Fertilization & Early Pre-Implantation/Pre-attachment Development

Question 1

what term do we use other than pre-implantation in our common domestic spp?

Answer 1

pre-attachment

Question 2

What is the structure of the salpinx?

Answer 2

infundibulum (w/ fingers over oviduct) to ampulla to isthmus

Question 3

What is mating?

Answer 3

• union of female & male gametes (requires mating behaviour; accomplishes delivery of sperm-containing semen to a mature ova or egg)
• in most vertebrates, MATING OCCURS @ PEAK OF FEMALE FERTILITY (estrus (oestrus) in most mammals)

Question 4

Facts about semen deposition?

Answer 4

• volumes are spp specific
• vaginal semen deposition (humans, rabbits, rodents, cows; in carnivores the cervix is open - Ca & Fe)
• uterine semen deposition (llamas, camelids, Eq, Sw, AI techniques)

Question 5

Are copulatory plus &/or gels present?

Answer 5

• cervical cap in rodents
• human semen coagulates but then liquefies w/in ~30 mins
• camelid semen also coagulates
• canine penis acts as plug

Question 6

How fast is sperm transported through the uterus?

Answer 6

• rodents & Sw: sperm reach oviducts w/in 30 mins of mating
• large and small ruminants: 8-10 hr are required after mating for sufficient sperm numbers in oviduct (strong uterine contractile activity during estrus)
• primates: sperm can reach oviducts w/in ~10 mins (uterus has waves of smooth muscle contraction in late follicular phase)
• sperm must undergo capacitation before fertilization

Question 7

What is capacitation?

Answer 7

• process of physiological alterations of the sperm so they are competent to fertilize the oocyte (REQUIRES the female reproductive tract)

Question 8

What do sperm undergo during capacitation?

Answer 8

• removal of the membrane cholesterol to improve oocyte binding
• increase in intracellular Ca2+ concentration
• increase in intracellular pH
• protein phosphorylation (esp tyrosine residues)
• hyperactivated motility (ex: asymmetrical beating)

Question 9

What are the functional affects of capacitation?

Answer 9

• penetrate cumulus cell matrix (un-capacitated sperm adhere to the outer edge of the oocyte)
• adherence to zona pellucida of oocyte
• undergo zona-stimulated acrosome reaction

Question 10

what is the function of the utero-tubal junction (UTJ)?

Answer 10

• after ejaculation, sperm become maintained in high numbers at the UTJ (beyond the cervix, UTJ is second major selective barrier w/ lots of folds)
• in domestic spp, mechanisms used in selection are largely unknown
• there is selection for live, motile, normal morphology, uncapacitated, & acrosome intact sperm
• removal of the genes that code for adhesion of the sperm head prevent fertilization from occurring

Question 11

How does the isthmus act as a reservoir?

Answer 11

• oviduct epithelial cells (OECs) are ciliated & non-ciliated
• sperm bind ciliated OECs that have specific glycoproteins
• sperm can be bound to such OECs particularly in the isthmus for 2-4 days in domestic spp (SUCH SPERM SEEM TO BE OF HIGHEST QUALITY - a functional sperm reservoir which can be provided @ time of ovulation)

Question 12

What are the effects of ovulation time?

Answer 12

• in most mammals, E2 concentrations in circulation & oviductal fluid are elevated during pre-ovulatory period (drop after ovulation) (PROMOTE SHORT TERM SPERM STORAGE)
• post-ovulation, progesterone levels rise in circulation & in oviductal fluid (CHEMOTACTIC SIGNAL FOR SPERM TO MOVE TO AMPULLA & UNDERGO CAPACITATION IN AMPULLA)

Question 13

What is fertilization?

Answer 13

• sperm contact & penetration (through cumulus cell layer (corona radiata))
• zona pellucida (ZP) binding & acrosome reaction (increased by zona binding)
• zona penetration (sperm penetration of zona)
• SPERM FUSION w/ oocyte mb and ooplasm
• CORTICAL GRANULE RELEASE from oocyte (“zona reaction” & “vitelline block”)
• PRONUCLEAR FUSION & initiation of metabolism

Question 14

How is the acrosome reaction initiated?

Answer 14

sperm plasma mb covering acrosome has 2 receptor regions
- ZONA BINDING REGION - ZBR that binds zona pellucida-3 glycoprotein on oocyte zona (ZP3) (attaches sperm to zona)
- ACROSOME REACTION PROMOTING REGION (ARPR) (binds ZP3 & starts acrosome reaction ex: fusion of membranes)

Question 15

What is the acrosome reaction?

Answer 15

• before acrosome reaction, membranes of sperm head are intact
• during reaction, overlying plasma mb fuses w/ outer acrosomal mb (contents released (ex: ACROSIN) & DIGEST ZONA PROTEINS & INCREASE SPERM BINDING TO ZONA)
• inner acrosomal mb & equatorial portion of sperm head increase binding to oocyte mb

Question 16

what happens post acrosome reaction?

Answer 16

• sperm penetrates the zona & into perivitelline space
• Oocyte produced cortical granules accumulate at periphery
• oocyte & sperm fuse @ equatorial region
• CORTICAL GRANULES RELEASE CONTENTS (mucopolysaccharides, proteases, plasminogen activator, acid phosphatases, & peroxidases; CHANGE ZONA TO PREVENT POLYSPERMY (ZONA BLOCK OR ZONA REACTION); CHANGE OOCYTE MB TO PREVENT SPERM ATTACHMENT (VITELLINE BLOCK))
• sperm nuclear contents decondense

Question 17

what is pronuclear fusion?

Answer 17

• nucleus of the sperm now forms a male pronucleus (both female & male pronuclei are haploid)
• pronuclei fuse & a zygote is created that is diploid
• DNA replication takes place, chromosomes condense, first mitotic division occurs
• male mitochondria including mtDNA are degraded leaving only maternal mitochondria

Question 18

What are cleavage divisions?

Answer 18

• mitotic cell divisions in early embryos. cells of the divisions are called blastomeres

Question 19

What deviations from normal cell division do cleavage divisions during preimplantation development have?

Answer 19

• rapid multi-cellularity
• no growth ex: nuclear to cytoplasmic ratio increases & cell size decreases
• shape is maintained
• asynchronous in mammals

Question 20

What is holoblastic?

Answer 20

division completely cuts through the embryo. this occurs in embryos w/ moderate (ex: amphibians) TO LITTLE/NO YOLK (EX: MAMMALS) & can be unequal or equal (rotational: cleavage will produce blastomeres at right angles to 1 another)

Question 21

What is rotational cleavage of the mammalian embryo?

Answer 21

• cleavage divisions are asynchronous in mammals (divisions of 2-cell to 4-cell can also appear as 3-cell to 5-cell under the microscope)

Question 22

What is pre-attachment development?

Answer 22

• in mammals, the period of embryonic development from fertilization to just prior to implantation or uterine attachment

Question 23

What is the activation of the embryonic genome?

Answer 23

• maternal transcripts & proteins stored in the oocyte play a major role in initiating early development. following embryonic genome activation, cellular machinery becomes that of embryo & maternal transcripts & proteins are degraded
• embryonic genome becomes activated during early cleavage stages

Question 24

What is the morphogenetic event I?

Answer 24

compaction
- unique event that begins after 3rd cleavage
- cells huddle closely together & form compact ball at 8-cell stage
- just prior to & during compaction, blastomeres become polarized
- outside cells form a tight permeability seal w/ tight junctions, desmosomes, & adherens junctions
- cells on the inside become connected by gap junctions
- 16-cell stage referred to as a morula
- most of the outer cells of the morulae are destined to become trophectoderm & then trophoblast (Ex: extraembryonic lineage)
- the inner cells will derive the embryo by first forming the inner cell mass (ICM)
- the 2 regions are distinct & synthesize different proteins

Question 25

What is the morphogenetic event II?

Answer 25

Cavitation
- fluid begins to accumulate in the compacted morulae forming small vesicles
- the vesicles coalesce into a blastocoel & the embryo becomes a mammalian blastula known as a blastocyst
- the enzyme Na, K - ATPase appears to be a primary driver of the process (pumping 3 Na+ into developing cavity per 2 K+ into the cell creating an osmotic gradient for water to flow into the blastocoel)

Question 26

What is the morphogenetic event III?

Answer 26

Blastocyst expansion
- the enzyme Na,K-ATPase again appears to be a primary driver of the process. ex: ouabain inhibits the enzyme & expansion
- requires a very efficient permeability seal that can be disrupted by drugs that breakdown the actin cytoskeleton or Ca2+ requiring adhesion molecules
- process is essential b/c it culminates in the 1st differentiation event: the differentiation of the ICM & the trophectoderm (1st epithelium) (ICM = embryo proper; trophectoderm = extra-embryonic lineages)

Question 27

What happens during preparation for implantation or attachment: hatching?

Answer 27

• w/ blastocyst expansion, hatching follows
• requires release of proteases (Ex: strypsin) to break down the matrices comprising the zona pellucida
• @ this time, ICM differentiates into: hypoblast & epiblast
• w/ formation of hypoblast & epiblast, the former ICM is referred to as a bilaminar disk

Question 28

What 3 forces is hatching governed by ?

Answer 28

• growth & fluid accumulation w/in the blastocyst
• production of enzymes by the trophectoderm cells to break down the matrices comprising the zona pellucida (ex: strypsin)
• contraction of the blastocyst

Question 29

Describe the blastocyst once hatched?

Answer 29

• blastocyst is now free-floating embryo w/in lumen of the uterus (totally dependent on the uterine environment for survival

Question 30

Why is the Eq zona pellucida & capsule the exception?

Answer 30

• in Eq, the embryo does not hatch from the zona pellucida b/c the ZP disintegrates around d7-8 post-ovulation
• @ ~ d6.5, the trophectoderm of the blastocyst begins to secrete a glycoprotein containing capsule beneath the ZP & the capsule remains until ~d22 of pregnancy