Fertilization and Pregnancy Fertilization Sperm arrive at the - - PDF document

Fertilization and Pregnancy

Fertilization

• Sperm arrive at the

uterotubule junction

– Within 20 minutes – By uterine contraction

• Fertilization takes place at the

ampulla-isthmus junction

– Gamete fusion results in formation of separate pronuclei. Male is larger and easier to see.

Preimplantation in the Mouse

• Day 0-2 (40-50 hours):

– Embryo in the ampulla – 1-2 cell

• Day 3 (30 hours):

– Embryo in the isthmus – Develops from 2 to 32 cell embryo – Entry into the uterus at the end of day 3

• Day 4: Embryo implants into the uterus

Entry into the Uterus

Hour/day Stage Mouse 72/3 32 cell, morula Hamster 72/3 8 cell Rat 96/4 8-16 cell Guinea pig 84/3.5 8 cell Rabbit 60/2.5 68-80 cell, morula

Implantation

• Timing (at least 1 day beyond entry):

– Day 4 in a mouse – Day 5 in a rat

• Timing can be influenced by presence of

nursing pups

• Embryo maintenance prior to implantation

is via secretions stimulated by P4.

Bruce Effect

• Mouse and gerbil; possibly the rat
• Pheromonal effect
• Exposure to stranger male leads to

interruption of pregnancy (abortion) due to increased DA

• Effective only prior to implantation (day 4)
• Effective only when dam is not lactating
• Effective only with prolonged exposure

Superovulation

• A method for timing ovulation and

increasing the number of eggs ovulated.

• Exogenous hormones

– PMSG—Pregnant mares serum gonadotrophin

• FSH-like, give ~4 pm

– hCG—Human chorionic gonadotrophin

• LH-like, give 46 hours later
• Mate if fertilized eggs are desired

Collection in the Mouse

• Collect eggs or embryos

according to desired stage

• f development

– Unfertilized: 0.5 days post

• vulation from ampulla

– 1-2 cell: 0.5 – 1.5 d, ampulla – 2-16 cell: 1.5-2.5 d, UT junction – Blastocyst: 3.5 d, uterus

Embryo Transfer

• Superovulate donor female and mate with

intact male.

• Mate a recipient female with a

vasectomized male

– Mating should be timed to coincide with the eggs developmental stage (obtained from superovulated donor female)

• Perform a surgery to expose the ovary.

Then insert the eggs into the oviduct or the upper portion of the uterus.

Uses of Embryo Transfer

• Rederivation of

contaminated lines allowing them to enter a barrier facility

• Rederivation of lines

that have been cryogenically preserved.

• Embryo manipulation

and formation of transgenic lines

Maintenance of Pregnancy Mouse Model

• Progesterone (P4)—Absolute requirement

– Negative feedback on GnRH – Promotes secretory activity – Inhibits myometrial contractions

Maintenance of Pregnancy

• Progesterone comes from the CL
• Prolactin (PRL)—1st luteotrophic hormone

– Maintains corpus luteum – Prolactin is inhibited by DA unless DA is inhibited

1. Critical levels of estradiol inhibits DA 2. Cervical stimulation sets up a long term neural reflex arc to inhibit DA and rescue the CL. (Required because rodents lack a luteal phase; estrous cycle is considered “incomplete.”)

DE PE E

ME

Copulation during behavioral estrus stimulates the cervix DA is again inhibited releasing PRL from inhibition. PRL maintains the CL which produces P4

Initiation of Pregnancy

GnRH LH PRL E2 2nd P4

E2 at 100 – 200 pg/ml

Inhibits DA Releasing PRL From inhibition

1st

DA

PRL P4 d10

Supraoptic nuclei Cervical Stimulation Short acting inhibition Long-lived reflex arc

DA PRL CL +

1st

2nd

P4

Maintenance of Pregnancy

E2

• Reflex Arc ends

Day 10 of gestation DA

PRL P4 FSH d10

By midpregnancy the long-lived reflex arc fades away releasing DA from inhibition.

PL

Supraoptic nuclei Cervical Stimulation Short acting inhibition Long-lived reflex arc

DA PRL CL +

1st

2nd

Placenta begins producing placental lactogen P4

3rd

Maintenance of Pregnancy

E2

• DA

Placental Lactogen

• 2nd Luteotrophic hormone

– Secreted by the placental-fetal complex – Takes over maintenance of the corpus luteum during the second half of pregnancy – Activities mimic PRL

• Luteotrophic
• Mammotrophic
• Maternal behavior

Maintenance of Pregnancy Guinea Pig Model

• Progesterone (P4)—Absolute requirement

– Negative feedback on GnRH – Promotes secretory activity – However, it is not the major hormone to inhibit myometrial contractions in the guinea pig. This is achieved by the hormone relaxin.

• PRL is not luteotrophic
• Estrous cycle is complete

– It has a luteal phase

PE ~1.5 days E ~12 hours ME ~2.5 days DE: luteal phase CL maintained ~ 13 days Ovulation PE

Reproduction in the Guinea Pig

• Normal cycle length is 15-17 days
• The guinea pig has a luteal phase

– The CL is functional for 13 days of each cycle (regression begins by day 10 to 12)

P4

PE E ME DE (luteal phase) ~ 13 days Ovulation PE

Reproduction in the Guinea Pig

• The CL is not maintained by PRL
• Possible luteotropic hormones include

FSH or a combination of hormones

PE E ME DE (luteal phase) ~ 13 days Ovulation PE

Reproduction in the Guinea Pig

• CL only requires 2 to 3 days of a luteotropic

hormone to stay through the luteal phase.

• CL regresses at the end of the cycle due to a

luteolytic factor

PE E ME DE (luteal phase) ~ 13 days Ovulation PE

Reproduction in the Guinea Pig

• Regression of the CL causes P4 to decrease

which releases GnRH from inhibition.

Pregnancy in the Guinea Pig

• If mating occurs, implantation takes place 6 –

8 days post-coitus, during the luteal phase.

• Plasma progesterone increases rapidly by

day 15 post-coitus and will peak at 5 times the concentration seen during the cycle.

– Progesterone continues to come from the CL – P4 also comes directly from the placenta (no PL) – Progesterone stays in the blood stream longer because its metabolic clearance rate slows. This is unique to the guinea pig and other hystricomorphs.

PE E ME DE (luteal phase) ~ 13 days Ovulation PE

Pregnancy in the Guinea Pig

Ovulation and mating E ME DE

Gestation D 13 D 70

P4 from CL

P4 from CL & placenta

Implantation d6-8

Pseudopregnancy (PSP)

• A response to a non-fertile mating
• Duration: About ½ of normal gestation
• Effect: Physiological changes of

pregnancy

– Interruption of the estrous cycle – Increased blood flow to uterus – Secretory activity in oviduct and uterus – Mammary gland development – Suppression of myometrial contractions

Pseudopregnancy

• Control

– PSP is initiated with cervical stimulation and continues as long as the long term neural reflex loop is in effect – PSP is terminated when the reflex loop ends because there is no placental lactogen

PRL P4 d10

Pseudopregnancy

E2

Stimulation of the cervix During an infertile mating Inhibits DA By mid-gestation the reflex loop fades away. With no placenta, there is no PL. The CL is resorbed.

DA

Embryo Transfer

• Superovulate donor female and mate with

intact male.

• Mate a recipient female with a

vasectomized male

– Mating should be timed to coincide with the eggs developmental stage (obtained from superovulated donor female)

• Perform a surgery to expose the ovary.

Then insert the eggs into the oviduct or the upper portion of the uterus.

PRL P4 d10

Pseudopregnancy

E2

Stimulation of the cervix During an infertile mating Inhibits DA By mid-gestation the reflex loop fades away. With no placenta, there is no PL. The CL is resorbed.

Species PSP (days) Gestation (days) Mouse 8 – 10 19 – 21 Rat 10 - 12 21 – 22 Hamster 8 – 10 16.5 Rabbit 15 - 19 30 - 32 Guinea pig None 60 - 70

DA

NO Pseudopregnancy in the Guinea Pig

Ovulation and mating E ME DE

Gestation D 13 D 70

P4 from CL

P4 from CL & placenta No pseudopregnancy in the guinea pig because, if pregnant, the placenta is producing P4 by the end of the normal cycle.

Implantation d6-8

PGF2α

P4 PL

Events Leading up to Parturition

Mouse Model

• Maturation of fetuses triggers regression of the CL
• PGF2α secreted by the uterus stimulates luteolysis
• PL synthesis from the placenta stops removing CL

maintenance.

• As the CL regresses, P4 levels drop off

Events Leading up to Parturition

• CL regresses, P4 levels decrease.
• GnRH is released from P4 inhibition, GnRH levels rise.
• FSH stimulates follicular development leading to increased E2.
• PGF2 a begins upregulation of oxytocin receptors.

.

PGF2α

P4 PL GnRH FSH E2 OTr

Events Leading up to Parturition

• PGF2 a binds to receptors in the CL and causes regression.
• PL levels decrease under control of maturing fetuses.
• As CL regresses, P4 levels decrease.
• GnRH is released from P4 inhibition, GnRH levels rise.
• FSH stimulates follicular development leading to increased E2.
• PGF2 a begins upregulation of oxytocin receptors.
• E2 (and no P4) stimulates uterine motility.
• Expansion of the myometrium stimulates secretion of oxytocin.
• OT increases strength, frequency, and synchronicity of uterine contractions.

PGF2α

P4 PL GnRH FSH E2 OTr

Birth

Oxytocin

• Source

– Synthesized in the paraventricular nuclei of the hypothalamus – Secreted from the posterior pituitary

• Stimulus: stretching of smooth muscle tissue

– Expansion of myometrium – Stimulation of the nipple line

• Functions

– Parturition: Increases strength and frequency of contractions

• Strongest uterotonic agent
• Helpful but not required

– Milk letdown: contraction of myoepithelial cells surrounding the alveoli to expel milk to the nipple

• Absolute requirement

Parturition

• Mice, rats, hamsters: 1-3 hours
• Guinea pigs & rabbits: 30 minutes
• However, stress can interrupt parturition
• Nursing doesn’t begin until all pups are

born.

Oxytocin

• Clinical uses

– May facilitate parturition in animals experiencing dystocia

• Not effective if dystocia is

not caused by weak contractions

• Has side-effects that could

be dangerous.

– Facilitates expulsion of retained placentas – Facilitates milk letdown for milk collection