Horse reproduction - Part 7

The endocrine and reproductive consequences of endotoxin-

mediated / inflammatory diseases

Prof. Huszenicza, GyulaDVM, PhD, DSc, Dipl. of ECAR

Szent István University,

Faculty of veterinary Science, Budapest

1) Endotoxin (LPS) / Peptidoglycans2) Pathophysiology (endocrine aspects)3) Consequences on reproductive functions:

• follicular growth / maturation, ovulation - during cyclicity - during the onset of cyclicity / at the expected time of

the 1st ovulation

• CL: - during cyclicity - in pregnants

• endotoxin ⇒ pregnancy • sow: MMA syndrome

The endocrine and reproductive conse-quences of inflammatory diseases

1) Endotoxin (LPS) / Peptidoglycans 2) Pathophysiology (endocrine aspects)3) Consequences on reproductive functions:


the 1st ovulation



The endocrine and reproductive conse-quences of endotoxin-mediated diseases

(Sandholm and Pyorala, 1995)Initiation of inflamma-Initiation of inflamma-tory process:tory process: . Host (PMN leukocyte) -Host (PMN leukocyte) - . pathogen interactionpathogen interaction

Questions:Questions: What kind of What kind of mediatorsmediators??

What of the What of the bacterial compoundsbacterial compounds can induce can induce their production? their production?

Macrophage Macrophage

MonocyteMonocyte

Pathogens:Pathogens:

- - Gram-negative (GN) microbesGram-negative (GN) microbes: : endotoxin (LPS), ...endotoxin (LPS), ... - Gram-positive (GP) microbes: peptidoglycan,

(exo)toxins, ...

(Sandholm et al., 1995; Sordillo and Delay, 1995)

Bacterial compounds:Bacterial compounds:Gram-negativesGram-negatives

Endotoxin (LPS): cell wall structure of Gram-negative (GN) bacteria

(Sandholm and Pyorala, 1995)

Endotoxin (LPS): cell wall structure of Gram-negative (GN) bacteria


Liberated after the death of bacteria

Most / all of the invading pathogens have died by time of first clinical symptoms

Sources of endotoxin loading1) Gram-negative sepsis

2) Iatrogenic

3) Gastrointestinalis tract (forestomack, large intestine, … )

4) Organic diseases caused by Gram-negative bakteria: - uterus: (acut putrid) endometritis

- mammary gland: GN mastitis

- …

Sources of endotoxin loading

Endocrine/ reproductive / clinical consequences

Differences in absorption / detoxification

1) Gram-negative sepsis

2) Iatrogenic

3) Gastrointestinalis tract (forestomack, large intestine, … )

4) Organic diseases caused by Gram-negative bakteria: - uterus: (acut putrid) endometritis

- mammary gland: GN mastitis

- …

Macrophage Macrophage

MonocyteMonocyte

Pathogens:Pathogens:

- Gram-negative (GN) microbes: - Gram-negative (GN) microbes: endotoxin (LPS), ...endotoxin (LPS), ... - - Gram-positive (GP) microbesGram-positive (GP) microbes:: peptidoglycan,peptidoglycan,

(exo)toxins, ...(exo)toxins, ...

(Salyers and Whitt, 1994; Sordillo and Delay, 1995)

Bacterial compounds:Bacterial compounds:Gram-positivesGram-positives

Inflammatory Inflammatory mediatorsmediators

Oxygen radicalsOxygen radicals (nitric oxide, NO)(nitric oxide, NO)Eicosanoids Eicosanoids

(PG-s, leukotrienes, tromboxanes)(PG-s, leukotrienes, tromboxanes)

ProteinsProteins

(TNF(TNFαα , , interleukins)interleukins)

(after Sandholm and Pyorala, 1995)

„„Classical”Classical” (histamine, (histamine, serotonin, kinins, serotonin, kinins,

complements)complements)


==

Inflammatory processInflammatory process

Local Local

General General }}


==

symptomssymptomsMastitisMastitis

(Endo)metritis(Endo)metritis

OthersOthers

(Postpartum) (Postpartum) E. coliE. coli mastitis mastitis

- Direct endotoxin effect: - Direct endotoxin effect: << 24 h 24 h

- Absorption: only mediators (endotoxin ???)- Absorption: only mediators (endotoxin ???)

- Detoxification: alveolar epithel (+ liver, but: function ? - Detoxification: alveolar epithel (+ liver, but: function ? ))

- Coinciding bacteraemia: never- Coinciding bacteraemia: never


Intensive Intensive LPS LPS cytokine cytokine release release

(Postpartum) (Postpartum) E. coliE. coli mastitis mastitis

- (Usually) severe clinical symptoms- (Usually) severe clinical symptoms

- Characteristic endocrine changes- Characteristic endocrine changes


Intensive Intensive LPS LPS cytokine cytokine release release

(Postpartum) (Postpartum) S. aureusS. aureus mastitis mastitis

- Usually: exacerbation / relapse of chronic - Usually: exacerbation / relapse of chronic infections infections

- Clinical symptoms: may be severe / fatal- Clinical symptoms: may be severe / fatal

- Endocrine changes ???- Endocrine changes ???

(Sandholm et al., 1995)

CytokineCytokine release: may release: may be intensivebe intensive

(Postpartum) mastitis caused by other(Postpartum) mastitis caused by other GP GP (environmental) pathogens (Str. ssp)(environmental) pathogens (Str. ssp)

- Local symptoms:- Local symptoms: may vary may vary

- General symptoms: usually are not - General symptoms: usually are not severe / never fatal severe / never fatal

- Endocrine changes: ---- Endocrine changes: ---

(Sandholm et al., 1995)CytokineCytokine release: usually release: usually not intensivenot intensive

Endotoxin: (acut putrid) endometritis

- At least 2/3 - 5/7 days after parturition !

- Absorption: endotox. + mediators + other toxic compounds

- Detoxification: limited (⇐ way of absorption + liver function )

- Coincindingly: perhaps also bacteraemia

Endocrine consequences

of experimental

(iv/ipt) endotoxin

administration

Oxygen radicals Oxygen radicals (nitric oxide, NO)(nitric oxide, NO)Eicosanoids Eicosanoids


ProteinsProteins


(after Sandholm and Pyorala, 1995)„„Classical” Classical” (histamine, (histamine,

serotonin, kinins, serotonin, kinins, complements)complements)

==

Mediator-induced endocrine changesMediator-induced endocrine changes

Proteins:Proteins:

- - TNFTNFαα -- interleukinsinterleukins

ACTH ACTH ⇒⇒ cortisol cortisol

(+ progesterone (+ progesterone ) )

Prolactin Prolactin / /

TSH TSH ⇒⇒ T4 / T3 T4 / T3

GH GH / / ⇒⇒ IGF-1 IGF-1 + IGFBP-2 + IGFBP-2

Insulin Insulin glukagon glukagon

Leptin Leptin / / ∅∅ ? ?

(Hirvonen et al., 1999; Kakizaki et al., 1999; Soliman et al., 2002;

Lehtolainen et al., 2003; Waldron et al., 2003)


TNFTNFααIL-1IL-1

IL-6IL-6

ACTHACTH

GlucocorticoidsGlucocorticoids


Proteins:Proteins:



(+ progesterone (+ progesterone ))


TSH TSH ⇒⇒ T4 / T3 T4 / T3




(Suzuki et al., 2001)


Proteins:Proteins:




Prolactin Prolactin

TSH TSH ⇒⇒ T4 / T3 T4 / T3




(Jackson et al., 1990)


Proteins:Proteins:




Prolactin (Prolactin (only in sowsonly in sows: : ) )

TSH TSH ⇒⇒ T4 / T3 T4 / T3




(Smith and Wagner, 1984 and 1985)


Proteins:Proteins:





TSH TSH ⇒⇒ T4 / T3 T4 / T3




( Lab rodents / primates: Wartofsky and Burma, 1982; Haastaren et al., 1994;

Rettori at al., 1994; Bartalena et al., 1998; Bertók, 1998

Cattle: Kahl et al., 2000)

--

HThHTh

++

TSHTSH

TT44 ⇒⇒ T T33

--

DopaminDopaminTRHTRH

++

HELHEL

Production:Production: Only in severe casesOnly in severe cases

TNFα / IL-1: may

inhibit the TSH

release from the

pituitary cells

( Haastaren et al., 1994; Rettori at al., 1994; Hashimoto et al., 1998; Bertók, 1998 )

Johnson és Becker 1987;

Brabant és mtsai, 1991

Dupre és mtsai, 2004

--

HThHTh

++

TSHTSH

TT44 ⇒⇒ T T33

--


++

HELHEL

Metabolism:Metabolism:

TT44 ⇒⇒ T T3 3

TT3 3 ⇒⇒ rTrT3 3

Outer-ring Outer-ring deiodination by deiodination by

type-I 5’Dtype-I 5’D

( Haastaren et al., 1994; Hashimoto et al., 1998; Kahl et al., 2000 )

InactivationInactivation

--

HThHTh

++

TSHTSH

TT44 ⇒⇒ T T33

--


++

HELHEL

Metabolism:Metabolism:

TT44 ⇒⇒ T T3 3

TT3 3 ⇒⇒ rTrT3 3

InactivationInactivation

Plasma: Plasma: TT4 4

TT3 3

rTrT3 3

Production:Production: Only in Only in severe casessevere cases


Proteins:Proteins:





TSH TSH ⇒⇒ T4 / T3 T4 / T3




( Lab rodents: Rettori et al., 1994

Cattle: Elsasser et al., 1995 and 1996; Briard et al., 2000; Nikolic et al., 2003;

Waldron et al., 2003)


Proteins:Proteins:





TSH TSH ⇒⇒ T4 / T3 T4 / T3




(McMahon et al., 1998; Steigner et al., 1999; Koshibiki et al., 2000;

Soliman et al., 2002; Waldron et al., 2003)


Proteins:Proteins:





TSH TSH ⇒⇒ T4 / T3 T4 / T3




(Lab rodents/primates: Bornstein et al., 1998; Mastronaldi et al., 2000;

Pig: Spurlock et al., 1998; Leininger et al., 2000;

Ruminants: Soliman et al., 2001 and 2002; Waldron et al., 2003)


Proteins:Proteins:


( Metabolism: shifted catabolic direction Metabolism: shifted catabolic direction

(( Anorexia) Anorexia)




TSH TSH ⇒⇒ T4 / T3 T4 / T3




(Houseknecht et al., 1998;

Waldron et al., 2003)

1) Endotoxin (LPS). Sources of endotoxin ✔ 2) Pathophysiology (endocrine aspects) ✔ 3) Consequences on reproductive functions:


the 1st ovulation




THECA CELLS

OOCYTE

GRANULOSA CELLS

BASEMENT MEMBRANE

BLOOD VESSELS

Oocyte quality (?)Mechanisms:Mechanisms:

- TNF- TNFαα , IL-1, IL-6 , IL-1, IL-6 (others ?)(others ?)

- Catabolic and/or - Catabolic and/or endocrine changes?endocrine changes?

- Endotoxin ?- Endotoxin ?

- Fever (= heat ?)- Fever (= heat ?)

- Blood supply / - Blood supply / coagulation ?coagulation ?

… … ??

(Oliver et al., 2000)

THECA CELLS

OOCYTE

GRANULOSA CELLS

BASEMENT MEMBRANE

BLOOD VESSELS

ESTROGEN

Endocrine regula-tion / activity

FSH

LH (basal + preovul.)

- TNF- TNFαα , (IL-1, IL-6?), (IL-1, IL-6?)

(- Nitric oxide(- Nitric oxide

- Catabolic - Catabolic changes?)changes?)

(Rettori et al., 1994; Oliver et al., 2000;

Battaglia et al., 2000; Suzuki et al., 2001;

Daniel et al., 2002)

(Peter et al., 1989;

Lopez-Diaz and Bosu, 2002)

Endotoxin (prooestrus)

(Suzuki et al., Dom. Anim. Endocr., 2001. 20. 267-278.)

Progesterone(P4)

Animals:

- Cyclic heifers (24-38 mo, 480-540 kg)

- Treated: n = 6; Control: n = 6

Intravenous LPS loading:

- at 48 after a luteolytic dose of PGF2α

- with 5 µg/kg E. coli O111:B4 (Sigma)

17β-estradiol (E2)

Progesterone(P4)

Cortisol

Animals:Animals:

- Cyclic heifers (24-38 mo, 480-540 kg)- Cyclic heifers (24-38 mo, 480-540 kg)

- - Treated:Treated: n = 6; n = 6; Control:Control: n = 6 n = 6

Intravenous LPS loading:Intravenous LPS loading:

- at 48 after a luteolytic dose of PGF2- at 48 after a luteolytic dose of PGF2αα

- with 5 - with 5 µµ g/kg g/kg E. coli O111:B4 (Sigma)(Sigma)

Endotoxin Endotoxin (prooestrus)(prooestrus)


n

g/m

l

ng/

ml

pg/m

l

ControlControl

Minutes after LPSMinutes after LPS

(Suzuki et al.,

Dom. Anim. Endocr.,

2001. 20. 267-278.)

Treated Treated (Heifer 2)(Heifer 2)



Basal LHBasal LH (ng/ (ng/ml)ml)

ControlControl TreatedTreated

Hours after luteolytic dose of PGF2Hours after luteolytic dose of PGF2αα

Preovulatory LH peakPreovulatory LH peak


Milk: PMilk: P44

Estrus: exam.+AIEstrus: exam.+AI

30 60 80 95-100 30 60 80 95-100 dayday

Vagin. + RPVagin. + RP

Plasma: Plasma: metab.metab.

3x / week (2-3 d apart)3x / week (2-3 d apart)

Exp. 3Exp. 3:: ≥≥ 2nd parity cows in 4 herds (n=335 2nd parity cows in 4 herds (n=335 ⇒⇒ 263) 263) . If in If in prev. lactprev. lact.: .: chr. recurrent mastitis chr. recurrent mastitis ∅∅ (SCC<400 th/ml) (SCC<400 th/ml)

current lact.: acute putid (endo)metritis ∅

CalvingCalving

(Jánosi et al, Acta Vet. Hung., 2003;

Huszenicza et al., Repr. Dom. Anim., in press.)

Milk: PMilk: P44

Estrus: exam.+AIEstrus: exam.+AI

30 60 80 95-100 30 60 80 95-100 dayday

Vagin. + RPVagin. + RP

Plasma: Plasma: metab.metab.

3x / week (2-3 d apart)3x / week (2-3 d apart)

Exp. 3Exp. 3:: ≥≥ 2nd parity cows in 4 herds (n=335 2nd parity cows in 4 herds (n=335 ⇒⇒ 263) 263) . If in If in prev. lactprev. lact.: .: chr. recurrent mastitis chr. recurrent mastitis ∅∅ (SCC<400 th/ml) (SCC<400 th/ml)

current lactcurrent lact.: .: acute putid (endo)metritis acute putid (endo)metritis ∅∅

Clin. exam. + ScoringClin. exam. + Scoring

Sampling for bact. Sampling for bact.

CalvingCalving

(Jánosi et al, Acta Vet. Hung., 2003;

Huszenicza et al., Repr. Dom. Anim., in press.)

14 28 42 day14 28 42 day

AcycliaAcyclia

PP44, nmol/l, nmol/l


PP44, nmol/l, nmol/l≤≤ 28 day: 50 %28 day: 50 %aab

48 %48 %bba

22 %22 %abab

a-a, b-ba-a, b-bP<0.05P<0.05

32.032.0±±13.4 day13.4 dayaa

n=175n=175

33.433.4±±16.0 day16.0 day

n=59n=59

n=27n=27

GN + NDP !!!GN + NDP !!!38.638.6±±11.8 day11.8 dayaa

CalvingCalving

***P<0.001; ***P<0.001; a-a, b-ba-a, b-bP<0.001P<0.001

CalvingCalving 20 40 60 80 20 40 60 80 dayday

Mastitis Mastitis

Follicle phaseFollicle phase

+ Ovulation + Ovulation

GN+NDPGN+NDPGPGP


Foll. phase: Foll. phase: >10 day>10 day

AllAll 24 24 13 13 289289

Of them: Elongated*** Of them: Elongated*** 0 0 54 % 54 % 4 %4 %

⇒⇒ Estrus***Estrus*** 17 % 17 % 8 % 8 % 64 % 64 %

Follicular phase, dayFollicular phase, day 7.9 7.9±±0.60.6aa 10.8 10.8±±3.13.1abab 7.2 7.2±±0.80.8bb

Dominant Dominant follicle follicle (DF)(DF)

GN mastitisGN mastitis

Endotoxin Endotoxin .

cytokines+NOcytokines+NO

- 5 5 10 day day


Mastitis Mastitis ⇒⇒ folicular maturation / ovulation folicular maturation / ovulation

InterpretationInterpretation

DF-1 DF-2


- 5 5 10 day day


??Endotoxin Endotoxin .




DF-1 DF-2


CL

Ovulation Ovulation ⇒⇒ CL CL

- 5 5 10 day- 5 5 10 day






DF-1 DF-2


CL


- 5 5 10 day- 5 5 10 day





Mastitis Mastitis ⇒⇒ folicular maturation / ovulation folicular maturation / ovulation (in acyclic cows)(in acyclic cows)

DF-1 DF-2


CL


Mastitis Mastitis ⇒⇒ folicular maturation / ovulation folicular maturation / ovulation (in cyclic cows)(in cyclic cows)

- 5 5 10 day- 5 5 10 day





0

1

2

3

0Calving

15 30 45 60 75 90Day

Cessation of cyclicity

Secondaryacyclicity: >21 days

RP/US:

0

1

2

3

0Calving

15 30 45 60 75 90Day

Short-lived (<10 days) CL

(sCL)

0

1

2

3

0Calving

15 30 45 60 75 90

Acyclicity: > 35-45 days

Negative energy balance, baseline LH pulsatility and 1st ovulation

Energy

balance (EB)

-5 0 +5 + 10

Days relative to the nadir of negative EB

1st ovulation

(Butler et al., 1981; Butler and Smith, 1989 ; Butler, 2000)

Mastitis: can/may override this mechanism (LPS / cytokines ⇒ follicular maturation / ovulation)

Energy

balance (EB)

-5 0 +5 + 10

Days relative to the nadir of negative EB

1st ovulation+ 7-10 days

0

1

2

3

0Calving

15 30 45 60 75 90


0

1

2

3

0Calving

15 30 45 60 75 90Day


(sCL)

0

1

2

3

0Calving

15 30 45 60 75 90Day



RP/US:

Presence of anovulatory cysts

Diagnosed : by (repeated) rectal palpationSize : > 25 mmPresent : for > 7-14 daysSimultaneously : no regular cyclicity / CL

Presence of anovulatory cysts

0

5

10

15

20

25

E.coliinfected

Notinfected

On days 28-35: E. coli

P<0.05

%

0

5

10

15

20

25

A. p

yo

ge

ne

s

A. p

yo

+ G

N-

an

ae

rob

s

GN

-an

ae

rob

s

No

ute

rin

ep

ath

.

On days 28-35: A. pyogenes and /or GN anaerobs

%

0

5

10

15

20

25

APE No APE

On days <14:acute putrid endometritis

%

1) Endotoxin (LPS). Sources of endotoxin ✔ 2) Pathophysiology (endocrine aspects) ✔ 3) Consequences on reproductive functions:

• follicular growth / maturation, ovulation ✔ - during cyclicity ✔ - during the onset of cyclicity / at the expected time of

the 1st ovulation ✔




Oxygen radicals Oxygen radicals (nitric oxide, NO)(nitric oxide, NO)Eicosanoids Eicosanoids


Proteins Proteins


(after Sandholm and Pyorala, 1995)„„Classical” Classical” (histamine, (histamine,

serotonin, kinins, serotonin, kinins, complements)complements)

==

Corpus luteumCorpus luteum

Eicosanoids:Eicosanoids:

- PG-s incl. - PG-s incl. PGF2PGF2αα

- leukotrienes- leukotrienes

- tromboxanes- tromboxanes

PGF2PGF2αα

Induced Induced luteolysis?luteolysis?

(Metritis: Peter and Bosu, 1987; Peter et al., 1987; Giri et al., 1990;

Gilbert et al., 1990; Kindahl et al., 1996)

Corpus luteumCorpus luteum

Eicosanoids:Eicosanoids:

- PG-s incl. - PG-s incl. PGF2PGF2αα

- leukotrienes- leukotrienes

- tromboxanes- tromboxanes

PGF2PGF2αα


Also in Gram-positive infection !

Prostanoids / PG F2alfa

PGFPGF22αα


Way of mediators to ovary

???

Systemic way

Utero-ovarian vascular Utero-ovarian vascular countercurrent diffusion countercurrent diffusion systemsystem

Oxytocin induced PGFM response in experimental

(Str. uberis) mastitis Animals:

Cows with no IMI on d ≈ 30 postpartum, in the CL phase

Experimental intramammary (IMI) infection:

With 5 ml of 5.05 x103 CFU µg/kg encapsulated S. uberis susp.

(Hockett et al., Anim. Reprod. Sci., 2000. 58. 241-251.) .

Treated: n = 5Control: n = 5

100 IU oxytocin (on d 4

postinf.)

Prostanoids / PG F2alfaSystemic way


Hypothetic model


???

PGFPGF22αα

TNFTNFαα

(after Okuda et al., 2002)

Prostanoids / PG F2alfa

… in accordance with LPS administration in cattle ...

Induced luteolysis:Induced luteolysis:

in cyclic cows: >4 d in cyclic cows: >4 d

in pregnants: in pregnants: < ≈ 90-120 d ???

PGFPGF22αα

TNFTNFαα

(Kindahl et al., 1996)

Systemic way


1) Endotoxin (LPS) / Peptidoglycans ✔2) Pathophysiology (endocrine aspects) ✔ 3) Consequences on reproductive functions:






***P<0.001; ***P<0.001; a-a, b-ba-a, b-bP<0.001P<0.001

CalvingCalving 20 40 60 80 20 40 60 80 dayday

GN+NDPGN+NDPGPGP

sCL: sCL: <10 day<10 day

Mastitis Mastitis

CL CL szőérés

+ Ovuláció

AllAll 24 24 30 30 211211

Of them: Luteolysis / sCL*** Of them: Luteolysis / sCL*** 8 % 8 % 47 % 47 % 4 %4 %

⇒⇒ Estrus***Estrus*** 0 0 7 % 7 % 62 % 62 %

CL phase, dayCL phase, day 16.9 16.9±±3.53.5aa 13.1 13.1±±4.54.5abab 17.1 17.1±±2.52.5bb


0

1

2

3

0Calving

15 30 45 60 75 90


0

1

2

3

0Calving

15 30 45 60 75 90Day



RP/US:

0

1

2

3

0Calving

15 30 45 60 75 90Day


(sCL)

Endotoxin-induced luteolysis in cyclic females

Day after ovulation

Cattle: > 4th

Sheep: > 4th

Goat: > 4th

Horse: > 5th

Pig: > 13th

(Domestic carnivores: ??? )

1) Endotoxin (LPS) / Peptidoglycans ✔ 2) Pathophysiology (endocrine aspects) ✔ 3) Consequences on reproductive functions:



• CL: - during cyclicity ✔ - in pregnants



Endotoxin-induced luteolysis in pregnant females

Day after conception

Cattle: < ≈ 90-120th

Sheep: < ≈ 50th

Goat: all time of pregnancy

Horse: < ≈ 35 - 40th

Pig: < ≈ 80th

(Domestic carnivores: ??? )




• CL: - during cyclicity ✔ - in pregnants



Endotoxin-mediated diseases /pregnancy

- CLpregn.

- Other effects: • cortisol • placenta• embryo foetus




• CL: - during cyclicity ✔ - in pregnants ✔

• endotoxin ⇒ pregnancy ✔ • sow: MMA syndrome


Sow: agalactia

Endotoxin-induced inflammatory changes and their endocrine consequences

Proteins:

TNFα interleucins

ACTH ⇒ cortisol

(progesterone )

TSH, T4 / T3; prolactin

(GH-induced) IGF-1 prod.

Insulin / ? Leptin ?

Thanks for Thanks for

your your

honoring honoring

attention ..attention ..

..

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Horse reproduction - Part 7

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