What is important about sperm's storage in cervical crypts?
1. Sperm is only released from crypts in absence of progesterone (so after CL has regressed/luteal phase is over & female is ready for another cycle)
2. Cervix selects against morphological abnormalities
What's important about sperm storage in the isthmus of the oviducts?
It seems to be able to delay maturation and release of sperm coincides with ovulation.
What happens during the acrosome reaction?
Sperm binds its acrosome site ZP3-binding proteins to the oocyte's outer layer, the zona pellucida.
Some 50,000 ZP3 binding molecules are activated to initiate the acrosome reax (fusion of sperm membranes to release enzymes):
1. Fusion of sperm's outer membrane with outer acrosomal membrane, creating pores for enzymes to escape.
2. Enzymes hyalurinase & acrosin released to digest hole in oocyte's zona pellucida (& thru perivitelline space).
3. Exposure of sperm's equatorial segment at sperm's "neck" & oocyte, which bind.
How would you assess both capacitation & the acrosome reaction of the sperm?
How does the assessment work?
Chlortetracycline (CTC) staining:
NB this is only used in labs.
Capacitation causes Δs in [Ca++]i (decrease)
CTC binds to [Ca++]i in sperm membranes causing fluorescence; if it fluoresces, then it hasn't capacitated yet or gone through acrosome reax; still lots of Ca++
Three classifications of sperm:
1/ Uncapacitated & acrosome-intact - ideal for AI!! - uniform fluorescence over whole head
2/ Capacitated & acrosome-intact - fluorescence-free band just behind sperm head
3/ Capacitated & acrosome-reacted - dull or absent fluorescence in head
How would you assess whether sperm have undergone the acrosome reaction only?
Fluorescent staining with arachis hypogea (peanut glutinin), aka FITC-PNA
PNA binds to outer acrosomal membrane:
Bright green, defined edges = acrosome intact
Fragmented, ruffling = acrosome damaged
Only equatorial staining, smaller = acrosome reacted
What happens once the sperm head enters the oocyte's perivitelline space?
Remember the perivitelline space is beneath the zona pellucida but outside of the oocyte plasma membrane and cytoplasm.
1. The sperm head is surrounded by microvilli coming up from the oocyte's plasma membrane.
2. Sperm's equatorial region fuses with oocyte's plasma membrane. Sperm becomes engulfed by the membrane and the oocyte's cortical granules are released into the perivitelline space, causing a block to polyspermy (no other sperm can enter).
3. Sperm nucleus, now engulfed fully in oocyte cytoplasma, decondenses its chromatin/nucleus to form the male pronucleus (1n)
4. Fertilization - the male & female pronuclei fuse to form zygote (2n)
What is zygote cleavage?
After fertilisation, zygote undergoes a series of mitotic divisions known as cleavage.
It goes from one-cell zygote to a two-cell embryo, after which it continues through more mitotic divisions.
Each of the two cells cells in the embryo is called a blastomere.
Blastomeres cleave to form first an 4, then 8, then 16-cell embryo, when it is called a morula (after the eight-cell stage).
What is a morula and why is it important for genetic testing even though it hasn't even implanted in the uterus yet?
A morula is an embryo that has gone beyond the eight-cell stage.
The 16-cell morula can be tested for genetic diagnosis of hair colour, sex, skin colour and health issues such as blindness or deafness.
At the morula stage, the cells separate into two distinct layers. What are they, and what do they become?
Inner Cell Mass (ICM) - becomes embryo
Peripheral cells, or trophoblasts - becomes placenta
What is the name given to the morula after it has formed two distinct cell layers, the ICM & the trophoblast layer?
What is the blastocoele and how is it formed? What does it become?
The blastocoele is an accumulation of fluid inside the blastocyst, formed by the pumping of sodium (and water) into the cell by the trophoblasts on the periphery.
The blastocoele together with the hypoblast layer of the blastocyst/implanted embryo become the yolk sac during gastrulation, which follows implantation.
How does the blastocyst implant?
It hatches out of the zona pellucida, becoming a free-floating embryo dependent on the uterus for survival.
What role do trophoblasts play in implantation of the embryo?
Trophoblasts invade the uterine epithelium, lose their plasma membranes and fuse with the endometrium. The degree of fusion varies, which is why there is variation between species in the types of placenta (haemochorial, epichorial, endochorial). Eg. horses form endometrial cups.
This trophoblast invasion & fusion with uterine endometrium forms syncitiotrophoblast - the early placenta.
The implanted blastocyst, an embryo, consists of an inner cell mass & a syncytiotrophoblast layer, which is fused with the uterine endometrium.
The inner cell mass differentiates into two cell layers. What are they?
Epiblast & Hypoblast
Where is the epiblast layer in the embryo, and what does it differentiate into?
The epiblast layer is located next to syncytiotrophoblast to maximise exposure to maternal nutrients.
It gives rise to all embryonic tissues.
Where is the hypoblast in the embryo & what does it give rise to?
The hypoblast is located between the epiblast layer and the blastocoele. It doesn't form anything as it hypoblast cells are later displaced by primitive endoderm cells that line the blastocoele & form the yolk sac.
What are the three germ layers that come from epiblast cells?
What is derived from the ectodermal layer of the epiblast during gastrulation?
All neural tissue and the epidermis
What is derived from the mesoderm layer of the epiblast during gastrulation?
Muscles, bones, organs
What is derived from the endodermal layer of the epiblast during gastrulation?
Lining of gut & derivatives