Target organs for ovarian steroids
uterine lumen Oviduct
myometrium endometrium
cervix Uterine Histoarchitecture
R.D. Geisert & L. Burdett Oviduct and endometrium respond differently to E2 and P4
Cross section
E2 most important for tubal secretions P4 most important for uterine secretions Progesterone Ovarian hormone Estrogen cycle
follicular development corpus luteum present/regresses
Uterine n
o
i
menstrual t
a
l
cycle
u
v
O
Menses Proliferative phase Secretory phase 0 1 2 3 4 5 6 7 8 9 10 1112 13 14 1516 1 7 18 1920 21 22 2324 25 26 27 28 Days of menstrual cycle Cells of the endometrium
Uterine n uterine lumen o
menstrual i
t a
cycle l Luminal epithelium
u v
O Uterine Glands (epithelium) functionalis
Stroma (fibroblasts /leukocytes)
basalis Blood vessels including spiral arterioles
Menses Proliferation Secretion myometrium 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Days of menstrual cycle Endometrial morphology changes as the cycle progresses
Proliferative phase Secretory phase Regeneration of endometrium: Differentiation: epithelial/stromal cells Development of spiral arterioles proliferation of all cell types Menstruation
The breakdown and shedding of endometrial tissue, accompanied by bleeding, that occurs at the end of each normal mentstrual cycle in the absence of conception.
Normal menstrual fluid loss <80ml (mean 40ml) (Hallberg ’66, Cole ’71)
Results from falling progesterone levels as the corpus luteum regresses.
Only in women, Old World primates, few bats
A consequence of the considerable preparation of the endometrium for implantation in these species
Uterine n o
menstrual i
t a
Menstruation cycle l
u
v O
. Breakdown of extra-cellular matrix (ECM) (incomplete, focal, variable)
. Bleeding occurs with simultaneous Menses Proliferation Secretion disruption of blood vessels & overlying 01 21 32 43 54 65 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Days of menstrual cycle epithelium Day 1
. Re-epithelialization of the endometrium occurs in some areas at Day 4 the same time breakdown of epithelial cells is in progress in other areas
. Cessation of bleeding needs timely and adequate repair Ludwig et al, 1990
Initiation of menstruation: early events
Probably reversible Non-reversible
Vasoconstriction Upregulation of matrix (increased degrading enzymes or prostaglandins, matrix metalloproteinases endothelin) (MMPs)
Increased inflammatory Increased leukocytes mediators
May be inhibited by Not inhibited by exogenous P4 exogenous P4
P4 withdrawal leukocytes
chemokines PGRA+
stromal/decidual cells activated leukocytes
PGRA+ proteases
proMMP active MMP
Epithelial cells TIMPs/2M MMP;Matrix Metalloproteinases; alpha 2 microglobulin; Tissue Inhibitors of MMPs ECM breakdown The need for endometrial remodelling
Progesterone Ovarian hormone Oestrogen
cycle
Uterine n
o
i
menstrual t
a
l
cycle
u
v O
Menses Proliferation Secretion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Days of cycle
hostile/neutral neutral receptive non-receptive d0-4 d6-10 d11-14 preovulatory post-ovulatory The ‘window of receptivity’ in women The short period of time during which the endometrium is receptive to implantation of a blastocyst
Approximately Days 6 to10 post-LH surge
Important changes
In epithelium – increases in secretory products, adhesion molecules, e.g., secreted phosphoprotein 1 (SPP1), integrin heterodimers such as αvβ3, insulin-like growth factor binding protein (IGFBP1)
In stroma – decidualization starts around spiral arterioles
ECM – edema
Increased vascular permeability
Changes induced by blastocyst (hCG and other signalling molecules) Maintenance of CL during early pregnancy
blastocyst ovary produces hCG CL
implants progesterone
endometrium Summary
P4 and E2 stimulate growth of the endometrium in anticipation of fertilization and implantation of the blastocyst
No fertilization, P4 & E2 inhibit secretion of LH and FSH
~8 days after ovulation (Day 22 of cycle) the CL begins to degenerate so concentrations of E2 and P4 in blood decrease
Hormone withdrawal results in molecular changes in cells of uterine endometrium leading to the onset of menstruation.
With low P4 and E2 levels, secretion of LH and FSH increase to drive follicular development and ovulation for the next cycle
In a fertile cycle, the blastocyst signals via hCG which prevents CL regression to ensure secretion of P4 required for pregnancy Overall summary
Estrous and menstrual cycles are driven by the same hormones and events, but have different detection systems
The main hormones driving the cycle are GnRH, LH, FSH, E2, P4 and inhibin
The main event is ovulation
The endometrium is the main target of E2 and P4 that prepare it for implantation of a blastocyst
In the absence of a blastocyst, the cycle must start again in women as menstruation is needed because some endometrial changes cannot be reversed Relaxin Peptide hormone discovered by Frederick Hisaw in 1926 •A heterodimer of two peptide chains of 24 and 29 amino acids linked by disulfide bridges; •Related to insulin. •Produced from prohormone (“pro-relaxin”) by splitting off one additional peptide chain Member of relaxin-like peptide family in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1 (RNL1), 2 (RNL2) and 3( RNL3) insulin-like peptides, INSL3, INSL4, INSL5 and INSL6 Functions of relaxin-3, INSL4, INSL5, INSL6 are not known.
SOURCE AND FUNCTIONS OF RELAXIN
Produced by CL, mammary gland, chorion and decidua
Present in milk of sows – “lactocrine” hormone
Produced in prostate, present in human semen and enhances sperm motility
. aid in parturtition by softening connective tissue (collagen) of cervix and pubic symphysis by altering collagen metabolism, inhibiting collagen synthesis, and enhancing collagen breakdown by increasing matrix metalloproteinases
enhances angiogenesis
renal vasodilator
In prepubertal pigs, evidence for stimulation of uterine development
In piglets, evidence for enhancing gut maturation and perhaps uterine development Receptors for Relaxin • Relaxin receptor LGR7 (RXFP1) and LGR8 (RXFP2) belong to G-protein-coupled receptor superfamily that contains a heptahelical transmembrane domain and a large glycosylated ectodomain, similar to receptors for LH and FSH.
• Relaxin receptors in heart, smooth muscle, connective tissue, and central and autonomous nervous system.
Intracellular mechanisms whereby relaxin and oxytocin regulate contractions of uterine myometrial cells
Sherwood, O. D. Endocr Rev 2004;25:205-234
Copyright ©2004 The Endocrine Society Fig. 7-3 Characteristics of Estrous and Menstrual Cycles
Species Estrus Estrous Cycle Time of Ovulation Cow (B. taurus) 12-18h 21 days 30h post LH surge Cow (B indicus) 6-12h 21 days 30h post LH surge Sow 36-72h 21 days (18-22) 40-44h post LH surge Ewe 12-24h 17 days 30h post LH surge Mare 5-7 days 21 days 48h before end of estrus Mouse 10h 4-5 days 2-4h after onset of estrus Rat 10-15h 4-5 days 204h after onset of estrus Human None 28 days 24-30h post LH surge Duration of Luteal Function Across Species
Weeks
Weeks
Days/Weeks
Weeks
Months
Physiological Review 79:263 Regulation of Rat Corpus Luteum during Pregnancy
PRL LH
PL A D-PRL PGF2 CL CL CL CL Regress
Progesterone
P 1 6 12 21 Day of Pregnancy Nocturnal surge is terminated by rising titers of estrogen secreted from developing follicles in the face of lowering progesterone secretion by the CL. The diurnal surge is terminated the withdrawal of progesterone. Mating in the rat induces two daily surges of prolactin (PRL), one at the end of the light period (diurnal) and the other at the end of the dark period (nocturnal).
These surges are last seen on Day 9 (diurnal) and 10 (nocturnal) of pregnancy. On Day 9, placental lactogen (PL) is secreted by the placenta which peaks on Day 12.
The PRL surges do not require ovarian steroids. These surges will endure for up to 14 days. A luteotrophic hormone of placental origin terminates the surges of PRL, while a shifting of the ratio of estradiol to progesterone at the end to the pseudopregnancy terminates the PRL surges in nonpregnant animals. 20-Hydroxysteriod Dehydrogenase 20-HSD
H3C H3C O OH Prolactin Inhibits Enzyme
20-HSD NADPH NADP
O Progesterone 20-dihydroprogesterone
Does not support pregnancy or decidualization in rodents Regulation of Rat Corpus Luteum during Pregnancy
PRL LH
PL A D-PRL PGF2 CL CL CL CL Regress
Progesterone
P 1 6 12 21 Day of Pregnancy Reproductive Biology and Endocrinology 2004, 2:22
Serum concentrations of progesterone and prolactin, and luteal 3betaHSD and 20alphaHSD activities in rats throughout pseudopregnancy Pseudopregnant rats were sacrificed at 18:00 h on each of the days of PSP studied. Trunk blood was obtained after decapitation and serum progesterone (Panel A) and PRL (Panel B) concentrations measured by RIA. The CL were isolated and 3betaHSD (Panel C) and 20alphaHSD (Panel D) activities were measured spectrophotometrically. Results are mean ± SEM of groups of 5–12 animals. Columns with different letters differ significantly. Prostaglandin F2 increases 20HSD expression
20-HSD mRNA levels L19 250 PGF2 20-dihydro 200 Progesterone Steroids Levels 150 20-HSD (ng/ml) 100 50 Progesterone 0 18 19 20 21 22 Days of pregnancy 20-Hydroxysteriod Dehydrogenase 20-HSD
H3C H3C O OH Prolactin Inhibits Enzyme
20-HSD NADPH NADP
O Progesterone 20-dihydroprogesterone
Does not support pregnancy or decidualization in rodents 20-Hydroxysteriod Dehydrogenase Expression is Inhibited by Prolactin
Protein mRNA
20HSD
PRL HYPOX
Albarracin and Gibori. Endocrinology 1991 129:1821. Cat (Queen)
Induced Ovulator Pregnancy – 63-65 Days Pseudopregnancy – about 40 Days CL Resistant to PGF until after Day 40 Secretion of PRL increases after Day 40 and considered luteotrophic Inhibition of PRL secretion causes abortion in last trimester CL, uterus and fetus express detectable levels of relaxin – uterine quiescence
Dog (Bitch)
Spontaneous Ovulator Pregnancy – 64-66 Days Pseudopregnancy – about 60-63 Days CL Resistant to PGF until after Day 40 Secretion of PRL increases after Day 30-40 and is considered luteotrophic Inhibition of PRL secretion causes abortion after about Day 30 Ovary and pregnant uterus express relaxin detectable for up to 60 days post-partum for uterine quiescence
Hormone Profile in Cyclic Ewes Endometrium
• Mucosa + submucosa – Epithelia • Lumenal – Glands • Glandular Epithelia – Blood vessels – Lymphatics – Stroma • Stratum Compactum • Stratum Spongiosum Responses to target cell to E2: 1. Histamine mobilization 2. Hyperemia a.growth of blood vessels b.vasodilation 3. Lysosome labilization (lysosomal membrane becomes more fragile. 4. in RNA and protein synthesis 5. in lipid metabolism because Ca mobilization and arachadonic acid production 6. in secretion-due to release of secretory vesicles (stimulus-secretion coupling)-some pancreatic proteins can be release this way. 7. precursor uptake-amino acid production and glucose 8. _mitotic activity 9. cell hypertrophy 10. in membrane excitability 11. in OXTR (parturition, luteolytic mechanism) 12. in Ca mobilization 13. water inhibition of tissue Responses to target cells to progesterone: 1. RNA and protein synthesis 2. growth of uterine glands 3. water inhibition 4. membrane potential (smooth muscle cell relaxed) LD50 -because can cause relaxation of the diaphragm and kill rat 5. phospholipid stores 6. PG synthesis (PG synthase and phopholipid stores must be present) 7. substrate (AA and glucose) uptake 8. mitotic activity Progestins Inhibit Endometrial Adenogenesis
Acyclic Infertile 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) Hormone Receptors During Development of the Endometrial Luteolytic Mechanism in Cyclic Ewes Day 9 Day 15 Progesterone Receptor (PGR)
Estrogen Receptor (ESR1)
Oxytocin Receptor (OXTR)
UTERINE MICROENVIRONMENT Components • Enzymes • Growth Factors • Adhesion Proteins • Cytokines • Hormones • Transport proteins
“Histotroph” G Johnson Secretions from luminal (LE) and glandular epithelia (GE) of endometrium and selective transport mechanisms Hormone Profile in Cyclic Ewes EVENTS OF PROESTRUS AND ESTRUS PROSTAGLANDINS AND LUTEOLYSIS cPLA2 Cox-1 cyclooxygenase 1 or COOH Phospholipids PTGS1 – prostaglandin synthase 1 Phosphotidyl choline Arachidonic Acid (5, 8, 11, 14-eicosatetraenoic acid) Phosphotidyl serine Cox-2 cyclooxygenase 2 or PGSG2 Phosphotidyl ethanolamine Prostaglandin synthase 2 Cox-1 Cox-2 7 4 2 9 O 8 6 5 3 COOH 10 14 18 20 15 16 O 12 11 13 17 19 Nuclear receptor OH PGG PPAR 2 COOH PPAR Cox-1 Cox-2 PPAR PGI Synthase TX Synthase O COOH COOH O O IP receptor O O TP receptor OH OH 2+ cAMP Ca + IP3 PGH2 TxA2 OH O PGD Synthase PGE Synthase PGF Synthase OH PGI2 OH O
DP receptor COOH COOH COOH FPA,B receptor 2+ cAMP Ca + IP3 O OH OH OH OH OH PGD PGDH PGE 9-keto-PGE PGDH 2 PGEM 2 reductase PGF2 PGFM
Relaxant Receptors Constrictor Receptors cAMP 2+ Ca / IP3 or cAMP EP1 EP2 EP3a EP3b EP3c EP3d EP4 Ca2+/IP3 cAMP cAMP cAMP cAMP cAMP/ cAMP IP3 Phosphoatidyl Choline Primate-intraovarian PGF is luteolytic hormone.
Pulsatile release of PGF required for luteolysis in most mammals.
Uterus is source of luteolytic PGF in subprimate mammals.
PROSTAGLANDIN SYNTHESIS
Uterine epithelia store phospholipids-(phosphotidyl choline, phosphotidyl inositol, phosphotidyl ethanolamine) and phosphotidyl serine that, when acted on by Phospholipase A2, yields arachidonic acid, the precursor for prostaglandin synthesis. 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 PGF2 pulses in ewes
Subluteolytic
Luteolytic Hormonal Regulation of OTR in Ovine Uterine Endometrial Epithelium Ovary Ovary
OT CL E2-17b OOTRR P4 + + - ER- - PR +
Endometrial Epithelium IFNT and Pregnancy Recognition Pituitary Ovary Ovary
C
OXT L
OXTR X 17b-E2 + X P4 + PTGS2
ESR1 - PGRPR Luminal ERX- Epithelium (LE) IRF2 +
IFNAR IFNT CONCEPTUS Fig. 9-5 Cyclic Pregnant
Geisert et al. 1990, J. Reprod Fert Suppl. 40:293 Geisert et al. 1990, J. Reprod Fert Suppl. 40:293 Conceptus estrogen production appears to reduce the levels of PGF2 in the uter-ovarian vein in pregnant animals in comparison to nonpregnant gilts.
However, the levels of PGF2 found in the uterine lumen during Days 12 through 18 are extremely elevated in pregnant compared to nonpregnant gilts Pregnant
Nonpregnant
Bazer et al. 1982, Control of Reproduction in the Pig, p227 Note the large increase in prostaglandin F in the blood of control, cyclic animals in contrast to estrogen treated
Frank et al. 1977, Prostaglandins 14:1183 Note that there is a large accumulation of Prostaglandin in the uterine lumen of pregnant gilts.
Frank et al. 1978, Prostaglandins 15:151 Treatment of cyclic gilts with estrogen (5 mg/day) from Day 11 to 15 results in pseudopregnancy in the pig.
Frank et al. 1977, Prostaglandins 14:1183
Maternal Recognition of Pregnancy in the Gilt
In the cyclic gilt, luteal regression begins on about day 15 with plasma progesterone concentrations declining to basal levels (1 ng/ml or less) by day 17 to 18.
Bilateral hysterectomy before day 16 results in prolonged luteal function for periods equal to or longer than 114 days. However, unilateral pregnancy fails in pigs by 21 days in the presence of a non-gravid uterine horn. One fourth of the one uterine horn approached the minimal quantity of uterine tissue necessary for bilateral luteal regression in the pig.
The results of studies indicating that concetuses must be present in both uterine horns (2 per horn) for pregnancy to be established indicate a combined and local uterro-ovarian pathway. In swine, PGF2 is converted into an inactive metabolite, 15 keto - 13, 14 dihydro-prostaglandin F2, by the lungs as it is in other species. However, only 18% of the PGF2 released is metabolized in one passage through the lungs in comparison to 99% in the lungs of the ewe. Therefore active PGF2 can reach the contralateral ovary by a systemic route in the pig thereby causing luteolysis on both ovaries. PGF2 is luteolytic in the pig when given after Day 12 of the cycle or pregnancy. The CL of the pig may remain refractory to PGF2 until LH begins to dissociate from the luteal cell receptors. It is thought that conformational changes within the luteal cells facilitate PGF2 binding. The PGF2 alters adenylate cyclase system to inhibit progesterone secretion and activate lysosomal enzymes to cause morphological regression of the luteal cells.
Maternal Recognition of pregnancy occurs on about Day 12 in the pig. Flushing of embryos from the uterine horns after Day 12 results in an extension of the diestrous period.
Conceptus estrogen synthesis (estrone, estradiol-17b and estriol), as well as possible effects of catechol estrogens, on Day 12 coincident with rapid elongation of the blastocyst is the maternal recognition signal. This production of estrogen is reinforced by a second period of synthesis and release between Day 15 to 18 which sustains pregnancy till term. Injection of exogenous estrogen on Day 11and 14 through 16 will stimulate pseudopregnancy (prolonged CL function in the absence of any embryo) for an equivalent or slightly longer period than pregnancy. The Theory of Maternal Recognition of Pregnancy in the Pig was First Proposed by Bazer and Thatcher (1977)
In nonpregnant pigs, progesterone enhances and/or induces PGF2 synthesis by the uterine endometrium and secretion is associated with the elevated plasma estradiol concentrations (ovarian) between Day 12 and 18 of the cycle.
Secretion of PGF2 in mid-luteal phase is primarily in an endocrine direction (uterine capillaries). The increased levels of PGF2 lead to luteolysis.
In pregnant pigs, progesterone stimulates PGF2 in the endometrium as in nonpregnant pigs. However, estrogen production by pig conceptuses (local uterine effect) alters direction of PGF2 movement so that it remains in the uterine lumen (exocrine secretion). Maintenance of PGF2 in exocrine direction would prevent PGF2 from entering the uterine venous drainage. Therefore, PGF2 synthesis is not inhibited but compartmentalized. It is also significant that histotroph (protein in lumen) is also maintained in an exocrine direction.
Temporal and tissue-specific expression of prostaglandin receptors
PGE: EP2, EP3, EP4 (not expressed in cow)
PGF: FP
Cyclooxygenases 1 and 2 in uterus and fetal membranes
Meidan R, Levy N. Endothelin-1 receptors and biosynthesis in the corpus luteum: molecular and physiological implications. Domest Anim Endocrinol. 2002;23:287-298
Endothelin-1 (ET-1) 21-amino acid peptide identified as a potent vasoconstrictor inhibits steroidogenesis in vitro and in vivo compliments luteolytic effects of PGF2 alpha
ET-1 receptors type A (ETA)
ET-1 gene is transcribed as prepro ET-1 (ppET-1)
Active form of ET-1 derived from inactive intermediate big ET- 1 by endothelin-converting enzyme-1 (ECE-1)
CL responsive to PGF2 alpha only when both ppET-1 and ECE- 1 genes are expressed for uninterrupted ET-1 biosynthesis.
Luteolytic Effects of PGF2alpha
Vasocontriction of blood vessels to CL
Inhibition of LHCGR and STAR Expression and Steroidogenesis
Recruitment of Immune Cells and Cytokines that Induce Cell Death
Endothelin Compliments PGF by Inhibiting Steroidogenesis and Acting as Vasoconstrictor Uterine Prostaglandins • Produced by endometrium – Arachidonic acid – Specific timing for pulsatile PGF release – estradiol and oxytocin – Uterine irritation/infection • Luteolytic PGF2-alpha (PGF) – Vascular spasm – Direct effect on luteal cells • CL sensitivity – Timing • Mare, cow, ewe, doe vs sow – Dose • Sow & ruminants vs mare – Vascular anatomy
Fig. 9-11 Fig. 9-12 Conceptus-Maternal Interactions
P. Sauders & F.W. Bazer