvin, bovilene, ETC

Explain prostaglandin protocol in cattle. What does it do?

Regress active CL

-Only effective days 5-17 (grey window days 5-7 b/c CL has to become sensitive to PGF2a)

In estrus 2-5 days after injection

-Heifers: 50 hrs

-Cows: 72 hrs b/c more metabolic strain

Single vs two injections of PGF2a in cattle?

Single injection to random animals

-60-65% will respond - may be in follicular or metestrus in luteal phase

Two injections to synchronized entire herd

-10-14 days after first injection

-All should have CL old enough to respond so 100%