Pituitary

• Give the target organ and function from the pituitary hormone?

  ACTH - Adrenal gland, stimulates glucocorticoid secretion for stress response

  TSH - Thyroid gland, stimulates thyroid hormone secretion to increase metabolic rate

  GH - Liver and adipose tissue, growth in childhood, lipid control, protein and carbohydrate metabolism

  LH + FSH - Ovary, testis, reproductive function

  PRL - Mammary gland, milk production
• Pros and cons of human cells for pituitary research?

  Healthy human pituitary not available, but can use tumour cells

  Can use cell lines (widely used) - originate from tumours in one particular cell type - however, don't know how good they are at modelling the primary healthy cell

  Don't have vasculature in cell lines (important feature of pituitary) - cannot investigate change in vasculature in response to changes in physiology etc, plasticity
• Overall challenges of anterior pituitary research?

  Difficult to study the release of one hormone in particular, since 5 different cell types in the gland each secreting different hormones, release of one may be influenced locally by release of others

  Hard to dissociate action of the pituitary hormone and the steroid hormone that it controls the release of - other factors may underly findings
• Pros and cons of rats as experimental models?

  Majority of studies use rats - means can compare work over time

  Pituitary gland 5mm across, can get ~2 million cells from rat pituitary, means only 6 rats needed for 1 experiment (conservative use of animals)

  Relatively easy to measure plasma hormone levels from rat circulation
• Pros and cons of sheep as experimental models?

  Good for modelling seasonal function of hormones - particularly reproductive ones, since sheep only fertile in winter

  Large gland, so easy to study
• Pros and cons of arctic grey squirrel/hamsters as experimental models?

  Used to study hormones in hibernation - study endocrine transitions - see rapid (overnight) changes in fertility as emerge from hibernation, change in gonadotroph secretion from almost nothing to significant pulsatile secretions
• Pros and cons of mice as experimental models?

  Used for transgenic approach - mouse straightforward for gene deletion and suppression, can target to specific cell types, label cells to see distribution - transgenic mice models support idea of network in pituitary function, coordination within the gland (fluorescently tag hormones, live imaging of cells, can watch how they communicate)

  Pituitary roughly half the size of rat's, mice smaller than rats so much harder to sample blood from, mice much aggressive in lab than rats - stress affects pituitary hormone release (suppresses gland)
• How dense is vasculature in anterior pituitary?

  Very - shown by scanning EM of pituitary stalk when perfused with resin, shows dense network of portal vessels and capillaries. Or show by injecting blood vessels with rhodamine, can label specific cell types with GFP, see that every cell right next to a capillary, and secretory granules concentrated at side nearer blood vessel

  Used for rapid circulation of hypothalamic hormones around gland to influence cells there, as well as rapid release of pituitary hormones into circulation and to target glands
• Draw and/or describe the hypothalmo-pituitary-gonadal axis

  Electrical pulses measured in GnRH neurons correlating to spikes of LH in periphery plasma

  Delete kisspeptin gene then infertile

  Feedforward and feedback control of hormone secretion

  
• Geoffrey Harris experiments, and continuation by Schally?

  Harris lesioned pituitary stalk, inserted hypothalamic extracts onto anterior pituitary, monitored that stimulated ovulation 24hr later. Then lesioned pituitary stalk, monitored results 1 month later, found atrophy of the pituitary, thyroid, and adrenal glands, and ovaries and testes - growth and maintenance of these relied on the hypothalamus, with target gland control via the pituitary, but didn't know hormones involved

  Schally identified GnRH as 1st hypothalamic hormone, and then TRH and CRH - did large scale protein biochemistry
• Where do GnRH neurons originate, describe Kallmans syndrome?

  GnRH hypothalamic neurons develop in the nose in the olfactory epithelium (olfactory bulb), migrate to the hypothalamic pre-optic area in childhood

  Mutations in genes for anosmin1 (KAL1 gene) cause infertility, lack of sexual development, lack of sense of smell - neurons haven't migrated to the hypothalamus - Kallman's syndrome

  In syndrome, olfactory bulbs and nerves absent, GnRH neurons trapped in nose as cannot migrate along nerves

  Induce puberty, restore fertility using treatment with GnRH pump implants (deliver every 2hrs)
• Role of FSH and LH in ovarian follicles?

  LH - acts on LH receptors on theca interna cells, stimulates androgen production via cAMP (androstenedione)

  FSH - acts on FSH receptors on granulosar cells, produces aromatase, converts androgens (diffused from theca cells) to estradiol (major female oestrogen hormone) and inhibin

  Codependent hormones, need both for fertility
• Describe the hormones involved in the follicular phase of the menstrual cycle?

  Follicular phase is day 0-14, ovulation at day 14

  Dominant hormone is FSH, rises from day 0-7, then rising oestradiol and inhibin (peptide) inhibit FSH release (negative feedback) - select a dominant follicle

  Rising oestradiol from dominant follicle stimulates preovulatory surge of LH and FSH, positive feedback of LH and FSH secretion - rising oestradiol sensitises pituitary to GnRH, surge of LH acts on receptors on granulosar cells of dominant follicle, causes ovulation and first meiotic division
• Describe hormones in luteal phase of menstrual cycle?

  After ovulation, LH and FSH fall, stimulate production of progesterone from follicle remains (reassembles as corpus luteum), reduces oestrogen production, lots of progesterone production, prepares female body for possible pregnancy (uterus wall more vascular and nutritious etc)

  If pregnancy has occurred then HCG acts similarly to LH, made by early embryo, and continues to stimulate corpus luteum, continues peak of progesterone, essential for support of embryo

  If no embryo then progesterone no longer secreted, decreases back to baseline
• How does LH induce ovulation?

  Causes breakdown of follicle wall (collagen breakdown and thus weakening of wall) through plasminogen formation (cAMP pathway)

  Also causes maintenance of positive intrafollicular pressure through prostaglandins increasing vascular permeability - creates driving force for extrusion of oocyte
• Why is paracrine signalling in pituitary hard to show?

  Need to purify two different cell types to study their interactions in isolation, which is difficult to prepare, since would normally use cell lines (tumour cells), and there is lots of evidence for paracrine signalling in tumour cells to cause cell differentiation, don't know if this signalling found in healthy cells

  Also don't know if interactions with other cells mediate paracrine functions between two cell types in the gland
• Pros and cons of cell line research?

  Cheap, cells easy to grow, easy to learn techniques used

  Cells all derived from tumours, vary to the extent they represent healthy cells (e.g use alpha T3 cells as a model for gonadotroph cells, but these don't make FSH or LH, or secrete anything - used as model GnRH signalling, as high number of GnRH receptors)
• Pros and cons of using primary pituitary cells?

  Need to digest pituitary glands with trypsin, collagenase, and DNAse to break into single cells, however this also digests the surface receptors, which are needed for paracrine functions - means that hormone secretion underestimated

  Also may be reduced secretion when isolated as not part of network
• Pros and cons of using pituitary segments?

  Not helpful for proving direct connection of two cell types as other cell types present influence - several hormones secreted at the same time

  Lots of prolactin secreted as no DA from hypothalamus, so no inhibition, this high concentration of prolactin in the solution may then have an impact on the other cells
• Monitoring cells using fluorescence?

  Can GFP tag a specific hormone, monitor over time and in response to specific stimuli - can use flow cytometer to detect colour

  Can use 2-photon microscopy to show arrangement of cells in network

  However, low yields of cells
• Formation of the pituitary gland?

  Rathke's pouch grows up from oropharyngeal ectoderm and forms the anterior pituitary

  Infundibular process grows down from forebrain vesicle to form the posterior pituitary

  Connection of these forms the intermediate lobe, intersperses with the anterior pituitary in humans

  Blood supply from internal carotid artery to posterior pituitary and hypothalamus, portal vessels to anterior pituitary