DE - Reproduction, Marie Nováková1 PHYSIOLOGY OF REPRODUCTION 2 Life is a dynamic system with focused behavior, with autoreproduction, characterized by flow of substrates, energies and information. 3 Reproduction in mammals (humans) 1) Sexual reproduction 2) Selection of partners 3) Internal fertilization 4) Viviparity 5) Eggs, resp. embryos – smaller, less, slow development, placenta 6) Low number of offspring, intensive parental care High investment, low-volume reproduction strategy ! Pregnancy (days) Mouse 20 Rat 23 Rabbit 31 Dog 63 Cat 65 Lion 107 Pig 114 Sheep 149 Human 260 - 275 Cow 285 Rorqual 360 Elephant (Indian) 609 4 Reproduction in humans – gender comparison 1) Both male and female are born immature (physically and sexually) 2) Sex hormones are produced in men also during prenatal and perinatal periods, not in women! 3) Reproduction period significantly differs – puberty, climacterical 4) Character of hormonal changes significantly differs – cyclic vs. non-cyclic 5 • Meiosis occurs only in germ cells and gives rise to male and female GAMETES • Fertilization of an oocyte by an X- or Y-bearing sperm establishes the zygote´s GENOTYPIC SEX • Genotypic sex determines differentiation of the indifferent gonad into either an OVARY or a TESTIS • The testis-determining gene is located on the Y chromosome (testis-determining factor, sex-determining region Y) • Genotypic sex determines the GONADAL SEX, which in turn determines PHENOTYPIC SEX (fully established at puberty) • Phenotypic differentiation is modified by endocrine and paracrine signals (testosteron, DHT, AMH) 6 AMH (MIH, MIF, MIS, MRF) – ANTIMŰLLERIAN HORMONE 1940, TGF-b, receptor with internal TK activity Source: Sertoli cells (5th prenatal week) or embryonal ovary (36th prenatal week) In adult women – granulosa cells of small follicles (NO in antral – under influence of FSH - and atretic follicles) Role in men: • Regression of müllerian duct • Marker of central hypogonadism Role in women: • Lower plasmatic levels (by one order), till climacterical • Estimation of ovarian reserve (AMH level corresponds to pool of pre-antral follicles) • Marker of ovarian functions loss (premature climacterical) • Diagnosing of polycystic ovaria syndrome TUMOUR MARKER 7 BIOSYNTHESIS OF STEROID HORMONES CHOLESTEROL PREGNENOLON DEHYDROEPIANDROSTERON PROGESTERON ANDROSTERON CORTIKOSTERON TESTOSTERON CORTIZOL ALDOSTERON ESTRADIOL ACTH LH LH AT II. FSH chol.desmolase 17-a-hydroxylase 3-b-dehydrogenase 21-b,11-b-hydroxylase ald.synthase aromatase cortex of suprarenal glands gonads PUBERTY DHT (5a-reductase)(inhibition-treatment of alopecia) (lack in hermaphrodites; inhibition-treatment of endometriosis) Impact of androgens on CNS! 8 GONADOLIBERIN (GnRH, GONADOTROPIN-RELEASING HORMONE) - Specific origin of GnRH neurons out of CNS - GnRH-I, GnRH-II, (GnRH-III) - Important up and down regulation (steroidal hormones, gonadotrophs) - Down regulation – malnutrition, lactation, seasonal effects, aging, continual GnRH - Up-regulation – effect of GnRH on gonadotrophs (menstrual cycle) Hypothalamo-hypophyseal axis - FSH, LH - Significance of GnRH pulse frequency (glycosylation) - Menstrual cycle, puberty and its onset Other functions and places of production - CNS – neurotransmitter (area preoptica) - Placenta - Gonads - Tumours (prostate, endometrium) Unknown function 9 - Inputs from various CNS areas (pons, limbic system) - Dominating inhibitory effect of sex hormones with exception of estradiol (negative-positive-negative feedback) - Kisspeptin in women - Inhibitory effect of PRL - Effect of circulating substrates (FA, Glu) - Leptin (NPY, kisspeptin) - Stress of various origin - Acute – MC impairment without effect on fertility - Chronic – impaired fertility, decreased levels of circulating sex hormones GONADOLIBERIN – REGULATION OF SECRETION 10 Pinilla et al., Phys Rev 92: 1235- 1316, 2012 CONTROL OF SEX HORMONES SECRETION 11 GONADOTROPHINS - FSH and LH - Glycoproteins - Heterodimer, different expression of subunits, glycosylation - Structurally close to hCG (placenta) Regulation of secretion - sex hormones, local factors – paracrine (activins, inhibins, follistatin) - (+) – glutamate, noradrenaline, leptin - (-) – GABA, opioids - Key role of kisspeptin, neurokinin B and substance P in GnRH secretion – FSH/LH - Estrogens, progesterone, androgens – direct influence on gonadotrophs, indirect influence through GnRH - Estrogens (-) – inhibition of transcription (a), kisspeptin – NEG - Estrogens (+) shift - Progesterone (-) – influences pulsatile secretion of GnRH - Testosterone, estradiol (-) – males, kisspeptin neurons and AR - Different half-life for circulating LH and FSH 12 ACTIVINS and INHIBINS Inhibins – dimeric peptides (a + 1 or two bA or bB) – circulating hormones produced by gonads – inhibin A – dominant follicle, corpus luteum – inhibin B – testes, luteal and early follicular phase of ovarian cycle Activins – dimeric peptides – dimers of b subunits – FSH stimulation – autocrine/paracrine factors – other tissues – growth and differentiation Follistatin – monomeric polypeptide – FSH inhibition - „supplementary “ regulation of FSH and LH secretion - activins = regulation of transcription, follistatin and inhibins = inhibition of activins through appropriate activin-receptor binding 13 FSH and LH - functions FEMALES FSH - Growth and development of follicular cell (maturation) - Biosynthesis of estradiol - Regulation of inhibin synthesis during follicular phase - Upregulation of LH receptors (preovulatory follicles) - Selection of dominant follicle - Recruitment of follicles for next cycle LH - Stimulation of estrogen synthesis at various levels (theca) - Oocyte maturation (preovulatory follicle) - Rupture of ovulatory follicle, ovulation - Conversion of follicle wall to corpus luteum MALES LH Intratesticular synthesis of testosterone (Leydig cells) FSH Spermatogenesis (Sertoli cells) 14 REGULATION OF SEX HORMONES SECRETION – simplified scheme HYPOTHALAMUS ADENOHYPOPHYSIS GONADS GnRH dopamine (PIF) endorphins noradrenalin FSH LH Activin Inhibin B n.arcuatus gonadoliberin (decapeptide) E P T PROLACTIN Pre-pubertal block of GnRH ??? Blocking the effects of gonadotrophins on gonads Down-regulation of LH receptors in testes and ovaria Fight or flight Exercise Nutritional state changes GABA, kisspeptin 15 LEPTIN A REPRODUCTION Activation of reproductive system does not depend on age, but on nutritional state of organism. LEPTIN: ob-protein, ob-gen, 7.chromosome „leptos“ = thin, slim polypeptide, 176 AA Bound in hypothalamus: n.paraventricularis, suprachiasmaticus, arcuatus a dorsomedialis Produced in: adipocytes, stomach, placenta, mammal epithelium (???) Leptin plasmatic levels are sex-dependent (less in males) and do not depend on nutritional state Leptin receptor: gene on 4.chromosome, 5 types of receptor, A-E Receptor B – effect in gonads and hypophysis Leptin is not only a factor of body fat amount, but affects also the regulation of neuroendocrine functions, including hypothalamo-hypophyseo-gonadal axis. 16 ncl. arcuatus area preoptica - reproduction ??? Critical amount of adipose tissue – leptin – hypothalamus – LHRH – puberty ??? 17 Effects of leptin on testes are not fully elucidated yet. Testosterone and dihydrotestosterone suppress production of leptin in adipocytes! REGULATION OF PUBERTY ONSET BY LEPTIN Critical body mass (critical nutritional state). Leptin plasmatic levels in pre-pubertal children are sex-independent. Pre-pubertal „leptin resistance“ (relative). In puberty, girls produce 2x more leptin per 1kg of adipose tissue than boys. 18 PROLACTIN - PRL - Protein - Lactotropic cells (only PRL) - Mammosomatotrophic cells (PRL and GH) - Hyperplasia – pregnancy and lactation - Expression regulated by oestrogens, dopamine, TRH and thyroid gland hormones - Polypeptide, circulating in 3 forms (mono-, di-, polymer) - Monomeric PRL – highest biological activity - Monomeric PRL further cleaved (8/16 kDA) - PRLR – mamma, adenohypophysis, suprarenal gland, liver, prostate, ovary, testis, small intestine, lungs, myocardium, SNS, lymphocytes Regulation of secretion - Pulsatile secretion: 4 – 14 pulses/day - Highest levels during sleep - Lowest levels between 10:00 and 12:00 - Gradual decrease of secretion during aging - TIDA cells – dopamine (-, D2R) - Paracrine – endothelin-1, TGF-b1, calcitonin, histamine (-) - TRH, oestrogens, VIP, serotonin, GHRH at higher concentrations (+) (Co-hormone) 19 MAIN FUNCTION: Milk production during pregnancy and lactation = „survival“ function Other functions – metabolic, synthesis of melanin, maternal behaviour Breast development a lactation - Puberty – mamma development under the effects of GH a IGF-1 - Effect of oestrogens and progesterone - Age of 8 – 13 - During pregnancy – proliferation of alveoli and proteosynthesis (proteins of milk and colostrum) - During the 3rd trimester – production of colostrum (PRL, oestrogens, progesterone, GH, IGF-1, placental hormones) - Lactation – increase in PRL post-partum, without sucking drop after approx. 7 days - Milk accumulation prevents further PRL secretion Reproductive function of PRL - Lactation = amenorrhea and secondary infertility - Inhibition of GnRH secretion PROLACTIN - functions 20 DOPAMINE (PIH, prolactin-inhibiting hormone) Characteristics - D2R (G protein inhibition, AC, cAMP decrease, inhibition of shaker type K+ channels, MAPK, PAK – proliferation!) - D1R (activation) Hypothalamo-hypophyseal axis - Inhibition of PRL (D2R) secretion – lactotropic cells - ! Lactotrophs with continual high PRL production - PRL secretion regulated also at adenohypophysis level (paracrine, autocrine) - Neuroendocrine regulation of PRL secretion – pregnancy, lactation, menstrual cycle, sensory inputs 21 - Important feedback mechanism (short loop) of PRL secretion regulation - Circadian rhythm (maximum in the morning) - Nipple stimulation (1-3 min, peak 10 – 20 min) DOPAMINE – REGULATION OF SECRETION PROLACTIN-RELEASING FACTORS (PRF) - TRH, oxytocin, VIP - under specific conditions ADH, ATII, NPY, galanin, substance P, GRP, neurotensin - prolactin-releasing peptide (PrRP) – stress, satiety (other parts of CNS) Enkephalin, dynorfin (m a k receptors) 22 Puberty • Adrenarche • Pubarche • Telarche • Menarche Pubertas praecox (central) Pseudopubertas praecox (peripheral) CRITICAL DEVELOPMENTAL PERIODS 1) Birth 2) Weaning 3) Puberty (adolescence) 4) Climacterical (menopause) Late puberty Critical body mass (critical amount of adipose tissue/nutritional state) 23 MALE REPRODUCTION SYSTEM 24 TESTOSTERON PRODUCTION: •Embryonic – sex differentiation, development of generative organs •Perinatal – descensus testis (?) •Fertile period – LH pulsation •Ageing – decrease of sensitivity to LH FSH LH SERTOLI CELLS LEYDIG CELLS INHIBIN B TESTOSTERON AMH ACTIVIN DHT ABG aromatase GnRH glycoproteins ENDOCRINE REGULATION OF REPRODUCTION FUNCTIONS IN MAN - - - PROLACTIN 5a-reductase 25 26 27 FEMALE REPRODUCTION SYSTEM 28 OOGENESIS DEVELOPMENT: 6-8 weeks GERMINAL EPITH. hormonally OOGONIA FOLLICLE independent mitotic division PRIMORDIAL 24 weeks OOCYTES I. 7 x 106 1. meiosis birth prophase 2 x 106 hormonally puberty OOCYTES II. 3 x 105 dependent haploid DOMINANT (GRAAF) (cyclic) 2. meiosis ATRETIC metaphase OVUM OVULATION 2. meiosis – end climacterical 0 29 CYCLIC CHANGES ovarian uterine GnRH FSH, LH estradiol basal temper. 0 4 14 28 MENS. PROLIPHER. SECRETORY PHASE + _ 0.5 – 0.75°C FOLICULLAR OV. LUTEAL PHASE 6-10x 2-3x progesteron + vagina/cervix uteri + mamma 30 OVARIAN CYCLE Germinal epithelium Primordial Primary Graaf Corpus haemorrhagicum C. luteum follicle 25m 150m up to 2 cm estradiol progesterone Oocyte-maturation inhibiting factor Vesicular follicle Luteinisation inhibiting factor (estrogens) (progestins) OVULATION methrorhagia 31 VESICULAR FOLLICLE PRIMARY FOLLICLE - FSH Growth acceleration of primary follicle – change into vesicular follicle: 1) estrogens released into follicle stimulate granul. cells UP REGULATION of FSH receptors and intrinsic positive feedback (higher sensitivity for FSH!!!) 2) UP REGULATION of LH receptors (estrogens and FSH) – another acceleration of growth due to „higher sensitivity“ to LH (positive feedback) 3) Increased estrogens and LH secretion accelerates growth of theca cells, secretion is increased explosive growth of follicle 32 DOMINANT FOLLICLE 1. High levels of oestrogens from the fastest-growing follicle 2. Negative feedback on FSH production from adenohypophysis 3. Gradual decrease in FSH secretion 4. „Dominant follicle“ continues in growing due to intrinsic positive feedback 5. Other follicles grow slowly and subsequently become atretic 33 MECHANISMS OF OVULATION LH PROGESTERON Hyperaemia of follicle Secretion of prostaglandins Weakening of follicle wall PROTEOLYTIC ENZYMES (collagenases from theca externa) Degeneration of stigma Rupture of follicle Release of oocyte Transudation of plasma into follicle Swallowing of follicle 34 EFFECTS OF OVARIAN HORMONES E P Secondary sexual signs + Adipose tissue: store (predilection), (critical amount) Bone tissue: absorption closure of fissures development of pelvis Total water retention: + + Sexual behaviour: + Ovaries: maturation of follicles Hysterosalpinx: motility motility Uterus: proteosynthesis proteosynthesis vascularisation and proliferation of endom. secretion of endom. glands motility glycogen motility Cervix: colliquation of „plug“ creation of „plug“ Vagina: cornification of epithelium proliferation of epithelium Mamma: growth of terminals growth of acines 35 CONTRACEPTION (BIRTH CONTROL) • RHYTHM METHOD • SPERMICIDE SUBSTANCES • COITUS INTERRUPTUS • CONDOM, PESSARY • IUD • HORMONAL CONTRACEPTIVES – risk of failure less than 1% • VASECTOMY AND LIGATION OF HYSTEROSALPINX Hormonal curettage (excochleation). Substitution therapy in climacterium. 36 HORMONAL CONTRACEPTION • block of ovulation by suppression of hypothalamic releasing hormones (block of preovulatory surge of LH) • changes of character of cervical plug (progestin thickens mucus) • changes of endometrium (suppression of its growth) • changes of hysterosalpinx motility 37 PREGNANCY, PARTURITION, LACTATION 38 HORMONAL PROFILE OF PREGNANCY I. II. III. trimester hCG P E PROL Relaxin hCS OX 0 10 20 30 40 weeks of pregnancy STH TSH ACTH INS KORT ALD T4 PTH Placental – maternal – foetal source of hormones Corpus luteum graviditatis hCG, hCS, E, PE, P, Relaxin (8th week!!!) luteinisation and luteotropic effects inhibition of myometrial contractions preparation of lactation growth hormone in pregnancy induction of parturition Placenta 39 RELATIONSHIP BETWEEN P:E IN PREGNANCY P E P > E E > P MOTHER PLACENTA FOETUS cholesterol pregnenolone DHEAS 16OH-DHEAS progesterone cortisol aldosterone DHEAS estradiol Estriol Foetoplacental unit Excretion of estriol in urine – index of foetal status 40 PHYSIOLOGICAL CHANGES DURING PREGNANCY Changes of reproductive organs • Uterus – Growth (from 60 g to 1000 g), change of position – Hyperaemia – Functional differentiation of myometrium • Cervix – Changes of colour, consistency; shortening – Hypertrophy a hyperplasia of glandules – mucus plug • Vagina – Changes of colour, increase of secretion • External genitals – Vascularization, vasocongestion (changes of colour) Somatic changes • Breasts – Growth – alveolar as well as ductal part – Enlargement and hyperpigmentation of mammillae and areolas • Skin – Increase in subcutaneous fat – Changes in connective tissue – Hyperpigmentation Endocrine and metabolic changes Immunological changes Psychic changes 41 ENDOCRINE and METABOLIC CHANGES DURING PREGNANCY Endocrine glands • Thyroid gland – Slight hypertrophy (E), increase in thyroxine production, in III. trimester BEE +25% • Parathyroid glands – Increase in production of parathormone • Adrenal glands – Increase in production of aldosterone • Pancreas – Hyperplasia of Langerhans islets Anterior pituitary gland Metabolism • Weight gain: 12-15 kg • Glycaemia – Glc – main energetic source for foetus – Prohyperglycemic state – Decrease of renal glucose reabsorption, increase in glomerular filtration - glycosuria – Gestational diabetes • Increased demand for Ca (1300 mg), P (1200 g) and Fe (18 mg/day) • Water retention: + 6.5 l 42 OXYTOCIN - Mechanoreceptors/tactile receptors - Magnocellular neurons (PVN, SON) - inhibition by endogenous opioids, NO, GABA - Autocrine (+ ZV) - Prolactin, relaxin (-), Estrogens (+) - OXT receptors (Gq/11) – effect of up/down regulation - Acts together with prolactin and sex hormones Functions - Lactation (under 1 min) - Parturition - rhythmical contractions of smooth muscles (gapjunction, stimulation of prostaglandin synthesis – extracellular matrix) - postpartum bleeding - uterus involution - Ejaculation (males) - Behavior Other functions and places of synthesis - CNS - Stimulation of ACTH secretion through CRH - Stimulation of ADH/induced vasoconstriction - Stimulation of prolactin secretion - Memory traces recollection inhibition - Maternal behavior 43 – 9 AA, differs from ADH in 3. a 8. AA – Precursor molecule is synthetized in the same location as ADH (nucleus paraventricularis) – Stimulus for synthesis: dilatation of birth path caused by pressure of foetus and stimulation of mechanoreceptors at breast nipple – Reflex release: during breast-feeding, orgasm – Main effects – on reproduction system: • Uterokinetic effects (induction of parturition), milk ejection, involution of uterus • In men: probably increases contractions of smooth muscle in ductus deferens – Regulation of water and mineral metabolism – natriuretic effect, potentiation of ADH effect – Effect on memory: opposite to ADH effect – inhibits forming of memory and its recollection – Note: Melanocytes inhibiting factor – from oxytocin, modulates certain types of receptors, modulation of melatonin effects (melatonin – epiphysis, together with glomerulotrophin and DMT, circadian/circannual biorhythms, controlled by hypothalamus, information from retina) OXYTOCIN 44 INDUCTION OF BIRTH P > E E > P maternal placental foetal CHOLESTEROL HT ACTH OXYTOCIN DHEA PREGNENOLONE CRH EP CORTISOL PG oxyt.rec. conexin gap-junctions collagenase afferentation from mechanoreceptors 100x Foetal respiratory insufficiency 45 LACTATION GnRH PIF PSF OX. GH FSH LH PROL. GCPH UTERUS involution MAMMA E HCSP ejection milk production stop of cycle Composition of milk: water (88%), fat (3,5%), lactose (7%), proteins (1%) trace minerals (Ca), vitamins, antibodies (hyperprolactinaemia) placental hormones 0.6-2 l/day 1 – 3 days after birth; initiated by decrease of oestrogens´ concentrations post partum