Reproduction

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Reproduction

Definition of hormones• synthesized and secreted by ductlessendocrine glands• minute quantities• directly into the blood vascular system• Classic endocrine glands:– pituitary, thyroid, parathyroid, adrenal,pancreas, ovary, testis, placenta, pineal gland


Definition of hormones• Reproduction– pituitary, pineal glands, gonads, placenta– adrenal and thyroid glands– uterus, hypothalamus


Extra hypothalamic centresEstrogensHypothalamiccentresInhibinGnRHAnteriorpituritaryOvaryFSH/LHMelatoninEstrogens/progesteroneEstrogens/progesteronePinealReproductiveendocrine1. Gonadotropin-releasinghormone2. Gonadotropichormone3. Sex steroid hormonesPGF2αUterus


Chemical classes of reproductivehormones• Peptide and protein hormones– Releasing hormones, Inhibitory hormones– Hypophyseal hormones– Neurohypophysealhormones– Placental gonadotropins– Subunits• Steroid hormones• Prostaglandins


The hormones secreted fromthe anterior pituitary


Peptide and protein hormones• Releasing hormones, (GnRH, TRH, CRH, PRH,GH-RH)– Peptide hormones– produced within the hypothalamusportal vein– Adenohypophysis• Inhibitory hormones– Growth hormone(GIH)– Prolactin(PIH)TSH ›Insulin fl, glucagon fl


Releasing hormones• Gonadotropin-releasing hormone (GnRH)– pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (MW of GnRH @ 1,182 )regulate the release of FSH and LH


hypothalamic-pituitary-ovarian axis


LH and FSH are released into thesystemic circulation, traveling tothe target reproductive organs(testes and ovary) where sexsteroid hormones are controlled.LH sends the same message andresults in increased testosteroneproduction by the testis due toLH effect on the Leydig cells ofthe testis.


Releasing hormones• Thyrotropin-releasinghormone (TRH)– pGlu-His-Pro-NH2(MW of TRH @ 362 )regulate the release ofTSH, GH, and prolactinPathways of thyroid hormone metabolism


Releasing hormones• Corticotropin-releasing hormone (CRH)– CRH is a 41-amino acid peptideregulate the release of ACTH


Peptide and protein hormones• Hypophysealhormones– Glycoprotein, FSH and LH, prolactin– MW of LH @ 30,000 (bovine, ovine, procine,equine)– MW of FSH @ 32,000 (ovine, equine)– MW of prolactin @ 23,000 (bovine, ovine,procine)• FSH, LH, TSH, ACTH – produced in basophiliccells• Prolactin– localized in acidophilic cells


• Prolactin– Luteotrophichormone (LTH): a corpus luteumstimulatingeffect in mouse, rat, dog and cat– 198 a.a, MW 23,300 and a 15-minute half-life insheep– increasing in lactation (mamotropin)– stimulating the pigeon crop gland to secret“crop milk”


Peptide and protein hormones• NeurohypophysealhormonesOxytocin, Vasopressin (synthesized in hypothalamus)Posterior pituitaryreleaseAxoplasmic fluidStimulation ofthe N cell


Peptide and protein hormones• Placental gonadotropins– PMSG (eCG, MW @ 70,000), a protein hormonein serum, produced by fetal trophoblasticcellsof pregnant mares• The function of endometrial cups begins on day 36until 120 days of pregnancy.– hCG(MW @ 36,000), a protein hormone,extracted from the urine of pregnant womenfrom 30-40 days of pregnancy– glucocortocoid, estrogen, progestin, relaxin…


Endometrial cupsEndometrial cups develop from cells of thechorionic girdle, circumferentially aroundthe conceptusat a point where themembranes of the allantoisand yolk sacmeet.


270 days1. The relationship of eCGand CL during equinepregnancy2. eCGhas both FSH-likeand LH-like activity, itprovides the stimulus forthe formation of accessoryCLsand maintain a plateauof plasma progesteronefrom about 50-140 days.


Clinical use of hCG1. One Step hCG Urine PregnancyTest2. hCG is also used to treat womenwith certain ovarian disorders.


Clinical use of hCG• Athletes use hCG to increase the bodynatural production of testosterone which isoften depressed by long term steroid use.• HCG therapy has been found to be veryeffective in the prevention of testicularatrophy and to use the body own biochemicalstimulating mechanisms to increase plasmatestosterone level during training.


Peptide and protein hormoneshCGhCG-subunit α: 96 a.asubunit β: N-terminal specificfor hCGLHLH - subunit α: 96 a.asubunit β: 119 a.aspecific for LHFSH - subunit α: 96 a.asubunit β: specific for FSHTSH - subunit α: 89 a.a in humansubunit β: 112 a.a in human


Peptide and protein hormones• Subunits, a, b– b-subunit, unique for each hormone within aspecies, determines the biological activity.


Chemical classes of reproductivehormones• Steroid hormones• derived from the precursor, cholesterol• MW < 500– Estrogens (estradiol)– Androgens (testosterone)– Progesterones (progesterone)


Ccholesterol27 H 46 O C 21 H 30 O 2C 19 H 28 O 2 C 18 H 24 O 2


Chemical classes of reproductive• Prostaglandinshormones– a group of 20-C unsaturated fatty acids– MW: 300-400– local hormones, short biological half-life with alocal action– found in mammalian tissues, uterus andprostata


peroxidaseBiosynthesis ofPG fromarachidonicacid


Synthesis of prostaglandin affected by estrogenEstrogen↑oxytocin receptors ↑InactivatedActivatedPhospholipase A2Phospholipase A2Progesterone↓Prostaglandin H2synthetasePhospholipidsArachidonic acidEstrogen↑ Progesterone↓:1. proestrus → estrus2. prior to parturitionProstaglandin H2PGE PGF PGD


PGE 2stimulatesFunction of prostaglandinsvascular dilationbronchiodilationgastrointestinal and uterine contractioninflammatory responsemimicsinhibitshormones that act through cyclic AMPplatelet aggregationPGF 2stimulates vascular constrictionbronchioconstrictionsmooth muscle contraction, e.g. intestinal tract, uterusPGI 2inhibitsplatelet aggregation (increases cAMP)vascular constrictionbronchioconstrictiongastrointestinal and uterine contraction


vascular uteroovarianpathways for PGF2αUterinebranch of OVOvarianbranch of OV


vascular uteroovarianpathways for PGF2αSystemic uteroovarianpathway: Local uteroovarianpathway: Combined systemic and localuteroovarianpathway:horse, rabbit sheep, cattle, (guinea pig, hamsters, swinerats)?


endometritis/pyometraClinical use of PGF2a


Clinical use of PGF2ainduction of estrus


Corpus luteum/progesteronePGF2a injection forinduction of estrusOnset of estrusOvulationOnset of estrusOvulationOnset of estrusOvulationNormal estrus cycleShortenestrus cyclewith PGF2a


Clinical use of PGF2ainduction of abortion/parturition


Clinical use of PGF2ainduction of abortion/parturition


Clinical use of PGF2ainduction of abortion/parturition


Clinical use ofPGF2ainduction of abortionendometritis/pyometramisallianceonly in the case of open pyometra!!


Hormone receptors• Steroid hormones– fat soluble, able to enter all cells– low concentration (estrogen: 10-150pmol/L in non-pregnant female)• Receptors enable the target cells-to concentrate the hormone within the cell-to elicit particular cellular responses-to create a biological response on specificbinding


The steroid hormone receptor ismade up of three domains:1. a modulating N-terminaldomain (transcriptionactivationdomain),2. a DNA-binding domain3. a C-terminal steroid-bindingdomain.


Hormone receptorsprotein-boundsteroid hormonereceptorssteroid-receptor complexTranslocation ofcomplex into nucleusnon-proteinboundhormonepassivediffusionRNAsynthesis↑proteinsynthesis ↑Target cellsE2: mammary glandP4: endometriumT: sertoli cell


Hormone receptorsProtein hormoneprotein-receptor complexreceptorsG proteincyclicAMPactions ofFSH and LHin gonadsTarget cellsproteinkinaseactions of cholesterol


Tissues responsive to androgens


Interconversion of steroids in targettissuestestosterone5α-reductase5α-dihydrotesterone (5α-DHT)Target cells


Progesterone conversion in the placentaCholesterolCortisol17α-HydroxylasePregnenoloneProgesteroneparturitionPGF2α↑17α-HydroxyprogesteroneAndrostenedioneEstroneTestosteroneEstradiol 17β


Synthesis and clearance of hormones• Synthesis and clearance are main thefactors, which influence the hormoneconcentrations.– The concentration of a hormone reflects its rateof synthesis or secretion.– metabolized by liver– detection of the body fluids (plasma, urine,saliva, feces…)reproductive condition, ex wild animals


Synthesis and clearance of hormones• under the control of gonadotropinsinpulsatile mode♂♀– The secretion of testosterone changes inpulsatilerate.– varying from 3.5 – 20 nmol/L within hours– The pulse rate of E2 and P4 remains stable.


Synthesis and clearance of hormones• Placenta gonadotropins (hCG, eCG)– produced in high concentration– longer half-life than the pituitary gonadotropins• FSH, LH ≒ 10-30 minutes• hCG ≒ 1.5 days• eCG ≒ 6 days• equine LH > other species


Synthesis and clearance of hormones• PGF2α– Half life15-keto-13,14-dihydro-PGF2α• primary PGF2α < 20 seconds• 15-keto-13,14-dihydro-PGF2α ≒ 8 minutes– 25 mg PGF2α, IM in cows• > 90% excreted in the urine and feces (2:1) over 48hours


Assay Methods• Quantification of specific hormones isnecessary for the endocrinologicalevents.– Weight-gain change– chemical determinations for steroid hormones– urine analysis


AgAbImmunoassaytechnique


Assay Methods• Immunoassay– Radio-immunoassay (Berson et al., 1959), Sen:10 -9– Enzyme multipledimmunoassay technique,Sen:10 -6– Fluoescineimmunoassay, Sen:10 -9– EIA (Engvall et al., 1970s), Sen: 10 -12– Chemiluminescenceimmunoassay, Sen10 -15


RadioimmunoassayBecause of the ease withwhich iodine atoms canbe introduced intotyrosine residues in aprotein, the radioactiveisotopes 125 I or 131 I areoften used as theradioactive antigen.


Sandwich EIA


competitive EIA


semi quantitative ELISA test


LuminolThe chemical luminol is oxidised byH 2 O 2 , to produce a molecule in anexcited state. When the excitedelectrons in the molecules fall back totheir ground states, light is given out.Excited StateGround State


Assay Methods• Immunoassay– Radio-immunoassay (Berson et al., 1959), Sen:10 -9• Competitive protein binding– EIA (Engvall et al., 1970s), Sen: 10 -12– Chemiluminescenceimmunoassay, Sen10 -15Diagnostic aidsTherapy-monitoring


Monitoring the seasonal estrus activity of mareNorthern semi-sphereSubtropical Taiwan


Physiology of reproductivehormones in the female• Estrous cycle• Control of the corpus luteum• Early pregnancy• Pregnancy and parturition


Endocrine control of cyclical reproductive activity


The duration of estrus, time of ovulation and duration of CL function in domestic animals


increasingday lightPhotoreceptorsin the eyesneurotransmissionPinealdecreasingday lightInfluence of thelength of day light onthe ovary function inmaresMelatoninMelatoninGnRHGonadotropineHypothalamusportal systemAnteriorpituitaryblood circulationOvariesGnRHGonadotropine


estrous behavior of mares


Large amounts of equine LH are released during an 8-to 9-dayperiod with ovulation occurring on the 3rd to 5th day.8-9 daysControl of the corpus luteum


P4LH ›+ feed back1. theca interna has LHreceptor and producestestosterone.2. granulosa cell has FSHreceptor which causescell to take testosteronefrom theca and convertit to estradiol.3. dominant follicle hasLH receptors ongranulosa which canrespond to LH to driveprogesterone production.Each granulosa cellhas approximately1500 FSH receptors


Ovarian steroidogenesisProduction Rate of Sex Steroids in Women at Different Stages of the Menstrual CycleSEX STEROIDS*EarlyFollicularDAILY PRODUCTION RATEPreovulatoryMidlutealProgesterone (mg) 1 4 2517Hydroxyprogesterone (mg)Dehydroepiandrosterone (mg)0.5 4 47 7 7Androstenedione (mg) 2.6 4.7 3.4Testosterone (mg) 144 171 126Estrone (mg) 50 350 250Estradiol(mg) 36 380 250FSH causes increasedaromatase activity, whichconverts testosterone toestradiol


ovulation• cumulus cells produce hyaluronicacid andproteins to cause cumulus expansion• increased blood flow to follicle (vascularpermeability↑)


ovulation• Triggers increase protein and steroidsynthesis, and cellular differentiation withinfollicle• Plasminogenis converted to plasmin byplasminogenactivator ( active collagenase↑).• Prostaglandin stimulates smooth muscle tocontract and force the ruptured follicle toempty.


Control of the corpus luteum• CL is responsible for the estrous cyclicity.• Uterus controls the lifespan of the CL in thecow, ewe, sow and mare.– Estrogens initiate uterus to synthesizes PGF2α.– Regression of CL is accomplished within 48 hrs.


Control of the corpus luteum– Estrogens endometrial oxytocinreceptorsinitiate the release of PGF2α, which causes thereaction of luteal regression.– In ruminants, releasing of PGF2α is induced byoxytocinsecreted by the CL.– In dog and cat, PGF2α CL?


Control of the corpus luteumLH stimulates the secretion ofprogesterone from small luteal cellswhile PGF2α inhibits secretion ofprogesterone from large luteal cellsand causes degeneration of thesecells.


Early pregnancy• Maternal recognition– modification of PGF2α by the luteotropicproducts from the conceptus and uterus• Trophoblastproteins (cow, ewe, doe)• Estrogens (sow, mare)• intrauterine mobility of the embryo (mare)• ? (bitch, queen)• antiluteolyticaction or luteotrophiceffect• to extend the luteal activity


Early pregnancy• Trophoblastproteins (cow, ewe, doe)– oTP-1 secreted between 10 and 21-24 days– bTp-1 secreted between 16-19 days– cTP-1 secreted on Day 17• to bind uterine oxytocinreceptors• to block the uterine PGF2α secretion


Trophoblastproteins• Ovine trophoblastprotein (oTP-1, IFN-τ)– MW 18,000, produced on Day 10– inhibiting the uterine oxytocinreceptors– inhibiting the synthesis of PGF2αPGF2↓, PGE2↑


Trophoblastproteinoxytocinreceptorfl


Early pregnancy• Conceptus-derived estrogens (sow, mare)– estroneand estradiol 17-β converted fromprogesterone on Day 11, Day14-18– rapid enlongationof the blastocyst– releasing of calcium, specific proteins• altering an endocrine of PG to an exocrine PG• metabolized to inactive PGFM• PGF2PGE2


The secretion of estrogens by the blastocyststimulates the release ofcalcium, specific polypeptides and proteins.1.altering the PGF2 from the endocrine to an exocrine2.intrauterine PGF2 metabolisedto inactive PGFM by fetal membranes


• There is extensive mobility of theequine conceptus within the horns anduterine body before fixation occursbetween days 16 and 18.• ?? Endometrial ProstaglandinSynthesis Inhibitor, (EPSI) fromendometrium


Time of maternal recognition of pregnancyDay ofrecognitionDay of definiteattachmentMare 14-16 36-38Cow 16-17 18-22Sow 12 18Ewe 12-13 16Goat 17


Corpus luteum& Pregnancy• P4 from CL: cow, sow, goat, dog and cat• P4 from CL + fetoplacentalunit: mare, sheep


Pregnancy1. The sheep placenta produces enough progesterone by day 60-70.2. Luteal progesterone is required throughout gestation in cattleand goats because their placenta secrete much smaller quantitiesof progesterone.


DihydroepiandrosteroneFetal-Placental Unit


ParturitionProgesterone flParturition within12 hours


Parturition


FetalmaturationDecrease inplacental P4Luteolysisof theCL of pregnancyDecrease inserum P4IncreasedmyometrialexcitabilityAdrenalineFetal hypothalamusFetal pituitaryACTHFetal adrenalAdrenal corticosteroidsFeto-placental estrogensCotyledons/myometriumRelease of PGF2aMyometrialcontractionsOxytocinPosterior pituitaryCortisol › flParturitionRelaxin?Relaxation andsoftening of cervixpelvic ligaments,perineumExpulsion of fetusStimulates vaginaand cervixAbdominalstraining


Parturition• Prostaglandins are soluble in fat and water,Prostaglandins• Actions:– smooth muscle contraction– luteolysis– softening of cervical collagen– developing gap junctionspass from cell to cell– movement of Ca 2+ between the myofibrils• PGF2α, PGE↑, PGI2↓


Smooth muscle cells


molecules (< 1000 daltons) ) that can passinclude calcium ions, cAMP, , ATP,glutathione, and large molecules like aminoacids and sugars etc.


Smooth muscle contraction• Muscle protein: myosin and actin– Calmodulinactivates MLC kinaseCalcium-CalmodulinMLC kinaseinactive myosinlight chain(M*-ATP) + A (M*-ADP-A) + Pi(M*-ADP-A) (M-A) + ADPactomyosincomplexcontractions


Smooth muscle contraction1. Calcium ions come from outside of the cell.2. Calcium ions bind to calmodulin-MLCKcomplex on myosin.3. The enzyme complex breaks up ATP into ADP and transfers the Pi directly to myosin.4. This Pi transfer activates myosin.5. Myosin forms cross-bridges with actin.6. When calcium is pumped out of the cell, the Pi gets removed from myosin by phosphatase.7. The myosin becomes inactive, and the muscle relaxes.


Smooth muscle contractionThe top view shows a relaxed smooth muscle cell. Note the focaldensities and the network of actin and myosin filaments.When contracted, the filaments slide together and pull the cell to amore rounded appearance.


Calmodulin mediates processes such asinflammation, metabolism, apoptosis,muscle contraction, nerve growth,immune response….Calmodulin is one of the primaryreceptors of intracellular calcium.4 Ca 2+ bind to calmodulin. At eachbinding site, Ca interacts with oxygenatoms, mainly glutamate and aspartaseside-chain carboxyl group.


Parturition• Actions of estrogens– contractile protein synthesis↑– receptors of oxytocinand PGF2α ↑– calmodulinsynthesis↑– MLC kinaseactivity ↑– gap junctions ↑


Parturition• Actions of progesterone– gap junctions↓– receptors of oxytocinand PGF2α ↓– synthesis of oxytocinand PGF2α ↓– calmodulinbinding↑


Parturition• Actions of oxytocin– dependent on progesterone↓, estrogens↑– stimulation of receptors in the anterior vaginaandcervix oxytocinrelease• increasing PG release• increasing Ca 2+ release


Clinical aspects of reproductiveendocrinology• Steroid hormone concentrations are lowerin animals than in human. (estrogens < 1/10)• Progesterone analysis give the most usefulinformation on the reproductive status.• Blood, milk, urine, saliva and feces aresources for endocrine information.


Clinical aspects of reproductive• Cow:endocrinology– P4 for early diagnosis of pregnancy• Negative forecast (98%) is better than positiveforecast (67-90%).– P4 for postpartum ovarian activity– Estronesulfate for diagnosis of pregnancy• from Day 120, 96% accuracy rate– Bovine pregnancy specific protein B for earlydiagnosis of pregnancy on Day 30


Sampling timeP 4 for postpartumovarian activity orearly diagnosis ofpregnancy


P 4 for postpartum ovarian activityFollicular cystOvarian cyst


P 4 for postpartum ovarian activityLuteal cyst


Diagnosis of the ovarian diseases in cattle12Persistent CL or luteal cyst10Progesterone concentration(ng/ml)8642Normal cycleSampling timeSampling timeSampling timeOvarianatrophy orfollicular cyst0-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1Days of estrus cycle


Treatment of bovine follicular cystGnRHHypothalamusLHHypophyseRegulation ofestrus cycleProgesteroneovulation stimulation or cyst luteinizationor ovulation of cyst


Monitoring the treatment of bovine follicular cystProgesterone concentration(ng/ml)121086420GnRH inj.PGF2a inj.Sampling timeSampling timeSampling timeGnRH inj.AI-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24Days after application of GnRH


Clinical aspects of reproductive• Sheep:endocrinology– P 4 for early diagnosis of pregnancy• P 4 in estrus: 0.15-0.25 ng/ml, in CL phase: 2-4 ng/ml,in pregnancy from 60 days: 12-20 ng/ml– Estronesulfate (fetoplacenta) for diagnosis ofpregnancy from 70 days (≒ 88%)– Pregnancy specific protein, ovine placentallactogenfor diagnosis of pregnancy


Clinical aspects of reproductive• Sow:endocrinology– P 4 for early diagnosis of pregnancy• P 4 in pregnancy: 20-50 ng/ml– Estronesulfate for early diagnosis of pregnancyfrom Day 20-26, 98% accuracy rate


Clinical aspects of reproductive• Horse:endocrinology– eCGfor diagnosis of pregnancy– P 4 for ovarian activity• Persistent CL exhibits low P 4 value• Presence of cyclic ovarian activity• Ovulation prediction (88%)– Estronesulfate for diagnosis of pregnancy– Testosterone for diagnosis of granulosacelltumor, cryptorchidism


• Dog:– P 4 for preovulatorybreeding– P 4 for diagnosis of remaining ovarian remnant– Testosterone for checking the secretory statusof the Ledig cells– Confirmation of sertoli cell tumors: LH↓, T↓(Cryptorchidism)– Relaxinfor confirmation of pregnancy status


• Cat:– P 4 for confirmation of ovulatory failure, P 4 ↓10days postbreeding (P 4 < 0.15 ng/ml )– Estrogen analysis (from feces) for ovarianfollicular activity, completeness of an OHE– Testosterone for evaluation of Ledig cellfunction in the testis– Relaxinfor confirmation of pregnancy status


General comments• Reference values developed by laboratoriesare depend on the type of assay used.– 1 ng/ml as the basal value of P 4• Harvesting serum soon after collection– RBC can metabolize P 4 to other steroids– Temperature↓, P 4 declineslowly– Skim milk for the immunoassay of P 4


Drycontraception

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