Biochimica et Biophysica Acta, 1097 (1991) 101-109 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 0167488991001285

101

BBAMCR 12878

Effect of luteinizing hormone releasing hormone pulse characteristics on comparative luteinizing hormone and follicle stimulating hormone secretion from superfused rat anterior pituitary cell cultures T h e r e s a A. K e l l o m a n d J a m e s L. O ' C o n n e r Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, GA (U.S.A.) (Received 20 August 1990)

Key words: LHRH; Amplitude; Duration; LH; FSH; Cell culture; Pituitary

We have shown that 4 ng luteinizing hormone releasing hormone (LHRED pulses induced significantly greater luteinizing hormone (LH) release from proestrous rat supeffused anterior pituitary cells with no cycle related differences in follicle stimulating hormone (FSH). Current studies gave 8 ng LHRH in various pulse regimens to study amplitude, duration and frequency effects on LH and FSH secretion from estrous 0800, proestrous 1500 and proestrous 1900 cells. Regimen I I~ave $ ng LHRH as a single bolus once/h; regimen 2 divided the 8 ng into 3 equal 'minipulses' given at 4 min intervals to extend duration; regimen 3 gave the 3 'minipulses' at 10 rain intervals, thereby further extending duration; regimen 4 was the same as regimen 2, except that the 3 'minipulses' were given at a pulse frequency of 2 h rather than I h. In experiment 1, all four regimens were employed at proestrus 1900. FSH was significantly elevated by all $ ng regimens as c ~¢ared to 4 ng pulses; further, 8 ng divided into 3 equal 'minipulses' separated by 4 min at I and 3 h frequencies (regih~ens 2 and 4) resulted in FSH secretion that was significantly greater than with either a single g ng bolus (regimen 1) or when the 'minipulses' were separated by 10 rain (regimen 3). In experiment 2, at proestrus 1500, FSH response to the second pulse of regimen 4 was significantly greater than in regimen 2; LH release was significantly suppressed at pulse 2 compared to regimen 2 accentuating divergent FSH secretion. At estrus 0800, FSH response to the second pulse of regimen 4 was significantly greater than in regimen 2 without suppression of LH. Therefore, even though the second pulse of regimen 4 significantly stimulated FSH at proestrus |900, 1500 and estrus 0800, FSH divergence was most marked at proestrus 1500. These data indicate a potential role for hypothalamic LHRH secretory pattern in inducing divergent gonadotropin secretion in the rat. Introduction

The release of both LH and FSH by LHRH has been demonstrated in vivo [1-4] as well as in vitro [5,6]. Numerous studies have demonstrated that pulsatile LHRH delivery is essential for maintenance of pituitary sensitivity to LHRH [7-9]. While there is general acceptance that LHRH releases LH, there is debate over the role of LHRH as the predominant factor in releasing FSH. A separate hypothalamic FSH-releasing factor has been postulated [10]; however, detailed characterization of such a factor has yet to be reported. Considerable divergence in secretion of LH and FSH occurs with various physiological states such as during

Correspondence: J.L O'Conner, Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, GA 30912, U.S.A.

early estrus [3-5,11], after ovariectomy [12-14], during puberty [15,16] and during lactation in the rat [6,17]. It has been proposed that LHRH pulsatility and anterior pituitary sensitivity to pulsatile LHRH change through the estrous cycle [1-4,18]. Since variations in LHRH pulse amplitude and frequency have been demonstrated in vivo to partially modulate the relative amounts of LH and FSH released in ovariectornized ewes [18] and Rhesus monkeys [19], it has been hypothesized that divergence may in part result from changing LHRH pulse patterns emanating from the hypothalamus. Because previous studies which have exposed superfused lamb [20] and rat [21,22] pituitary cells to LHRH pulses of varying amplitudes and durations have reported LH secretion only, there have been no reports directly testing this hypothesis in an in vitro system. The current studies utilized superfused anterior pituitary cells derived from rats at three time points during the ovulatory

102 cycle to determine if defined LHRH pulse regimens can induce divergent LH and FSH release and if this effect is cycle related. Materials and M e t h o d s

Animals. Anterior pituitaries were recovered from female Spraguc-Dawley rats (Harlan, Inc.) determined to be in specific stages of the estrous cycle by daily examination of vaginal cytology. Anterior pituitaries were collected at proestrus 1500, 1900 and estrus 0800. Only those animals displaying two consecutive 4-day cycles were used. The animals were maintained on a 14:10 (light :dark) lighting schedule. The protocol was reviewed by the Institutional Committee on Animal Use. Cell dispersal. The cell dispersal procedure has been described previously [23], Briefly, cells were cultured in complete growth medium (CGM) which was composed of Dulbecco's modified Eagle's medium (DMEM, Gibco No. 430-3000EA) without Phenol red and was supplemented with 5~ horse serum (Sigma H7889) and 5~ fetal bovine serum (Sigma F3010). Phenol red was eliminated from the present studies because our laboratory [24] and others have demonstrated the potential estrogenic activity of such preparations. Sera were treated with dextran-coated charcoal [25]. Trypsin (Gibco No. 610-5090) action was terminated by addition of 1 ml CGM plus 0.1~ soybean trypsin inhibitor/pituitary (Sigma No. T-9128). Cells were centrifuged at 500 × g for 10 rain, resuspended in CGM, mixed with Cytodex Ill beads (Sigma C3275) and cultured for 48 h prior to being placed in columns [26]; previous studies in this laboratory have demonstrated that cycle related responsiveness is maintained up to 72 h in culture and is maximum at 48 h [27].

Superfusion system. Five anterior pituitary glands were used per column and six columns were run per cycle stage. We have described the physical configuration of this system previously [26,27]. t~ead attached cells were loaded into columns and superfused with DMEM; serum was eliminated from this medium due to the potential for degradation of L H R H [28]. A flow rate of 0.125 m l / m i n was used and 1 ml fractions were collected. L H R H regimens. Previous studies [24] in this laboratory have demonstrated that hourly 4 ng L H R H pulses resulted in maximum in vitro LH release at proestrus 1900, whereas cycle related differences in FSH release were not seen. In order to learn more about the role of pulse amplitude, duration arid frequency in FSH secretion, four regimens (Table I) were designed incorporating key characteristics of in vivo LHRH pulses [29-33]; regimen 1 delivered 8 ng LHRH as a single 100 #1 bolus once/h; regimen 2 divided the 8 ng dose of regimen 1 into 3 equal 'minipulse' doses administered at 4 min intervals, thereby maintaining the 8 ng mass of regimen 1 while extending the duration of exposure; regimen 3 administered the 3 LHRH 'minipulses' at 10 min intervals, thereby extending the duration of exposure beyond that of regimen 2; regimen 4 was the same as regimen 2 except that the 'minipulses' were administered at a pulse frequency of 2 h rather than 1 h. All LHRH pulses were delivered in 0.01 M phosphate buffered saline +0.1~ gelatin [PBS+ gel]. Elution profiles for regimens 1, 2 and 3 are provided in Figs. 1A, 1B and 1C, respectively; the elution profile for regimen 4 is the same as regimen 2. Evaluation of the elution profiles of regimens 3 and 4 indicated that delivery of LHRH in such manner resulted in a sustained broad peak and an increased duration of exposure. Recovery of LHRH in all regimens was 91-95~.

TABLE I

LHRH pulse regimens employed to study the effects of LHRH pulse characteristics on comparative LH and FSH secretion from superfused anterior pituitary cel/s

All LHRH pulses were delivered in 100 1~1 volumes of 0.01 M phosphate-buffered saline+0.1~ gelatin. All regimens contained a 100 ng pharmacological LHRH bolus at the beginning of the final h in the column; the data from this bolus were not included with the statistical evaluation of the other pulsatile responses. Regimen

ng LHRH injectedinto superfusioncolumn in 100 ttl 2 3

hour: 1

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6 0

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0

8

8

2

0

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100

0

0

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0

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100

0

0

4

0

2.7-2.7-2.7 4 rain apart

0

2.7-2.7-2.7 4 rain apart

0

100

103

Description of experiments performed Experiment 1: LHRH pulses of increased amplitude or

proestrus 1500 and estrus 0800; no further studies were pursued with regimens 1 and 3. Proestrus 1500 was chosen because the primed anterior pituitary has not yet begun maximum FSH secretion at this time [34]; estrus 0800 was chosen because 3H serum levels are well recognized to be maximally divergent at this time in vivo [351. Analysis of data. LH and FSH values obtained from individual columns were divided by D N A values obtained from the same columns. Elution profiles expressed as ng LH or F S H / n g D N A were plotted for each column and episodes of pu!satile LH or FSH release on each individual profile were identified by the PC Pulsar computer prog,'am [36,37]. The criteria use to identify pulses were chosen such that, with a one point pulse, the LH or FSH concentration was required to be 3.8 standard deviations (S.D.) greater than the baseline LH or FSH concentration (G1 = 3.8); the criteria for 2, 3, 4 and 5 point pulses were G2 = 2.6, G3 = 1.9, G4 =

duration and reduced frequency at proestrus 1900. Experiment 1 applied 8 ng LHRH (as regimens 1, 2, 3 and 4) to superfused anterior pituitary cells derived from proestrous 1900 rats; this is a stage at which LH responsiveness was greatest with 4 ng pulses of previous studies [24]. Statistical comparisons were then drawn between responsiveness to the 4 ng pulses of previous studies and the 8 ng doses of the present studies. Based on the results from these data, additional studies were conducted with regimens 2 and 4 at proestrus 1500 and estrus 0800.

Experiment 2: LHRH pulses of increased amplitude or duration and reduced frequency at proestrus 1500 and estrus 0800. Because the studies of experiment 1 indicated that regimens 2 and 4 consistently induced the highest release of FSH from proestrc'as 1900 cells, studies with these two regimens were extended to include

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Fig. 1. FSH responses (ng F S H / n g DNA) at proestrus 1900 to LHRH pulse regimens 1-4 utilizing superfused anterior pituitary cells derived from five 60-70-day-old rats. The graphed data represent the mean (n) plus the S.E. ( + ) of six individual superfusion columns; the mean minus the S.E. is not presented in these figures in order to preserve visual clarity. Average area (mm 2 :t:S.E.) is indicated under the elution profiles. The cross-hatched peaks indicate elution profiles for LHRH, the profile of regimen 4 (panel D) is the same as regimen 2. In this and all subsequent figures, each fraction is equal to 7.5 min; therefore, to convert fraction number to time, multiply by 7.5. v, pulsatile response as detected by the PC Pulsar computer program [36,37].

104 Results

1.5 and G5 = 1.13, respectively. These criteria identified pulses which were also recognized upon visual inspection of plotted data. In order to statistically evaluate the data, areas beneath pulsatile responses were measured (in mm2) with a Jandel digitizer and software (Corte Maderao CA). Significant differences in pulsatile response to different regimens were identified with ANOVA and Duncan's Multi Range test. DNA assay and RIA. With the completion of LHRH regimens, cells were removed from microcarrier beads with col!agenase (Sigma CO130); the cells were then frozen, homogenized and centrifuged. The resulting supernatant and pellet were assayed for total DNA using a modification of the Hoechst 33258 (Sigma B2883) assay of Labarca and Paigen [38] as previously described [39]. Both LH and FSH in collected fractions were estimated by RIA utilizing reagents obtained from the National Pituitary Hormone Distribution Program [23,26]; assay data were expressed in terms of RP-1 gonadotropin standards.

Experiment 1: Proestrous 1900 responsiveness to LHRH pulses of increased amplitude (regimen 1), increased duration (regimens 2 and 3) and reduced frequency (regimen 4) In previous studies [24] with 4 ng LHRH at proestrus 1900, FSH secretion at pulse 1 and 2 had been 80 ( + 13) and 89 (_+16), respectively (data not shown). When compared to these 4 ng responses, all regimens employing 8 ng LHRH (regimens 1-4) stimulated significantly greater FSH release at pulse 1 (Fig. 1A and C; P < 0.05; Fig. 1B and D; P < 0.01); at pulse 2, with the exception of regimen 3, all 8 ng regimens resulted in significantly greater I:SH release in comparison to 4 ng pulses (Fig. 1B and D; P < 0.01; Fig. 1A, P < 0.05). In previous studies with 4 ng LHRH at proestrus 1900, LH secretion at pulse 1 and 2 had been 908 (+49) and 851 ( + 115), respectively (data not shown). In comparison, only the first pulse of regimens 3 and 4 resulted in significantly greater LH release (Fig. 2C and D; P