Ventromedial hypothalamus as a target for oestradiol action on proceptivity, receptivity and luteinizing hormone surge of the ewe

241

Brain Research, 546 (1991) 241-249 (~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116506Q BRES 16506

Ventromedial hypothalamus as a target for oestradiol action on proceptivity, receptivity and luteinizing hormone surge of the ewe D. Blache, C.J. Fabre-Nys and G. Venier Centre de recherche INRA/CNRS Tours, Unitd de physiologie de la reproduction des mammifOres domestiques, Nouzilly, Monnaie (France) (Accepted 6 November 1990) Key words: Oestradiol; Hypothalamus; Stereotaxic implantation; Receptivity; Proceptivity; Luteinizing hormone; Sheep

Most of the literature suggests that in sheep as in rodents nervous structures involved in female sexual behaviour are not necessarily identical to those involved in the LH surge. In rodents, oestradiol triggers female sexual behaviour by acting on a restricted area of the mediobasal hypothalamus whereas the concomitant induction of the preovulatory LH surge is at least partially under the control of more anterior structures. The central sites of oestradiol action, however, remained poorly defined in sheep. To provide this definition, 37 ovariectomized ewes were stereotaxically implanted unilaterally or bilaterally with a guide cannula in preoptic area (POA), anterior, mediobasal, lateral, or posterior hypothalamus (AH, MBH, LILT, PH). Experiments were made during the breeding season (Br) and the anoestrous period (An: unilat only) and females were primed with a peripheral treatment of progesterone and a dose of 17 fl-oestradiol subthreshold for both the LH surge and sexual behaviour, lntracranial implants (i.d. = 0.45 mm) of crystalline E2 were lowered 16 h after progesterone removal and left in the brain for 48 h. Whereas POA implants never had any significant effects on either the behaviour or the LH surge, all MBH implants caused receptivity (11 bilat, 5 unilat Br and 5 unilat An). Bilateral MBH implants also induced proceptivity in 9 of 11 ewes and increased the LH levels in 7 of them. These proportions do not differ significantly from those observed after a 25/~g peripheral injection of E2. Unilateral MBH implants had no significant effect on proceptivity and LH increase but oestrous behaviour was induced by some implants placed laterally to the MBH (2/5 recept and 3/5 procept). The quality of oestrous behaviour after brain implants was not significantly different from that observed after peripheral steroid treatment, showing that all brain areas needed for full expression of oestrus have been stimulated. The amplitude of the LH increase, however, was lower after implantation than after i.m. injection of the steroid; we propose that this is due to an insufficient stimulation of the hypophysis. These results show that in the ewe, a restricted area in the mediobasal hypothalamus is an important site of oestradiol action to trigger both sexual behaviour and the LH surge. INTRODUCTION F e m a l e sexual behaviour, in m a n y m a m m a l i a n species, d e p e n d s on p r o g e s t e r o n e and oestradiol. F o r sexual b e h a v i o u r to occur in sheep, oestrogen must follow a p e r i o d of e x p o s u r e to p r o g e s t e r o n e but the latter must no longer be present. Thus oestrous b e h a v i o u r can be induced in o v a r i e c t o m i z e d ewes by r e p e a t e d t r e a t m e n t with p r o g e s t e r o n e ( m i n i m u m 3 days) followed by administration of oestradiol 4°'41. These behavioural changes are c o n c o m i t a n t with the e n d o c r i n e events leading to ovulation, although it is i m p o r t a n t to note that in sheep p r o g e s t e r o n e has no facilitative effect on the preovulatory L H surge which, unlike sexual behaviour, is trigg e r e d by oestradiol alone 12'43. H y p o t h a l a m i c and p r e o p t i c structures are at least partly responsible for controlling these events. Much of o u r current k n o w l e d g e a b o u t the neural circuits involved suggests that in the sheep, as in the rat, the essential structures for female sexual b e h a v i o u r are not necessarily

identical to those r e q u i r e d for the L H surge 25'35. Electrolytic lesions 11'16'36'37 as well as a n t e r o d o r s a l deafferentation of the m e d i o b a s a l h y p o t h a l a m u s 24'34'48 o r histological localization of L H R H cells 5'29 led to the hypothesis that in ewes the p r e o v u l a t o r y L H surge is triggered by L H R H neurons, situated in a n t e r i o r structures, whereas sexual b e h a v i o u r d e p e n d s on n e u r o n s l o c a t e d in m o r e m e d i o b a s a l h y p o t h a l a m i c structures. T h e target cells for oestradiol action in the sheep brain r e m a i n p o o r l y defined. Using intracranial implants of the steroid, Signoret 45 and D o m a n s k i et al. 17 f o u n d that oestradiol stimulates sexual b e h a v i o u r when placed in either the m e d i o b a s a l o r a n t e r i o r h y p o t h a l a m u s . These data suggest that the target a r e a for oestradiol induction of female sexual b e h a v i o u r is larger in sheep than in rodents 3. F o r the L H surge also, brain sites for oestradiol effects are still a m a t t e r of d e b a t e 26. T h e r e is g o o d evidence that oestradiol acts at least in part on the pituitary, where it increases the responsiveness of gonadotrophs to G n R H 9"27'38. H o w e v e r , the increase in

Correspondence: C.J. Fabre-Nys, Station de Physiologic de la Reproduction des mammif~res domestiques, INRA, Nouzilly, 37380 Monnaie, France.

242 a m o u n t o f G n R H m e a s u r e d in p o r t a l b l o o d b e f o r e t h e L H s u r g e also i n d i c a t e s a c e n t r a l site o f a c t i o n 6'7'1°'3°'33. The

i d e n t i f i c a t i o n o f this site h a s b e e n

sought

using

i m p l a n t a t i o n t e c h n i q u e s in o n l y o n e s t u d y 31 a n d this d i d not

provide

responding

clear evidence to

oestradiol

about

and

hypothalamic

thereby

triggering

sites LH

release. T h e a i m o f t h e p r e s e n t s t u d y was t o e l u c i d a t e t h e c e n t r a l sites o f a c t i o n o f o e s t r a d i o l , o n b o t h f e m a l e s e x u a l behaviour and LH surge, by direct application of microq u a n t i t i e s o f o e s t r a d i o l i n t o d i s c r e t e t a r g e t a r e a s in t h e hypothalamus and preoptic area of ovariectomized ewes. B e c a u s e s e n s i t i v i t y to t h e t r i g g e r i n g e f f e c t o f o e s t r a d i o l on oestrous behaviour

declines during the anoestrous

s e a s o n 22, e x p e r i m e n t s w e r e p e r f o r m e d b o t h d u r i n g t h e breeding season and the anoestrous period.

found to release 6.5 × 10 7/~mol in distilled water for 48 h. In 3 females CSF and blood were collected at 1,2,4,6,24 and 48 h after unilateral implantation of tritiated oestradiol. All 5 ewes were then killed, the brain and pituitary gland removed rapidly, and blood and urine samples collected. The hypothalamus was dissected and frozen in liquid nitrogen vapours. Samples of 1 mm 2 were cut for 5 mm from the implant site. The hypophysis was also cut into 1 mm thick slices. Samples of cortex, cerebellum, amygdala and liver were collected and weighed. Each tissue sample was homogenized in a vial containing 0.5 ml absolute ethanol and kept for 1 h at 50 °C. Ten ml of scintillation fluid were added to each vial to allow radioactivity to be counted. 200 jd of CSF and 1 ml of plasma were assayed for radioactivity. Results were expressed as c.p.m./mm 3 for the hypothalamus, c.p.m./100 mg for the other parts of brain and liver and c.p.m./ml for liquid samples (Table I). No radioactivity was found in the plasma or cerebrospinal fluid collected at various time during implantation. After 48 h of implantation of 1% tritiated oestradiol, radioactivity was below the detection threshold of the counter for samples obtained from the infundibulum, pituitary, cortex, cerebellum, amygdala, liver or urine. Around the implant, radioactivity decreased by more than 90% in the first mm and no radioactivity was detected at more than 1.5 mm of the tip of the implant.

MATERIALS AND METHODS Animals 53 fie de France and Prdaipes du Sud ewes (2-9 years old) were used as experimental animals. They were ovariectomized at least 3 weeks before the beginning of the experiment. Four other ewes, ovariectomized as adults, and 4 intact males, were used as animal stimuli during the behavioural tests. They were housed in unisex groups, in indoor pens, under natural photoperiod. They were fed throughout the year, between 07.00 and 09.00 h with a constant diet of straw, lucerne and maize pellets and mineral supplement. They had free access to water. Brain surgery Ewes were fitted with guide-cannulae using a technique which combines the stereotaxic method with lateral and dorsofrontal radiography 2~. Ewes, fasted for at least 24 h, were anesthetized by injection of Pentothal-Atropin and were maintained under fluothan (3% in 02). The head was fixed in a stereotaxic apparatus (Prdcision Cindmatographique) and 1 ml radio opaque liquid (Lipiodol Guerbet) was injected into the lateral ventricle. Stainless steel guide-cannulae (i.d. = 0.8 mm, o.d. = 1.2 mm) were implanted 5 mm above the target site under X-ray control. We used the massa thalamica as the internal landmark in the anteroposterior and vertical orientation and the third ventricle for laterality towards the mid- saggital plan (Fig. 1). Ewes were implanted bilaterally and unilaterally in the preoptic area (POA), anterior hypothalamus (AH) and ventromedian hypothalamus (VMH) and only unilaterally in lateral hypothalamus (LHT) and posterior hypothalamus (PH). Screws were fixed in the bone around the guide cannula. Dental cement was applied to hold the guides rigidly in position, and the guide was plugged. Implants made of a stainless tube (i.d. = 0.45 mm, o.d. = 0.7 mm) were filled with crystalline E2 or cholesterol by tamping the tip of the tube into the pure molten substance. After crystallization, the outer surface of the tube was carefully cleaned with ethanol to remove any hormone residue. The presence of oestradiol as a flat surface at the tip was verified under a dissecting microscope. Just before use, implants were placed in formol vapours for at least 4 h to ensure sterility. Steroid diffusion Using the same stereotaxic technique as described above, 5 ewes received bilateral guide cannula or a unilateral guide cannula plus a cannula in the lateral ventricle. Bilateral or unilateral implants of 1% of tritiated oestradiol (100 Ci/mmol, Amersham), diluted in 17 fl-oestradiol, were left in the brain for 48 h. Similar implants were

Histology At the end of the experiments the animals were killed and the head were perfused, via the carotid arteries with 1.5 l of 4% paraformaldehyde. The brain were removed and postfixed in the same fixative during at least 8 days. Frozen coronal sections (50/~m) were cut and stained by the Kliiver and Barrera method 2s or thionine staining. The site of implantation was determined using the Richard Atlas 39 and our unpublished data. Treatments Experiments were made during the breeding season (two groups of 11 and 16 ewes studied from October to January, unilateral and bilateral implantation, respectively) or during the anoestrous period (one group of 10 ewes studied from April to June, unilateral implantation). All ewes had between 3 and 8 successive 15 days artificial oestrous cycles consisting of a peripheral priming treatment with progesterone and oestradiol followed by the intracerebral oestradiol or control treatment (for protocol see Fig. 2). All ewes which showed a sign of illness during the course of the experiment, were immediately killed; therefore the number of cycles was not constant for all ewes. Behavioural measures Oestrous behaviour was quantified for each female individually, using the procedure standardized and described by Fabre-Nys and Venier ~9. Receptivity was measured by counting the frequency of immobilization (characteristic of oestrus) shown by a ewe in response to the courtship of the male during a test period. A 'receptivity index' (Rt) was calculated as the percentage of immobilizations in the total number of courtship interactions. Responses of the ewe to male mounting were also recorded. Each test usually lasted 2 min, but was extended to a maximum of 15 min if less than 10 interactions were recorded. A ewe was deemed 'receptive' when her RI was > 50%, or if she accepted mounting. To measure proceptivity, ewes were given the choice of interacting with either a group of 3 females or a group of two males and one female kept in pens 2 m apart. Each test lasted for 3 min and ewes which stayed just in front of the male pen, trying to make contact with this group for more than 60 s during the test period were described as proceptive. Control data were obtained by testing the same animals before progesterone removal and measuring the time spent in contact with the female group (baseline). The duration of proceptivity for responsive ewes was defined as the period during which time spent interacting with the male group was in excess of the baseline.

243

1Ver, tgt

Fig. 1. Upper panel: lateral and dorsofrontal ventriculograms of ewe brain after an injection of Lipiodol in the lateral ventricle. Lower panel: schematic drawings of (a) lateral ventriculogram; (b) dorsofrontal ventriculogram. Abbreviations: Ver. tgt, vertical tangent; Hor. tgt, horizontal tangent; Lv, lateral ventricle; IIh, third ventricle; Ir, infundibulary recess; Mr, mammillary recess; Mi, mass intermedia.

During each cycle made during the breeding season, ewes were tested for proceptivity and receptivity once before progesterone withdrawal and during 32 h (proceptivity) or at least 48 h (receptivity) at 8 h interval starting 8 h after E2 administration. During anoestrus, only receptivity was measured and at the same rhythm as during the breeding season with the exception of the positive control cycle during which ewes were tested only twice daily. Luteinizing hormone assays Jugular blood was collected for 44 h every 1 or 4 h starting 4 h before the time of E2 administration during each cycle. Plasma was separated and stored at -20 “C until assayed. Concentrations of LH were measured in duplicate aliquots of 100 ~1 plasma by a double antibody radioimmunoassay as described by Martin et a13’. The interassay and intraassay variation was 15% and lo%, respectively, for LH values of 2.5, 4.5, 10, 15, 20 and 35 @ml. Our knowledge of the LH profile in systemic blood during the oestrous cycle of

intact ewes, or after injection of 25 peg of E2 allows us to distinguish between two kinds of LH increase. These are: (1) ‘preovulatory surge’ when at least one sample reached a level above 20 @ml, 12 h or more after the E2 administration, and twice the amplitude of the mean of LH levels during the period before E2 administration; (2) ‘marked increase’, when blood concentrations stayed above 10 @ml for at least two consecutive samples and were twice the mean of the LH levels during the period before E2 administration. The onset and the end of the LH surge were defined as the time at which LH concentration increased above or decreased below the mean of LH levels recorded during the period before E2 injection. Statktical analysis The data were analysed in groups according to the histological localization of the implants. All behavioural data were analysed using non-parametric tests”. Fisher’s exact probability test was used

244 E2 central implant : Exp Cycle No injection : Neg Cont 1 Cholesterol centrol implont : Neg Cont 2 >k 8 I~g E2 I.M. : Pos Cont 1 • • 2 cm silostic implant : Pos Cont 1 >I< INTRAVAGINAL PROGESTERONE

25 ug E2 I.M. : Pos Cont 2 •

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'SUBCUTANEOUS SILASTIC IMPLANT OF OESTRADIOL

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48

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HOURS

Fig. 2. Experimental protocol. In each cycle, all ewes received a priming treatment (low priming) consisting in 8 days of intravaginal progesterone (Ovigestone Intervet) and a subcutaneous silastic implant of 17 fl-oestradiol (Dow Coming; i.d. = 3.35 mm; o.d. = 4.65 mm;

length = 1 cm, left in the leg for all the experiment). In some of the cycles made during anoestrus, an additional I cm oestradiol implant was added during progesterone treatment (high priming). 16 h after progesterone removal ewes received oestradiol via intracerebral implantS or peripheral administration. Central implants were left in the brain for 48 h. *, during anoestrous season only; • during breeding season only. [] made in 8 ewes different from implanted ewes. • made in 9 bilateral implanted ewes. Pos, positive; Neg, negative; Cont, control.

to compare the proportion of animals responding to the treatments. Mean levels of sexual activity were compared using the M a n n Whitney U-test (independent data) or the Wilcoxon test (data on the same ewes). LH data were analysed using t-test.

Breeding season Sexual behaviour Receptivity

RESULTS Implantation sites are summarized in Fig. 3.

TABLE I

Repartition o f radioactivity in tissues after central implantation of tritiated oestradiol (a) Around the implantation site Distance to the tip o f implant +3mm

(b) Other tissue

c. p. m. /mm 3 (mean + S. E. M. )

(mean + S. E. M. )

18.1 + 1.7

plasma

14.8+ 0.8

+2 mm

16.6 + 2.0

c.p.m./ml CSF c.p.m./ml

16.3 + 2.0

+ 1 mm

47.5 __+12.5

0 mm

590 + 88

- I mm

558 + 61

- 2 mm

36.6 + 7.2

Median eminence

19.0 + 1.0

Hypophysis

22.0 + 2.0

Urine c.p.m./ml Liver c.p.m./100 mg Cortex c.p.m./100 mg Amygdala c.p.m./100 mg Mesencephaion c.p.m./100 mg Noise*

30.0 + 5.0 16.0 + 2.3 45.5 + 6.8 35.3 + 6.8 26.5 + 4.8 15.4 + 0.7

* Average of c.p.m, counting in 10 vials containing 0.5 ml of absolute ethanol and 10 ml of scintillation fluid.

. T h e proportion of ewes showing recep-

tivity after the various treatments is shown in Fig. 4a. All the oestradiol implants in the ventromedial part of the hypothalamus, either unilateral (5/5) or bilateral (11/11), caused receptivity. Thus the efficiency of this treatment was similar to that of 25 #g or 8 #g of the steroid given peripherally. Two of the 5 ewes implanted unilaterally in the lateral hypothalamus (1.5 m m away from the ventromedial location) were also receptive. By contrast, the implants had no effect when located in the preoptic area or the anterior hypothalamus (bilateral P O A 0/5, unilateral P O A 0/5, unilateral A H 0/4, P < 0.05) or in the posterior hypothalamus (unilateral 0/3). Similarly no ewe was receptive after the priming treatment alone. The latency of receptivity was significantly shorter after peripheral injection of oestradiol than after unilateral or bilateral intracerebral implantation (P < 0.05, Fig, 4b). Ewes implanted bilaterally were receptive for a longer time than ewes treated peripherally or implanted unilaterally (P < 0.05, Fig. 4b). The quality of the receptivity (mean RI) was similar in the group implanted bilaterally and in the group injected peripherally but was signifi, cantly lower after unilateral implantation (P < 0.05, Fig. 4b). P r o c e p t i v i t y . Between 50 and 60% of the ewes were proceptive after peripheral treatment. This proportion was not significantly different after bilateral implantation in the V M H (80%, P > 0.05) which resulted in a

245

BREEDING SEASON Bilateral Implantation Unilateral Implantation

ANOESTROUS SEASON Unilaferal Implontation 18 ~

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Fig. 3. Localization of implantations on schematic saggital representation. © implantation site inducing ~ receptivity, ~ proceptivity, • both and A increase of LH (reconstructive view). but only one of 5 females was p r o c e p t i v e after implantation in V M H or in A H and n o n e in H P and P O A groups. T h e characteristics of the proceptivity - latency, d u r a t i o n and m a x i m u m time n e a r the males - were

significantly higher p r o p o r t i o n of positive responses than did P O A implants ( P < 0.05, Fig. 5a). Unilateral implants induced proceptivity less regularly (Fig. 5a). T h r e e out of 5 ewes were proceptive in the L H T group,

a) Proportion of responding ewes

b) Quality of the response LATENCY

PERIPHERAL BILATERAL UNILATERAL TREATMENT IMPLANTATION IMPLANTATION Hours

DURATION

MEAN RI

p<~:.~SJ ~<0;05

,

100

RI

p
1 O0

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60

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50

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50

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25

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VMH POA

PH

LHT

VMH AH POA

8ug25ugVMHVMHLHT bl unl

81KI251~)VMHVMHLI'IT bl unl

8~i 25~i~H bl

80 60 40

20 0

VMH LHT unl

Fig. 4. Effect of oestradiol treatments on receptivity during breeding season. *, P < 0.05 vs VMH bilateral group; $, P < 0.05 vs VMH unilateral group.

246

a) Proportion of responding ewes PERIPHERAL TREATMENT

BILATERAL

b) Quality of the response

UNILATERAL

LATENCY

MEAN OF TIME NEAR THE MALE

DURATION

IMPLANTATION IMPLANTATION

Y.

Hours

p
1O0

sec

i

140

25 p<0.05 I--1

20

75

120 1O0

l I I I l l l

50

25

l / I / / l l

15

80 6O

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PRIMING8 ug 25 gg TREAT,

VMH POA

0

PH LHT 'VMH All POA

0 8ug 251=jVldH LHT bi

8~g 25MgVMH LHT b|

8 ~ 25UgVMH LHT b1

Fig. 5. Effect of oestradiol treatments on proceptivity during breeding season. *, P < 0.05 vs 25/~g group.

those in the POA (P < 0.05, Fig. 6a). The proportion of positive responses was not different from that observed after peripheral treatments (bilateral VMH, 7/11; 8/~g, 6/6; 25/~g, 8/8) although the number of typical 'surges' was lower than after 25/~g (4/11 vs 7/8, P = 0.035). After unilateral implantation some females of the VMH, LHT and PH groups of E2 presented a marked increase of LH levels but never above 20 ng/ml. POA and AH unilateral implantation had no effect. As with the behavioural responses, the latency of the LH increase was longer after central implantation than

similar after bilateral implantation in the VMH and in the control group receiving 25/~g of E2 peripherally (Fig. 5b). However, after unilateral implantation in the LHT proceptivity appeared later than after peripheral treatment (P < 0.05) and was less intense than after peripheral treatment or bilateral implantation in VMH (P < 0.05, Fig. 5b).

Luteinizing hormone Bilateral implants in the VMH more often induced an increase in plasma LH (surge or marked increase) than

a)

b) Quality of the response

Proportion of responding ewes PERIPHERAL TREATIdENT

BILATERAL IMPI.~ATION

LATENCY

UNILATERAL IMPLANTATION

%

Hours

" "/

AMPLITUDE

p<0.05 r

100

DURATION

~

ng/ml

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LH surge Marked increose

p
i

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Fig. 6. Effect of oestradiol treatments on the increase of L H during breeding season. *, P < 0.05 vs 25 # g group.

" Id un! I ~

247 after peripheral injection of 25/zg of E2 (P < 0.05) but there was no significant difference when compared to 8 /zg E2 injection (P = 0.13)(Fig. 6b). The duration of LH surges was similar although peak amplitude were lower after implantation than after peripheral injection of 25/~g oestradiol (P < 0.02) (Fig. 6b).

Non-breeding season Because of the lack of seasonal effect on the LH surge (mentioned by Goodman et al. 23) and the presence of behavioural responses with unilateral implants bilateral implants were not tested during this period.

Sexual receptivity Unilateral implants after the priming treatment used during the breeding season (8 days of PG + 1 cm silastic implant of E2) caused receptivity in only two ewes (one V M H and one POA). This proportion was increased to 5 receptive ewes out of 5 when the priming treatment was increased (2 cm silastic implant during the progesterone treatment) and all the ewes were implanted with oestradiol in the VMH. Only one of them responded to cholesterol implantation. Implants situated in other locations were ineffective (POA, n = 4; LHT, n = 1). The characteristics of the receptivity observed in VMH implanted ewes during anoestrus were not significantly different from those observed in the same group during the breeding season (latency = 20 + 8 h, duration = 24 + 10 h, mean RI = 66 + 13%).

Luteinizing hormone During this experiment, for the groups as a whole, there was no significant increase in mean LH levels, except during the control cycle with 2 cm silastic implant of E2 (8/9 ewes responding). DISCUSSION Our experiments provide the first evidence for the ewe that oestradiol acts, at least in part, in a restricted area in the ventromedial region of the hypothalamus to stimulate both oestrous behaviour and LH secretion. This area corresponds to a zone of high neuronal cell density and is probably equivalent to the ventromedial nucleus (VMN) of the rodent 4'15. The earlier ovine studies probably failed to pinpoint this restricted zone because larger implants were used and kept in the hypothalamus for longer periods, than in our experiments 16'4s. Our data concur with results in other species 3 and with the site where Thi6ry 47 found 34% of neurons activated by intravenous oestradiol injection. Neighbouring structures, such as the infundibular nucleus, could have been

stimulated by some of our implants. But leakage of oestradiol to more remote structures such as the hypophysis or the anterior hypothalamus was unlikely. Oestrous behaviour and LH secretion were induced by some implants placed lateral or posterior, but never rostral, to the ventromedial region. This shows that anterior structures, known to be involved in the control of the LH surge from lesions or deafferentation studies 11'16'24'36'37'48 do not constitute a main site for oestradiol action. It also suggests that neurons in the lateral part of this region are of particular importance with respect to the induction of periovulatory events in the ewe. This part of VMN has been pointed out as the most sensitive site for oestradioi action on sexual behaviour in the female rat 2'42 and is particularly rich in oestradiol receptors 8'14'49. The failure of rostral sites to be active also yields information about the spread of hormone and confirms the conclusions made from the experiment involving measurement of tritiated oestradiol at progressive distance from the implantation site. The quality of the behaviour induced was identical to the quality of behaviour of intact 2° or ovariectomized ewes treated peripherally (control groups). This indicates that we stimulated all the brain areas needed for full expression of female sexual behaviour. This was not the case for the LH surge. The low amplitude of the LH increase we observed after intracerebral implantation suggests that steroid stimulation was not sufficient for induction of a normal LH surge. In our experiments, the hypophysis has been stimulated by the priming treatment but certainly not as it would have been after a peripheral injection. This could account for the lower amplitude of the LH surge (see ref. 27). The longer latency to the onset of oestrous behaviour, or the LH surge after brain implantation, when compared with the time taken after systemic injection of oestradiol can be explained by the probable slow diffusion of the hormone to neurons distant from the immediate area of the implantation site. Duration of oestrus is often longer after implantation and this can be explained by a longer duration of exposure to oestradiol (Fabre-Nys, unpublished results). As expected from the work from Fletcher and Lindsay 22, the priming dose needed to observe sexual behaviour after implantation during anoestrus is higher than during the breeding season. Because only unilateral implants have been used, it is not possible to conclude about steroid action on LH surge during anoestrus. For oestrous behaviour, the area in which oestradiol is active is similar during the two periods but we cannot know whether the decrease in sensitivity is localized in the mediobasal hypothalamus or somewhere else. Furthermore, it is interesting that our implants trigger both receptivity and proceptivity. Oestradiol is known to

248 affect lordosis, at the level of the hypothalamus 35. A l t h o u g h our criterion of receptivity does not involve a m o t o r response to tactile cues as lordosis does, but a m o r e general response from a distance to male behaviour, our results are in a g r e e m e n t with this view. Sites of steroid action on proceptivity, a p a r a m e t e r more relevant to appetitive aspects of sexual behaviour, are less clear. A c c o r d i n g to Barfield and Chen / and Rubin and Baffield 42 oestradiol also stimulates the hopping and darting b e h a v i o u r of rats by acting on the VMN. Takahashi et al. 46 consider that female hamsters present all the patterns associated with n o r m a l sexual behaviour only when both the V M N and the preoptic area or a n t e r i o r h y p o t h a l a m u s are stimulated. In any case it is difficult to be sure that the patterns observed reflect the level of sexual arousal. In o u r experiments, choice of the proximity to the male group is stimulated by oestradiol in the m e d i o b a s a l hypothalamus. This behaviour cannot be explained only by changes in responsiveness to male cues, because one intact male alone is not attractive for oestrous ewes 26. It cannot be explained either by facilitation of a specific m o t o r p a t t e r n but indicates change related to 'sexual motivation'. This shows that the m e d i o b a s a l h y p o t h a l a m u s in ewes is involved in both appetitive and c o n s o m m a t o r y aspects of female sexual behaviour. Of course, for sexual behaviour as well as for L H surge this does not m e a n that the m e d i o b a s a l h y p o t h a l a m u s is

the only site of oestradiol action. Results p r e s e n t e d here have been o b t a i n e d with p e r i p h e r a l oestradiol priming treatment. It is possible that the site for this priming effect is different from the m e d i o b a s a l h y p o t h a l a m u s and it would be interesting to know if we could replace the priming t r e a t m e n t , or enhance the quality of the L H surge by placing the implants in m o r e than one site, for example in the p r e o p t i c area and mediobasai hypothalamus. F u r t h e r m o r e , stimulation observed in our experiments could result from an indirect effect of oestradiol responsive neurons on o t h e r structures. A n a t o m i c a l links have been identified in rodents, b e t w e e n neurons in the mediobasal h y p o t h a l a m u s bearing oestradiol receptors and the preoptic area or the a m y g d a l a 1'13. Changes in L H secretion or sexual b e h a v i o u r could be partly due to such indirect pathways and involve for female sexual behaviour m o r e general changes in perception or e m o t i o n a l state as has been shown in male rats TM. In conclusion o u r experiments show that in sheep the m e d i o b a s a l h y p o t h a l a m u s is a p r i m a r y site of oestradiol action on both the L H surge and the different p a r a m e t e r s of female sexual behaviour. Links with other structures known to be involved in the occurrence of these p h e n o m e n a remain to be identified.

ABBREVIATIONS

i.m. intramuscular RI receptivity index GnRH gonadotrophs-releasing hormone PRIMING TREAT. cycle with priming treatment alone Ver. tgt vertical tangent Hor. tgt horizontal tangent Lv lateral ventricle IIIv third ventricle Ir infundibulary recess Mr mammillary recess Mi mass intermedia

VMH POA PH LH LHRH LHT MBH AH BIL UNI

ventromedian hypothalamus preoptic area posterior hypothalamus luteinizing hormone luteinizing hormone-releasing hormone lateral hypothalamus mediobasal hypothalamus anterior hypothalamus bilateral implantation unilateral implantation

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