Growth and Development Peri Implantation Embryo

Fuller W. Bazer

Department of Animal Science and Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, Texas, U.S.A.

Greg A. Johnson

Veterinary Integrative Biosciences and Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, Texas, U.S.A.

Thomas E. Spencer

Department of Animal Science and Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, Texas, U.S.A.

INTRODUCTION

Guillomot et al.[1] indicated that the phases of implantation in mammals include: 1) shedding of the zona pellucida; 2) pre-contact and blastocyst orientation; 3) apposition; 4) adhesion; and 5) endometrial invasion. In contrast to rodents and humans, true endometrial invasion does not occur in ruminants for which the sheep will serve here as the prototypical ruminant.[2-4]

in the embryonic disc appears at this stage and somites appear soon thereafter. The conceptus is initially localized to the uterine horn ipsilateral to the corpus luteum, but elongates into the contralateral horn on day 17. Elongation of the conceptus is critical for central implantation and for production of interferon tau (IFN-t), the signal for pregnancy recognition. Pregnancy recognition signals ensure maintenance of a functional corpus luteum for production of progesterone, the hormone of pregnancy.

IMPLANTATION (SEE FIG. 1)

Shedding of the Zona Pellucida (Phase 1)

Morula (16 32 cells) stage sheep embryos enter the uterus from the oviduct on day 4 post-mating (day 0 = estrus/mating), reach the blastocyst stage on day 6, and "hatch'' from the zona pellucida between days 8 and 9. Loss of the zona pellucida allows the trophectoderm to expand and make contact with the endometrial lumenal epithelium (LE). On day 8, the spherical blastocyst is 200 mm in diameter with about 300 cells. By day 10, it is 400 900 mm in diameter and contains «3000 cells. After day 10, the blastocyst or conceptus (embryo and associated membranes) develops into a tubular and then to a filamentous form.

Pre-contact and Blastocyst Orientation (Phase 2)

Between days 9 and 14 of pregnancy, there are no definitive cellular contacts between the trophectoderm and the LE, but sheep conceptuses become positioned and immobilized in the lumen of the uterus for rapid elongation of the trophectoderm. On day 11, the spherical, then tubular, and finally filamentous conceptus elongates to 25 cm or more by day 17. The primitive streak

Apposition (Phase 3)

The trophectoderm is in close association with the LE followed by unstable adhesion and then onset of apposition accompanied by a reduction of the apical micro-villi covering the trophectoderm between days 13 and 15 in sheep. The LE undergoes similar modification for closer association with the trophectoderm in some species. The apposition of the trophectoderm involves interdigitation of cytoplasmic projections of the tro-phectoderm and the LE beginning at the inner cell mass and spreads along the filamentous conceptus toward the ends of the trophectoderm. Uterine glands of sheep are also sites of apposition as the trophecto-derm develops finger-like villi that penetrate into the mouths of the uterine glands between days 15 and 18 and then disappear by day 20 of pregnancy. These villi appear to anchor the peri-attachment conceptus and absorb secretions of uterine glands. These villi are also present in cattle, but not in goats.

Adhesion (Phase 4)

The conceptus trophectoderm is firmly adhered to the LE on day 16 in caruncular and intercaruncular areas of the endometrium and adhesion is completed on

Fig. 1 Implantation involves five potential phases that involve increasingly complex interactions between the trophectoderm and the uterine endometrial epithelium and stroma.[1'10]

day 22 of pregnancy in sheep. IFN-t gene expression in mononuclear trophectoderm cells begins for pregnancy recognition during elongation. Trophoblast giant binucleate cells (BNCs) differentiate from the mononuclear trophoblast by day 16, but only mononuclear trophoblast cells adhere to endometrial LE. The BNCs produce hormones such as placental lactogen and progesterone that regulate maternal physiology. Tropho-blast BNCs arise from mononuclear trophectoderm cells by consecutive nuclear divisions without cytokinesis, migrate through the apical trophectoderm tight junctions of the chorion, and flatten as they become apposed to the apical surface of the LE. The BNCs then fuse apically with the LE and form syncytia of trinucleate cells, thereby assimilating and replacing the endometrial epithelium. Subsequently, the tri-nucleate cells enlarge by continued BNC migration and fusion to form syncytial plaques linked by tight junctions that are limited in sheep to 20 25 nuclei. The syncytial plaques eventually cover the caruncular surface and aid in the formation of placentomes. Indeed, BNCs migrate and fuse with uterine epithelial cells or their derivatives throughout pregnancy. The uterine LE persists but is modified to a variable degree, depending on species, by the migration and fusion of fetal BNCs with the endometrial LE. The sheep placenta is synepitheliochorial, being neither entirely syndesmochorial without uterine epithelium, nor completely epitheliochorial with two apposed cell layers for which the only anatomical interaction is interdigitated microvilli as in the pig.

UTERINE SECRETIONS AND CONCEPTUS DEVELOPMENT

All mammalian uteri contain endometrial luminal and glandular epithelia that synthesize and secrete or transport a complex array of enzymes, growth factors, adhesion proteins, hormones, transport proteins, amino acids, ions, and other substances referred to collectively as histotroph. Evidence from human, primate, and subprimate species indicate an unequivocal role for uterine histotroph in conceptus survival, development, production of pregnancy recognition signals, implantation, and placentation. Ewes that lack uterine glands (UGKO ewes) produce insufficient histotroph and are unable to support conceptus development to day 14 of gestation.[5] Defects in conceptus survival and elongation in UGKO ewes are not due to alterations in expression of steroid receptors, mucin glyco-protein one, adhesive integrins on the endometrial LE, or to the responsiveness of the endometrium to IFN-t, the pregnancy recognition signal, but likely are due to deficiencies in secreted adhesion molecules such as osteopontin, galectin-15, and glycosylated cell adhesion molecule one, which are secreted by the LE and GE.[3A6]

PREGNANCY RECOGNITION SIGNALS (SEE FIG. 2)

Primates

The conceptus trophectoderm secretes chorionic gonadotrophs (CG), the luteinizing hormone-like hormone that acts directly on CL to insure maintenance of its structure and function for continued secretion of pro-gesterone.[7] Secretion of CG is detectable by the eight-cell stage of development of human embryos, but secretion of CG increases during implantation, trophectoderm outgrowth, and pregnancy recognition.

Ruminants

The antiluteolytic signal for pregnancy recognition in ruminants is IFN-t secreted by the trophectoderm during the peri-implantation period to abrogate the luteolytic mechanism by inhibiting transcription of the estrogen receptor alpha and oxytocin receptor genes.[8'9] This prevents pulsatile release of luteolytic prostaglan-din F2-alpha (PGF) by uterine epithelia to protect corpus luteum function. In addition, IFN-t increases expression of a number of interferon-stimulated genes including interferon-stimulated gene 15 (ubiquitin cross-reactive protein), major histocompatibility complex-2, and galectin-15 that may affect the conceptus.

Pigs

Estrogens, produced by pig conceptuses between days 11 and 12 and then between days 15 and 25 of gestation, are the antiluteolytic signals for recognition of pregnancy.[8,9] The theory of estrogen-induced maternal recognition of pregnancy in pigs is based on the following evidence: 1) the uterine endometrium secretes luteolytic PGF; 2) pig conceptuses secrete estrogens which are antiluteolytic; 3) PGF is secreted toward the uterine vasculature (endocrine) in cyclic gilts to induce luteolysis; and 4) secretion of PGF in pregnant gilts is into the uterine lumen (exocrine) where it is sequestered and metabolized to prevent luteolysis. The transition from endocrine to exocrine secretion of PGF between days 10 and 12 of pregnancy is coincident with initiation of estrogen secretion by pig conceptuses that results in a transient release of calcium into the uterine lumen within 12 hr and is followed by an increase in endometrial receptors for prolactin.

Horses

The equine conceptus inhibits uterine production of luteolytic PGF as pregnant mares have little PGF in uterine fluids, low concentrations of PGF in uterine venous plasma, and no episodic pattern of release of PGF into peripheral plasma. Also, endometrial production of PGF in response to cervical stimulation and exogenous oxytocin is low or absent in pregnant mares. Estrogens secreted between days 8 and 20 of gestation by equine conceptuses may have a role in preventing luteolysis, but this has not been established. Further, equine conceptuses secrete several proteins (400, 50, and 65kDa) between days 12 and 14 of pregnancy, which may be involved in pregnancy recognition signaling.[8]

Inner Cell Mass

Inner Cell Mass

Fig. 2 The conceptus trophectoderm secretes signals for pregnancy recognition that include estrogens in pigs and interferon tau in ruminants. These hormones act on the uterine endometrium to prevent release of luteolytic prostaglandin F2 alpha so that progesterone secretion by the ovarian corpus luteum is maintained and the uterine glands secrete histotroph necessary for concep tus development.

CONCLUSIONS

The peri-implantation conceptus secretes a pregnancy recognition signal for establishment of pregnancy and initiates implantation. These two critical events must be successful if a successful pregnancy is to be maintained for birth of a viable offspring.

REFERENCES

1. Guillomot, M.; Flechon, J.E.; Leroy, F. Blastocyst development and implantation. In Reproduction in Mammals and Man; Thibault, C., Levasseur, M.C., Hunter, R.H.F., Eds.; Elipses: Paris, 1993; 387 411.

2. Bazer, F.W.; First, N.L. Pregnancy and parturition. J. Anim. Sci. 1983, 57 (suppl. 2), 425 458.

3. Burghardt, R.C.; Johnson, G.A.; Jaeger, L.A.; Ka, H.; Garlow, J.E.; Spencer, T.E.; Bazer, F.W. Integrins and extracellular matrix proteins at the maternal fetal interface in domestic animals. Cells Tissues Organs 2002, 172, 202 217.

4. Spencer, T.E.; Johnson, G.A.; Bazer, F.W.; Burghardt, R.C. Implantation mechanisms: insights from the sheep. Reproduction 2004, 128, 657 658.

5. Gray, C.A.; Burghardt, R.C.; Johnson, G.A.; Bazer, F.W.; Spencer, T.E. Evidence that an absence of endometrial gland secretions in uterine gland knockout (UGKO) ewes compromises conceptus survival and elongation. Reproduction 2002, 124, 289 300.

6. Gray, C.A.; Adelson, D.L.; Bazer, F.W.; Burghardt, R.C.; Meeusen, E.N.; Spencer, T.E. Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in trophectoderm. Proc. Natl Acad. Sci. USA 2004, 101, 7982 7987.

7. Stouffer, R.L.; Hearn, J.P. Endocrinology of the transition from menstrual cyclicity to establishment of pregnancy in primates. In Endocrinology of Pregnancy; Bazer, F.W., Ed.; Humana Press: Totowa, NJ, 1998; 35 58.

8. Bazer, F.W.; Ott, T.L.; Spencer, T.E. Endocrinology of the transition from recurring estrous cycles to establishment of pregnancy in subprimate mammals. In Endocrinology of Pregnancy; Bazer, F.W., Ed.; Humana Press: Totowa, NJ, 1998; 1 34.

9. Spencer, T.E.; Burghardt, R.C.; Johnson, G.A.; Bazer, F.W. Conceptus signals for establishment and maintenance of pregnancy. Ann. Reprod. Sci. 2004, 82 83, 537 550.

10. Guillomot, M.; Flechon, J.-E.; Leroy, F. Blasto-cyst development and implantation. In Reproduction in Mammals and Men; Thibault, C., Levasseur, M.C., Hunter, R.H.F., Eds.; Ellipses: Paris, 1993; 396.

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