What are the three main products of spermatogenesis and what are their chromosomal numbers?
Spermatogonia → Spermatocytes → Spermatids
Spermatogonia = 2n (lots of mitosis create lots of 2n spermatogonia)
Spermatocytes = 2n
Spermatids = 1n (x4)
Where does mitosis of spermatogonia (2n) occur in the testes?
Basal membrane of seminiferous tubules.
Where do the first and second rounds of meiosis occur in the testes?
Both meiosis 1 & meiosis 2, which result in spermatocytes (2n) & spermatids (1n) respectively occur in the adluminal compartment of the seminiferous tubules.
What are the three main STAGES of spermatogenesis?
1. Mitotic division - spermatogonia (2n)
2. Meiotic division - spermatocytes (2n) & spermatids (1n)
3. Cytodifferentiation - spermatozoa (1n)
What happens during the third stage of spermatogenesis, cytodifferentiation?
1. Think CYTOplasm - cytoplasm is condensed for elongation of spermatid into spermatozoa. Material removed by Sertoli cells.
2. Different genes are packaged for delivery to oocyte.
Where are Sertoli cells located and what roles do they play in spermatogenesis?
They're located in the seminiferous tubules.
1. They are responsible for providing proteins to spermatocytes via gap junctions during meiotic-divisions stage.
2. They remove cytoplasm during cytodifferentiation from spermatids as they elongate into spermatozoa.
3. They have receptors for testosterone from Leydig cells to convert testosterone to dihydrotestosterone, which enters testicular fluid and affects male erection, puberty & traits, or enter circulation to negatively feedback on FSH & LH
4. Convert some testosterone to oestrogen.
5. Secrete fluid in which spermatozoa are transported from seminiferous tubules to rete testes & epididymis.
Why would you assess testes function?
The testes are the site of spermatogenesis.
Assessments of whether the testes are functioning properly are used to test for:
Delayed or precocious puberty
Decreased sex drive
What are the two different types of tests that assess for testes function?
1. Blood test for testosterone production
2. Blood test for gonadotrophins
In a blood test for testosterone, what would you be looking for?
Very high plasma concentrations of testosterone could indicate testicular or adrenal tumours in men, potential polycystic ovarian syndrome (PCOS) in women.
Once the sperm is in the head of the epididymis, what happens?
1. Fluid from sertoli cells resorbed
2. Thus sperm are concentrated 100-fold
3. Sperm subsequently transported by muscles in epididymis
What happens in the corpus of the epididymis?
Modification of environment & maturation of sperm (basically - addition of surface molecules: proteins & carbohydrates):
Sperm structure changes:
– Loss of surplus cytoplasm (removal of cytoplasmic droplet)
– Condensation of nuclear chromatin by disulphide bridges
– Addition of surface glycoproteins stabilise plasma membrane
Sperm membrane changes:
– Membrane fluidity and lipid composition change: Cholesterol selectively metabolised shifting balance towards diaglycerol and unsaturated fatty acids; membrane made more resistant, more permeable to water, stronger against pH changes, depending on ratio of fatty acid and cholesterol
– Depression of metabolic activity to prolong life of cell
– Increased dependence on external fructose for energy production
- cAMP content of tail increases allowing acquisition of motility
What are the two main ways of assessing the plasma-membrane integrity of sperm in ejaculate?
1. Fluorescent staining with DNA-specific probes SYBR-14 and/or Propidium Iodide (PI) stains & flow cytometry
2. Fluorescence-Activitated Cell Sorting (FACS)
Describe how flow cytometry with DNA-specific probes SYBR-14 & Proprium iodide (PI) assess the integrity of sperm cell membranes?
What are the advantages?
Flow cytometry laser excites fluorescent particles in sperm causing them to emit light
Fluorescent detectors assess # of sperm passing through laser beam emitting light of specific wavelength, thus enabling you to determine proportion of SYBR-14/ PI stained sperm
SYBR-14 is a small molecule that stains intact plasma membranes and fluoresces green. PI is a big molecule that stains breached/degenerated plasma & fluoresces red.
Advantage of this technique is it evaluates thousands of sperm in real time.
Describe how Fluorescence-Activated Cell Sorting (FACS) can assess the integrity of sperm cell membranes, as well as sort semen?
FACS is a specialised type of flow cytometry.
It sorts sperm into subpopulations based on fluorescent labelling, one cell at a time
- FACS machine assigns electrical charge to sperm fluorescing at specified wavelength
- Sperm then sorted by electrical charge
- Specific probes for viability and chromosomes (sex)
Viability - green, positively charged sperm migrate to anode (with intact plasma membranes); red, negatively charged sperm migrate to cathode (damaged), sorted away from good sperm.
Sexed semen: stained sperm sorted by XX (female) and XY (male) chromosomes (X is so much bigger)
How would you test sperm's DNA integrity (vs. plasma integrity)? What is it assessing?
Comet Assay - assesses fragmentation of sperm DNA
How does a Comet Assay work?
What are the disadvantages?
Single-cell gel electrophoresis assay:
- Sperm placed in agarose gel & lysed
- Gel electrophoresis
- Intact DNA is too large to move down gel
- Fragments of damaged DNA will move
- Florescent staining of DNA to assess fragmentation
Not very precise: assay destroys each sperm after evaluation, so process only assesses one sperm from which to extrapolate / infer assessment of
What are the four accessory glands that produce seminal plasma?
What makes up the seminal fluid produced by accessory glands?
Glycoproteins (decapacitation factors) - to coat plasma membrane, to protect zona pellucida-binding proteins
Fructose & Sorbitol (SV & ampulla) – energy substrate
Citric acid (SVandprostate) – stops cell coagulation and formation of seminal ’stones
Buffers (SV) – neutralise acid pH of vaginal fluids
Ascorbic acid – reducing agent to protect against sperm oxidation at ejaculation
Prostaglandins – stimulate muscle contractions in female tract