SUGER 🍦🍧🍨🍩🍪🎂🍭🍬🍫 Flashcards
What proteins contribute to polycystic kidney disease?
Polycystin-1 causes PKD1
Polycystin-2 causes PKD2
How much of the cardiac output does the kidney receive?
Each kidney receives 10% of the cardiac output
Not just to meet their metabolic requirements, but to filter and excrete the metabolic waste products of the whole body
Key volumes of the kidney
Cardiac output - 5 L/min
Renal blood flow- 1 L/min
Urine flow- 1 ml/min
What do afferent and efferent mean?
afferent away
efferent towards
Factors determining filtration
Pressure
Size of the molecule
Charge
Rate of blood flow
Protein binding
How does pressure affect glomerular filtration?
Favours filtration:
Glomerular capillary blood pressure (PG)
Opposes filtration:
Fluid pressure in Bowman’s space (PBS)
Osmotic forces due to protein (πG)
How does size affect glomerular filtration?
Small molecules and ions up to 10kDa can pass freely
e.g. glucose, uric acid, potassium, creatinine
Larger molecules increasingly restricted
e.g. plasma proteins
How does charge affect glomerular filtration?
Fixed negative charge in GBM (glycoproteins and proteoglycans) repels negatively charged anions
e.g. albumin, phosphate, sulfate, organic anions
How does protein binding affect glomerular filtration?
Albumin has a molecular weight of around 66kDa but is negatively charged ∴ cannot easily pass into the tubule
Filtered fluid is essentially protein-free
Tamm Horsfall protein in urine produced by tubule
Affects substances that bind to proteins e.g. drugs, calcium, thyroxine etc
Glomerular filtration rate equation
Glomerular filtration rate = filtration volume per unit time (minutes)
GFR = KF (PG - PBS) - (πG)
KF is the filtration coefficient
Net filtration is normally always positive
Units are ml/min/1.73m2
What is GFR determined by?
-Net filtration pressure
-Permeability of the filtration barrier
-Surface area available for filtration (approx. 1.2-1.5m2 total)
GFR is not measured directly- how is it measured?
Calculated by measuring excretion of marker (M)
CM = UMV/PM
V = urine flow rate (ml/min)
UM = urine concentration of marker
PM = plasma concentration of marker
Properties of a good marker to measure GFR
Properties of a good marker:
freely filtered
not secreted or absorbed
not metabolised
∴ All the M that is filtered will end up in the urine, no more (as it is not secreted) and no less (as it is not reabsorbed)
Normal GFR
Has to be above 90
normal is 125ml/min
Why is creatinine used?
- Muscle metabolite
- Constant production
- Freely filtered
- Not metabolised
Although tubular secretion which is not the best as should not be secreted or absorbed
Things affecting creatinine
Gender
Height
Age
muscle damage
muscle mass
Supplements/ medications
Weight
Renal tubular handling
Outline cystatin c as a marker for measuring GFR
Cystatin C
Non-glycosylated protein produced by all cells
Properties of a good marker:
freely filtered ✓
not secreted or absorbed ✗ (reabsorbed)
not metabolised ✗ (metabolised)
Influenced by thyroid disease, corticosteroids, age, sex and adipose tissue
Inulin as a marker for measuring GFR
Inulin (gold standard)
Properties of a good marker:
freely filtered ✓
not secreted or absorbed ✓
not metabolised ✓
51Cr EDTA
99mTc-DTPA
Radioisotopes
Iohexol
Pressure regulation in the kidneys
Aim to maintain renal blood flow and GFR over defined range 80-180 mmHg
Protects against extremes of pressure
Independent of renal perfusion
Outline renal autoregulation of pressure
Myogenic mechanism
Tuboglomerular feedback
Outline myogenic mechanism
- Intrinsic ability of renal arterioles
- Able to constrict or dilate
- only pre glomerular vessles
- opposite for low bp
How does the myogenic mechanism work?
↑BP → stretches blood vessel wall → opens stretch-activated cation channels → membrane depolarisation → opens voltage-dependent calcium channels → ↑ intracellular calcium → smooth muscle contraction → ↑ vascular resistance → minimises changes in GFR
↓BP causes the opposite
ONLY PRE-GLOMERULAR RESISTANCE VESSELS
Outlinw tuboglomerular feedback
Juxtaglomerular apparatus
Stimulus NaCl concentration
Influences AFFERENT arteriolar resistance
Outline neural regulation of glomerular regulation
Sympathetic nervous system:
-Vasoconstriction of AFFERENT arterioles
-Important in response to stress, bleeding or low BP
Outline hormonal regulation of glomerular filtration
Renin-Angiotensin-Aldosterone System (RAAS)
Atrial Natriuretic Peptide (ANP)
Outline the Renin-Angiotensin-Aldosterone System (RAAS):
Renin released from JGA
Initiates cascade
Aldosterone influences Na reabsorption at distal tubule which influences blood volume and pressure
Outline the role of Atrial Natriuretic Peptide (ANP) in glomerular filtration
Released by atria
Stimulus of blood volume
Vasodilation of AFFERENT arterioles
What does the RAAS system do?
Negative feedback mechanism
Stabilises RBF and GFR
Minimises impact of changes in BP on Na excretion
Without renal autoreg - increase in BP leads to increase GFR and losses
How do intrarenal baroreceptors affect glomerular filtration?
- Respond to changes in pressure in glomerulus
- Influence diameter of AFFERENT arterioles
Outline the affect of extracellular fluid volume on glomerular filtration
Changes in blood volume
Resultant hydrostatic pressure
Effect of Blood colloid osmotic pressure
on glomerular filtration
Oncotic pressure exerted by proteins
Effect of inflammatory mediators in glomerular filtration
Local release of prostaglandins, nitric oxide, bradykinin, leukotrienes, histamine, cytokines, thromboxanes
What causes vasodilation of afferent arteriole?
Prostaglandins
Nitric slide 50
slide 51
slide 52
slide 53
Outline glomerular nephritis
Umbrella term
Causes: infection (bacterial/viral), autoimmune disorders, systemic diseases
Presentation: haematuria, proteinuria, hypertension, impaired kidney function
Outline nephrotic syndrome
Umbrella term
Increased permeability of glomerular filtration barrier
Presentation: triad of oedema + proteinuria + low albumin
Outline IgA neuropathy
Deposition of IgA antibody in the glomerulus
Resultant inflammation and damage
Cause: immune-mediated
Presentation: haematuria, potentially following resp/GI infection
Outline membranous neuropathy
Thickening of GBM
Most common cause of nephrotic syndrome in adults
Cause: primary or secondary
Presentation: proteinuria (often leading to nephrotic syndrome)
Outline diabetic neuropathy
Prolonged exposure to high blood glucose
Presentation: initially asymptomatic then progresses to proteinuria, hypertension and reduced kidney function
Outline minimal change disease
Type of nephrotic syndrome, only in children
Only visible under electron microscope
Outline Alport syndrome
Genetic disorder affecting GBM (X linked or autosomal recessive)
Progressive kidney damage
Potentially includes hearing loss and eye abnormalities
Define acidosis
Disorder tending to make blood more acid than normal
Define alkalosis
Disorder tending to make blood more alkaline than normal
Define acidemia
Low blood pH
Define alkalemia
High blood pH
Factors affecting pH
Metabolic component
intrinsic acid
extrinsic acid
1more
resp CO2 component
CO2 bicarbonate
What is Stewart’s strong ion difference?
Principle: pH and HCO3- are dependent variables governed by:
pCO2
Concentration of weak acids (ATOT)
ATOT = Pi + Pr + Alb
Strong ion difference (SID)
SID = Na+ + K+ + Mg2+ + Ca2+ – Cl- – other strong anions (eg lactate, ketoacids)
Strengths and problems with Stewart’s strong ion difference
Strengths:
Identifies the factors controlling pH
Problems:
Calculation can be very problematic
Probably adds little in practice
How do you diagnose an acid base disorder?
Disorders can be divided into acidoses – disorders that tend to make the blood acid and alkaloses
Disorders can be respiratory (ie driven by changes in CO2 excretion) or metabolic (ie driven by changes in acid load, acid excretion or bicarbonate recycling)
Different disorders can co-exist (mixed patterns)
What is measured in arterial blood gas?
pH
pO2
pCO2
Std HCO3-
Std Base excess
May include other measures (eg lactate, Na+, K+)
What is standard bicarbonate for?
What it would be if CO2 was normal- allows you to ignore the resp component of acid base in the blood
Outline standard bicarbonate
Measures of metabolic component of any acid-base disturbance
Absolute bicarbonate is affected by both respiratory and metabolic components
Standard bicarbonate is the bicarbonate concentration standardised to pCO2 5.3kPa and temp 37
Bicarbonate and std bicarbonate are calculated not actually measured
What is base excess?
Quantity of acid required to return pH to normal under standard conditions
Standard base excess corrected to Hb 50g/L
Can be used to calculate bicarbonate dose to correct acidosis 0.3xWtxBE (but not generally used in practice)
Base excess is negative in acidosis, can be referred to as base deficit
How do we interpret acid-base status?
2 major approaches:
Henderson
Stewart’s theory (strong ion difference)
What are the clinical features of acidosis
long term- stunted growth and muscle wasting
Clinical features: Sighing respirations (Kussmaul’s resps), tachypnoea
Compensatory mechanism: Hyperventilation to increase CO2 excretion
Outline investigation using the anion gap
Difference between measured anions and cations
Anion gap = [Na+] + [K+] – [Cl-] – [HCO3-]
Normal 10-16
What causes a wide anion gap?
Lactic acidosis, ketoacidosis, ingestion of acid, renal failure
What causes a narrow anion gap?
GI HCO3- loss: diahorrea, fissure, renal tubular acidosis,
Outline causes of metabolic alkalosis
Alkali ingestion
Gastrointestinal acid loss: Vomiting
Renal acid loss: Hyperaldosteronism, hypokalaemia
Compensatory mechanism of metabolic alkalosis
Hypoventilation (but limited by hypoxic drive), renal bicarbonate excretion
Outline respiratory acidosis
CO2 retention, leading to increased carbonic acid dissociation
Causes: Any cause of respiratory failure
Compensatory mechanism: Increased renal H+ excretion and bicarbonate retention (but only if chronic)
Outline respiratory alkalosis
CO2 depletion due to hyperventilation
Causes: Type 1 respiratory failure, anxiety/panic
Compensation: Increased renal bicarbonate loss (if chronic)
Questions to ask in ABG interpretation
What is the pH?
What is the respiratory component (ie pCO2)?
What is the metabolic component (std HCO3-, base excess)?
Which component is congruent with the pH?
Renal function numbers
Renal blood flow- 1250ml/min
Renal plasma flow- 700ml/min
Glomerular filtration rate- 120ml/min
Urine flow rate- 1ml/min
Outline the proximal tubule
Active reabsorption of multiple solutes
Metabolically active cells – lots of mitochondria
Sodium gradient generated by Na/K ATPases
Vulnerable to hypoxia and toxicity
Proximal tubular disorders
Reabsorbed solute Disorder
Glucose Renal glycosuria
AAs Aminoacidurias (eg
cystinuria)
Phosphate Hypophosphataemic rickets
(eg XLH)
Bicarbonate Proximal renal tubular
acidosis
Multiple Fanconi syndrome
Outline renal glycosuria
Defect: Sodium glucose transporter 2 (SGLT2)
Mechanism: Failure of glucose reabsorption
Clinical features: Incidental finding on testing, benign
SGLT2 inhibitors (eg empagliflozin) now established as treatments for type 2 diabetes
Wider use in heart failure and CKD
Outline Aminoaciduria: Cystinuria
Defect: Sodium glucose transporter 2 (SGLT2)
Mechanism: Failure of glucose reabsorption
Clinical features: Incidental finding on testing, benign
SGLT2 inhibitors (eg empagliflozin) now established as treatments for type 2 diabetes
Wider use in heart failure and CKD
Treatment of aminoaciduria: cystinuria
High fluid intake: High urine flow, lower concentration
Alkalinise urine: Increases solubility of cystine
Chelation: Penicillamine, captopril
Management of individual stones (percutaneous treatment, surgery etc)
Outline Hypophosphataemic rickets
Commonest form is X-linked hypophosphataemic rickets (XLH)
Defect: PHEX – zinc dependent metalloprotease
PHEX mutation results in increased FGF-23 levels, leading to decreased expression and activity of NaPi-II in proximal tubule
Clinical features and treatment of Hypophosphataemic rickets
Clinical features: Bow legged deformity, impaired growth
Treatment: Phosphate replacement
Outline proximal (type 2) renal tubular acidosis
Defect: Na/H antiporter
Mechanism: Failure of bicarbonate reabsorption
Clinical features: Acidosis, impaired growth
Treatment: Bicarbonate supplementation
Outline the disorder affecting carbonic anhydrase
Genetic defects in carbonic anhydrase produce a mixed proximal/distal renal tubular acidosis
Inhibited by acetazolamide – mild diuretic effect and induces a metabolic acidosis
Used to treat altitude sickness – allows more rapid compensation of respiratory alkalosis
Outline Fanconi syndrome
- Mechanism: Generalised proximal tubular dysfunction, possibly due to failure to generate sodium gradient by Na/K ATPase
- Clinical features: Glycosuria, aminoaciduria, phosphaturic rickets, renal tubular acidosis
- Causes: Genetic (eg cystinosis, Wilson’s disease), myeloma, lead poisoning, cisplatin
- Not to be confused with Fanconi anaemia
What does the loop of henle do?
Generates medullary concentration gradient
Active Na reabsorption in thick ascending limb
Loop of Henle disorders: Barrter’s syndrome
Defect: NKCC2, ROMK, ClCKa/b, Barrtin
Mechanism: Failure of sodium, potassium and chloride cotransport in thick ascending limb. Salt wasting, hypokalaemic alkalosis due to volume contraction, failure of voltage dependent calcium & magnesium absorption
Clinical features of Barrter’s syndrome and what it is similar to
Clinical features:
Antenatal: Polyhydramnios, prematurity, delayed growth, nephrocalcinosis
Classical: Delayed growth, polyuria, polydipsia
Similar to effects of loop diuretics (eg furosemide, bumetanide)
What does the distal tubule and collecting duct do?
Distal tubule and cortical collecting duct allow “fine tuning” of sodium reabsorption, potassium and acid-base balance
Collecting duct mediates water reabsorption and urine concentration
Distal tubular & collecting duct disorders
Gitelman’s syndrome
Distal (type 1) renal tubular acidosis
Disorders resembling hyperaldosteronism
Type 4 renal tubular acidosis
Nephrogenic diabetes insipidus
Outline Gitelman’s syndrome
Gitelman’s syndrome
Distal (type 1) renal tubular acidosis
Disorders resembling hyperaldosteronism
Type 4 renal tubular acidosis
Nephrogenic diabetes insipidus
Outline the actions of aldosterone
Steroid hormone – predominantly acts on transcription
Increase expression of ENaC, Na/K ATP-ase
Mineralocorticoid receptor also activated by cortisol
Cortisol entry to renal tubular cells prevented by 11-beta hydroxysteroid dehydrogenase
Outline distal (type 1) renal tubular acidosis
Defect: ? Luminal H+ ATPase or H+/K+ ATPase
Mechanism: Failure of H+ excretion and urinary acidification
Can be genetic or acquired (eg Sjogren’s syndrome, chronic pyelonephritis, drugs – amphotericin)
Outline the disorders that cause aldosterone levels to be out of control
Excessive aldosterone activity produces sodium retention, hypertension and hypokalaemic alkalosis
Excessive aldosterone production may be primary (eg Conn’s syndrome) or secondary (eg renal artery stenosis)
Several disorders can produce a “hyperaldosteronism” phenotype with high blood pressure, hypokalaemia and alkalosis
Outline Glucocorticoid remediable aldosteronism
Defect: Chimeric gene – 11beta hydroxylase and aldosterone synthetase
Mechanism: Aldosterone is produced in the adrenal in response to ACTH, so levels inappropriately high
Treatment: Suppress ACTH using glucocorticoids
Outline Liddle’s syndrome
Defect: Activating mutation of ENaC
Mechanism: Sodium channel always open so constant aldosterone like effect
Treatment: Amiloride (blocks ENaC)
Outline Syndrome of Apparent Mineralocorticoid Excess (AME)
Defect: 11-beta hydroxysteroid dehydrogenase
Mechanism: Cortisol not broken down in the renal tubules, therefore activates mineralocorticoid receptor.
Treatment: Spironolactone (mineralocorticoid receptor antagonist)
Outline Hyperkalaemic distal (type 4 ) renal tubular acidosis
Defect: Low aldosterone levels
Mechanism: Reduced generation of electrochemical gradient, resulting in failure of H+ and K+ excretion
Common in elderly patients with diabetes
Treatment: Diuretics or fludrocortisone
Outline Nephrogenic diabetes insipidus
Defect: Vasopressin V2 receptor or aquaporin 2 water channel
Mechanism: Failure of water reabsorption in the collecting duct, resulting in inability to concentrate urine
Clinical features: Polyuria, polydipsia, hypernatraemia
Describe the make up of the pancreas
- Formed of small clusters of glandular epithelial cells
- 98-99% of cells are clusters called acini
Outline the exocrine activity of the pancreas
Exocrine activity performed by acinar cells
Manufacture and secrete fluid and digestive enzymes, called pancreatic juice, which is released into the gut
Outline the endocrine activity of the pancreas
- Endocrine activity performed by islet cells
- Manufacture and release several peptide hormones into portal vein
60-70% beta cells- insulin
rest alpha cells which secrete glucagon
and delta cells which secrete somatostatin
Outline the communication between the alpha and beta cells of the islets of langerhans
Paracrine ‘crosstalk’ between alpha and betacells is physiological, i.e., local insulinrelease inhibits glucagon
What are the peptides secreted by the islets of langerhans?
Insulin
Glucagon
Somatostatin
Pancreatic polypeptide
Ghrelin
Outline insulin
polypeptide, 51 amino acids
Reduces glucose output by liver, increases storage of glucose, fatty acids, amino acids
Outline glucagon
29 amino acid peptide
Mobilises glucose, fatty acids and amino acids from stores
Outline somatostatin
Somatostatin secreted from d cells – inhibitor
Outline pancreatic polypeptide
Pancreatic Polypeptide – inhibit gastric emptying
Outline ghrelin
Ghrelin – stimulates release of glucagon
What does insulin do?
Suppresses hepatic glucose output
- Decreases Glycogenolysis
- Decreases Gluconeogenesis
Increases glucose uptake into insulin sensitive tissues
- Muscle – glycogen, and protein synthesis
- Fat – fatty acid synthesis
Suppresses
- Lipolysis
- Breakdown of muscle (decreased ketogenesis)
What is the function of glucagon?
Counterregulatory
Increases hepatic glucose output
- Increases Glycogenolysis
- Increases Gluconeogenesis
Reduces peripheral glucose uptake
Stimulates peripheral release of gluconeogenic precursors (glycerol, AAs)
- Lipolysis
-Muscle glycogenolysis and breakdown
Other counterregulatory hormones (adrenaline, cortisol, growth hormone have similareffects to glucagon and become relevant in certain disease states, including diabetes
Outline insulin secretion by the beta cells
Glucose entry via GLUT2 glucosetransporter
Glucokinase does glucose metabolism which produces ATP which stimulates the
—-> look at it again- slide 12
Outline the role of proinsulin in insulin release
- Proinsulin contains the A and B chains of insulin (21 and 30 amino acid residues respectively), joined by the C peptide.
- Disulfide bridges link a and B chains
- Presence of C peptide implies endogenous insulin production
Outline the biphasic insulin release
- B-cells sense rising glucose and aim to metabolise it
- First phase response is rapid release of stored product
- Second phase response is slower and as it is the release of newly synthesised hormone
Insulin receptors on plasma membrane-n high affinity
Signalling cascade
stimulates GLUT4 vesicles which mobo
15
Outline glucose homeostasis
Glucose levels should remain constant
Liver glycogen is a short-term glucose buffer
What happens if blood glucose is too high?
> 6 is too high
SHort term response- Make glycogen (glucose to glycogen= glycogenesis)
Long term response- Make triglyceride lipogenesis slide 16
Outline glucose sensing
- Primary glucose sensors are in the pancreatic islets
- Also in medulla, hypothalamus and carotid bodies
- Inputs from eyes, nose, taste buds, gut all involved in regulating food
- Sensory cells in gut wall also stimulate insulin release from pancreas - incretins
Does it matter where you get the glucose from?
- Primary glucose sensors are in the pancreatic islets
Also in medulla, hypothalamus and carotid bodies - Inputs from eyes, nose, taste buds, gut all involved in regulating food
Sensory cells in gut wall also stimulate insulin release from pancreas - incretins
Outline incretins
K and L cells in gut
secrete glp 1 and gip
How are post prandial glucose levels regulated?
Increase of insulin-> rising plasma glucose stimulates beta cells to secrete incuijn
GLP1- glucose dependent and short half life
dipeptidyl peptidase IV cleaves GLP-1
Half life is 1-2 mins
DPPIV prevents hypoglycaemia
Outline regulation of CHO metabolism
- In the fasting state, all glucose comes from liver
- Breakdown of glycogen
- Gluconeogenesis (utilises 3 carbon precursors to synthesise glucose including lactate, alanine and glycerol) - Glucose is delivered to insulin independent tissues, brain and red blood cells
- Insulin levels are low
- Muscle uses FFA for fuel
- Some processes are very sensitive to insulin, even low insulin levels prevent unrestrained breakdown of fat
Outline the regulation of CHO metabolism post prandial
- After feeding (post prandial) - physiological need to dispose of a nutrient load
- Rising glucose (5-10 min after eating) stimulates 5-10 fold increase in insulin secretion and suppresses glucagon
- 40% of ingested glucose goes to liver and 60% to periphery, mostly muscle
- Ingested glucose helps to replenish glycogen stores both in liver and muscle
- Excess glucose is converted into fats
- High insulin and glucose levels suppress lipolysis and levels of non-esterified fatty acids (NEFA or FFA) fall
What is diabetes mellitus?
A disorder of carbohydrate metabolism characterised by hyperglycaemia
What causes diabetes
Mutations to Kir 6.2
Sulphonylureas
Describe pathogenesis of diabetic ketoacidosis
Outline gametogenesis
-The process by which gametes are produced in the reproductive organs (gonads)
of an organism.
-Gametes are fundamental for sexual reproduction and genetic diversity.
follicule 1 oocyte
Steps of folliculogenesis
Primordial follicle-> Primary follicle -> Developing follicles -> Mature (graafian) follicle + secondary ovum -> ruptured follicle + liberated ovum -> early corpus luteum -> corpus luteum -> corpus albicans
What is oogenesis?
Oogenesis Begins in fetal life, with significant milestones at puberty and ceasing at menopause
Describe the stage of oogenesis that happens during foetal life
Oogonium (diploid) divides by mitosis to form 2 daughter oogonium
These then grow and form primary oocytes
Describe the stage of oogenesis that happens after puberty
Primary oocyte (diploid) undergoes meiosis I to form the first polar body and a secondary oocyte (haploid)
What covers the oocyte?
Oocyte initially covered in cumulus cells
then when that is gone it is covered in a layer of proteins called the zona pellucida
What are the two female reproductive hormones that are produced by the pituitary gland
FSH- stimulates maturation of the oocyte
LH- stimulates the release of an oocyte
What is the duration of the menstrual cycle?
Around 28 days
What does FSH do?
FSH goes to ovary and stimulates production of follicles and stimulates oestrogen production
What does oestrogen do?
Oestrogen stimulates proliferates endometrium cells so the endometrium gets thicker
What are the first 14 days categorised as?
Proliferating phase
What happens at around day 14 of the menstrual cycle?
Surge of LH
stimulates ovulation release of oocyte
What happens on day 20?
Empty follicle converts to corpus luteum and corpus albicans
What phase is it between days 14 and 28?
cyclical phase
progesterone is high
Describe the relationship between GnRH, FSH and Oestrogen
GnRH- starts
Stimulates pituitary to produce FSH
FSH affects oestrogen
Oestrogen increases eggs and uterus
Describe the relationship between LH and Progesterone
LH surge
stimulates ovulation
corpus luteum
progesterone produced
Days 1-7 of menstrual cycle
menstruation
Days 8-11 of menstrual cycle
Lining of womb thickens in preparation for the egg
Day 14 of menstrual cycle
ovulation
Days 18-25 of menstrual cycle
If fertilisation has not taken place the corpus luteum fades away
Days 26-28 of menstrual cycle
The uterine lining detaches leading to menstruation
What is spermatogenesis?
Spermatozoa being produced in the testis
What is ejaculate made of?
-Ejaculate is a mixture of spermatozoa and seminal
plasm
Outline the testes
Oval organ, 4 cm long x 2.5 cm in
diameter
* Covered anteriorly by a saclike extension
of the peritoneum (tunica vaginalis) that
descended into the scrotum with the testes
* Tunica albuginea = white fibrous capsule
Outline the compartments of the testes
septa divide the organ into compartments containing
seminiferous tubules where sperm are produced
Outline leydig cells
clusters of cells between the seminiferous
tubules and source of testosterone
AKA interstitial cells
produce sperm
Outline sertoli cells
s promote sperm cell development
* blood-testis barrier is formed by tight junctions between
sertoli cells; separating sperm from immune system
Where do seminiferous tubules drain into?
eminiferous tubules drain into network called rete testi
Outline male inguinal and scrotal region
*Pendulous pouch holding the testes divided into 2 compartments
by median septum
*Testicular thermoregulation is necessary since sperm are not
produced at core body temperature - need to be 35 not 37
Outline mitosis
Mitosis produces 2 genetically identical daughter
cells (occurs in tissue repair & embryonic growth)
Outline meiosis
Meiosis produces gametes haploid cells required
for sexual reproduction
– 2 cell divisions (after only one replication of DNA)
– meiosis keeps chromosome number constant from
generation to generation after fertilization
– meiosis occurs in seminiferous tubules of males
What are the 2 cell divisions of meiosis?
Meiosis produces gametes haploid cells required
for sexual reproduction
– 2 cell divisions (after only one replication of DNA)
* meiosis I separates homologous chromosome pairs2
haploid cells
* meiosis II separates duplicated sister chromatids4 haploid
cells
– meiosis keeps chromosome number constant from
generation to generation after fertilization
– meiosis occurs in seminiferous tubules of males
What are the 2 types of spermatogonia daughter cells?
Type A
Type B
Describe type A spermatogonia
Type A remain outside blood-testis
barrier & produce more
daughter cells until death
Outline type B spermatogonia
type B differentiate into
primary spermatocytes
* cells must pass through
BTB to move inward
toward lumen - new tight
junctions form behind
these cells
* meiosis I 2 secondary
spermatocytes
* meiosis II 4 spermatids
What is spermiogenesis?
Spermiogenesis is transformation of spermatids into
spermatozoa
– sprouts tail and discards cytoplasm to become lighter
Number of sperm produced
-300 to 600 sperm are made
per gram of testis per second.
-50g x 50 min x 60 sec x 500
sperm =
75,000,000 spermatozoa
Describe the hormonal regulation of sperm production
GnRH-
ptuitary produces FSH and LH
FSH induces surge if spermatogenesis
LH acts on leydig cells
leydig cells produces testosterone inhibits gnrh
Outline the head of the sperm
Head is pear-shaped front end
– 4 to 5 microns long structure
containing the nucleus, acrosome
and basal body of the tail flagellum
* nucleus contains haploid set
of chromosomes
* acrosome contains enzymes
that penetrate the egg
* basal body
Outline the tail of the sperm
Tail is divided into 3 regions
– midpiece contains mitochondria
around axoneme of the flagellum
(produce ATP for flagellar
movement)
– principal piece is axoneme
surrounded by fibers
– endpiece is axoneme only and is
very narrow tip of flagellum
Outline the efferent ductules
– 12 small ciliated ducts collecting sperm
from the rete testes and transporting it
to the epididymis
Outline the epididymis
– 6 m long coiled duct adhering to the
posterior of testis
– site of sperm maturation & storage
(fertile for 40 to 60 days)
Outline the ductus (vas) deferens
– muscular tube 45 cm long passing up
from scrotum through inguinal canal to
posterior surface of bladder
– widens into a terminal ampulla
Outline ejaculatory duct
– 2 cm duct formed from ductus deferens
& seminal vesicle & passing through
prostate to empty into urethra
What are the accessory glands?
Seminal vesicles
Prostate gland
Bulbourethral gland
Outline main components of semen
-2-5 mL of fluid expelled during orgasm
– 60% seminal vesicle fluid, 30% prostatic & 10% sperm and
trace of bulbourethral fluid
* normal sperm count is 50-120 million/mL (< 25 million/mL is
associated with infertility)
* sperm serve to digest path through cervical mucus and to
fertilize egg
Outline other components of semen
– fructose provide energy for sperm motility
– fibrinogen
– clotting enzymes convert fibrinogen to fibrin causing semen to
clot
– fibrinolysin liquefies semen within 30 minutes
– prostaglandins stimulate female peristaltic contractions
– spermine is a base stabilizing sperm pH at 7.2 to 7.6
Outline the role of sex chromosomes
- Our cells contain 23 pairs of chromosomes
– 22 pairs of autosomes
– 1 pair of sex chromosomes (XY males: XX females) - males produce 50% Y carrying sperm and 50% X carrying
- all eggs carry the X chromosome
- Sex of the child is
determined by the type
of sperm that fertilizes
the mother’s egg
Sperm transport in female reproductive tract
*A small number of
spermatozoa reach to the
upper part of female
reproductive tract
*Both Sperm motility and
female reproductive tract
movement are responsible
for sperm transport
*Cervical mucus
Penetration test
Outline capacitation
*Capacitation was first discovered by Chang and
Austin independently (1950).
*The final maturational stage of spermatozoa that
takes place in the female genital tract, before
spermatozoa gain the ability to fertilize oocyte.
*It is one of the most investigated areas of
andrology and one of the least understood areas
of andrology.
Decsribe acrosome reaction
Happens when sperm is in contact with the zona pellucida
zp3 starts
then zp2
What is an embryo morula?
The morula is a globular solid mass of 16-32 blastomeres formed by cleavage of the zygote that precedes the blastocyst
Different types of lab processes to do with stem cells and gametes
*Artificial Insemination (AI)
*Embryo Transfer (ET)
*In Vitro Fertilization (IVF)
*Intra-Cytoplasmic Sperm Injection (ICSI)
*Somatic Nuclear Transfer (Cloning)
*Stem Cell Therapy (Regenerative Medicine)
*IPS Cells (Induced pluripotent Stem Cells)
Outline day 1 of fertilisation
Oocyte activation is key and is triggered by a sperm protein called Phospholipase C zeta (PLCz).
This ‘activates’ the egg to release calcium from internal stores and this rise in calcium facilitates fertilisation.
Oocyte activation is essential for the transformation of the decondensed sperm nucleus in to pronucleus.
4-7 hours after gamete fusion the two sets of haploid chromosomes form the female and male pronucleus (23 chromosomes each)
Pronuclei are equal size and contain nucleoli
In IVF multinucleate oocytes can be identified - polyspermic
What happens in syngamy?
- Male and Female pronucleus migrate to centre (cytoskeletal system plays important role)
- Haploid chromosomes pair and replicate DNA in preparation for the first mitotic division
- The pronuclear membranes breakdown
- The mitotic metaphase spindle forms
- 46 Chromosomes organise at the spindle equator
Describe day 2 cleavage
Approx 24 hours after fertilisation the ooplasm divides in to two equal halves
If one or more of the PN fail to decondense and move in to one of the blastomeres, diploid or triploid mosaics may occur
What is cleavage and what does it do?
Cleavages are timed from sperm entry by an oocyte program that also regulates ‘house keeping’ activities in embryos.
Successive cleavages result in an increase in cell number – essential to provide sufficient cells for differentiation.
Describe genetic control in the embryo
Prior to 4-8 cell stage the developmental control depends on maternally-derived stores of RNA laid down during oogenesis.
Activation of the embryonic genome and start of embryonic transcription occurs in a 4-8 cell embryo.
Developmental arrest can occur.
Describe day 3 cleavage
Early cleavage stage embryos are ‘totipotent’ – the nuclei of individual blastomeres are each capable of forming an entire foetus.
Describe day 4 compaction
- Cells flatten
- Maximise intracellular contacts
- Tight junctions form
- Polarisation of outer cells
- Morula – 16 cells
Descriebe day 5 cavitation and differentiation
- Tight junctions occur between outer cells – forms the trophectoderm
- Fluid filled cavity expands
- Sodium pumped in which pulls water in by osmosis
Now >80 cells
50-66% comprise trophectoderm, rest is ICM
Pluripotent
What do trophectoderm cells do?
Trophectoderm cells pump fluid in to the embryo to form the blastocoel cavity
Describe day 5/6 expansion
- Cavity expands
- Diameter increases
- ZP thins
Outline day 6+ hatching
- Blastocyst expansion and enzymatic factors cause the embryo to hatch from the ZP.
Essential for implantation - TE – extraembyronic
- ICM - embryonic
When is the embryo transplanted into the uterus?
Day 5
Describe the energy metabolism and requirements of the early pre-implantation embryo
ATP turnover low
ATP / ADP ratio is high
Energy metabolism characterised by consumption of pyruvate
Glucose uptake and utilisation is low
Describe the Energy metabolism and requirements in the Blastocyst stage
Metabolic activity rises sharply
ATP / ADP ratio falls, reflecting an increase demand for energy e.g for protein biosyntheses and ion pumping associated with blastocoel cavity.
Glucose is the predominant exogenous energy substrate
Genetic control of the embryo at different stages
early cleavage- maternal
blastocyst- embryonic
What is exogenous nutrients in vivo supplied by?
Cumulus cells
Fallopian Tube secretions e.g. calcium,
sodium, chloride, glucose, proteins.
Uterine secretions e.g. iron, fat soluble vitamins, glucose
How is the fallopian tube adapted to provide the right nutrients to the embryo?
Concentrations of nutrients vary along the tract to provide the embryo requirements at the right time
Growth factors and cytokines
important in embryonic growth and differentiation
Insulin-like growth factor - IGF–I and IGF–II increase cell numbers in blastocyst
Leukaemia inhibitory factor (LIF) enhances embryo-endo interaction
Cellular differentiation – 10 days
After implantation embryogenesis continues with the next stage of gastrulation when the three germ layers of the embryo form in a process called histogenesis
The 3 germ layers form: ectoderm, mesoderm and endoderm (three overlapping flat discs)
It is from these three layers that all the structures and organs of the body will be derived
Changes to the uterus for implantation
- Endometrial cell changes to help absorption of uterine fluid – bring the blastocyst nearer to the endometrium and immobilises it.
- Changes in thickness of endometrium and its blood supply development
- Formation of the decidua
Implantation window = 4 days (6-10 days postovulation)
Outline the implantation process
Well defined starting point
Gradual process over several weeks
No universal agreement when process is completed
Relationship between size of implantation site and thickness of endometrium
small size of implantation site compared to thickness of endometrium
Outline the regulation of implantation
After embryo hatched the embryonic and maternal cells enter into a complex dialogue
High degree of preparation and coordination required
Controlled cascade of trophoblast proliferation, differentiation, migration
and invasion
Mechanisms of embryo implantation
The cross talk between endometrium and the developing embryo is mediated by substances including:
Hormones (sex steroids)
Cell adhesion molecules
Proteases
Cytokines, Growth Factors
Also genetics
5 genes up regulated during implantation window (Haouzi et al 2009)
3 phases of embryo implantation
Apposition
Attachment
Invasion
Describe apposition
Unstable adhesion of the
blastocyst to the uterine lining
Synchronisation of embryo
and endometrium (decidua)
Hatched blastocyst orientates via embryonic pole (always attaches at the area above the ICM)
Receptive endometrium (implantation window day 19 – 22)
Describe Attachment (adhesion)
Stable/stronger adhesion
penetrate with protrusions of the trophoblast cells (microvilli)
Massive communication between the blastocyst and endometrium conveyed by receptor-ligand interactions
Apical surfaces of the endometrial epithelial cells express variety of adhesion molecules (integrin subunits)
Trophoblastic cells also express integrins
Attachment occurs through the mediation of bridging ligands that connect with integrins on their surfaces
Describe invasion (penetration)
Trophoblast protrusions continue to proliferate and penetrate the endometrium
cells differentiate to become syncytiotrophoblast
The trophoblast surrounding the ICM = cytotrophoblasts.
Highly invasive - trophoblast quickly expands and erodes into endometrial stroma.
Invasion is enzymatically mediated
Syncytiotrophoblast erodes endometrial blood vessels
Eventually syncytiotrophoblast comes into contact with maternal blood and form chorionic villi – in initiation of the formation of the placenta
Blood filled lacunae form (spaces filled with maternal blood). Exchange nutrients and waste products.
What happens to the invasiveness of the trophoblast?
After first few days of implantation, the trophoblast changes character to become less invasive
killer cells don’t attack it
Outline decidual reaction
Promotes placental formation
stromal cells adjacent to the blastocyst differentiate into metabolically active, secretory cells or Decidual Cells (under influence of progesterone)
Secretions include growth factors/proteins to support growth of implanting blastocyst in the initial stages before the placenta is fully developed.
Endometrial glands enlarge and local uterine wall becomes highly vascularised.
The decidual reaction is not required for implantation e.g. ectopic implantation can occur anywhere in the abdominal cavity.
What changes happen in the decidual reaction
These changes include swelling of stromal cells due to accumulation of glycogen and other nutrients. These nutrients help the embryo survive the initial days before the placenta is fully developed, which then establishes a channel for fetus nutrient exchange.
When does the decidual reaction happen and where?
The decidual reaction is seen in very early pregnancy in the generalized area where the blastocyst contacts the endometrial decidua. It consists of an increase in secretory functions of the endometrium at the area of implantation, as well as a surrounding stroma that becomes edematous.[1]
Role of progesterone in implantation
- Modifies the distribution of oestrogen receptors
- Stimulates secretory activity
- Stimulates stromal oedema
- Increases volume of blood vessels
- Primes decidual cells
- Stabilises lysosomes
- Might be an immunosuppresent
- May stimulate growth factors and binding proteins
- May regulate the formation of reactive oxygen species (reducing oxidative stress)
Describe maternal recognition of the embryo
Embryo is antigenically different from the mother
At the same time as the decidual reaction, leukocytes in the endometrial stroma secrete interleukin-2 which prevents maternal recognition of the embryo as a foreign body during the early stages of implantation
uterine natural killer cells
What is Human Chorionic Gonadotropin?
produced in the human placenta by the syncytiotrophoblast
hCG-a Synthesised in cytotrophoblast cells
hCG-b synthesised in syncytiotrophoblast cells of placenta
Rising hCG-b levels from day 7-8 signify onset of implantation
What is the role of hCG?
Essential to sustain early pregnancy
ensures the corpus luteum continues to produce progesterone throughout the first trimester of pregnancy (prevents menstruation).
Interacts with the endometrium via specific receptors
Immunosuppressive – has highly negative charge, may repel the immune cells of the mother & protect the foetus.
Outline hCG Measurement in early pregnancy
hCG to double every 1.3 days in the first 10-12 days of a normal singleton pregnancy
A short doubling time signifies a healthy pregnancy
What might slow rate of increase of HCG show?
Early abortion
Ectopic pregnancy
Delayed implantation
Inadequate trophoblast
Embryos are highly sensitive to the environment – essential to optimise conditions to enable successful program
Use a sequential culture medium – different composition at different stages
What does an embryo need in days 1-3
Day 1-3
Water, salts &ions
Pyruvate, lactate, protein
and
No/low Glucose
Non essential amino acids
What do embryos need in day 3+
Day 3+
Water, salts &ions
Pyruvate, lactate, protein
and
Glucose
Essential and non essential amino acids
Vitamins
Describe 1 step culture system
Culture day 1-6 in same media one media
Let the embryo choose principal
useful in uninterrupted systems like time lapse
Why? Reduce stress, less disturbance, Increase embryo viability
Factors affecting embryo growth
maternal factors
embryonic factors
lab conditions
Maternal factors
Follicle environment
Oocyte maturity (hCG trigger 36hours before egg collection)
