Reproductive Physiology Flashcards
Epigenetic factors in pregnancy
Nutrition Psychological stress Physical stress Immunological stress Alcohol Smoking Circadian rhythms IVF??
What’s the pulse generator of the HPG axis
GnRH from the hypothalamus
Can be modulated by stress, diet, puberty, exercise and other environmental factors
What is Capacitation
Mammalian sperm can’t fertilise oocytes when freshly ejaculated they must undergo capacitation. They acquire the capacity to fertilise by changes in lipids, proteins in the membrane and this requires the removal of seminal fluid components so can’t occur immediately. Usually occurs in the uterus and oviduct. May take 5-7 hours in humans
Role of the sperm
Egg activation - including changes to egg coverings to prevent polyspermy, release of oocyte from neurotic arrest and activation of egg metabolism
Supplies paternal half of genes
When do sex organs begin to develop
Week 5 of development
Sexual development in utero overview
Male - XY, testis develop and Wolffian duct promoted forming epididymis and vas deferens etc.
Female - XX, ovaries develop and Müllerian duct promoted forming uterus and Fallopian tubes
Determination of gonadal sex
The presence of the SRY gene on the Y chromosome determines the male sex - no matter how many X chromosomes present the SRY gene will produce male characteristics
Some females XX have no SRY yet still develop male characteristics XX males may have small parts of Y chromosomes and XY females have deletions of small parts of Y chromosome losing the SRY gene.
Several other pathways can act around the SRY gene causing intersex cases
Adult sexual behaviour is dependent on
Hormones - testosterone, oestradiol etc exposure during short, critical early stages of development as well as throughout life
Males and females when adults don’t respond in the same way to hormones.
Days 1-5 is critical for testosterone exposure in male sexual behaviour in rats.
Sexually dimorphic structures of CNS
Sexually dimorphic nucleus of preoptic area, spinal nucleus of bulbocavernous in spinal cord are different in males and females and are determined by exposure to steroids during short, critical early stages of development
Sex change in some fish species
Larger of the fish will become the male while the others become females - places glass in between fish to determine if signals were conveyed but still same result so concluded that visual cues were responsible for sex change!
How to investigate HPG axis pulses
Lesions between hypothalamus to pituitary - using electrodes running Hugh current through destroys bits of tissue tracts from one to the other.
Once no endogenous GnRH is present and results noted then exogenous GnRH can be administered to see if effects are reversed.
LH response to different GnRH pulse frequencies
One pulse per hour - high levels of LH
Give an hour - knock out of LH, levels drop completely
Many other doses and frequencies tried
Pulse frequency concluded to be essential for correct response for LH secretion - altering GnRH frequency alters response in pituitary and therefore too slow or too quick reduced response.
Brain lesioned where GnRH pulse generator is, leading to a subsequent fall in LH (and FSH)
Restoration of the GnRH pulse restores pulsation of LH and FSH
A continuous pulse of GnRH diminishes LH and FSH pulsing
This is due to desensitisation
Important clinically in:
Precocious puberty
IVF
LH secretion at puberty
Pre pubertal - low constant fluctuations of LH
Mid - night time increases in LH pulses
Late - pulses of LH throughout the day
GnRH neurones in development
GnRH neurons are unique to mammalian nervous systems as it arises outside the brain in the nasal placode and migrates into the brain during development.
Kallmann syndrome is a rare genetic condition characterised by a failure to progress through puberty. It is also accompanied by a lack of sense of smell as the GnRH neurons arise from the olfactory bulb.
GnRH neuron morphology is unlike any other neuron in the CNS. The neurons project one or both of the long dendrites to the median eminence. Here they break up into short axon terminals to enable GnRH secretion into the portal vasculature.
Operating characteristics of GnRH /LH drive to gonads
System is suppressed (in both sexes) by negative feedback of gonadal steroids
‘positive feedback’ system generates the preovulatory LH surge
Oocyte is 100 microns
High levels of E2 induce +ve feedback which induces ovulation by inducing the LH surge
Follicle ruptures and oocyte is released (ovulation)
The system is a sexually differentiated response:
Days 1-5 are critical periods in mice for hormone exposure to induce sexually differentiated response
Control male mice do not have an LH surge following treatment with oestradiol
Male mice with testes removed at birth do show an LH surge in response to oestradiol
Male mice with testes removed and treated with testosterone in days 1-5 do not have an LH surge in response to oestradiol
Rats mate at night as it is the most opportune time for fertilisation to occur
Female rats on a 12h day/night cycle have an LH surge at 2PM and ovulate at 2AM
Tested by treating ovariectomised rats with oestradiol ONCE, LH surges will follow daily for 10 consecutive days
Therefore, the LH surge is dependent on:
E2 feedback
Sexual differentiation of the brain
Input from the circadian rhythm
GnRH does not have a receptor for oestradiol, so how does E2 contribute to the LH surge?
KISSPEPTINS
Kisspeptins are ligands for GPR54
Mutations in this receptor or knockouts in Kiss lead to infertility
Two kinds:
Arcuate kisspeptin neuron: primary control of pulsatile secretion
Preoptic area kisspeptin neuron: drive preovulatory LH surge
How does this relate?
GnRH stimulates the pituitary, causing the LH surge.
The ovaries then release steroid hormones (i.e. E2)
E2 in high doses upregulates Kisspeptin expression
The Kisspeptin neurones project to GnRH neurones, which also have Kisspeptin (GPR54) receptors
Kisspeptin induces a further LH surge which leads to ovulation by activating GnRH neuron electrical activity
To block the LH surge, Kisspeptin anti-serum is used. In addition, Kisspeptin has many other roles, this is just one of them.
Seasonal changes in testes diameter
Summer: High LH, FSH Large testes High testosterone In Winter: Low LH, FSH Testes regulated Low testosterone
Factors of energy balance/nutritional status affecting reproductive status
Low weight (fat mass) Lactation Restricted food/eating disorders/diet Hyperactivity (jogging, running, dancers, athletes) Psychological stress
PCOS and nutrition
Reproductive disorder
Characterised by:
Hyperandrogenism
Chronic anovulation
Polycystic ovaries
Metabolic disturbances
50% of women with PCOS are overweight or obese and have insulin resistance
PCOS shows that reproductive status affects metabolism and fat AND VICE VERSA
This is due to steroids affecting fat mass and distribution in humans
Demonstrated during menopause, where menopausal women put on weight due to oestrogen withdrawal
Menopausal women move from Gynoid to Android fat distribution (subcutaneous fat redistributes to visceral areas)
During adolescence, boys gain primarily lean muscle and little fat, whereas girls gain little lean muscle but substantial fat
Puberty HPG changes
During normal puberty, GnRH pulses from the hypothalamus triggers the anterior pituitary to increase LH and FSH secretion, which triggers the ovaries/testes to release steroid hormones which have a negative feedback effect on the hypothalamus
In the case of hypothalamic lesions or tumours, this system is compromised, and subsequently precocious puberty (early puberty) can start
Puberty and nutrition
LH pulsatility is disturbed with reduced energy intake
The age of menarche (first period) has declined since the 1830s in Western countries, influenced by lifestyle and diet choices
Musicians, thought to be more sedentary lifestyle-wise have a younger onset of menarche whilst ballet dancers with strict exercise regimen and diet have a later onset of menarche.
Height increases with age of menarche, but menarche onsets at a mean weight of 47kg on average for girls according to the graphs, therefore indicates that menarche requires energy reserve to occur
Menstrual cycle and exercise
In some elite squads, menstrual disturbances approach 100%
Amenorrhea (no period) increases with amount of exercise
LH pulsatility is suppressed in runners with amenorrhoea as compared to sedentary controls
Untrained women underwent a 3 month training programme leading up to 10 mile/day for 5 days/week and over 50% showed disturbances in menstrual function (short luteal phase)
May also depend on type of exercise:
Running (and other weight bearing sports associated with leanness) have a high incidence of menstrual irregularities (25%-79%)
Cycling/swimming (non weight bearing, strength based and non diet restricted) have a low incidence of menstrual irregularities
This shows that it’s likely that body composition plays a role in menstrual regulation in addition to activity
Body composition does not account for all of this: Body composition doesn’t change within a day yet LH pulses were drastically affected
So there must be another signal
Reproductive status and energy balance
LH pulsatility is suppressed with reduced energy intake (low BMI), but high BMI also causes infertility and disturbed LH pulsatility.
Steroids and nutrition
Steroids affect fat mass and distribution, appetite and energy balance in animal models
Female mice given an ovariectomy increase weight gain and food intake
Effects reversed by administration of oestradiol
Female mice with no oestrogen receptors in the CNS also respond as if ovariectomised
Shows that effects are partially mediated by CNS neurons
There are many acute and longer term potential signals, but a key one of interest is Leptin
Leptin and reproductive status
Leptin deficient ob/ob mouse
Mouse is fat and also infertile
Administer Leptin and obesity is reversed and fertility restored
Leptin is derived from white adipose tissue, and its secretion is proportional to total adipose stores
% white fat in body measured by leptin in blood
Leptin acts on many targets in the body (immune system, CVS, bone etc) but most importantly it is one of the signals to the hypothalamus reflecting energy balance
In the case of leptin deficiency:
Despite fat stores, cannot produce leptin
Leptin deficiency
Feedback into appetite and metabolism causes increased appetite and reduced energy expenditure
Causes obesity
Patients with leptin deficiency:
Females: Cycles with luteal phase defect
Males: Low testosterone, hypogonadal (absent puberty)
After treatment:
Females: Regular ovulatory cycles and weight loss
Males: Pubic and axillary hair, growth of testes and penis, improved muscle strength, weight loss
Fasted male monkeys (suppressed LH pulsatility) administered leptin, which resumes their LH pulsatility
Kisspeptin neurones which project to GnRH neurons express the leptin receptor while GnRH neurones do not.
Leptin associated with hypothalamic amenorrhea (menstruation stops for months due to hypothalamic issues)
Low leptin levels = hypothalamic amenorrhea
Treatment with leptin restores ovulatory cycles
But treatment takes a long time to have an effect because leptin is only one important signal for GnRH secretion: here is where AMPK comes in
AMPK and energy status
this is a protein kinase is activated by ATP deficiency
Mediates communication of cellular energy status
Undernutrition stimulates an increase in AMPK in arcuate nucleus kisspeptin neurons
Kiss1 decreases, which decreases GnRH
Leads to a delay in puberty
Knocking out AMPK in the arcuate nucleus kisspeptin neurons reduces the effect of undernutrition
AMPK affects:
Muscle (glucose uptake and FA oxidation)
Heart (Glucose uptake, FA oxidation, glycolysis)
Hypothalamus (food intake)
Liver (FA synthesis, gluconeogenesis)
Adipose Tissue (FA synthesis, lipolysis)
Pancreas (insulin secretion)
If GnRH stimulates both LH and FSH secretion which come from gonadotrophs why do their serum profiles differ
LH pulses are discrete fluctuations and FSH are flatter.
There’s a single cell type within the Pituitary producing both LH and FSH.
Differential secretion is therefore determined by regulating synthesis
They’re glycoproteins heterodimers comprised of alpha subunit and unique beta subunit which determines the biological specificity. It’s the rate limiting step for production of mature gonadotropin
GnRH stimulates synthesis of both hormones and pulse frequencies control the ratio. Slower decreases LH and increases FSH, and vice versus.
There are several other hormones of HPG axis that also differential regulate gonadotrophin synthesis and secretion for example activins, inhibins, follostatin, sex steroids, bone morphogenetic protein.
There’s specific negative feedback into gonadotrophs by the protein hormone inhibin which specifically downregulates FSH release but not LH by reducing FSH beta mRNA and FSH protein. Inhibin is secreted by testicular Sertoli cells and ovarian granulosa cells.
Melatonin and reproduction
Melatonin is released at night during sleep. Injecting melatonin at the right time can change the duration of the signal. Long day (summer) Shorter nights, so shorter melatonin signal Reproduction is switched ON Long day (winter) Longer nights, longer melatonin signal Reproduction is switched OFF Long day (summer) Artificially injecting melatonin increases melatonin signal despite short night Reproduction is switched off
GnRH neurones
GnRH neurons are located in the preoptic nucleus in rodents and in the arcuate nucleus in humans and primates, and descend into the median eminence
The neurons are covered in spines
Spines are the site synapses come into the neuron
Many spines mean that the neurons can receive a lot of input
They travel huge distances and are found in bundles
Bundling increases the opportunity for the integration of activity
why does LH pulsing increase during LH surge
Amplitude of LH pulsing increases during the surge
This is due to an increase in the sensitivity of the receptors
MUA
Multi unit activity - measuring the activity of multiple neurones
Transfection of hypophysiotropic GnRH neurones with ChR2
ChR2 helps to identify/target specific neurons when introduced into cells (by shining a blue light on them)
Kisspeptins and reproduction
Kisspeptin is the most potent stimulator of GnRH neurons
Kisspeptin neurones may act as central processors for relaying signals to GnRH neurones (NO KISS = NO FERTILITY)
In mice, kisspeptin exerts a potent activational effect on GnRH neurones
In rodents, there is a population of kisspeptin in AN and another in AVPV
These are side by side in mice and rats so easy to stimulate both populations
Optogenetic activation of GnRH neurones in vitro with pulses of blue light
ChR2 expressing neurons activated with pulses of blue light given at 5, 10 and 20 Hz for repeated 1-s on (blue bar) and 9-s off time periods
Histograms show the mean and SEM evoked spike fidelities at the different stimulation frequencies
Spike fidelity of 100% means that every blue light stimulation generates an action potential
Study does not show what actually initiates pulse frequency (oscillation)
However, when using ChR2 to activate arcuate KISS neurones, you evoke pulsatile LH secretion in freely behaving mice, and archaerhodopsin has the opposite effect
role of ARC kisspeptin/neurokinin B NKB/Dynorphin
KNDy network projects to preoptic area in rodents and releases glutamate there, but role of glutamate is unclear
Kisspeptin/NKB/Dyn neurons in the ARC form a neural circuit by their collaterals and dendrites
Neuron activated - releases NKB
Essentially excites the whole network and activates it
Initiates pulse of kisspeptin on to GnRH neuron
GnRH then releases a pulse
At the same time, Dynorphin is released with Kisspeptin, acting on the presynaptic element to inhibit the release of NKB
This stops kisspeptin release and therefore GnRH release, causing a pulse
Within the neural circuit, NKB/NK3 signaling plays the role of accelerator where as Dyn/KOR signalling serves as a brake on activation of kisspeptin/NKB/Dyn neurons
Through the reciprocal actions of NKB/NK3 and Dyn/KOR signaling, rhythmic oscillation of neural activity is generated in kisspeptin/NKB/Dyn neurons
This in turn induces pulsatile kisspeptin release at the median eminence, which leads to pulsatile GnRH release into the portal circulation
ARC kisspeptin/NKB/DYN neurons thus act as the GnRH pulse generator through coordinated interaction between three peptides
In primates, there is a positive feedback effect on oestrogen in the arcuate nucleus
GnRH and kisspeptin neurones are in the same place, therefore likely that kisspeptin neurones are being stimulated experimentally as well as GnRH
KNDy neurones And LH secretion
KNDy neurones exhibit periodic synchronisations in intracellular calcium concentrations tightly correlated with pulsatile LH secretion in gonadectomised male mice
Ca oscillations associated with LH pulses
This is strong evidence for kisspeptin neurons in the AN being the pulse generator
Introduction of viruses that visualised Ca signalling, so the presence of an action potential meant that there was Ca signalling
Model one predictions - Continuous optogenetic activation of KNDy neurones in vivo initiates and increases LH pulse frequency in oestrous mice.
Increased basal activity starts oscillation
Increased frequency of basal activity increases oscillations
2nd threshold - system turns off
Can shift these peaks by frequency of pulsation
HPA and HPG axis interaction
Stress HPA axis - CRH from hypothalamus drives cortisol secretion in stress and this In turn suppresses the HPG axis
Evidence - conditions such as functional hypothalamic amenorrhea, resumption of normal menstrual cycles and gonadotrophin secretion associated with normalisation of blood cortisol levels.
Many environmental stressors cease reproductive function - exercise, excessive eating, anorexia, brain injury, PTSD etc.
Men have much more sensitive HPG switching systems and need less stress in order to turn off reproductive function, women have much more robust systems in place due to having less oocyte reserve etc. More damage is caused by preventing female reproductive cycles
Monkey studies have indicated that stress resilient animals range from highly resilient to stress sensitive - highly resilient groups have High serotonin levels, oestradiol, with low CRF, GABA
IVF not immune to these stresses in fertility.
Fertility rates proven to increase in stress free fun environments, termed medical clowning. Or counselling sessions found useful in pre IVF sessions in 91% of patients so it’s recommended a necessary part of IVF planning.
