Growth and Development Pre Implantation Embryo

Fuller W. Bazer

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

Greg A. Johnson

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

Thomas E. Spencer

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

INTRODUCTION

In most species, ovulated ova are at Metaphase II of meiosis and remain so until fertilized within the oviduct some 5 30min after ovulation. Within 11 22 hr after fertilization, meiosis is completed and the female and male pronuclei, each with its 1N or haploid compliment of chromosomes, fuse to establish the 2N or diploid status with pairing of maternal and paternal chromosomes. The one-cell fertilized ovum or zygote then undergoes cleavage to form a two-cell embryo by about 26 hr after fertilization. Ova may be either meiotically immature or chromosomally abnormal, which contributes to the 25 40% pre-implantation embryonic mortality in most mammalian species.

EMBRYONIC DEVELOPMENT

Following fertilization, most mRNA encoding proteins in the zygote is of maternal origin, but as the embryo advances in development, it becomes less dependent on maternal mRNA as more mRNA transcripts representing its own genome are expressed.[1] This maternal to embryonic transcript transition occurs at the 4-cell stage in pigs and 8- to 16-cell stage in sheep and cat-tle.[2] Advancing embryonic development results in genes being silenced, so that the genome loses "totipo-tency'' or its ability to orchestrate development of a normal individual. However, the cloning of the sheep Dolly taught us that the nuclear genome in adult cells can be reprogrammed to restore totipotency following somatic cell nuclear transfer, although less efficiently than cloning by blastomere nuclear transfer using 8- to 16-cell sheep and cow embryos.[3]

Genome imprinting refers to the requirement for an embryo to have both maternal and paternal chromosomes (genomes) to develop normally. This is because of preferential expression of maternal genes in the embryonic disc for development of the embryo/ fetus, while paternal genes are expressed preferentially in the trophectoderm for development of a functional placenta. Imprinted genes include those for insulin-like growth factor II from the maternal genome and insulinlike growth factor II receptor from the paternal genome, both of which are essential for the development of a normal conceptus.

After fertilization, embryos remain in the oviduct and enter the uterus at 48 56 hr in pigs, 72 hr in ewes, 72 96 hr in cows, and 144 hr in mares. Unfertilized equine ova are not transported from the oviduct into the uterus, perhaps because of the lack of production of prostaglandin E2. However, unfertilized ova of sheep, pigs, and cows are transported into the uterus. Embryos of most species fail to develop beyond the early blastocyst stage if confined to the oviduct owing to the absence of critical factors needed for development or the presence of embryotoxic factors.

The early cleavage stages of mouse embryonic development require expression of many genes including 1) phosphoproteins known as cyclins that regulate cell division; 2) heat shock proteins for genome activation during the maternal mRNA to embryonic mRNA transition; 3) lamins A and B for formation of the nuclear membrane after pronuclei fuse; 4) laminins which are extracellular matrix proteins that promote cell adhesion; 5) uvomorulin for compaction and tight junction formation for blastomeres of morulae to become polarized, and form a hollow center, the blas-tocoel, into which water and nutrients are pumped;

6) gap junctions for cell cell communication; and

7) sodium-potassium ATPase for active transport of water, electrolytes, and essential nutrients.[3] Blastocyst formation is a key stage in early embryonic development when cells segregate into the embryonic disc, trophectoderm, extraembryonic endoderm, and blasto-coel necessary for continued development and differentiation to a conceptus [embryo and associated extraembryonic membranes (Fig. 1)].

Beginning at the two-cell stage of embryonic development, many growth factors are expressed that influence cellular proliferation and differentiation including transforming growth factor-a and -p, insulin-like growth factor-I and -II, fibroblast growth factor-7, epidermal growth factor, and insulin. Proto-ncogenes and viral-like genes incorporated into the embryonic genome are also expressed and include c-mos that regulates meiosis, c-fos that is a transcription factor, c-erb that encodes epidermal growth factor receptor, c-fms that encodes colony stimulating factor-1 receptor, and oct-4 that encodes for germ line differentiation.^

As pre-implantation embryos develop, increased carbon dioxide production reflects changes in metabolic activity, and there is increased uptake of precursors for RNA (uridine), proteins (amino acids), and glycoproteins (glucosamine) as well as water and glucose for general metabolism.

BLASTOCYST DEVELOPMENT AFTER HATCHING FROM ZONA PELLUCIDA[1]

Before blastocysts develop into a conceptus, they "hatch'' from the zona pellucida. The trophectoderm expresses plasminogen activator that converts plasminogen to plasmin which is a protease that degrades the zona pellucida to allow the blastocyst to emerge and continue development in cows (Day 9), mares (Day 8), ewes (Day 7), pigs (Days 6 7), mice (Day 4), and humans (Days 7 8).

Pig embryos are 0.5 1-mm diameter spheres when they ''hatch'' from the zona pellucida, increase in size by Day 10 of pregnancy (2 6 mm), and undergo a morphological transition to large spheres (10 15-mm diameter) and then tubular (15 mm x 50 mm) and filamentous (lmm x 100 200 mm) forms on Day 11. During the transition from tubular to filamentous forms, pig conceptuses elongate at 30 45mm/hr remodeling of trophectoderm. However, hyperplasia of trophectoderm is responsible for subsequent growth and elongation of pig conceptuses to 800 1000-mm length by Day 15 of pregnancy. Rapid elongation of

Stage Blastocyst Pig

Fig. 1 Embryonic development during the pre implantation period begins with the zygote and continues with early cleavage stage embryos, and blastocysts which transition from spherical to tubular and filamentous forms. The polar bodies (extruded extra sets of chromosomes following meiosis and fertilization), blastomeres, zona pellucida, inner cell mass or embryonic disc, trophectoderm, and blastocoel are illustrated.

Fig. 1 Embryonic development during the pre implantation period begins with the zygote and continues with early cleavage stage embryos, and blastocysts which transition from spherical to tubular and filamentous forms. The polar bodies (extruded extra sets of chromosomes following meiosis and fertilization), blastomeres, zona pellucida, inner cell mass or embryonic disc, trophectoderm, and blastocoel are illustrated.

pig conceptuses is accompanied by estrogen production by trophectoderm, a marked increase in free calcium in the uterine lumen, and increases in prostaglandins F2a and E2, plasminogen activator, hyaluronic acid, and hyaluronidase associated with tissue remodeling.

Sheep blastocysts are spherical on Days 4 (0.14mm) and 10 (0.4mm), elongate to filamentous forms between Days 12 (1.0 mm/33 mm) and 15 (1/150 190 mm), and extend through the uterine body into the contralateral uterine horn by Days 16 17 of pregnancy. Cow blastocysts are spherical on Days 8 9 (0.17 mm diameter), oblong or tubular on Days

12 13 (1.5 3.0mm diameter), tubular to filamentous between Days 13 14 (1.4/10 mm) and 17 18 (1.5/160 mm) before occupying two-thirds of the gravid uterine horn by Days 18 20, and extending into the contralateral uterine horn by Day 24. Conceptus development during the pre-implantation period for goats, water buffalo, and camelidae is similar to that for sheep and cows. Equine conceptuses do not undergo elongation, but remain spherical until around Day 50, because of the prominent fluid-filled yolk sac established on Day 12 of pregnancy. Elongation (porcine, bovine, and ovine) or expansion (equine) of the conceptus is a prerequisite for central implantation.

Females of the family camelidae are induced to ovulate by mating.[4] Ovulation occurs 24 26 hr after mating in llama and alpaca and 30 48 hr postmating in camels. Camels ovulate from the right and left ovaries with equal frequency; however, all conceptuses develop in the left uterine horn, as the right uterine horn is incapable of supporting conceptus development beyond Day 50 of pregnancy.

INTRAUTERINE MIGRATION OF BLASTOCYSTS

Intrauterine migration and spacing between embryos is critical to embryonic survival, especially in litter-bearing species like pigs.[5] Pig embryos are located in the uterine horns, just below the oviduct, on Days 5 6 after mating, migrate toward the uterine body where embryos from the two uterine horn mix by Day 9, and then migration and spacing ends by Days 11 12 as conceptuses become filamentous. Intrauterine migration and spacing is modulated by peristaltic contractions of the myometrium in response to estrogens, histamines, and prostaglandins secreted by pig conceptuses. Intrauterine migration of blastocysts is rare in ewes and cows with a single ovulation; however, it occurs in sheep following multiple ovulations. Transuterine migration of equine blastocysts occurs approximately

13 times per day between Days 10 and 16 of gestation before fixation of the conceptus to uterine endometrium on Day 16.

IMPLANTATION

There are three classifications for implantation: 1) central (noninvasive) in livestock; 2) eccentric (invasive) in rodents; and 3) interstitial (invasive) in primates. The phases of implantation involving interactions between trophectoderm and uterine lumenal epithelium include 1) shedding of the zona pellucida, 2) precontact and blastocyst orientation, 3) apposition, 4) adhesion, and 5) endometrial invasion in rodents and primates.[6,7] Implantation in livestock is central with increasing trophectoderm-uterine epithelial cell apposition and adhesion without permanent erosion of uterine luminal epithelium.

CONCLUSIONS

Embryonic development during the pre-implantation period is characterized by the onset of expression of its own genome, differentiation into blastocyst stage, and conceptus in preparation for implantation. The pre-implantation period is critical to embryonic survival and subsequent establishment and maintenance of a successful pregnancy.

REFERENCES

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

2. Menezo, Y.; Renard, J.P. The life of the egg before implanation. In Reproduction in Mammals and Man; Thibault, C., Levasseur, M.C., Hunter, R.H.F., Eds.; Elipses: Paris, 1993; 349 368.

3. Wilmut, I.; Beaujean, N.; DeSousa, P.A.; Dinnyes, A.; King, T.J.; Paterson, L.A.; Wells, D.N.; Young, L.E. Somatic cell nuclear transfer. Nature 2002, 419, 583 587.

4. Skidmore, J.A. The main challenges facing camel reproduction research in the 21st century. Reproduction 2003, 61 (Suppl.), 37 47.

5. Senger, P.L. Pathways to Pregnancy and Parturition; Current Conceptions; Washington State University Research and Technology Park: Pullman, Washington, 2003.

6. 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.

7. Bazer, F.W.; Johnson, G.A.; Spencer, T.E. Growth and development: mammalian conceptus periimplantation period. In Encyclopedia of Animal Science; Pond, W.G., Bell, A.W., Eds.; Marcel Decker, Inc.: New York, 2005.

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