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Oviduct and Pre-Implantation Growth and Development

Intra-Uterine Migration and Spacing of

Definitions • Trophectoderm: Cells forming the outer layer of a which provide nutrients to blaostocyst and later participate in forming the or outer layer of cells of the . • : Inner layer of trophectoderm to and external to the wall of the blastocyst; considered a trophoblastic stem cell as layer surrounding the blastocyst remains while daughter cells differentiate and proliferate to function in multiple roles as: 1) cytotrophoblast and syncytiotrophoblast.

1. Syncytotrophoblast 2. Cytotrophoblast 3. Endothelial Cells 4. Smooth Muscle Cells (tunica muscularis) 5. Spiral Artery 6. Endovascular cytotrophoblast

Definitions (Continued) • Syncytiotrophoblast: multi-nucleated epithelial syncytrium covering highly vascular embryonic placental villi, which invade the wall of the uterus to establish nutrient circulation between the embryo and the mother. • : Evagination of foregut of the embryo that provides nourishment and is site of development of circulatory and hematopoietic systems. • : evagination of hind gut of embryo for holding allantoic fluid and supports vasculature of chorioallantoic placenta. Definitions (Continued) • Chorion: membrane formed by extraembryonic and (sommatopleure) that surrounds the embryo/fetus and other placental membranes; emerge from the chorion to invade the ; transfer of nutrients from maternal to fetal blood. • : membrane that immediately surrounds the embryo/fetus and fills with amniotic fluid to provide a protective environment for the developing embryo.

PRE-IMPLANTATION

Morula To Blastocyst Transition Polarization Of Trophectoderm – Tight and Gap Junctions Outgrowth Of Trophectoderm

Arg Leu

Gwatkin, 1966; Martin and Sutherland, 2001 1- Zona Pellucida 2- Trophectoderm 3-Extra-embronic 4-Blastocoel 5. or

Conceptus Development in Sheep

D12 D13

7,889 UNDERGRADUATE D14 D15 2,859 GRADUATE

D16

Cell Proliferation and Migration mRNA translation Cytoskeletal Changes in Trophectoderm

Extra-Embryonic

Sheep, Goat, Cow Pig and Horse

Cat, Dog Primates, Rodents

Uterine Microenvironment Histotroph Components • Enzymes • Growth Factors • Adhesion Proteins • Cytokines • Hormones • Transport proteins

• NUTRIENTS G Johnson Secretions From Luminal (LE) And Glandular Epithelia (GE) Of Endometrium And Selective Transport of Nutrients and Proteins

Down-Regulation of Progesterone Receptors in Uterine Epithelia by Progesterone: A Prerequsite for Implantation in Mammals

• Decline in anti-adhesive Muc-1 • Increase in certain adhesive integrins • Change in patterns of epithelial gene expression

THE OVIDUCT: TRANSPORT OF SPERM AND OVUM AND EMBRYO • EGG TRANSPORT IN THE OVIDUCT Different between species ► DISCONTINUOUS PROGRESSION (cow, ewe, monkey, baboon, human, guinea pig) Due to a prolonged stay at the Ampullary-Isthmic Junction Antiperistaltic contractions maintain egg for 2-3 days Stronger, peristaltic contractions then transport egg to isthmus and on into uterus Does not matter whether egg is fertilized or not

► SLOW, CONTINUOUS PROGRESSION (rabbit, pig, rodent) RABBIT Time (h) Event 0 Ovulation induced by hCG 18-24 All eggs found in ampulla and A-I junction 36 Eggs at A-I junction and distal isthmus 48 Eggs equally distributed between distal and proximal isthmus 60 2/3 eggs in proximal isthmus 63 90% eggs in proximal isthmus 72 All eggs in uterus ► FERTILIZED PROGRESSION (mare) Only fertilized eggs pass into uterus in mares (early pregnancy recognition) > Fertilization specific changes in ZP (mucopolysaccharide coat) > Embryo secretes prostaglandins (PG) E and F that are hypothesized to promote embryo migration through oviduct as well as in the uterus > Infusion of PGE2 into oviducts of pregnant mares hastened transport of oocytes and into the uterus > Rat and hamster - fertilized eggs passage more rapidly and synchronously > Fertilized eggs may secrete motility regulatory factor(s) such as platelet activating factor (PAF) or prostaglandins > Cigarette smoke interferes with oocyte and embryo transport ► ROLE OF STEROIDS IN OVIDUCT MOTILITY

Estradiol - accelerates egg passage if administered after ovulation, but causes a prolonged blockage of the eggs if administered at the time of ovulation > ovum transport is accelerated in superovulated animals > may involve membrane estrogen effects rather than nuclear Progesterone - shortens duration of tubal ovum transport if administered at ovulation Steroids affect smooth muscle contraction activity ROLE OF OVIDUCTAL SECRETIONS IN DEVELOPMENT OF FERTILIZED EGG > Physiological or biological role: OBLIGATORY vs FACILITORY ???? > Culture of in various media leads to loss in viability or to a block in development - Block occurs at stage when embryonic genome becomes functional - Prepubertal oviduct can ensure activation of embryonic genome > Oviductal secretions, oviductal-specific proteins and oviductal epithelium enhance fertilization and embryonic development > Oviduct sustains and enhances fertilization and early -stage embryonic development Provides optimal environment in terms of temperature, pH, osmotic pressure, nutrients, oxygen tension and other factors

Molecules found in the oviduct (Kane et al., 1997; Buhi et al., 2001)

Amino acids, Glucose and fructose, Lipids, Albumin

Oviductal proteins Protease inhibitors (TIMP-1, PAI-1) Retinol binding protein (RBP) Osteopontin (OPN) Large number of growth factors Transferrin

Porcine oviduct secretory proteins (POSPs) (Buhi et al., 2001)

-Estrogen enhances synthetic activity

-Infundibulum and ampulla affected by estrogen, but not isthmus

-Synthetic capacity: Infundibulum & ampulla > isthmus (2-3X)

-Oviduct synthesized proteins are similar in each species

-Major secretory product is a chitinase family member (cleaves chitin = N- acetylglucosamine polymer)

- Binding of POSPs can be detected on the ZP and Recent evidence in the pig indicates that pOSPs decrease the rate of polyspermy observed in IVF

- Ligation of oviduct causes degeneration of blastocysts in rat and ewe

- Tubal pregnancies only exists in humans

INTRAUTERINE MIGRATION AND SPACING > Morulae differentiate into blastocysts 2-4 days postovulation and then enter uterus to become blastocyts (exception in domestic animals is the horse) > Myometrial contractility Increase in myometrial electrical or contractile activity Day 9 to 12 - sow 10 and 20 hours on Day 4 - rat > RAT Uterine motility before 5th day is involved in equidistribution of embryos Catecholamines Treatment of rats with α1 receptor blockers renders myometrium electrically quiescent and disorganizes embryo distribution Prostaglandins (PGs) Inhibitor of PG synthesis administered on Day 5 prevents regular distribution of implantation sites

Estrogens Increase in myometrial electrical activity on Day 4 concomitant with increase in uterine E2 concentration

Estrogen may directly stimulate myometrial contractility or increase myometrial sensitivity to catecholamines

Administration of RU486 to rabbits or rats during passage of morulae into uterine horns causes accumulation at cervical extrimity and their expulsion

> PIG In the pig, Day 10 to 12 embryos migrate through both uterine horns. Usually end up with 5 embryos in each horn. After migration, get spacing.

Blastocyst migration and spacing Days 3-8 embryos in upper 1/3 of horn Days 9-10 mixing and migration of embryos Day 11 equal spacing

Blastocyst produces estrogen which acts on uterine epithelium to release calcium Calcium may affect myometrium to cause contractions

Blastocyst also produces PGF and PGE - may also act on myometrium

Estrogen acts on uterine epithelium to cause release of histamine Histamine acts on myometrium

EXPERIMENTAL EVIDENCE

Pope et al., Biol Reprod 1982; 27:575-579; J Anim Sci 1982; 55:1169-1178.

> Beads containing estrogen or cholesterol were inserted into uterine lumen on Day 7 On Day 12, beads impregnated with estrogen were found to migrate farther than those impregnated with cholesterol

> Day 6 Px - subserosa of each uterine horn tip was injected with vehicle, 8 mg cromolyn sodium or 8 mg cromolyn sodium plus 1 mg histamine > Found that cromolyn sodium treatment alone restricted D10 embryos to tip of uterine horn, but Day 12 embryos overcame restriction. > Injection of histamine prevented actions of cromolyn sodium treatment

Both E2 and histamine are involved in intrauterine migration of porcine embryos

Uterine flushings from pregnant horns contained substance that, in part, mimicked the effects of embryo on in vitro myometrial contractility

Flushings containing Day12 embryos overcame in vitro inhibitory effects of indomethacin on myometrial contractility > HORSE

Blastocyst capsule has a mucin coat around it (MUC1)

As long as capsule present, blastocyst is motile.

Blastocysts migrate between horns (>12-15 times/day)

Makes contact with uterine epithelium

Embryo migration part of pregnancy recognition mechanism

Stays mobile until Day 18 and then undergoes fixation (removal of MUC1) Horse Embryo

DAY 12

• Edg7 (endothelial differentiation, lysophosphatidic acid G- protein-coupled receptor 7) – Also known as Lpa3 – Preference for unsaturated LPA • Knocked out Edg7 (Lpa3) • Grossly normal – litter size reduced by 50% – Pregnancy prolonged (1.5 days)

ROLE OF CYTOKINES AND SECRETORY PROTEINS IN EARLY PREGNANCY (Tables 1, 2, 3 and 4 from Kane MT et al. Human Reproduction Update 1997; 3:137- 157)

Growth Factors and Cytokines • Can easily demonstrate a variety of growth factors and cytokines in both preimplantation embryo and endometrium of ruminants • Many of these growth factors are expressed by both the oviduct and endometrium throughout early pregnancy • May be important to support growth and differentiation of the peri-implantation conceptus • Potential for endocrine, paracrine and autocrine interactions

Growth factor effects on preimplantation embryo development • Growth factors can affect embryo development in vitro (most work in mouse) • However, GF addition to embryo cultures has not become a standard practice Endometrial Secretions • Classically termed histotroph • Uterine gene expression is dependent on progesterone • Composition of histotroph Glucose and fructose - C6 sugars - Essential for RNA and DNA synthesis - Omission of glucose from culture medium of cow and mouse blastocysts prevents further development - Neoglucogenesis is low or nonexistent to satisfy embryos COH requirements Amino acids - Increases considerably during luteal as compared to follicular phase - Gly, Ser, Ala - very high concentrations (~10 mM) Gly - purine base synthesis Albumin Transferrin Unique proteins Ruminants - endometrial protein synthesis is greatest in ipsilateral horn to CL Rabbit - uteroglobin or blastokinin Uteroglobin - Day 3 to 4 until Day 9 or 10 Concentration increases until makes up half of total proteins Binds steroids, mainly P4, and passes into blastocyst Sow - uteroferrin Uteroferrin - purple colored, iron binding protein, 35 kDa Member of Tartrate Resistant Acid Phosphatase family Secreted by uterine glandular epithelium (GE) and transported across placenta Transports iron Also has hematopoietic growth factor activity Secreted by uterine GE of mare, cow and sheep and human placenta Other proteins Plasmin/trypsin inhibitors Lysozyme - inhibitor of bacterial infection Large number of enzymes

Role of uterine proteins in pre-implantation embryo development ? Required for blastocyst elongation in ruminants Delayed implantation suggests that uterine milieu can regulates embryonic development: Estrogen induces secretion of histotroph and increases expression of nutrient transporters for glucose and amino acids Microinjections of progesterone into uterus increases total lipid content of embryo and increase in dry matter percentage Low percentage of pregnancies in women after IVF/ET may be attributed to inadequate intra-uterine milieu due to high steroid levels induced by superovulation hormone regimen, particularly estrogen that increases receptors for progesterone in uterine epithelia.

Greg A. Johnson Uterine Microenvironment Histotroph Components • Enzymes • Growth Factors • Adhesion Proteins • Cytokines • Hormones • Transport proteins

• NUTRIENTS G Johnson Secretions From Luminal (LE) And Glandular Epithelia (GE) Of Endometrium And Selective Transport of Nutrients and Proteins Uterine Factors and Conceptus Growth • Peri-implantation – Survival and Growth Dependent on Histotroph • Histotroph is nutrition from secretions produced by uterine epithelia

• Post-implantation – Growth Dependent on Histotroph and Hematotroph • Hematotroph is nutrition from maternal blood that is transferred to the placenta for the fetus

– Histotroph Production is Modulated by Ovarian and Placental Hormones

Architecture of the Uterus

UTERINE SECRETIONS Components • Enzymes • Growth Factors • Adhesion Proteins • Cytokines • Transport proteins • Sugars • Amino acids

“Histotroph” Greg A. Johnson

Postnatal Development of the Sheep Uterus • Gland development or adenogenesis is a postnatal event

• Events

(1) Bud formation (2) Tubulogenesis (3) Coiling & Branching Morphogenesis

• Critical period that is susceptible to programming by hormones - Progestins - Estrogens Uterine Gland Knockout (UGKO) Sheep Model • Inappropriate exposure of neonatal ewes to progesterone from birth to PND 56 permanently ablates endometrial gland development – Bartol et al., J Animal Sci 1988; 66:3000 – Spencer et al., Endocrinology 1999; 140:4070 – Gray et al., Biol Reprod 2000; 62:448

• Differentiation of other female reproductive tract structures is not affected by neonatal progestin exposure – Gray et al., Mol Reprod Dev 2000; 57:67 – Gray et al., Biol Reprod 2000; 62:448

• Function of the brain or ovary in the adult is not perturbed by neonatal progestin exposure – Gray et al., Biol Reprod 2000; 62:448 Norgestomet Ablates Uterine Adenogenesis

Bartol et al., J Reprod Fertil Suppl 1999; 54:287 Early Pregnancy Events in Sheep

IFN t production Entry into Shedding of

by

uterus zona pellucida

Fimbria

position position Conceptus Oviduct Utero- tubal Embryo Implantation

junction migration

30 190 mm mm Defect in 425µ X

Conceptus 150µ 170µ 205µ 340µ UGKO Ewes Development Development

0 4 8 12 16 20 Days Post - mating

Gray et al., Biol Reprod 2001; 64:1608 Gray et al., Reproduction 2002; 124:289 Day 14 Conceptus Morphology

NORMAL Histology Elongated (97 mm)

Tubular

(10 mm) UGKO No Conceptus Uterine Lumenal Proteins or Histotroph on Day 14 Post-mating PREGNANT UGKO

500 mg protein with silver stain detection Cyclic Pregnant UGKO Integrin av and Muc-1 Expression

ß3 on Day 14

• No differences a 5 were detected in LE expression of these as well as ß5 many other genes (ERa, PR, OTR, enJSRVs) Muc-1

Gray et al., Reproduction 2002; 124:289 Secreted Adhesion Proteins in the Uterine Lumen Osteopontin (SPP1) Gray et al., Reproduction 2002; 124:289 Day 14 Normal Day 14 UGKO

Glycosylated Cell Adhesion Molecule (GlyCAM1) Day 14 Normal Day 14 UGKO Genomics and Proteomics

Normal UGKO

Gray et al., Biol Reprod 2006; in press Early Pregnancy Events in Sheep

IFN t production Entry into Shedding of

by trophoblast

uterus zona pellucida

Fimbria

position position Conceptus Oviduct Utero- tubal Embryo Implantation

junction migration

30 190 mm mm 425µ

Conceptus 150µ 170µ 205µ 340µ Development Development

0 4 8 12 16 20 Days Post - mating Implantation Events Universal event in rodents, pig, ruminants, ferret, and human

• Decline in anti-adhesive Muc-1 • Increase in certain adhesive integrins • Change in patterns of epithelial gene expression PR Regulation in Endometrium

• PR down-regulation

 Required for IFNt induction of IFN- stimulated gene (ISG) expression in stroma and GE

 Changes patterns of gene expression in endometrial luminal epithelium (LE) and glandular epithelium (GE) Progesterone Regulation of Blastocyst Development • Hypothesis: – Administration of progesterone to pregnant ewes during the pre-implantation period of early embryo development will enhance conceptus development by stimulating uterine secretions

• Rationale: – Hatched blastocysts will not develop further in culture and require the uterus (Fléchon et al., Reprod Nutr Dev 1986; 26: 1017) – Early progesterone treatment of beef cattle enhanced conceptus development (Garrett et al., J Reprod Fert 1988; 84:437) – Early rise in progesterone is beneficial for pregnancy in dairy cattle (Mann and Lamming, 1999; Reproduction 2001; 121:175) – Experimental Design

Day 0 1.5 8 12

CO Corn Oil (1 ml) P4 Progesterone (25 mg per day) P4 Progesterone (25 mg per day) +RU RU486 Circulating Progesterone

CO P4

4 3.5 3 2.5

2 ng/ml 1.5 1 0.5 0 0 1 2 3 4 5 6 7 8 9 10 11 12 Day Post-estrus Day 12 Conceptus Morphology

Control Progesterone

Spherical or Tubular Filamentous No conceptuses in P4+RU486 Galectin-15 mRNA in Endometrium

Endometrial Galectin-15 mRNA Levels

400000

350000

300000

250000

200000

150000

100000 Galectin-15 mRNA (RU+SE) mRNA Galectin-15

50000

0 CO P4 P4+RU Progesterone Receptor

CO P4

P4+RU IgG Galectin-15 mRNA in Endometrium Embryo Diameter

282+64 mm

636+64 mm

Greg A. Johnson

NO!

PREGNANCY RECOGNITION SIGNALING MAINTENANCE OF PREGNANCY

YES! Pregnancy Recognition Signaling Common Feature in Mammals: CL Maintenance as P4 is the Hormone of Pregnancy! Antiluteolytic IFNT Estradiol/PRL

Luteotrophic CGB PRL Lactogenic Hormones

P. Sauders & F.W. Bazer INTERFERONS AND PREGNANCY

IFND IFND IFNT IFNG IFNG??

BIOACTIVITIES Antiviral Immunosuppressive Antiproliferative ******** IFNA IFNA Antiluteolytic (Ruminants) IFNW IFNB Angiogenic (Rodents) IFNG?? IFNG

Hormonal Profile in Cyclic Ewes EFFECTS OF PROGESTERONE ON UTERINE ENDOMETRIAL EPITHELIA

1. Increases cell stores of phospholipids and triglycerides

2. Increases Prostaglandin Synthase 2 that converts Arachidonic Acid to Prostalandins

3. Permissive to secretion of PGF2a

4. Inhibits OXTR synthesis (initially)-period of P4 block 5. Basal and OXTR-induced Phospholipase C activation (activate PKC and Ca/CAM kinase which are cell signaling pathways for OXT)

6. Inhibits expression of ER

7. Inhibits PGR expression after 10 to 12 days EFFECT OF E2 ON LUTEOLYTIC MECHANISM OF EWES

1. Induces premature uterine secretion of PGF2a

2. Increases endometrial expression of OXTR OXTR Gene does not have Estrogen Response Element, but ER-Estrogen binds to SP1 transcription factor and acts via SP1 Binding Sites in OXTR Gene

3. Increase Phospholipase 2 that stimulates Arachidonic Acid release and turnover from phospholipids and triglyceride pools to yield arachidonic acid

4. Cellular Ca++/CaM kinase interacts with PLA2 OXYTOCIN AND LUTEOLYTIC MECHANISM IN EWES

OXY from CL and Posterior Pituitary stimulates frequency and/or amplitude of PGF pulses

Platelet Activating Factor (PAF) affects interpulse frequence of PGF pulses

Ewes require 5 pulses of PGF in 24 h to regress CL. High concentrations at peak of a pulse may affect low affinity FP unique to small luteal cells and low concentrations may affect large luteal cells with high affinity receptors for PGF2a.

Ewe and cow: OXY production: Days 0-3 transcription (may be in response to LH surge Days 4-7 translation Days 8-14 storage of OXY-Neurophysin Days 15-16 release of OXY-Neurophysin as secretory granules move to surface of large luteal cells for release in pulses regardless of whether the ewe or cow is cyclic or pregnant.

In pregnant ewe-basal PGF is higher than in cyclic ewes, but the absence of pulsing prevents luteolysis. OTHER INFORMATION ON CONTROL OF LUTEOLYSIS DURING ESTROUS CYCLE

Pulsatile release of PGF2a.

Why there is a 5-6 hour interval between pulses is not clear.

Speculation: 1) time for different OXT-Neurophysin granules to migrate to periphery of large luteal cells for release represents a refractory period

2) periods of PGF receptor downregulation;

3) molecules such as Platelet Activating Factor inhibits OXY stimulation of OXTR;

4) some molecule coordinates secretion of OXT from both Posterior Pituitary and CL

Indomethacin and Banamine are examples of Prostaglandin Synthase 2 inhibitors If you inject an animal with OXT and measure PGF2a: Day 3 – luteolytic pulses of PGF2a

Days 4 or 5 to 14 - Progesterone block to ESR1 and OXTR expression, so no response.

Days 14-16 - P4 downregulates PGR, so ESR1 and OXTR increase in LE and GE so that uterine epithelia become responsive to luteolytic effects of E2 and OXT

PR antagonist blocks PR downregulation and extends length of estrous cycle by delaying increases in ER and OXTR and luteolysis

Administration of P4 early in the estrous cycle shortens the estrous cycle due to early downregulation of PR and increases in ESR1 and OXTR and luteolysis

Luteolysis depends on ESR1 and OXTR expression by LE and GE (ductal GE specifically), not SC or deep GE.

For pregnancy in ruminants, interferon tau blocks transcription of ER and so no Estradiol-induced OXTR expression. McCracken Hypothesis (Anim Reprod Sci 1984; 7:31-55)

LE/GE Pregnancy inhibits endometrial production of luteolytic pulses of PGF2a in sheep

Subluteolytic

Luteolytic Hormone Receptors During Development of the Endometrial Luteolytic Mechanism in Cyclic Ewes Day 9 Day 15 Progesterone Receptor (PGR)

Estrogen Receptor a (ESR1)

Oxytocin Receptor (OXTR)

Wathes & Hamon, J Endocrinol 1993; 138:479 Spencer & Bazer, Biol Reprod 1995; 53:1527 Conceptus Development in Sheep

D12 D13

7,889 UNDERGRADUATE D14 D15 2,859 GRADUATE

D16

Cell Proliferation and Migration mRNA translation Cytoskeletal Changes in Trophectoderm Flush Pregnant Uterus Recover and Incubate With MEM Conceptus

Conceptus Incubation Conceptus Incubation 100 µCi S35-Met 50 µCi L-C14-Leu 100 µCi H3-GlcN 100 µCi H3-GlcN MEM MEM 95% O2 and 5% CO2, 30 h 95% O2:5% CO2, 30 h Rocking Platform

Interferon Tau: The Pregnancy Recognition Signal in Ruminants Embryo Transfer Experiments

Trophoblastin

Protein X

Ovine Trophoblast Protein-1

Type I Trophoblast Interferon

IFNT OVINE INTERFERON TAU  Secreted by mononuclear trophectoderm cells from Days 10-21 of pregnancy

 19 kDa with 3-4 isoelectric variants of pI 5.3-5.7, nonglycosylated

 Type I IFN family (IFNA, IFNB, IFNW, IFND)

 Secreted at 1 x 109 antiviral units per day by Day 15 and 16 conceptuses

 Antiviral, antiproliferative and immunomodulatory activities of other Type I IFNs

 Unique as pregnancy recognition signal in ruminants IFNT and Pregnancy Recognition Pituitary Ovary Ovary

CL

OXT

OXTR X 17b-E2 + X P4 + PTGS2

Uterine ESR1ER- a - PGRPR LE/sGE X IRF2 +

IFNAR IFNT CONCEPTUS