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 P 4 .

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 of development – Unfertilized: 0.5 days post ovulation 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 (P 4 )—Absolute requirement – Negative feedback on GnRH – Promotes secretory activity – Inhibits myometrial contractions

Maintenance of Pregnancy • Progesterone comes from the CL Prolactin (PRL)—1 st 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.”)

Initiation of Pregnancy E2 at 100 – 200 pg/ml Copulation 2 nd during behavioral estrus 1 st stimulates the cervix Inhibits DA Releasing PRL DA is again inhibited From inhibition releasing PRL from inhibition. PRL maintains the CL LH which produces P 4 P 4 PRL DA E 2 GnRH DE PE E ME

Maintenance of Pregnancy Cervical Stimulation 2 nd Reflex Arc ends Day 10 of gestation Long-lived reflex arc Supraoptic nuclei - - 1 st DA - Short acting inhibition PRL + P4 CL PRL P 4 E 2 DA d10

Maintenance of Pregnancy Cervical Stimulation 2 nd By midpregnancy the long-lived reflex arc fades away releasing DA from inhibition. Long-lived reflex arc 3 rd Placenta begins producing placental lactogen Supraoptic nuclei - - 1 st DA - Short acting inhibition PRL + P4 CL PRL P 4 E 2 PL FSH DA d10

Placental Lactogen • 2 nd 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 (P 4 )—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

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) Ovulation P 4 ME PE E DE: luteal phase PE ~2.5 ~12 CL maintained ~ 13 days ~1.5 days hours days

Reproduction in the Guinea Pig • The CL is not maintained by PRL • Possible luteotropic hormones include FSH or a combination of hormones Ovulation ME PE E DE (luteal phase) PE ~ 13 days

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

Reproduction in the Guinea Pig • Regression of the CL causes P 4 to decrease which releases GnRH from inhibition. Ovulation ME PE E DE (luteal phase) PE ~ 13 days

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 – P 4 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.

Pregnancy in the Guinea Pig Ovulation ME PE E DE (luteal phase) PE ~ 13 days Implantation d6-8 Ovulation and mating P 4 from CL P 4 from CL & placenta D 13 Gestation DE E ME D 70

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

Pseudopregnancy Stimulation of the cervix By mid-gestation the reflex loop fades away. During an infertile mating With no placenta, there is no PL. Inhibits DA The CL is resorbed. P4 PRL E 2 DA d10

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.

Pseudopregnancy Stimulation of the cervix By mid-gestation the reflex loop fades away. During an infertile mating With no placenta, there is no PL. Inhibits DA The CL is resorbed. Species PSP Gestation (days) (days) Mouse 8 – 10 19 – 21 Rat 10 - 12 21 – 22 P4 PRL Hamster 8 – 10 16.5 E 2 Rabbit 15 - 19 30 - 32 Guinea pig None 60 - 70 DA d10

NO Pseudopregnancy in the Guinea Pig No pseudopregnancy in the guinea pig because, if pregnant, the placenta is producing P 4 by the end of the normal cycle. Implantation d6-8 Ovulation and mating P 4 from CL & placenta P 4 from CL D 13 Gestation DE E ME D 70

Events Leading up to Parturition Mouse Model •Maturation of fetuses triggers regression of the CL  PGF 2 α secreted by the uterus stimulates luteolysis  PL synthesis from the placenta stops removing CL maintenance. PGF 2 α •As the CL regresses, P 4 levels drop off P 4 PL

Events Leading up to Parturition •CL regresses, P 4 levels decrease. •GnRH is released from P 4 inhibition, GnRH levels rise. •FSH stimulates follicular development leading to increased E 2 . • PGF 2 a begins upregulation of oxytocin receptors. . PGF 2 α P 4 OTr FSH E 2 PL GnRH

Events Leading up to Parturition •PGF 2 a binds to receptors in the CL and causes regression. •PL levels decrease under control of maturing fetuses. •As CL regresses, P 4 levels decrease. •GnRH is released from P 4 inhibition, GnRH levels rise. •FSH stimulates follicular development leading to increased E 2 . • PGF 2 a begins upregulation of oxytocin receptors. •E 2 (and no P 4 ) stimulates uterine motility. PGF 2 α •Expansion of the myometrium stimulates secretion of oxytocin. •OT increases strength, frequency, and synchronicity of uterine contractions. P 4 OTr FSH E 2 PL GnRH Birth