Fertilization (cell bio)

Question 1

Oocyte developmental events

• Most mammals are born with oogenesis at ? oocyte stage
• Oocytes get arrested until puberty in ?
• Primary oocyte (Meiosis I)
  
  • Duplicates the ? (which phase?) -
    Chromosomal complement is 4n
  • ? over
• Meiosis resumed at ovulatory surge of ?
• Meiosis I results in a ? oocyte
  (2n) and a ? body (will degenerate)

Answer 1

• Most mammals are born with oogenesis at primary oocyte stage
• Oocytes get arrested until puberty in prophase I

(recall in spermatogenesis - no formation of polar bodies, only oogenesis)

• Primary oocyte (Meiosis I)
  
  • Duplicates the DNA (interphase) -
    Chromosomal complement is 4n
  • crossing over
• Meiosis resumed at ovulatory surge of LH
• Meiosis I results in a secondary oocyte
  (2n) and a polar body (will degenerate)

Question 2

Postnatal oocyte development

Secondary oocyte (Meiosis II)
* The secondary oocyte is the cell that is formed by meiosis I or II?? in oogenesis. Thus, it has only one of each pair of homologous chromosomes (after crossing over). However, each chromosome still has two chromatids, making a total of 46 chromatids (2N).
* The secondary oocyte continues the ? stage of meiosis (meiosis II), **however get arrested at ? **; until they meet the ? in the fallopian tube.

• ? occurs during second Meiotic division
• IMP!!! -> Meiosis II is completed at the time of ?.
• Results in an ? (n) and a polar body (will degenerate)

Answer 2

Postnatal oocyte development

Secondary oocyte (Meiosis II)
* The secondary oocyte is the cell that is formed by meiosis I in oogenesis. Thus, it has only one of each pair of homologous chromosomes (after crossing over). However, each chromosome still has two chromatids, making a total of 46 chromatids (2N).
* The secondary oocyte continues the second stage of meiosis (meiosis II), **however get arrested at metaphase II **; until they meet the sperm in the fallopian tube.

• ovulation occurs during second Meiotic division
  ** Meiosis II is completed at the time of fertilization **
• Results in an ovum (n) and a polar body (will degenerate)

Question 3

Ovulation

(oocyte gets arrested then oocyte gets ovulated)

• Release of the ? from the follicle
• Oocyte and cumulus mass moved into the ? of uterine tube by ?
• Meiosis continues to ? meiotic metaphase (blocked there unless fertilized)
• Fertilization occurs in ** ? ** of uterine tube
• long or short? window for fertilization (6-12 h)

Answer 3

Ovulation

(oocyte gets arrested then oocyte gets ovulated)

• Release of the oocyte from the follicle
• Oocyte and cumulus mass moved into the infundibulum of uterine tube by fimbria
• Meiosis continues to second meiotic metaphase (blocked there unless fertilized)
• Fertilization occurs in ampulla of uterine tube
• short window for fertilization (6-12 h)

after that the oocyte dies however only one species is diff. everybody else is exactly the same but dogs, cats and dogs specifically the female ovulate and oocyte BEFORE the release of the FIRST POLAR BODY.

Question 4

Dogs

Oocyte ovulate at ?
Before completion of ?

Maturation finishes in the ?

so oocytes are ovulating in prophase I, and get arrested there and are already ovulating in prophase I. and all this maturation after that will happen in the oviduct. that’s y one hormone that can be measured to pick the phase when u want the breed the dog is:

progesterone because they need to ovulate before that oocyte is viable for fertilization. They need to take at least three or four days until this oocyte completes meiosis I and starts meiosis II.

so the oocyte is mature enough for fertilization and if the oocyte in dogs isn’t fertilized then the dog wasn’t bred and it will get arrested in metaphase II (the same).

we want complete fertilization after meiosis II.

Answer 4

Dogs

Oocyte ovulate at prophase I
Before completion of meisis I

Maturation finishes in the oviduct

Question 5

Fertilization - Sperm transit

• Sperm gain ability to fertilize during ? transit
• Sperm move from site of ? (insemination x ?) to uterine tube within minutes/hours
  
  • ? of female tract
• Many millions, or billions, of sperm ejaculated
• About a thousand reach the ? tube
• Only ? present for fertilization

Sperm reservoir
* ** ? of ? tube ***
* Near uterotubal junction
* Sperm adhere to ? epithelium
* Intimate junction with cell membrane ?
* Adherent sperm have a ? life span
* Sperm are released at time of ?
* Unknown mechanism

Answer 5

Fertilization - Sperm transit

• Sperm gain ability to fertilize during epididymal transit
• Sperm move from site of deposition (insemination x natural mating) to uterine tube within minutes/hours
  
  • contractions of female tract
• Many millions, or billions, of sperm ejaculated
• About a thousand reach the uterine tube
• Only 100s present for fertilization

Sperm reservoir
* ** isthmus of uterine tube ***
* Near uterotubal junction
* Sperm adhere to tubal epithelium
* Intimate junction with cell membrane interaction
* Adherent sperm have a prolonged life span
* Sperm are released at time of ovulation
* Unknown mechanism

• for fertilization, these females need to be bred/inseminated - deposition of semen in the reproductive tract and diff. species will have semen deposition in diff. portions of the reproductive tract
• and insemination will also allow us to deposit semen in different portions of their reproductive tract.
• Most of these species, their semen, is ejaculated at the cranial vagina, so the semen is to move through the cervix -> uterus -> oviduct and then reaches ampulla of oviduct which is where fertilization happens
• specifically in horses and donkeys, ejaculation happens inside the uterus; its intrauterine ejaculation.
• smtms some females have long periods of estrus so they will accept a male for long period of time and oocyte isn’t ready for ovulation yet.
• there is a portion of the oviduct that’s called isthmus, that’s the first portion of oviduct and is closer to the uterus.
• And this portion is called the sperm reservoir because we know that the sperm they can attach to the cells in the isthmus and undergo arrest - no choice as it’ll die without isthmus, sperm has no cytoplasm so must be arrested in the isthmus

Question 6

oviduct is the wiggly line under ampulla - ampulla: where fertilization happens

infundibulum: portion closer to the ovary that is responsible for catching the oocyte.

Question 7

Sperm Capacitation

• Sperm become capable of undergoing ? ? -> most imp. step before fertilization
• Biochemical (non-morphologic) changes required in ? tract before sperm are capable of ?
  
  • Removal of seminal plasma components, interaction with female secretions
• ?
• Changes in membrane ?, glycosaminoglycans (membrane fluidity)
• Oocyte receptor ?

Answer 7

Sperm Capacitation

• Sperm become capable of undergoing acrosome rxn -> most imp. step for fertilization and for it to gain the ability to fertilize the ovum.
• Biochemical (non-morphologic) changes required in female tract before sperm are capable of fertilization
  
  • Removal of seminal plasma components, interaction with female secretions
• Hypermotility
• Changes in membrane cholestrol, glycosaminoglycans (membrane fluidity)
• Oocyte receptor expression
• so sperm gets ejaculated in the cranial vagina or uterus and migrate to oviduct and got sum rest in the isthmus.
• then the oocyte gets ovulated and there are signalling from the oocyte which tells the sperm that the sperm needs to go to the ampulla but this sperm is not able to fertilize the oocyte.
• but the sperm is not able to fertilize the oocyte right now. recall, oocyte has cumanefeous oferus? and zona pellucida and sperm needs to cross all those cells. and for that sperm needs to go through sperm incapacitation and acrosomal rxn
• THUS SPERM W/ NO OR DAMAGED ACROSOME WON’T BE ABLE TO FERTILIZE THE OOCYTE as it won’t be able to cross zona pellucida.
• And the most important thing associated with sperm capacity is that they gain hypermotility and they only gain this after sperm capacitation.
• the sperm that have free sperm capacitation die as hypermotility will cause damage to sperm cell if they don’t reach the oocyte in time.

They must first undergo a series of biochemical and physiological changes, collectively known as capacitation. Capacitation is a significant prerequisite to fertilization. During the process of capacitation, changes in membrane properties etc, induce multiple signaling events in the female reproductive tract - These, in turn, stimulate the acrosome reaction and prepare spermatozoa for penetration of the egg ZONA PELLUCIDA prior to fertilization

Question 8

Acrosome reaction

** Essential for ? – allows sperm ? into the oocyte **
** ? dependent **

• Usually occurs upon contact of sperm with ? (?)
  
  • In at least some species during
    ? penetration
• Allows fusion of sperm plasma membrane with outer ? membrane
• Extensive ? over surface of sperm and acrosome
• Allows orderly release of ? enzymes

Answer 8

Acrosome reaction

** Essential for fertilization – allows sperm penetration into the oocyte **
** calcium dependent **

• Usually occurs upon contact of sperm with zona pellucida (oocyte)
  
  • In at least some species during
    cumulus penetration
• Allows fusion of sperm plasma membrane with outer acrosomal membrane
• Extensive vesiculation over surface of sperm and acrosome
• Allows orderly release of acrosomal enzymes
• from the lateral side we can tell that the sperm is flat
• DNA is stored in head of sperm cell, the midpiece of the sperm cell contians mitochondria and acrosome is around the head of the spermatozoa.

Question 9

Fertilization

Penetration of zona pellucida
* Sperm migrate between ? cells
* Attach to and migrate through ? ?
* Fusion of sperm and ? membrane
* Zona pellucida has 3 major proteins

• ZP3 is specific binding site for sperm
• Only acrosome-intact sperm bind to zona
• Sperm motility and ? required for ? penetration (e.g. acrosine)

Answer 9

• Sperm migrate between cumulus cells
• Attach to and migrate through zona pellucida
• Fusion of sperm and oocyte membrane
• Zona pellucida has 3 major proteins * ZP3 is specific binding site for sperm
• Only acrosome-intact sperm bind to zona
• Sperm motility and enzymes required for zona penetration (e.g. acrosine)
• • acrosoma will fuse with the head of the sperm cell’s PM and will release a bunch of granules which are imp. for sperm to cross the cumulus cells (shown in pic on the next slide) and the zona pellucida.
• when these granules can digest the zona pellucida and the cumulus cells, the sperm will be able to cross this and bind zona pellucida of the oocyte
• once the sperm cell fuses with the zona pellucida bc of the acrosomal rxn the sperm cell just releases DNA at this moment.
• so only portion that comes from our father is the DNA and mitochondria and others come from mom so lil bit more from mom.
• right after the sperm release the zona pellucida kind of solidifies and this is called “blocks to polyspermy”.

Question 10

Fertilization Gamete fusion

• Oocyte membrane (vitelline membrane) is less species-specific in binding than ZP
• Completion of ? reaction is required before sperm can fuse with oocytes
• Fusion involves post-? sperm membrane (equatorial region)
• Becomes fused (“intermingled”) with ?

Blocks to polyspermy
* The zona pellucida, becomes ? upon fertilization.
Extensive reorganization of ZP structure
* Driven by oocyte cortical ? released into perivitelline space after the releasing of soluble factors and ? by the sperm.

Answer 10

Fertilization Gamete fusion

• Oocyte membrane (vitelline membrane) is less species-specific in binding than ZP
• Completion of acrosomal reaction is required before sperm can fuse with oocytes
• Fusion involves post-acrosomal sperm membrane (equatorial region)
• Becomes fused (“intermingled”) with oolema

Blocks to polyspermy
* The zona pellucida, becomes impermeable upon fertilization.
Extensive reorganization of ZP structure
* Driven by oocyte cortical granules released into perivitelline space after the releasing of soluble factors and DNA by the sperm.

• right after the sperm release the zona pellucida kind of solidifies and this is called “blocks to polyspermy”. y imp? because we have 1 haploid cell that is the oocyte and 1 haploid cell that is the sperm cell.
• Each one carries 23 chromosomes (humans), after they fuse one normal embryo with 46 chromosomes or 23 pairs - normal cell.
• If for any reason another sperm comes and also releases its DNA (23 more chromosomes) so this embryo dies thus blocks to polyspermy (solidification of zona pellucida)

Question 11

Fertilization

• After sperm penetration into the oocyte
• Oocyte completes ?
  
  • Expels ? polar body
• Remaining maternal (haploid) chromosomes are enclosed in ** ? **
• Sperm ? envelope disintegrates
• DNA decondensation
• New envelope: ** ? ? **
• Male and female pronuclei migrate to cell center
• Nuclear envelopes disperse
• Intermixing (fusion) of ? occurs
• Chromosomes aggregate in which phase of meiosis? of first cleavage division
• Proximal centriole of sperm forms one of the zygote’s ?

Answer 11

Fertilization

• After sperm penetration into the oocyte
• Oocyte completes meiosis
  
  • Expels second polar body
• Remaining maternal (haploid) chromosomes are enclosed in ** pronucleus **
• Sperm nuclear envelope disintegrates
• DNA decondensation
• New envelope: ** male pronucleus **
• Male and female pronuclei migrate to cell center
• Nuclear envelopes disperse
• Intermixing (fusion) of chromosomes occurs
• Chromosomes aggregate in prophase of first cleavage division
• Proximal centriole of sperm forms one of the zygote’s centrioles
• so we have one oocyte with one polar body and then oocyte got arrested in metaphase II (means the species alr hit puberty) , during this time the sperm got activated and acrosomal rxn
• then it crosses the cumulus and then the zona pellucida and release the DNA
• then block of polyspermy takes place where zona pellucida solidifies and then the male pronucleus forms and after the oocyte releases the second polar body
• DNA is alr there and in the end oocytes release the second polar body and finishes meiosis II; after that male and female nucleus fuse and they will form the zygote
• the zygote then divides; once it reaches 16 cells that is last state at which the cell is TOTIPOTENT! and after the 16th stage is morula!

Question 12

Migration to uterus

• Early cleavage occurs in uterine tubes
• Zygote reaches uterus in about #? d in cows and most other species, but takes longer in horse (#? d) and dog (#? d)
• # ?-cell/compact morula stage (cows, sheep)
• ?/early blastocyst (mares and dogs)
• MARES
• Only ? zygotes transported
  through ? tubes (oviductal papilla)
• Zygote probably responsible for transport by secretion of hormone? (this hormone only produced by viable ones so that y able to move)

Answer 12

Migration to uterus

• Early cleavage occurs in uterine tubes
• Zygote reaches uterus in about 4 d in cows and most other species, but takes longer in horse (6 d) and dog (8 d)
• 16-cell/compact morula stage (cows, sheep)
• morula/early blastocyst (mares and dogs)
• MARES (horse)
• Only fertilized zygotes transported
  through uterine tubes (oviductal papilla)
• Zygote probably responsible for transport by secretion of hormone PGE2 (this hormone only produced by viable ones so that y able to move)
• so this oocyte released from ovary and got fertilized in the ampulla and now needs to move as pregnancy occurs in uterus and not in oviduct.
• how it happens? so most species will move around day 4 after ovulation and they just move regardless of fertility or not; if they are embryos or non-fertile oocytes etc.
• EVERYTHING WILL MOVE TO UTERUS. however in horses, only viable embryos move to the uterus, and they take a little bit longer around day six after ovulation
• the non-viable ones get stuck in oviduct
• in DOGS EVERYTHING MOVES AS WELL, but they take longer only bc the female dog ovulates an immature oocyte. so it takes around 3-4 days for the oocyte to finish meiosis I and II if fertilized and then more 4-5 days for oocytes to move to uterus.
• in equine species, the hormone PGE2 is really important and only viable embryo can produce this hormone hence why they are the only one to be able to mvoe to uterus.

Question 13

Early embryo development

• Several meiotic or mitotic? divisions
• Early divisions occur without increase in cell ?
  
  • “?” (or “? Division”)
• Metabolic support provided by
  ? secretions
• Zygotic protein synthesis begins at 2 to 16-cell stage depending on species (? cells)

Answer 13

Early embryo development

• Several mitotic divisions
• Early divisions occur without increase in cell mass
  
  • “cleavage” (or “reduction Division”)
• Metabolic support provided by
  maternal secretions
• Zygotic protein synthesis begins at 2 to 16-cell stage depending on species (TOTIPOTENT cells)
• We have the embryo moving up to 16 cells TOTIPOTENT and after that, this embryo will be a MORULA.
• and trophoblast produces sodium inside of the embryo and the inner cell mass starts pumping water and then this embryo will become after that a blastocyst.
• until the stage of morula the embryo carries the zona pellucida and remember until MORULA the embryo DOESN’T GROW IN SIZE and it only DIVIDES as it’s stuck inside zona pellucida until morula stage.

pic: ootid -> zygote -> 2-celled embryo -> morula -> early blastocyst -> hatching blastocyst.

Question 14

Hatched Blastocyst

The ? hatches out from the zona pellucida (beginning of ?)

• Increase ? in the blastocele
• Proteolytic enzymes from the ?
• ? contraction

** Most species – ?-? days
Mares – ?-? days* -
however, equine embryos have a ? surrounding the ZP, therefore, the ? shape is maintained **

Answer 14

Hatched Blastocyst

The blastocyst hatches out from the zona pellucida (beginning of implantation)

• Increase fluid in the blastocele
• Proteolytic enzymes from the trophoblast
• blastocysts contraction

** Most species – 7-10 days
Mares – 9-10 days* -
however, equine embryos have a capsule surrounding the ZP, therefore, the rounded shape is maintained **

• at this stage of blastocyst, the embryo will hatch from zona pellucida as it needs space to grow and will not be in zona pellucida anymore.
• when embryo hatch from zona pellucida it starts to grow in size and becomes a blastocyst.
• at this stage ruminants embryo loses this and also loses its shape so not rounded anymore
• but in horse, in this stage produces another sturc. called protien capsule that maintains the roundedness of the embryo
• so in horses the hatching happens lil bit later i.e. around 9 - 10 after ovulation and is aimed to already produce the capsul So the embryo hatch inside of that capsule and maintain the rounded shape.
• in other species hatch earlier and there’s no capsule so they lose round shape and ruminants the embryo elongates which is imp. for maternal recognition of pregnancy.

Question 15

Important Early Events

• Development inside zona ? (ZP)
• ? from ZP
• ? recognition of pregnancy

Formation of extraembryonic membranes
* Yolk sac – nutrition and sex differentiation

• Amnion – amniotic ? is important for ? protection, ?, temperature changes, ? movement – mostly water from other cells
• Allantois - collect secretions from the ? (fetus) that’ll be around of that (also in contact with placenta) and helps with ? exchange
• Chorion (outermost fetal membrane) – external layer of the ? that attaches with the ? - nutrition of the embryo

Answer 15

Important Early Events

• Development inside zona pellucida (ZP)
• hatching from ZP
• maternal recognition of pregnancy

Formation of extraembryonic membranes
* Yolk sac – nutrition and sex differentiation

• Amnion – amniotic fluid is important for mechanical protection, hydration, temperature changes, fetus movement – mostly water from other cells
• Allantois - collect secretions from the embryo (fetus) that’ll be around of that (also in contact with placenta) and helps with gases exchange
• Chorion (outermost fetal membrane) – the external layer of the placenta that attaches with the uterus - nutrition of the embryo
• After this time there is a fetus maturation
• We have the yolk sac at the beginning that’s important for the nutrition of this
  and for sex differentiation

Question 16

Maternal recognition of pregnancy

Critical process in which mother “recognizes” she is ?

Major consequence is to prolong period of function of ?

Transition from ? to pregnant state

Different process in each species
Two major strategies

Anti-luteolytic
Diversion of PGF2α secretion
Inhibition of PGF2α secretion

Luteotropic
Maintain CL (e.g. hCG in human)

Answer 16

Maternal recognition of pregnancy

Critical process in which mother “recognizes” she is pregnant

Major consequence is to prolong period of function of corpus luteum

Transition from cycling to pregnant state

Different process in each species
Two major strategies

Anti-luteolytic
Diversion of PGF2α secretion
Inhibition of PGF2α secretion

Luteotropic
Maintain ? (e.g. hCG in human)

• maternal recognition of pregnancy will avoid luteolysis bc of the anti-luteolytic effect OR delays/inhibites the production of prostaglandin or changes the course of that prostaglandin produced by the endometrium
• or in primates there is production of chorionic gonadotropin (hCG) which acts as a luteotropic agent so increase the development of the CL, 2 months into a pregnancy.

Question 17

Maternal recognition of pregnancy

• Pregnant and non-pregnant cycle similar for first ? days
• Mechanism not completely understood in ?

Embryo mobility - horses
Interferon tau (IFNT) - ?
Estrogens - pigs
Chorionic gonadotropin - ?

Answer 17

Maternal recognition of pregnancy

• Pregnant and non-pregnant cycle similar for first 14-16 days
• Mechanism not completely understood in mares

Embryo mobility - horses
Interferon tau (IFNT) - ruminants
Estrogens - pigs
Chorionic gonadotropin - primates

• In horses. We know that the maternal recognition of pregnancy takes place because of embryo mobility so they aim to produce capsule - round shape and bc of shape embryo literally moves into uterus
• HYPOTHESIS is embryo needs to reach all cells in the endometrium to avoid that the endometrium will open the receptors of oxytocin - so the embryo needs to move.
• So in ruminants including cows there is a production of interferon tau (IFNT) by the embryo as it needs to elongate,

so they aim to elongate -> will reach all the surface of the endometrium and produce just this interferon TAU that’ll avoid the endometrium wall opening their receptors for oxytocin -> thus avoiding the production of prostaglandin and luteolysis.

• And also in pigs, we know the estrogens are important and in primates, the choriono gonadotropin.

Question 18

Skim it !

Maternal recognition of pregnancy in ruminants

• After hatching from zona pellucida blastocyst elongates
• Contacts most of uterine luminal epithelium
• Trophoectoderm (future “placenta”) produces Interferon tau (IFNτ)
• IFNτ
• Inhibits oxytocin receptor synthesis * Inhibits prostaglandin F2α
• Stimulates endometrial glands

Question 19

Maternal recognition of pregnancy in swine

• ? produces Estradiol

Estradiol changes ? of PGF2α secretion by ?
* Endometrial-derived PGF2α is directed away from the uterine vasculature (endocrine secretion) and sequestration into the uterine ? (exocrine secretion)

• PGF2α is ? in lumen
• PGF2α is metabolized to its ? 13, 14-dihydro-15-keto prostaglandin F2α metabolite

Answer 19

Maternal recognition of pregnancy in swine

• blastocyst produces Estradiol

Estradiol changes direction of PGF2α secretion by endometrium
* Endometrial-derived PGF2α is directed away from the uterine vasculature (endocrine secretion) and sequestration into the uterine lumen (exocrine secretion)

• PGF2α is inactivated in lumen
• PGF2α is metabolized to its inactive 13, 14-dihydro-15-keto prostaglandin F2α metabolite
• how it happen in pigs? embryos produce estrogens.
• These estrogens produced by the embryos or just change the course of prostaglandin does not inhibit the receptors for oxytocin
• So there is production of prostaglandin by the endometrium in pigs BUT instead of prostaglandin going to the bloodstream, and cause luteolysis, these prostaglandins go to uterus and the estrogen produced by the fetus will metabolize the prostaglandin
• and so this prostaglandin is inactivated and does not cause luteolysis of CL so ESTROGEN produced by the fetus is the way that maternal recognition of pregnancy happens in pigs species.

Question 20

Maternal recognition of pregnancy in horses
Embryo mobility

• Fertilization in ? tube
• Viable embryo enters uterus around #? day after fertilization (first maternal
  recognition of pregnancy)
• Transport depends on ?
• Equine embryo migrates through both ? horns and body ? times a day
• Up to #? horn changes per day
• Movement is mediated by ?
• But simulated (fake) vesicles move much less
• Some signal from ? (PGE)?
• Movement is maximal around days ?-?
• Prevention of movement results in pregnancy loss - therefore MOVEMENT MUST OCCUR

(And then we just talk about these is the embryo anything mobility horses that we know is important for maternal recognition.)

Answer 20

Maternal recognition of pregnancy in horses
Embryo mobility

• Fertilization in uterine tube
• Viable embryo enters uterus around 6 day after fertilization (first maternal
  recognition of pregnancy)
• Transport depends on PGE2
• Equine embryo migrates through both uterine horns and body several times a day
• Up to 18 horn changes per day
• Movement is mediated by uterus
• But simulated (fake) vesicles move much less * Some signal from embryo (PGE)?
• Movement is maximal around days 10 – 12
• Prevention of movement results in pregnancy loss