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Biphasic response in the secretion of gonadotrophin-releasing hormone in ovariectomized ewes injected with oestradiol

In ovariectomized ewes, an injection of oestrogen initially inhibits the tonic secretion of LH, and then induces a large release of LH similar to the preovulatory surge in intact ewes. The pattern of hypothalamic secretion of gonadotrophin-releasing
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  Biphasic  response  in  the  secretion  of gonadotrophin-releasing hormone  in  ovariectomized  ewes injected  with  oestradiol A.  Caraty,  A.  Locatelli  and  G.  B.  Martin Station  de  Physiologie  de  la  Reproduction,  Institut  National  de  la  Recherche  Agronomique,  37380  Nouzilly, France *MRC  Reproductive  Biology  Unit,  Centre  for  Reproductive  Biology,  Chalmers  Street,  Edinburgh  eh3  9EW (G.  .  Martin  is  now  at  CSIRO Division  of Animal  Production,  Private  Bag,  Wembley,  Western  Australia 6014,  Australia) received  4 May  1989 ABSTRACT In  ovariectomized  ewes,  an  injection  of  oestrogen initially  inhibits  the  tonic  secretion  of  LH,  and then induces  a large  release  of  LH  similar  to  the  pre- ovulatory  surge  in  intact  ewes.  The  pattern  of hypothalamic  secretion  of  gonadotrophin-releasing hormone  (GnRH)  into  the  pituitary  portal  blood during  this  biphasic  response  to  oestrogen  was  investi- gated  in  conscious,  unrestrained,  ovariectomized adult  Ile-de-France  ewes  during  the  breeding  season. The  ewes  were  ovariectomized  and  implanted  with cannulae  for  portal  blood  collection  on  the  same day. Seven  days  later,  portal  and  peripheral  blood  samples were  collected  simultaneously  every  5  min  for  25  h. The  ewes  were  injected  with  oestradiol-17\g=b\ (25  \g=m\g i.v. and  25  \g=m\g i.m.)  6\m=.\25 h  after  the  start  of  sampling. GnRH  and  LH  were  measured  by  radioimmunoassay in  portal  and  jugular  plasma  samples  respectively. A  clear  pulsatile  pattern  of  LH  secretion  was observed  before  the  oestradiol  injection  in all ewes,  followed  by  the  typical  biphasic  decrease (negative  feedback)  and  increase (positive  feedback) in  mean  concentrations.  The  sampling period  was divided,  for  analysis,  into  pretreatment,  negative  feed- back and  positive  feedback  phases.  Before  injection with  oestradiol,  the  GnRH  pulses  were  clearly  defined in  portal  blood  and  always  synchronized  with LH pulses  in  the  peripheral  circulation.  The  frequency was  5\m=.\9  \m=+-\ 0\m=.\6 pulses/6  h  (mean  \m=+-\ s.e.m.),  and the amplitude  was  31\m=.\6\m=+-\7\m=.\6 pmol/l.  During  negative feedback,  both  the  frequency  (4\m=.\2  \m=+-\ 0\m=.\5 pulses/6  h, P<0\m=.\01) and  amplitude  (15\m=.\2  \m=+-\ 4\m=.\6 pmol/l,  P<0\m=.\05) of  the  GnRH  pulses  decreased.  During  positive  feed- back,  there  was  a large  surge  in  the  concentration of  GnRH,  due  primarily  to  an  increase  in  pulse frequency  (11\m=.\0\m=+-\1\m=.\3 pulses/6  h,  P<0\m=.\01). A  change in  pulse  amplitude  was  not  detected,  but  there  was  a large  increase  in  the  basal  level  of  GnRH  (P<0\m=.\05). As  a  consequence  of  the  changes  in  frequency  and amplitude  of  the  pulses,  the  mean  levels  of  GnRH before  injection  with  oestradiol  (5\m=.\3  \m=+-\ 1\m=.\0 pmol/l) differed  (P<0\m=.\05) from  those  during  negative (3\m=.\8\m=+-\0\m=.\5  pmol/l)  and  positive  (18\m=.\9\m=+-\4\m=.\7 pmol/l) feedback  phases. These  results  show  that  the  biphasic  pattern  of  LH secretion  induced  by  oestrogen  injection  in  short-term ovariectomized  ewes  is  caused  by parallel  changes  in the  secretion  of GnRH  as  well  as changes  in  pituitary responsiveness  to  GnRH.  An  abrupt  increase  in  the frequency  of  GnRH  pulses  appears  to  be a key  com- ponent  of  the positive  feedback  mechanism whichelicits  the  oestradiol-induced  surges  of  both  GnRH and  LH. Journal  of Endocrinology  (1989)  123,  375\p=n-\382 INTRODUCTION In  ovariectomized  ewes,  exogenous  oestrogen  elicits  a decrease  in  luteinizing  hormone  (LH)  secretion  fol¬ lowed  by  an  increase  (Pelletier  &  Signoret,  1969; Radford, Wheatley & Wallace,  1969;  Goding,  Blockey, Brown  et  al.  1970;  Scaramuzzi,  Tillson,  Thorneycroft &  Caldwell,  1971).  This  biphasic  pattern  is  due  to negative  and  positive  feedback  exerted  by  oestradiol on  the  hypothalamus  and  pituitary  gland,  and  similar changes  are  observed  during  the  follicular  phase  of the oestrous  cycle  of  the  intact  ewe  (Wallace,  Martin  & McNeilly,  1988).  The  exact  roles  of the  hypothalamus through  the  changes  in  gonadotrophin-releasing  hormone  (GnRH)  secretion,  and  of  the  pituitary gland  through  changes  in  the  responsiveness  to GnRH,  have  long  been  the  subject  of speculation  (for review  see  Martin,  1984).  Coppings  &  Malven  (1976) showed  that  both  the  negative  and  positive  feedback phases  are partly  mediated  by  changes  in  pituitary  re¬ sponsiveness.  It  was  later  shown  that  oestradiol  did not  appear  to  reduce  pulse  frequency  during  the breeding  season  (Goodman,  Bittman,  Foster  & Karsch,  1982)  but did  reduce  LH pulse  amplitude, suggesting  that  the  negative  feedback  was  entirely due  to  reduced  pituitary  responsiveness.  Both  of these  conclusions  can only  be  confirmed  by  direct comparison  of  the  patterns  of  secretion  of  GnRH and  LH.With  regard  to  the  positive  feedback  phase,  the importance  of  hypothalamic  input  has  been  clearly demonstrated  in  experiments  using  neurophysiologi- cal  techniques  (Jackson,  Kuehl,  McDowell  & Zaleski,  1978;  Thiéry,  Pelletier  &  Signoret,  1978; Thiéry  &  Pelletier,  1981)  and  immunization  against GnRH  (Fraser,  Clarke  &  McNeilly,  1981).  How¬ ever,  the  exact  nature  of  the  contribution  by  the hypothalamus  is  still  not  clear,  despite  the  recent development  of  techniques  for  measuring  GnRH secretion  directly.  Clarke  &  Cummins  (1985) reported  an  increase  in  GnRH  pulse  frequency  at the  time  of  the  surge,  suggesting  an  active role  of the  hypothalamus  in  the  induction  of  the  surge. The  effect  was  not  observed  in  all  ewes,  but  the finding  is  supported by  observations  of  high  fre¬ quency  LH pulses  during  the  LH  surge  (Martin, Thomas,  Terqui  &  Warner,  19876)  by experiments showing  that  a  sudden,  large  injection  of  GnRH  is necessary  to  produce  an  LH  surge  of  normal amplitude  in  ewes  in which  GnRH  secretion  is blocked  (Kaynard,  Malpaux,  Robinson  et  al.  1988), and  by  the  theoretical demonstration  that  the  high concentrations  of  LH typical  of  the  surge  are  diffi¬ cult  to  achieve  unless  the  frequency  of  pulses  is exceptionally  high  (Martin  et  al.  \9ilb).  However, when  Schillo,  Leshin,  Kuehl  &  Jackson  (1985) attempted  to  measure  GnRH  secretion  during  the LH  surge  in  ovariectomized  ewes,  using  the  push- pull  technique,  they  found  no  effect  of  oestrogen treatment.  It  is  thus  still  possible  that  the  LH  surge is  achieved  simply  by changing  the  responsiveness of  the  gonadotrophs  to  a  constant  tonic  input  from the  hypothalamus.  We  therefore  designed  a study to  test  whether  hypothalamic  GnRH  secretion  plays an  active  role  in  the  biphasic  pattern  of  LH  se¬ cretion  induced  in  the  ovariectomized  ewe  by  ex¬ ogenous  oestrogen.  We observed  GnRH  secretion in the  pituitary  portal  blood  of  conscious,  unre¬ strained,  ovariectomized  ewes  before  and  after treatment  with  oestrogen. MATERIALS  AND  METHODS Animals  and  surgery Six  mature  Ile-de-France  ewes  were  used  in  October for  this  study  (the  normal  breeding  season  extends from  September  to  February).  They  were  ovariecto¬ mized and  implanted  with  cannulae  for  portal  blood collection  on  the  same day,  using  the  method described  by Caraty  &  Locatelli  (1988).  Anaesthesia for  surgery  was  induced  with  pentothal  (Abbott,  BP 2406,  Angers,  France;  10  mg/kg  body weight)  and maintained  with  halothane  (Coopers  Vétérinaire  S.A., BP  142,  Meaux,  France;  4-5%  in  oxygen,  700  ml/  min).  To  implant  the  cannula  for  portal  blood  collec¬ tion,  the  head  of the  animal  was  firmly  held  on  a  metal frame  at  an  angle  of  about  45°.  A  triangle  of  skin between  the  top  of the  orbits  down  to  within  10  cm  of the nasal  tip  was  incised  and  displaced  to  one  side.  A smaller  triangle  of bone  (about  1  cm  inside  the  margin of  the  skin  triangle)  was  removed and  immersed  in saline  until  the  end  of  the  operation.  The  septum  of the  frontal  sinus  and the  back  of  the nasal  cavity were gently  removed  until  the  anterior  border  of  the sphenoid  bone  was  exposed.  Using  a high  speed  drill (12  mm  diameter)  a  tunnel  was  drilled  in  the  sphenoid bone.  The  position  of this tunnel  was  limited  on  either side  by  the  olfactory  bulb,  and  above  by  the  optic chiasma.  When  the  cartilaginous  band  of the  sphenoid bone  was  reached,  the  operation  was  then  performed with  the  aid  of  an  operating  microscope.  The  bone covering  the  pituitary  fossa  was slowly  removed  using the  drill  and  a part  of the  dura  mater  (in  the  shape  of a small  square)  was  detached  from  the  anterior  surface of  the  pituitary  gland  to  expose  the  network  of hypo¬ physial  portal  vessels.  This  hole  was  made  2-3  mm below  a  level  indicated  by  the  protusion  of  the  optic chiasma.  The  apparatus  for  portal  blood  collection was  constructed  from  a  12  gauge  needle  (upper  can¬ nula)  and  a  15  gauge  needle  (lower  cannula)  joined with  dental  acrylic.  The  tip  of  the  upper  cannula  was placed  2  mm  ahead  of the  tip  of the  lower  cannula  and the  two  tips  were  surrounded  by  a  polyethylene  cone (8  mm diameter).  The  apparatus  was  introduced  into the  tunnel in  the  splenoid  bone  and the  tip  of  the upper  cannula  was  pushed  directly  towards  the  portal vessels  until  the  polyethylene  cone  reached  the anterior  surface  of  the  pituitary  gland.  The  space  in the  splenoid  bone  was  then  filled  with  dental  acrylic. The  bone  plate  was  replaced,  the  skin  was  sutured and the  needles  were  filled  with  heparinized  saline  (100  IU/  ml)  and  capped.  The animals  were  monitored  very closely  during  the  postoperative  recovery  period,  and then  allowed  7  days  to  recover completely  from  the anaesthesia  and  to  allow  healing  of  the  tissue.  The cannulae  were  flushed  daily  with  heparinized  saline  (lOOIU/ml).  Each  ewe  received  an  indwelling  cath¬ eter  in  each  jugular  vein  the  day  before  blood  col¬ lection.  Samples  were  taken  simultaneously  from two  animals  placed  side  by  side  in  two  adjacent pens  on  the  floor,  without  any  other  form  of  re¬ straint.  Food  and  water  were  available  ad  libitum and  the  animals  could  see  each  other. On  the  day  of sampling,  the  ewes  were  given  an  initial  dose  of heparin  (25  000  IU)  and  peripheral  blood  was obtained  from  one  of  the  jugular  catheters  by  con¬ necting  it  to  a peristaltic  pump  and  a  fraction  col¬ lector. The  collector  pooled  blood  over  5  min before  changing  the  tube.  Additional  doses  of heparin  (12  500IU)  were  given  every  30  min through  the  second  jugular  catheter.  About  1  h  after the  start  of  peripheral  blood  collection,  a  needle with  a  sharp,  flattened  end  was  introduced into  the upper  portal  cannula  and  four  to  five  small  lesions (2-3  mm deep  and  distributed in  a  circle)  were  made in  the  hypophysial  portal  network. Blood  from these  lesions  was  removed  through  the  lower  can¬ nula,  again using  the  peristaltic  pump and  fraction collector. When  the  portal  blood  flow  was  satisfac¬ tory  (at  least  0-7  ml/5  min),  samples  were  collected every  5  min,  simultaneously  with  peripheral  blood samples,  in  glass  tubes  containing  100  µ bacitracin solution  (1  mmol/1;  Sigma,  La  Verpilliere,  France) and  maintained  in  an  ice  bath  until  centrifuged. Each  ewe  was  injected  with  50  pg  oestradiol-17ß (25  pg  i.v.  and  25  pg  i.m.)  6  h  25  min  after  the  be¬ ginning  of  peripheral  blood collection  and  blood collection  was  continued  for  a  further  18-19  h.  Por¬ tal  blood  volume  was  checked  for  each  sample  and both  peripheral  and  portal  blood  samples  were  cen¬ trifuged  (0°C,  4800 g,  20  min)  and the  plasma  was frozen  (—15°C)  until  assayed.  Care  was  taken  to maintain  portal  blood  samples  near  0  °C  during  all manipulations.  The  day  after blood  collection,  the animals  were  killed  by  decapitation  and  the  pitu¬ itary  glands  were  removed and examined  to  confirm the  site  and the  shape  of  the  lesions  in  the  portal network  (Caraty  &  Locatelli,  1988). Hormone  assays Plasma  concentrations  of GnRH  were  measured  after methanol  extraction  using  the  method  described  by Caraty,  Locatelli  &  Schanbacher  (1987).  Known quantities  of  synthetic  GnRH  were  added  to per¬ ipheral  plasma  samples  containing  similar  quantities of  bacitracin  and  submitted  to  similar  extraction procedures  to  allow calculation  of  recovery  (usually 70-80%).  The  plasma  concentrations  were  corrected for  these  losses.  The  sensitivity  of  the  assays  was 0-2  pmol/tube  of synthetic  GnRH  (UCB-Bioproducts, Brussels,  Belgium)  and  the  maximal  intra-assay coefficient  of variation  was  4%.  All  samples  from  one ewe  were  measured  in  the  same assay. Luteinizing  hormone  was  measured  in  duplicate aliquots  of  plasma  by  a  specific  radioimmunoassay described  previously  (Pelletier,  Gamier,  de  Reviers  et al.  1982).  Samples  collected  during  the  LH  surge  were diluted  1:10.  The  sensitivity  of  the  assay  was  0-1  pg/1. The  standard  preparation  (CY1051; given  by  Dr Combarnous,  INRA,  Physiology  of  Reproduction, Nouzilly,  France)  has  an activity  of  2-5  IU  NIH-LH- Sl/mg.  The  intra-assay  coefficient  of  variation  was 8%.  All  samples  from  one  ewe  were  measured  in  the same  assay. Analysis  of the  dataThe  GnRH  and  LH  data  were  analysed  with  the algorithm  'Munro'  (Martin,  Taylor  &  McNeilly, 1987a),  a  modified  version  of  'Pulsar'  (Merriam  & Wächter,  1982).  The  G  parameters  (the  number  of standard deviations  by  which  a  peak  must  exceed  the baseline  in order  to  be  accepted)  were  2,  1-5,  11,  0-8 and  0-6  for G,-G5,  these  being  the  requirements  for LH  pulses  composed  of  one  to  five  samples  which exceed  the  baseline  respectively.  For  GnRH  pulses, the  values  were  3-98,  2-40,  1-68,  1-24  and  0-93 respectively. The  Baxter  parameters  describing  the  parabolic relationships  between  the  concentration  of  hormone and the  standard  deviation  (assay  variation)  aboutthat  concentration  were  0089  (b,,  the  y  intercept), 000 (b2,  the   coefficient)  and  0-00009 (b3,  the  x2 coefficient)  for  the  LH  assay,  and  0-22368,  0-057  and 000015  respectively  for  the  GnRH  assay. The  pulse  frequency,  mean  nadir  and  pulse  ampli¬ tude  were  calculated  for  each  profile  and  were  used  in the  analysis  of  treatment  effects.  The  onset  of  the  LH surge  was  defined  as  the  onset  of  the  first  LH  pulse that  preceded  a  sustained  rise  in  LH  secretion  (con¬ centrations  do  not return  to  the baseline  within  2  h). The  period  (about  25  h)  of  blood  sampling  was divided  into  three  sections:  PI,  before  oestradiol administration;  P2,  negative  feedback  on  LH  secre¬ tion,  from  the  time  of  oestradiol  administration  until the  onset  of  the  LH  surge;  and  P3,  positive  feedback on  LH  secretion,  from  the  onset  of the  LH  surge  until the  end  of  the  blood  sampling.  Data  from  these periods  were compared  using  the  paired  /-test. RESULTS Portal  and  peripheral  blood  were  collected  success¬ fully  from  five  of  the  six  animals  used  in  the experiment  (surgery  was  unsuccessful  in  one  animal).Peripheral  blood  was  collected  throughout  the  25-h  experimental  period  and  portal  blood  for  2215,  22-15, 23-00  and  2310  h  in  four  ewes  and  in  the  fifth  ewe peripheral  and  portal  blood  were  collected  for  10-75 and  6-90  h  respectively,  before  blood  collection  was abandoned  because  of damage  to  the  portal  cannulae. Because  of  variations  in  the  shape  of  the  pituitary gland  the  place  and  shape  of  the  lesions  differed between  animals  leading  to  large  (100-150  pm diameter)  or  small  (20-30  pm  diameter)  portal  blood vessels  being  cut  (usually  the  proportion  of portal  vas- culature  left  intact  was  in  the  range  of 60-80%).  Thus, portal  blood  flow  varied  between  animals  (0-8-20  ml/  5 min),  but  was reasonably  constant  throughout  the experiment  for  a  given  animal,  except  for  the  last 8-10  h  when  the  volume  of  portal  blood  increased  by 30-60%  in  three  ewes. Secretion  of LH Plasma  profiles  of GnRH  and  LH covering  about  25  h are  shown  for  three  representative  ewes  in  Fig.  1. Typically  for  short-term ovariectomized  ewes,  a clear  pulsatile  pattern  of  LH  secretion  was  observed until  the  oestradiol  was  injected.  Immediately  after oestradiol  treatment,  there  was  a  decrease  (negativefeedback)  followed  by  an  increase  (positive  feed¬ back)  in  LH  secretion.  In  the  four  ewes  for  which  the entire  biphasic  pattern  was  observed,  the  LH  surge began  11-5  ±0-4 h  after oestradiol  injection  (range 10-4-12-2  h). As  shown  in  Fig.  2,  the  negative  feedback  phase (P2)  was  characterized  by  a  lower  pulse  frequency (.P<0-01),  pulse  amplitude  (P<001),  nadir  (P<001) and  mean  concentration  (P<00\)  compared  with  PL During  positive  feedback,  the  values  for  pulse  ampli¬ tude  (P < 005),  the  nadir  concentration  (P < 001 )  and the  mean  concentration  (P<00\)  were  all  greater than  the values observed  at  either  PI  or  P2.  On  the other  hand,  the  frequency  of  the  pulses  increased above  the  frequency  observed  during  P2 (i,<0-01), but  the  difference  between  PI  and  P3  was  not statistically  significant. Secretion  of  GnRH Before the  oestradiol  injections,  GnRH  secretion  was clearly pulsatile  and  all  the  GnRH  pulses  observed coincided  with  an  LH  pulse  in  the  peripheral  circu¬ lation.  Only  one  LH  pulse  was  not  associated  with  a GnRH  pulse.  Compared  with  this  period,  the  negative feedback  phase  revealed  reductions  in  pulse  frequency (P<001),  pulse  amplitude  (P<005)  and  the  mean concentration  (P<005).  In  contrast,  there  was  a slight  increase  in  the  nadir  (P<00\).  During  this period,  37  GnRH  and  23  LH pulses  were  observed  in the  five  ewes,  so  the  coincidence  was  less  marked.  A large  increase  in  GnRH  secretion  was  observed  in all  four  ewes  sampled  during  positive  feedback.  The frequency  of  the  pulses  (P<0-01)  and the  nadir (P<0-05)  increased above  levels  observed  before oestrogen  treatment,  but  pulse  amplitude  did  not change  significantly  from  the  levels  observed  during negative  feedback.  Again,  we  observed  more  GnRH pulses  (52)  than  LH pulses (45). When  the  values  for  GnRH  concentration  were corrected  for  rate  of  portal  blood  flow,  the  rate of  secretion  of  GnRH  was  also  found  to  change after  oestrogen  treatment.  The  rate  (mean  + s.e.m.) was  significantly  lower  during  negative  feed¬ back  (5-8  + 0-7pmol/5 min)  than  before  oestradiol administration  (7-0±0-8,  P<00\)  and  was  signifi¬ cantly  (P<0-01)  higher  during  positive  feedback (39-7  +  8-4  pmol/5  min)  than  at  any  of the  other  times. DISCUSSION The  administration  of  oestrogen  induces  a  biphasic pattern  in  the secretion  of  GnRH into  hypophysial portal  blood  of  conscious,  unrestrained,  ovariecto¬ mized  ewes.  This  pattern,  in  which  a  decrease (negative  feedback)  is  followed  by  a preovulatory- type  surge  (positive  feedback)  parallels  the  changes  in the  pattern  of  secretion  of  LH  into  the  peripheral circulation,  reported  by  Pelletier  &  Signoret  (1969). Moreover,  the  pattern  is  similar  to  that  of LH secretion  over  the  follicular  phase  in  intact  ewes (Wallace  et  al.  1988). The  secretion  of  both  GnRH  and  LH  was clearly pulsatile  before  the  administration  of  oestradiol,  and each  GnRH  pulse  in  portal  blood  preceded  an  LH pulse  in  jugular  blood.  The  strong  cause-effect relationship  between  GnRH  and  LH pulses,  described initially  by  Clarke  &  Cummins  (1982),  was  thus reproduced  in  the  present  study.  By  combining  this relationship  with  the  differential  effects  of  oestrogen on  the  characteristics  of  the  pulses  of  the  two  hor¬ mones,  we  can  determine  the  sites  at  which  oestradiol acts  to  exert  negative  and  positive  feedback in  this model. There  was  a rapid  reduction  in  LH pulsatility immediately  after oestradiol  administration  suggest¬ ing  that  oestradiol  initially  caused  a  reduction  of pituitary  responsiveness  to  GnRH  and  was  an import¬ ant component  of  the  negative  feedback  effect  of  the steroid  on  LH  secretion  as  reported  by  Coppings  & Malven  (1976),  Caraty,  Martin  &  Montgommery (1984)  and  Clarke  &  Cummins  (1984).  On  the  other hand,  the  pulses  of  GnRH  continued after  the  injec¬ tions  of  oestradiol  but,  after  some  delay,  there  was  a clear  reduction  in  both  frequency  and  amplitude, suggesting  that  oestradiol  was  exerting  feedback  at the level  of the  hypothalamus.  A  similar  response  was  12 111 8 6 4 2 (I 35 m^ma^JUvmI  » vA^^JlxJVA^^'V «  30 E  25 J « 20 J  15 D. a  10 3 OX)  c 3  J 0 20-, (c)  /iAMjAk^ 50 40 30 20 10 0 60 o so   o. 40 I Oí •30 O   20  j   10  2 I—   0. L(l 15   ío-l 5 0 0  4  8  12  16  20 24 Time  from  start  of  jugular  samples  (h) figure  1.  Secretory  profiles  for  gonadotrophin-releasing  hormone  (GnRH)  in  hypophysial portal  blood  (solid  line)  and  LH  in  jugular blood (·) in  three  representative  ovariectomized ewes  (a,  b  and  c)  sampled  continuously  for  25 h.  Oestradiol-17ß  (25  pg  i.v.  and  25  pg  i.m.) was injected  after  6-25  h  (solid  arrowheads)  and  the  preovulatory  surge  (open  arrowheads) began about  12  h  later. observed  in  the  ovariectomized  rat  (Sarkar  &  Fink, 1980),  but  not  in  the  ewe  (Clarke  &  Cummins,  1985), perhaps  because  the  animals  used  in  this  study  had been  ovariectomized  for  a  long  time.  It  has  often  been suggested  that  oestradiol  is  unable  to  reduce  pulse frequency  during  the  breeding  season,  except  in  the presence  of  progesterone  (for  reviews  see  Karsch, 1980;  Martin,  1984),  but  these  studies  were  limited  by the  use  of  LH  pulse  frequency  to  estimate  GnRH pulse  frequency  and  by  restricted  ranges  of  doses  of oestrogen.  High  doses  of  oestradiol  could  not  be tested because  the  responsiveness  of  the  pituitary
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