Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongationC.A. Gray, R.C. Burghardt, G.A. Johnson, F.W. Bazer and T.E. Spencer

Team OneCarolyn Allen Donna CovingtonAzure Faucette Megan Minten

Morphology and Histology of the Mature Ovine Uterus

Functions: Sperm transport, luteolysis and control of cyclicity, environment for preattachment embryo, maternal contribution to the placenta, expulsion of the fetus and fetal membranes

Structure:• Bicornuate (two uterine horns and small uterine body) • Layers Perimetrium (Serosa) Myometrium (Muscularis) Endometrium (Mucosa and Submucosa)

Target for:• Oxytocin, Estradiol, Progesterone

Surface molecules:• Non-adhesive factors (e.g. Muc-1), adhesion molecules (e.g. Integrins)

Senger P.L., Pathways to Parturition

Morphology of the Mature Uterus

• Continuous with the mesometrium of the broad ligament

• Consists of endothelial cells and connective tissue

• Consists of 2 layers of smooth muscle

1) Longitudinal layer (outer) 2) Circular layer (inner)

•Provides motility, is responsible for uterine tone when estradiol is high, and aids in expulsion of fetus and fetal membranes at parturition

Perimetrium (Serosa) Myometrium (Muscularis)

Outer layerInner layer

MyometriumPerimetrium

Morphology of the Mature Uterus

Endometrium (Mucosa + Submucosa)• Stroma: Stratum compactum (dense stroma) Stratum spongiosum (less dense stroma)•Luminal Epithelium (LE)•Glands/Glandular Epithelium (GE)•Lymphatics, blood vessels

EndometriumStratum Compactum

Stratum Spongiosum

Luminal Epithelium

Glands/Glandular Epithelium

Endometrium

Myometrium

Histology of the Ovine Uterus

Endometrium

Histology of the Ovine Cyclic and d14 Pregnant Uterus: Intercaruncular and Caruncular Areas

Caruncular Area• Devoid of glands• Vascularized• Maternal side of palcentome

Intercaruncular Area• Vast number of glands

d14 Pregnant

CyclicL

Gss

sc

M

ss

sc

Gray et al., 2002

Endometrial adenogenesis in the uterine wall of most species

Gray C A et al. Biol Reprod 2001;65:1311-1323

©2001 by Society for the Study of Reproduction

Comparative time line of the seminal events of endometrial gland morphogenesis in the

sheep, pig, rodent, and human

Gray C A et al. Biol Reprod 2001;65:1311-1323©2001 by Society for the Study of Reproduction

Spencer, TE et al. Endocrinology 1999,Vol. 140, No. 9:0013-7227

•L – Lumenal Epithelium•G – Glandular Epithelium

•SC – Stratum compactum•SS – Stratum spongiosum

Interactive mechanisms involved in endometrial adenogenesis

Gray C A al. Biol Reprod 2001;65:1311-1323et

©2001 by Society for the Study of Reproduction

Spencer, TE et al. Endocrinology 1999,Vol. 140, No. 9:0013-7227

•L – Lumenal Epithelium

•G – Glandular Epithelium

•SC – Stratum compactum

•SS – Stratum spongiosum

•V – blood vessel

•M - Myometrium

Ruffled L

Epigenetic Effects of Progesterone on Uterine Phenotype

•Progesterone:

•C-21 steroid hormone•Important hormone for the estrous cycle and menstrual cycle (human)•required to maintain pregnancy•embryogenesis

Progesterone

Neonatal development

• Dramatic remodeling occurs-1st few months- “critical period”• Glands emerge from internodular, luminal epithelium during

fetal development• Introduction of progesterone has been shown to have a

lasting effect on tissue form and function• Can cause permanent changes to the organization of the

target tissue• Dependent upon:

– Steroid involvement– Dosage– Duration of exposure

Epigenetic Effects of Progesterone

• In the neonatal ovine uterus, key growth factors regulate morphogenesis:– FGF-10: stimulates proliferation and differentiation of the placenta– FGF-7: mediates the development of uterine blood vessels– HGF: stimulate epithelial morphogenesis (establishment of

pregnancy, conceptus implantation and placentation).

• Progestin: inhibits the expression of both the growth factors and their receptors.

• In addition, progestins disrupt the expression of ER (ER is expressed in emerging, proliferating and developing glandular epithelium and stroma cells.

Estrous Cycle

ProestrusEstrus

MetestrusDiestrus Estrus

5 10 15 20

E2

P4

FSH

LH

Rel

ativ

e B

loo

d H

orm

on

e C

on

cen

trat

ion

s

Estrus: E2 peakLH surge induces ovulationFSH suppressed surge by inhibin and E2

P4 minimal

Metestrus:P4 increasingE2 dropsLH pulsatileFSH low

Diestrus:P4 increases and plateaus, PGF released and P4 starts decliningE2 minimalLH pulsatileFSH low

Proestrus:E2 risesP4 dropsLH increasesFSH increases

PGF

Day of Estrous (relative to estrus)Senger P.L., Pathways to Parturition

Estrous Cycle

• Gray et al., 2000 demonstrated UGKO ewes had variable interestrous interval differing from control ewes (16-18 day) ranging from 17-43 days– No difference between UGKO and control ewes in response to PGF

injection

Pregnancy in UGKO Ewe

• Peri-implantation pregnancy loss was observed in the UGKO ewe model Gray et al., 2000; Gray, Bazer et al., 2001; Gray, Taylor et al., 2001

– Normal, hatched blastocysts found in uterine flushings at day 6 and 9

• Gray et al., 2002 hypothesized that the peri-implantation defect in UGKO ewes was due to an absence of endometrial glands and secretory products. Alternatively the defect could be the result from deficient expression of LE adhesion molecules or inability of endometrium to respond to IFN-– Determine differences between control and UGKO ewes in:

• Distribution of Muc-1 and integrin subunit• Abundance of osteopontin and GlyCAM-1• Endometrial expression of IFN--stimulated genes

Pregnancy in UGKO Ewe

• UGKO ewes were found to have:– no conceptus (n = 5/12); growth-retarded, tubular conceptus (n =

6/12); or fragmented, filamentous conceptus (n = 1/12)– No difference in expression of Muc-1 and integrins αv, α5, β3 and β5

– IFN stimulated genes expressed– Osteopontin was absent in uterine flushes, GlyCAM-1 was low or

undetectable

• CONCLUSIONS:– Peri-implantation conceptus survival and growth is altered by the

absence of endometrial glands and their secretions, including molecules involved in cell-cell adhesions such as osteopontin and GlyCAM-1 Gray et al., 2002

Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongationC.A. Gray, R.C. Burghardt, G.A. Johnson, F.W. Bazer and T.E. Spencer

Team OneCarolyn Allen Donna CovingtonAzure Faucette Megan Minten

Questions