M. Aslam  ( Department of Physiology, Army Medical College, Rawalpindi. )
M.A. Hameed  ( Department of Physiology, Army Medical College, Rawalpindi. )
S. Nicholson  ( Upnited Medical Schools of Guy’s and St. Thomas’s Hospital, London (UK), )
M.T. Jones  ( Upnited Medical Schools of Guy’s and St. Thomas’s Hospital, London (UK), )

Anterior pituitary gland fragments removed from female rats at various days of the oestrous cycle were perifused into small Biogel columns. After 2 hours stabilisation, the tissues were exposed at the beginning of each of five hours of perifusion to a volley of six 1-mi pulses of LHRH (10M1 8, given 4 min.apart. Pituitary glands removed during oestrus or met-oestrus showed little change in the sensitivity to pulses of LHRH, LH release being similar in response to all volleys. Pituitaries removed during di-oestrus showed a priming response to the second volley of LHRH but this was followed by desensitisation there­after. In contrast, tissues removed at various times during pro-oestrus and exposed to LHRH pulses of different frequencies exhibited only progressive sensitisation. In conclusion, sensitivity of pituitary gland to LHRH varies throughout the oestrous cycle which plays an important role in the control of episodic secretion of LH (JPMA 38: 237, 1988).

Since the introduction of neuro-hormonal theory ¹, it is being increasingly evident that LHRH is secreted episodically in the hypothala­mo-hypophyseal portal system for delivery to the anterior pituitary gland². Furthermore, it is now established that rhythmicity of menstrual or oestrous cycle in mammals is the consequence of a reciprocal inter-relationship between hypotha­lamus, pituitary and gonods. ^3-4 Various workers have characterised the secretory pattern of LH in vivo throughout the oestrous cycle in the female rat. We have investigated the effects of giving volleys of pulses of LHRH in vitro to the perifused pituitaries removed from female rats on specific days of the cycle.

Experimental Animals
All animals used were 150-250 gram female Wistar-derived rats and were housed on 14h light: 10h dark schedule (lights on 0700-2 100h). They had access to food and water ad libitum. Only animals which had exhibited two consecutive 4-day oestrous cycle as shown by the daily vaginal smearing were selected for these experi­ments.
Perifusion of Pituitary Tissue
The method for perifusion of anterior pituitary gland fragments (1mm3) in a small Biogel column was as described previously. ⁵ Pituitaries were removed from rats during oestrus, met­oestrus, di-oestrus and pro-oestrus. The tissues were packed into the columns and perifused at a rate of 0.5m1/min. With Kreb’s bicarbonate medium at 37C° containing 11mM glucose and gassed with 95% 02: 5% CO2. After 2 hours equii,bration period, the tissues were exposed at the beginning of each of 5 hours of perifusion to a volley of six 1min. pulses of lOM-8 synthetic LHRH (Hoechst, Hounslow, Middx, UK), pulses being 4 min. apart. Pituitary perifusates were collected in 2 min, (lml) fractions and analysed for rat LH (rLH) by radioimmunoassay (RIA)
Radioimmunoassay of Rat LH
The rLH from pituitary perifusate was measured⁶ by a double antibody RIA kit supplied by the National Hormone and Pituitary Pro­gramme (University of Maryland School of Medi­cine, USA) NIADDK. The concentration of rLH was expressed in sensitivity terms of NIADDK rLH-RP-2 as the reference preparation. The sensitivity of this assay was 0.39mg/mI. The intra-assay variation was found to be 6.6% at a level of 0.78ng/ml and 8.7% at a level of 50 ng/ml, and the inter-assay variation at same levels was found to be 8.0% and 123% respectively, where % variation is sem/mean for 5 estimations.

Pattern of tissues removed during Oestrus and Met-Oestrus
Pituitaries removed during oestrus or met-oestrus showed no specitic pattern of LH rlease in response to the repeated volleys of LHRH. Each volley caused a similar fold increase in LH secretion (4.5 fold increase for oestrous tissue, 5 fold increase for met oestrus) above the corresponding basals(Figure 1 a & b).


Di-Oestrus and Pro-Oestrus
For pituitaries removed during di-oestrus, release of LH in response to the second volley of LHRH was significantly greater (P<0.005 one way ANOVA) than to the first, that is, sensitisa­tion occurred. Thereafter, the responses gradually declined (difference between responses to second and third volleys, P < 0.01), such that less LH was released in response to the fifth volley than to the first (desensitisation occurred
In contrast, the pateern of response of tissues removed either in the beginning (l000h), middle (1400h) or end (1700h) of the pro-oestrus (Figure 2 a)

showed no evidence of desensitisation, amount of LH secretion increased in response to successive volleys to LHRH (sustained priming effect of the releasing hormone). This change in LH release with time in each case was significant (P<0.01, one way ANOVA). This was a specific response to LHRH as hourly stimulations of pro-oestrous pituitaries with volleys of 48mM Potassium (K+) ions according to exactly the same protocol produced no evidence of changing responsiveness (Table).


Effect of different modes of LHRH Pulses on Pro­Oestrus
LHRH pulses were given according to three different protocols to tissues removed during pro­oestrus. Firstly, the protocol used in the above experiments was that of volleys of six Imin. pulses of LHRH, given 4min. apart of the beginning of each hour of perifusion. The.. response to this regimen of LHRH pulses has already been shown in Secondly, to ascertain whether the tissue became prefractory. to LHRH, short pulses (1mm) were given at different (20mm) intervals over a long time-course, and thirdly, much longer (5min) pulses with longer rest periods in between (35min) were given. LH release in response to these single LHRH pulses (as opposed to volleys) is shown in Figure 3.


There was no evidence of desensitisation to 1mIN. pulses with 20 mIN. interval in between, given over 6 hours (18 pulses), LH release in res­ponse to each pulse being slightly greater than that to the preceding one. The response to 5mIN. pulses of LHRH separated by 35min. rest periods was also similar showing a gradual rise in LH release.
Expression of data and statistical analysis
As each column contained tissues from 6 different animals and the values obtained from experiment to experiment were consistent, obser­vations for a given set of conditions were repeated 3-5 times (n=5), equivalent to the response of tissues obtained from 18-30 animals. Values and bars represent mean ± sem but in some figures, sems less than 10% of the mean have been omitted for clarity.
The data from these experiments were expressed in terms of total LH released during each hour after the onset of a volley of LHRH pulses (that is, release per 30 fractions). Temporal differences across several groups were analysed by one-way analysis of variance (ANOVA). Dif­ferences were considered significant if P values from the test were less than 5%.

This pituitary characteristics of changing LHRH responsiveness and LHRH self-priming are components of a system which produces the cyclicity of LH release during the oestrous cycle and the pro-oestrous LH surge. In these experi­ments, pituitanes removed in the beginning, middle or at the end of pro-oestrous phase showed a sustained priming effect to the volleys of LHRH 300 over 5 hours of perifusion.
These results are in contrast with those of Waring and Turgeon⁷ who showed that the 200priming effect of LHRH (2xl0min. pulses with a 2 hour interval in between) in vitro was significantly greater in superfused pituitary quarters removed on the afternoon of pro-oestrus before the onset of LII surge than of those taken out in the morn­ ing of pro-oestrus. Whereas others⁸ have reported priming effects of LHRH when anterior pituitary 0f ragments taken from pro-oestrous rats were challenged with pulses of LHRH (2x30min. pulses with on interpulse interval of 2 hours) in vitro. However, they found gradual desensiti­sation of pituitary dispersed cells prepared from pro-oestrous rats on continuous exposure to the releasing hormone for 5 hours.
To ascertain whether the tissues became refractory, LHRH pulses were given to pituitary fragments removed from pro-oestrous rats accord­ing to the protocol described by various wor ­kers^9-10 over a long time-course. There was no evidence of desensitisation with both the frequen­cies of LHRH in vitro on pro-oestrus. The fact that very similar amounts of LH were recorded over the 40 mIN. following a 5mIN. pulse as during the 20mIN. after a 1mIN. pulse suggests that the tissue became refractory to the effect of this concentra­tion of LHRH within 5mIN.
Since pro-oestrus in rat is not a long phase¹¹ (12h), it was thought that LHRH pulses given particularly with long intervals may not truly reflect the pituitary responsiveness peculiar of this phase of the cycle. However, pituitaries removed during oestrus, the phase which follows pro-oestrus, showed no sensitisation to the releas­ing hormone. Pituitaries taken even during met­oestrus exhibited little change in the responsive­ness, not even priming, to repeated LHRH stimula­tions in vitro. In another study, ¹²  a lesser concen­tration of LHRH-receptors in the pituitary gland on oestrous than that on pro-oestrus was found in the rat. In the present study, the inability of LHRH to prime the pituitaries in vitro on the day of oestrous and met-oestrus may be related to the prior LH release during pro-oestrous with partial depletion of glandular content or to the long interval since the occurrence of high oestro­gen titres.¹³
Prolonged sensitisation was a specific response to LHRH as hourly stimulation of spro­oestrous tissue with volleys of pulses of 48mMK+ ions to exactly the same protocol produced no evidence of changing sensitivity, the magnitude of LH release being similar to all volleys of K+ ions. The LU release induced by the raised C ions in the perifusion medium is presumably the result of depolarisation of the plasma membrance of the gonadotrophs which causes an elevation of cyto­solic calcium leading to the release of readily releasable pool of the hormone. ¹⁴
The exact mechanism of sustained priming to LHRH is unknown. However, several studies suggest that LHRH may induce the formation of additional specific high affinity plasma membrance receptors on the gonadotrophs’14 or this may increase the synthesis of releasable pool of LH. ¹⁵ Recently, protein kinase C & phosphatidylinositol have reported to be involved in the priming effects of LHRH. ¹⁶
In this study, tissue removed on di-oestrous showed the well-known self-priming effect in response to the second volley of LHRH followed by subsequent desensitisation. A similar study’¹⁷  carried out using static incubation of hemipitui­taries from di-oestrous rats showed a rise in LH release in response to the second hour of exposure to U-IRH in vitro, but thereafter the LH release became smaller. It has been suggested that down-regulation by LHRH of the number of its recep­tors or receptor-occupancy are the basis of this phenomenon of desensitisation.¹⁸
The data discussed here, show that sensiti­sation followed by subsequent desensitisation of anterior pituitary tissue in response to repeated pulses of LHRH in vitro is a characteristic of di­oestrous. However, on pro-oestrous, when, the pre-ovulatory LH surge takes place, there occurs only progressive sensitisation, specific to LHRH. This suggests that there is a basic difference in the response of anterior pituitary gland to LHRH on various days of the oestrous cycle and this presumably plays a role in maintaining the LU surge.

1. Harris, G.W. The induction of avulation in the rabbit by electrical stimulation of the hypothalamo­hypophysial mechanism. Proc. Roy. Soc., 1973; 122: 374.
2. Sarkar, D.K. and Fink, G. Luteinising hormone releasing factor in pituitary stalk plasma from long-term ovariectomised rats; effects of steroids. J.EndocrinoL, 1980;86 :511.
3. Butcher, R.L., Collins, WE. and Fugo, N.W. Plasma concentration of LH, FSH, prolactin, progesterone and stradiol-1 7 throughout the 4-days estrous cycle of the rat., Endocrinology, 1974;94: 1704.
4. Gallo, R.V. Pulsatile L H release during periods of low level LH secretion in the rat estrous cycle. Biol. Reprod., 1981;24: 771.
S. Aslam, M., Hameed, M.A., Nicholson, SA. and Jones, M.T. Perifusion of pituitary gland a methodologic analysis, (Submitted for publication).
6. Monroe, S.E., Parlow, A.F. and Midgley, A.R. Radioimmunoassay for rat luteinizing hormone. Endocrinology, 1968;83: 1004.
7. Warling,D.W. and Turgeon, J. Luteinising hormone  releasing hormone induced luteinising hormone secretion in vitro; cycle changes in responsiveness and self-priming.. Endocrinology, 1980; 106: 1403.
8. Evans, W.S., Uskaviteh, D.R., Kaiser, D.L., Hell mann, P., Borges, J.L.C. and Thorner, M.O. The self-priming effect of gonodotrophin releasing hormone on luteinising hormone release; observa­tions using rat anterior pituitary fragments and dispersed cells continuously perifused in parallel. Endocrinology, 1984; 114 : 861.
9. Buckingham, J .C. and Cover, P.O. Changes in the responsiveness of perifused rat adenohypophysial cells to repeated stimulation with luteinizing hormone-releasing hormone. A cta Endocrinol., 1986;\\ 113: 479.
10. Edwardson, JA. and Gilbert, D. Application of an in-vitro perifusion technique to studies of luteinizing hormone release by rat anterior hemi­pituitaries; self-potentiation by luteinizing hor­mone releasing hormone. J. Endocrinol., 1976; 68:197.
11. Fox, R.R. and Laird, C.W. Sexual cycles, in repro­duction and breeding techniques for laboratory animals. Edited by Hasez. Philadeiphin, Lea & Febigar, 1970, p. 107.
12. Clayton, R.N. and Catt, K.J. Gonadotrophin­releasing hormone receptors; characterization, physiological regulation, and relationship to repro­ductive function. Endocr. Rev., 1981; 2 : 186.
13. Schwartz, N.B. A model for the regulation of ovulation in the rat. Recent Progr. Horm Res 1969; 25:1.
14. Mitchell, R.H., Jaffeiji, S.S. and Jones, ML. The possible involvement of phosphatidylinositol breakdown is the mechanism of stimulus response coupling at receptors which controls cell-surface calcium gates, in functions and biosynthesis of lipids. Edited by Bazen et aE. New York., Plenum Press, l977,p.447.
15. Smith, P.S. Release of luteinizing hormone and follicle stimulating hormone and high potassium concentration. Endocrinology, 1985; 116: 1826.
16. Noor, Z. Integration of phosphoinositide tutnover, calcium mobilisation and protein kinase C activation in GnRH action. J. Endocrinol., 1986; III (Suppl) Abs.4.
17. Conn, P.M., Hsueh, AJ. and Crowley, W.F. Jr. Gonadotrophin-releasing hormone; molecular and cell biology, physiology, and clinical applications. Fed. Proc., 1984;43 :2351.
18. Witcher, J.A., Nearhoof, K.F. and Freeman, M.E. Secretion of luteinizing hormone (LH) and pituitary receptors for LH — releasing hormone as modified by the proestrous surge of progesterone. Endocrinology, 1984; 115: 2189.