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ELECTROMYOGRAPHIC STUDIES ON THE OVINE UTERUS
A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF PHILOSOPHY AT MASSEY UNIVERSITY
Li Yuemin
_January
1991TABLE O F CONTENTS
ELECTR O MYOGRAPHIC STUDIES ON THE OVINE UTERUS
A b s tra ct . . . ii
Lis t o f Ta b l e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
List o f Figures . . . vi
Acknowl e dgmen ts . . . v i ii I. INTRODUCTION . . . 1
11 . LITERATURE REVIEW . . . 3
I I . 1 The Repro d uc t i ve Cyc l e o f S h e e p . . . 3
Il . l .A. The b reeding seaso n . . . 3
Il . l . B . Th e o estro u s cycle i n ewes . . . 5
Il . l . C. Pregn ancy a n d part u r i t i o n i n ewes . . . 7
Il . l . D .The post- partu m period i n ewes . . . 9
11 . 2 Myo m e tri a l Acti v i ty . . . 1 3 I I . 2 . A . Stru c t u re a n d o rgan isation o f t h e myom etrial cont r act i l e u n i t . . . 1 3 I I . 2 . B . Speci a l featu res o f t h e u l trastructure o f t h e fu n c t ional u n i t s o f the myometriu m . . . 1 4 (a) The c o n tract i l e apparat u s . . . 1 4 (b) P lasm a membrane . . . 1 4 ( c ) The sarcoplasmic reticu l u m (SR) . . . 1 5 (d) Th e g a p j u nctions . . . 1 5 I I . 2 . C . Myo metrial con traction . . . 1 6 I I . 2 . D . E l e c t r o p h y s i o l o g y o f m y o m e t r i a l con t racti l i ty . . . 1 6 11 .3. Effe c t s of Horm o n e s on Emg Activity . . . 19
11 . 3 . A . 11 . 3 . B . 11 . 3 . C . 11 . 3 . D . 11 . 3 . E. 11 . 3 . F. O e strogen . . . 1 9 Progesterone . . . 22
Oxytoci n . . . 24
Prostagl an d i n s . . . 28
Relaxin . . . . . . . . . . . . . . 30
Vasoactive i n t es t i n al pepti d e (VIP) . . . 30
11 .4. Uterine Moti l i ty S t u d i e s with Pa r ticu lar Reference to the Ewe . . . • . . • • . . . . . . . . • . . . . • . . . . . . . . . . . • . . . . . 3 1 11 . 4 .A. Methods o f studying u te r i n e motility . . . . . . . 3 1 11 . 4 . B . Uterin e motil ity observations recorded from t h e uterus o f t h e i n t act ewe . . . . . . . . . . . . . . 33
Ill.
11 ! . 1 . 111 . 2 . 11! . 3 . 111 .4.
I I I . 5 . I I I . 6 .
11 . 4 . C . Uter i n e motility observations recorded fro m
t h e u terus o f t h e ovariecto m i s e d ewe . . . 4 1 GENERAL MATERIALS AND M ETHODS . . • . . . . . . . . . . . . . 4 8 A n i m al . . . 4 8 Electrodes ... . ... . Su rgery a n d an aesthesia . . . . . . . .
Recordi ngs . . . . Th e Electromyograms . . . . Q u a n t itative a n alysis o f the Electromyograms . .
48 48 54 5 4 60
IV. R E S U LTS . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 IV. l . Myometrial E m g Activity i n t h e Inta c t Ewe
d u ri n g A n o e s tr u s . . . . 62 IV.l .A.
I V. LB .
IV.l . C .
IV.l . D .
I V. LE.
I V. l . F.
Th e resting emg of t h e u t e r u s .
Th e e ffect o f exogen o u s o xytoci n on t h e restin g e m g o f the u t e r u s
d u ri n g anoestru s . . . . Th e e ffect o f exogeno u s o estrogen on t h e resting emg of t h e uterus
62
. . . 62
d u ri n g anoestrus . . . 62 Th e e ffect of oxytocin on o estrogen
i n d u c ed emg a c t i v i ty d u r i n g
a noestrus . . . 62 Th e effect of PG F21X on o estrogen
i n d u c ed emg a c t i v i ty d u r i n g
anoestrus . . . . . . . . . . . . 66 Th e chan ges in myometrial activity
�hen st i m u l ated by a secon d
i n jection o f oestrogen . . . . 66 IV. 2 . Myometrial Acti vity i n t h e Ovariecto m i zed Ewe d u ri n g t h e
A n o e s tr u s S e a s o n .. . ... . .. . ... . . . .. . . .. . . 7 2 I V. 2 .A. Specific features of the design o f
t h i s experiment . . . . . . . . . . . . . . . . 72 I V. 2 . B . The gen eral patterns o f electrical activity . . . 73
(a) Resting emg activity a n d t h e e ffects of exogeno u s oxytoci n a n d PGF21X
on it . . . . . . . . . . . . . . . . . 73 (b)
(c) (d)
O estrogen i n duced e m g activity i n the ovariectomized e w e and t h e e ffects o f oxytocin a n d P GF21X o n i t . . . . Th e e ffects o f progesterone o n oestrogen i n duced e m g activity Th e reduced responsiven ess of t h e uterus i n regime two o f t h e experiment . . . .
73 77
83
(e) Th e d irection o f propagation o f e m g
action pot e n t i a ls . . . . . . . . . . . . . . . . . . . . . . . . . 84
I V. 2 . C . S o m e q u a n t i t a t iv e a s p e c t s of t h e e l e c t r o myograms recorded from t h e ovariectom ized ewes . . . . 85
(a) A m p l i t u de . . . . . . . . . . . . . . . · . . . . . . . . . . . . . . . 85
(b ) B u rst frequency . . . . . . . . . . . . . . . . . . . . . . . . . . 85
(c) Spikes per burst . . . . 85
(d) M o t i l i ty in dex . . . 85
IV.3. The E m g Acti v i ty o f the Ute r u s o f the Cycl i n g Ewe ... 93
V. DISCU S S ION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 4 V. 2 . Emg Activity d u r i n g Anoestrus and i n t h e
Ovar i ec t o m ized Ewe . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 04 V.2 . Th e D irection o f Pro pagation of t h e Act i o n
Poten t i a l s . . . 1 06 V.3 . Th e I n fl u en c e o f O estrogen . . . 1 10 V.4 . Th e I n fl u e n c e o f Progestero n e . . . . 1 1 2 V.S . Uterine r e fractoriness . . . . 1 1 6
REFERENCES ... 1 1 7
APPE NDIX I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 0
ii
A B S TRACT
There are many repo r t s in t h e l i t er a t u re, u s i n g a variety of t e c h n i qu es , i n dicating t h at h ormonally i n du ced m ovem en t s o f t h e u terus play a m aj o r r o l e i n t h e reproduction process. Th e pu rpose o f t h is study w a s to explore some of t h ese p h enomena by measuring electro-myographical responses from t h e u terus o f i n t act a n d ovariectom i zed ewes b o t h b e fore a n d during t h ei r n o r m a l b reeding seaso n .
Six h e a l t h y non-pregn a n t Rom n ey ewes o f abou t 5 0 kg body w e i g h t were selected for the i nvestigations which were carried out b etween September 1 987 and February 1 988.
Electrodes, u sually in gro u p s of t h ree, were surg ically impl a n t e d a t pre
s elected s i t es (cervix, u t er i n e b o dy, u t erine h o r n , oviduct) on t h e reproductive t ract o f the ewes, leads plugged i n t o a un iversal AC a m p l i fier, a n d the o u t p u t recorded on a fo ur c h a n n e l i n k wri t i n g c h a r t r ecorder.
Four o f t h e six ewes were ovariectomized at the time o f implan t a t i o n o f t h e electrodes. I n dwelling silastic cath eters were inserted into t h e external j ug u l ar veins o f t h ese ewes to e n a b l e oxytocin to be injected wi t h o u t disturb i ng t h e animal. A l l ewes were h o u sed i n dividu ally i n doors i n p e n s t h a t all owed t h em to moy e freely during reco r d i n g session s . Recordi n gs b eg an abo u t 1 0 days after su rgery h ad been carried o u t .
Th e electromyographs (emgs) were described u si ng generally accepted terms such as the presence o f spikes, t h e i r ampl i t u de, t h e presence o f b u rsts a n d of episodes o f activity, w h e t h e r electrical activity was co-ordi nated o r u n co
ordinat ed, and t h e direction o f propagat i o n o f t h e activity. A m o t il i ty i n dex (MI) was derived as a m e t h o d of measuring electrical activity of t h e u te r u s over prolonged periods o f t i m e .
D uring t h e anoestro u s p e r i o d i n b o t h i n tact a n d ovariectomized e w e s a n emg pattern o f myo electrical complexes c h aracterised by alternating p h ases o f co
ordina t ed b u rsts o f electrical activity a n d qu i escent periods were ob served.
Th i s b asic pattern of activi ty, and respon s iv e n e ss to u t erine s t im u lan t s s u c h a s oxytocin and Glandin N (a P GF2oc an alogu e), was greater i n t h e i n t ac t
iii
ewes a s compared to t h e ovariec t o mized ewes a t t h i s t i m e . A l t h o u gh s u ch a d i ffer e n c e c o u l d be anticipated, a clear answer as to w h e t h er i t i s a general p h e n o m e n o n , or whether seaso n al d i ffere n c es exist, cou ld not be det e r m i n ed b ec a u se o f t h e l i m i t ations i n design o f t h i s s t u dy.
A d i s t i n c t pattern of emg activity was also i d e n t i fied d u r i n g d i ffer e n t ph ases of t h e o estrou s cycle in the ewe d u r i n g the n a t u ral b r e e d i n g season . Th is p a t t ern cou l d b e m imicked by a d m i n i s tr a t i o n of the exogen o u s steroid h o r m o n es oestrogen and progestero ne i n the ovariect o m ized ewes. Fou r days a fter daily oes tradiol- 1 7b i njec t i o n s (50J..Lg s . c . ) t h e emgs of t h e ovariec t o m ized ewes s h owed a marked i n crease in ampl i t u d e and i n b u rst frequency a n d a c o n s i d erably increased respo nse to oxytocin a n d Gla n d i n N . W h e n t h i s was followed by progesterone a d m i n i st r a t i o n (50mg. s.c.), even with o estrogen i nj e c t i o n s c o n t i n u ing, i n h i b i t ion of activi ty a n d redu ced respon siveness to oxy t o c i n and Glan din N was e q u a l ly m arked.
W i t h drawal o f progesteron e , b u t with co n t i n u i ng oest rogen a d m i n istratio n , r e s u l t e d i n a recovery fro m t h e i n h i b i t i o n a n d a respo n se t h a t w a s even greater t h an before the progestero n e h a d been given . Th i s s u ggests both an i n h i b i t i n g and a poten t i a t i n g action o f progestero n e o n u t eri n e electrical act ivity, a fin di n g which adds some suppo rt to Csapo's c l assical w i t h d r awal of the 'progeste ron e block' as one o f t h e pre-requ isi tes fo r i n i t i at i o n of normal p ar t u r i t ion in sh eep. _
W h i l e o estrogen h as a clear role t o play as a s t i m u l a t o r o f electrical activity it a l so seem s c apable of exh i b i t i n g a b iph asic resp o n s e with a period of depressed a c t ivity occurri n g befo r e the pos i t ive stimu l u s occurs. W h e t h e r t h i s i s a fu nction of dose o r s o m e o t h er factor c o u l d n o t b e e s t ab l i s h e d i n t h i s s t u dy . I t s poten t i a ti n g effec t on t h e action o f both oxytocin a n d G l a n d i n N i n t h e s e experiments adds fu r t h er sign i ficance t o t h e a t t e n t io n t h a t s h o u l d b e p a i d t o t h e reproductive s t a t u s o f t h e a n i m a l when c l i n i c a l u se i s m a d e o f t h es e s u bstances.
Th e d i rection of propagation of action pot e n tials recorded in t h ese s t u di e s d ep e n d ed o n t h e general l evel o f emg activity. W h e n t h is was l o w t h e directi o n o f propagation w a s cervico- t u b a l , when h i g h , t h e proportion o f a c t i o n poten ti a l s i s i n both d i rections fro m t h e t u b a l e n d o f t h e u terus. A
iv
c l e a r u n derst a n d i n g of such a m e c h a n i sm , a n d i t s sig n i fi c a n c e, m u s t await fu r t h er s t u dy.
Tab l e 1 :
Tab l e 11 :
Tab l e I l l :
Tab l e I V:
V
LIS T O F TAB LES
Th e effect o f oxytocin both o n spo n t an e o u s a n d
oestradiol i n duced e m g activity (Ewe 329) . . . 67
Th e e ffec t of oxytocin on spo n t aneo u s a n d oestradiol i n d u c ed e m g activity 1 3 days a fter l a s t oest radiol i nject i o n o f ewe 329 (compare w i t h
Tab l e I ) . . . 69 Th e d i fferences i n emg activity b etween a n i m a l s
du r i n g stage 2 o f regime o n e . . . 9 1
Th e effect o f oxytocin a n d PGF2cx: o n t h e oest radiol i n d u c e d electrical act ivity du r i n g stage
2 of regi m e one . . . 92
Fig. 1 :
Fig. 2 :
Fig. 3 :
Fig. 4 ( a & b ) :
Fig. 5 :
Fig. 6 : Fig. 7 :
Fig. 8:
Fig. 9:
Fig. 1 0:
Fig. 1 1 :
Fig. 1 2 :
Fig. 1 3 :
Fig. 1 4 :
Fig. 1 5 :
vi
LIST O F F IGURE S
Th e pos i t i o n s of electrodes s u t u r e d a t pre- selec t e d
sites o n t h e u teru s a m o n g 6 ewes . . . 50 Active a n d qu iesce n t periods of emg d u r i n g
a noestr u s . . . 56 An example o f the m e t h o d o f m easu r i n g the
du rat i o n o f bu rsts, the i n t erval b etween b u rsts, t h e b u rst frequ ency, spike frequ ency a n d am p l i t u d e o f
t h e spike . . . 5 7
A n example showi n g t h e propagative direc t i o n o f
bursts a n d episodes . . . 5 8
A n example o f co-o r d in at e d a n d u n co-o r d i n a t e d patterns of elect rical a c t iv i ty . . . .
An exam ple of calc u l a t i n g t h e m o t i l i ty i n dex . Oxytocin effect o n
durin g a n oestrus
spo n t a n eo u s emg activity
Oestradiol benzoate b o t h i n h i b i t s a n d s t i m u l ates
5 9 6 1
. . . 63
myomet r i a l activity in ewe 329 d u r i n g a n o e s t r u s . . . 64 The e ffect of oxytoci n on oestradiol i n du ce d emg
activi ty . . . 65 Th e effect of PGF2oc on oestradiol i n d u c e d emg
activity. during anoestrus . . . 68 Characteristics o f t h e emg recorded 13 days a fter
the i n i t i a l injec t i o n o f oes trogen . . . . . . 70 Th e effects o f oestrogen and oxytocin on
ampl i t u de, burst fre q u e n cy and m o t i l i ty i n dex during two expe r i m en t s on i n t a c t anoestro u s ewe
329. . . . . 7 1
Th e pattern o f emg a c t iv i ty i n ovariectomized ewe
247 d u r i n g the a n o es t r o u s season . . . 74
Th e pattern of emg activity i n d u c e d by o e s t r a d i o l - 1 7b (50JLg g i v e n s.c. daily) on ovariectomized ewes
during the anoestrou s season . . . .. . 75
Th e effect s o f oxyto c i n (500 m u given i.v.) on t h e oestrogen i n d uced e m g activity o f ovariecto mi z e d
ewes d u r i n g t h e anoestrous season . . . 76
Fig. 1 6a :
Fig. 1 6b :
Fig. 1 6c :
Fig. 1 6d:
Fig. 1 7 :
Fig. 1 8 :
Fig. 1 9 :
F i g . 2 0 :
Fig. 2 1 :
Fig. 2 2:
Fig. 2 3 :
F i g . 2 4 :
Fig. 2 5 : Fig. 2 6 : Fig. 2 7 : F i g . 2 8 : F i g . 2 9 : F i g . 3 0 :
vii
Th e d evelopment o f i n hi b it i o n a fter progestero n e
a d m i n istration . . . 7 8 Th e effect of oxytoc i n a n d P GF2<x: w h e n t h e
uterus i s i n h ibited by progest e r o n e . . . 7 9
T h e e m g pattern observed d u r i n g the recovery
from progesteron e i n h ib it i o n . . . 8 1 Th e pattern of oestrogen i n d u c e d emg i n ewe 1 0
before progesteron e admi n i stration (A) a n d i n t h e same ewe 1 1 5h after a large dose o f progesterone ( 1 00mg given i . m . ) had b e e n g iven when th e ewe h a d recovered from t h e progestero n e tr eatm ent
a d m i n istered during regime o n e (E) . . . 82 Th e difference i n amplit u d e a n d b u rst frequency
b etween 4 electrode positio n s o n t h e reproduct ive trace
Em g amplitu des d u r i n g treatment i n regime o n e .
di fferent stages o f
Di fferences i n bu rst fre q u e n c y at each tr eatm ent stage of regime one
Nu mber of spikes i n a b u r st during the period o f
. . . 8 6
. . . 87
. . . 88
oestradiol i n duced emg activity i n regi me one . . . 89 Th e n u mber of spikes in a bu rst d u r i n g each
treatm ent stage of regime one . . . 90
Th e motility i n dex du r i n g stages 2 a n d 4 o f reg i m e
o n e . . . 94 Th e c h aracteristics of emg act ivity during st ages 2,
3 and 4 of both reg i m es . . . . . . . Ewe 661 during dioestrus
Ewe 661 during pro-oest r u s .
95
96 . . . 97 Ewe 661 during oest r u s . . . . . . . 98
Ewe 661 du ring metoestru s/early dioestru s 1 00
Ewe 661 during early dioestr u s . . . . 1 0 1
Ewe 6 6 1 recorded at m id-oe strus 8.4. 88 102
Ewe 6 6 1 recorded at l ate o estr u s 9.4.88 103
viii
ACKNOWLEDGMENTS
I take t h i s opportun ity to t h a n k t h e m an y people who h ave g iv e n me encou rage m en t , h elp and advic e d u r i n g my M . P h i l . stu dy .
M y s i n c e r e t h an ks g o to m y su pervisors Prof. E . D . Fielden a n d D r . D . H . C a r r for t h ei r u n fa l t e r i n g e n th u siasm a n d g u i da n ce d u r i n g t h e proj e c t . Th eir e n c o u r agem e n t , e n t h u siasm, constructive suggestions and assistance t h r o u gh o u t the project were as important as t h ei r advice on the fi n al draft of t h i s thesis.
My t h a n k s a l so go to the Tech n i cal Sta ff o f the Department o f P h ysio logy and Anatomy, particu l arly that of Debbie Anthony for h el p i n preparation of figu r es, J . C . Pedl ey for making the recor d i n g faci l i t ies u sed in t h is study ava i l ab l e to m e , N . I . B roomfi e l d for givi n g v a l u able advice d u r i n g t h e cou rse o f the exp e r i m e n t s and helping m e i n the u se o f the word processo r , and I r e n e H a l l for assistance during the surgical procedures.
I a m also i n debted to Mr Cedric B a r n ett and Rose Law for their w i l l i n g h el p with t h e sh eep, a n d with obtai n i n g t h e drugs a n d disposables u se d . T. Law's assistance w i t h t h e photography of all emg records i n gratefu l ly a c k n owledged.
F i n a l ly I w i l l for ever ,appreciate the su pport and forb earance o f m y fam i ly a n d of Xinj i a n g S h i h ezi Agricu ltu ral College, w i t h o u t whose h elp t h i s s t u dy wo u l d n o t h ave b ee n po ssib le.
