LECTURE 28 - fertilisation and contraception

Question 1

What is the role of the Bulbourethreal Gland (Cowper’s gland)?

Answer 1

• pre-ejaculate
• lubrication
• neutralisation

Question 2

What is ovulation?

Answer 2

Cumulus-oocyte complex is picked up by ciliated fimbriae on the end of the uterine tube

Question 3

How is sperm transported in the female tract?

Answer 3

• ~200 million sperm deposited in upper vagina
• seminal plasma = short term buffering
• cervical mucus is least vicious during the 9-16 days of menstrual cycle
  ~100,000 sperm enter the uterus
  ~1000 sperm enter the uterine tube with 50% chance of choosing uterine tube with oocyte

Question 4

Where does fertilisation occur and when can it occur?

Answer 4

• occurs in ampulla region of uterine tube
• sperm remain capable of fertilisation for ~5 days within female tract
• oocyte remains viable for ~24 hours
• ovulation prediction kits (OPSKs) - looking for LH as huge surge occurs before ovulation

Question 5

Describe the sperm interaction with egg vestments

Answer 5

1. remote detection of oocyte-cumulus complex
2. penetration of cumulus
3. zona binding
4. acrosome reaction
5. zona penetration

Question 6

Explain penetration of the cumulus

Answer 6

• approx. 3,000 cells embedded in gelatinous matrix (hyaluronic acid)
• closely apposed cells form tight, organised layers
• sperm penetrate and can disperse the cumulus

Question 7

What is the zona pellucida?

Answer 7

• extracellular protein matrix which surrounds all mammalian eggs
• 4 glycoproteins in humans (ZP1-4)
• important for sperm-egg binding and induction of acrosome reaction (AR)
• persists post-fertilisation

Question 8

What is the acrosome reaction?

Answer 8

• permits zona penetration
• exposes new membrane for oocyte fusion
• only capacitated sperm can undergo AR
• hyper activated motility

Question 9

What is fusion?

Answer 9

• sperm penetrates ZP and occupies the perevitelline space
• equatorial segment of sperm head fuses with oocyte plasma membrane
• sperm nucleus is encased by a vesicle composed of internalised oocyte membrane
• large increase in the free [Ca2+] - sweeps across egg from point of sperm fusion

Question 10

What is izumo?

Answer 10

• sperm membrane receptor for fusion
• detectable on sperm surface only after AR
• KO completely abolishes fusion

Question 11

What is Juno?

Answer 11

• receptor for Izumo on the oocyte plasma membrane

- KO abolishes function

Question 12

How are oocytes activated?

Answer 12

• within 1-3 mins of fusion - large rise in [Ca2+] which sweeps across egg from point of sperm entry - lasts 2-3 mins
• followed by Ca2+ oscillations every 3-15 mins which may last hours
• Ca triggered by phospholipase C zeta - sperm specific PLC

Question 13

How does oocyte activation cause the release of meiotic block?

Answer 13

• M-phase Promoting Factor (MPF) = cyclin-dependent kinase (cdk1) plus cyclin B - blocks metaphase to anaphase transition
• MPF is stabilised by Cytostatic Factor (CSF)
• raised Ca levels suppress CSF activity and destroy cyclin B by activating anaphase-promoting complex/cyclosome (APC/C) - causes expression of ubiquitin (E3) ligase which targets cyclin B for destruction and once lost meiosis can continue
• as well as this, cohesion protein complexes (hold sister chromatids together - oppose pulling force of microtubules
• scc1 subunit of cohesin is cleaved by separase
• securin - inhibits separase activity until ubiquitinated by APC/C (caused by increased Ca levels)
• resumption of cell cycle in the oocyte and completion of meiosis II

Question 14

How does black to polyspermy occur?

Answer 14

Polyspermy = more than one sperm fertilises an egg 
Fast block
- electrical 
- membrane depolarisation
- takes minutes

Slow block

• cortical reaction = cortical granules fuse and release a mixture of enzymes (inc. proteases) which diffuse into zona pellucida => induces zona reaction
• triggered by increased [Ca2+]
• takes 1hr+

Question 15

What is the Zona reaction?

Answer 15

• the alteration in the structure of the zona pellucida catalysed by proteases from cortical granules
• cleavage of ZP2 by ovastacin protease
• sperm can no longer bind or penetrate

Question 16

What happens as a result of loss of Juno?

Answer 16

• Juno protein is shed from oocyte plasma membrane with the cortical granules
• undetectable within 40 minutes of fusion
• no further sperm can fuse

Question 17

What do the sperm and oocyte contribute?

Answer 17

Sperm

• haploid male genome - sex of the baby
• centriole - the oocyte does not have one, forms spindle for first cell division

Oocyte

• haploid female genome
• cytoplasm
• all organelles
• mitochondria; maternally inherited

Question 18

What is the zygotic/ pronucleate (2PN) stage of fertilisation?

Answer 18

• decondensation of sperm DNA (protamine/ histone exchange)
• male and female pronuclei replicate their DNA
• pronuclei migrate towards each other
• guided by sperm aster - microtubules radiating from centrosome, essentially capture other pronucleus

Question 19

What is syngamy?

Answer 19

• combination of male and female genetic material
• occurs 18-24 hours after fusion
• pronuclear membrane break down
• chromatin intermixes
• nuclear envelope reforms around zygote nucleus
• cleavage begins - end of fertilisation/ beginning of embryogenesis

Question 20

What are cleavage stages?

Answer 20

• occurs over next two weeks after fertilisation
• zygote cleaves to form 2 blastomeres (cells of embryo)
• 4-cell, 8-cell stages: pre-implantation genetic testing (PGT) - embryo transfer occurs in IVF
• morula: 16-32 cells - solid ball near end of uterine tube
• no cytoplasmic synthesis so blastomere size decreases with each division
• ZP still in place around zygote

Question 21

What is blastocyst formation?

Answer 21

• late day 4/5
• blastocoel = fluid filled cavity
• distinct ICM and single-layered trophoblast
• embryonic (next to mass) and abemryonic pole
• embryo transfer in modern IVF occurs at this stage because embryonic genome activated and past stage of totipotency

Question 22

What is hatching?

Answer 22

• late day 6 onwards
• blastocyst expands out of hole in ZP - abemryonic pole
• implantation

Question 23

Describe the structure of the endometrium

Answer 23

• thick muscular myometrial later
• uterine lining = endometrium made of 2 layers
  1. Basal layer - attached to myometrium; remains intact during menstruation
  2. Functional layer - undergoes proliferation then shedding (menstruation). reconstituted out of the underlying basal layer

Question 24

What is the follicular phase of menstruation?

Answer 24

• follicular = proliferative
• after menstruation, endometrium is very thin and consists only of few layers of cells (basal layer)
• in the first ~14 days, endometrial cells proliferate due to a rise in oestrogen

Question 25

What is the luteal phase of menstruation?

Answer 25

• luteal = secretory phase
• after ovulation, ovaries produce progesterone
• progesterone stimulates synthesis of secretory material by the glands - rich in glycogen, glycoproteins and amino acids => provides nutrition for blastocyst
  => receptive endometrium that is ready for implantation

Question 26

What are the 5 classes of contraception?

Answer 26

1. hormonal
2. barrier
3. IUDs
4. permanent
5. natural

Question 27

What are the hormonal methods of contraception?

Answer 27

• mimic hormonal levels during luteal phase - feedback inhibition on HPG axis
• constant exposure to progesterone suppresses ovulation
• progesterone also causes thickening of the cervical mucus and decreases endometrial receptivity
• oestrogen exerts addition -ve feedback and induces PR expression increasing effect of progesterone
• -> improves cycle control - irregular bleeding

Question 28

What are the barrier methods of contraception?

Answer 28

• prevent pregnancy by blocking the egg and sperm from meeting
• barrier methods have higher failure rates than hormonal methods due to design and human error
  Examples
  Condom
• most common and most effective (85-98%)
• used in prevention of pregnancy and spread of STIs

Diaphragm and cap

• latex barriers placed inside vagina before intercourse
• spermicidal jelly before insertion
• effectiveness = 84-94%

Question 29

What are intrauterine devices (IUDs)?

Answer 29

• placed in uterus - copper (presence increases spermicidal effect)
• lasts 5-12yrs (LARC = long-acting reversible contraception)
• extremely effective without use of hormones >99%
• release of leukocytes and prostaglandins by endometrium - hostile to both sperm and embryos
• post-fertilisation mechanisms contribute to effectiveness (emergency contraception option 2)
• side effects = heavy periods

Mirena IUS

• lasts 5 years
• acts as IUD plus continuously releases small amounts of progestin (atrophy of endometrium)
• thickening of cervical mucus, may suppress ovulation
• reduction in menorrhagia and dysmenorrhea
• 99.9% effective

Question 30

What are the permeant methods of contraception?

Answer 30

• permanent sterilisation
• Female = uterine tubes (99.5%)
• Male = vasectomy - vas deferens (99.8%)

Question 31

What are the natural methods of contraception?

Answer 31

• rhythm method (menstrual cycle ~75%)
• fertility awareness method (temp, cervical mucus and position: 75-95%)
• coitus interruptus/ withdrawal (73%)
• natural family spacing
  
  • lactational amenorrhea
  • prolactin
  • 6 months post part, breaks feeding exclusively with gaps
  • up to 98% effective but unpredictable
• abstinence (100%)