Pulsatile GnRH-stimulated LH release from the human fetal pituitary in vitro: sex-associated differences.

An in-vitro perfusion system was utilized to examine LH release from human fetal (19-24 weeks gestation) anterior pituitaries during repetitive GnRH stimulations. Pituitaries (five male, four female) were dissected into halves, and one hemipituitary of each pair was stimulated with 10-min pulses of 1 nM GnRH administered at 60-min intervals over 24h, whereas the matching hemipituitary received pulses of medium alone. Basal (no GnRH stimulation) LH release from female hemipituitaries was significantly (P less than 0.01) greater than from male hemipituitaries, and the amplitude of LH release associated with GnRH pulses was sixfold greater (P less than 0.001) with female hemipituitaries. Furthermore, the magnitude of LH release associated with individual GnRH pulses was significantly (P less than 0.001) enhanced during the course of female hemipituitary perfusions, but not during perifusion of male hemipituitaries. These studies demonstrate that LH secretion by the female, but not male, mid-gestational human fetal pituitary is increased in response to a physiological pattern and interval of repeated pulsatile GnRH stimulation in vitro.

Pulsatile gonadotropin-releasing hormone release from the human mediobasal hypothalamus in vitro: opiate receptor-mediated suppression.

An in vitro perifusion system was used to investigate pulsatile gonadotropin-releasing hormone (GnRH) release from the fetal (20-23 weeks of gestation) and adult human mediobasal hypothalamus (MBH). Fetal human MBHs released GnRH in discrete pulses, with a periodicity of approximately 1 h. Adult human MBHs also released GnRH in a pulsatile manner, with a periodicity of 60-100 min. The calcium-dependent pulsatile GnRH release from fetal human MBHs was suppressed by addition of morphine (10 microM) to the perifusion medium, and this suppression was reversed by addition of the opiate receptor antagonist naloxone (10 microM). These results indicate that the human hypothalamic GnRH pulse-generating mechanism is located entirely within the MBH, and that this pulse generator can maintain intrinsically pulsatile GnRH release independent of all innervation from outside this site. Our data also demonstrate that human hypothalamic pulsatile GnRH release can be suppressed by an opiate receptor-mediated mechanism located within the MBH.

Pulsatile beta-endorphin release from the human pituitary in vitro.

An in vitro perifusion system was used to characterize spontaneous immunoreactive beta-endorphin (i beta-END) release from 10 human fetal (20-23 weeks gestation) and 2 human adult anterior pituitaries. Spontaneous i beta-END release from fetal anterior pituitaries was pulsatile, with a mean (+/- SE) pulse interval of 9.1 +/- 0.5 minutes, pulse amplitude of 120.8 +/- 46.1 pg with nadir to peak increment of 106.0 +/- 32.9%, and overall release rate of 209.7 +/- 65.0 pg/2 minutes. Blockade of calcium activity with 10 microM verapamil and 4 mM EGTA suppressed the frequency and amplitude of the spontaneous pulsatile i beta-endorphin release (n = 2). Administration of 2 nM human CRF for 20 minutes at the end of 2 perfusions induced 205 and 883% increases of i beta-END release over the preceding basal levels. Administration of 2 nM CRF for 50 minutes at the end of another perifusion led to a greater and prolonged increase (maximum 4620% relative to the immediately preceding basal level) in i beta-END release. Addition of 56 mM KCl during the last 20 minutes of this prolonged CRF stimulation further increased i beta-END release (to 7680% relative to the baseline preceding the CRF stimulation). Each of 4 quarters of adult anterior pituitaries (2 quarters each from 1 male and 1 female) also released i beta-END in a pulsatile fashion, with a pulse interval of 11.8 +/- 2.0 minutes, pulse amplitude of 7.4 +/- 0.8 ng with nadir to peak increment of 51.4 +/- 15.3%, and overall release rate of 21.7 +/- 2.9 ng/2 minutes. These studies demonstrate that i beta-END release from the isolated human anterior pituitary in vitro is characterized by high-frequency pulses, independent of hypothalamic stimulation. This spontaneous calcium-dependent pulsatile i beta-END release apparently reflects the activity of an intrapituitary pulse-generating mechanism.

Intrinsic pulsatility of luteinizing hormone release from the human pituitary in vitro.

An in vitro perifusion system was used to investigate the spontaneous luteinizing hormone (LH) release from 10 human fetal (21-23 weeks of gestation) and 1 adult female pituitaries. The pattern of LH release from fetal pituitaries (n = 6) exhibited a remarkable pulsatile character with a mean (+/- SE) pulse interval of 12.7 +/- 1.7 min. The mean pulse amplitude was 5.2 +/- 0.9 mIU with a nadir to peak increment of 69.5 +/- 6.4%. The mean LH release rate was 12.3 +/- 3.3 mIU/2 min. Blockade of calcium activity with 0.1 mM verapamil and 4 mM EGTA suppressed the frequency (from 1 pulse/12-20 min to 1 pulse/50-100 min) and amplitude (from 5.4-5.7 mIU to 1.4-2.1 mIU) of this spontaneous pulsatile LH release (n = 2). Administration of 8 nM gonadotropin-releasing hormone induced 255 and 954% increases in LH secretion (n = 2). Each quarter of an adult human pituitary also secreted LH in a pulsatile fashion, with a pulse interval of 15.2 +/- 5.6 min, a pulse amplitude of 5.4 +/- 0.6 mIU, a nadir to peak increment of 67.5 +/- 5.2%, and an overall release rate of 14.8 +/- 0.9 mIU/2 min. These studies demonstrate that LH release from the isolated human pituitary in vitro is characterized by high-frequency/low-amplitude pulses, independent of hypothalamic stimulation. Accordingly, this spontaneous calcium-mediated pulsatile LH release apparently reflects the activity of an intrinsic intrapituitary pulse-generating mechanism.

Intrinsic pulsatility of ACTH release from the human pituitary in vitro.

An in-vitro perifusion system was used to investigate spontaneous ACTH release from human fetal (21-23 weeks gestation) and adult pituitaries. The pattern of ACTH release from fetal pituitaries (n = 7) exhibited a remarkable pulsatile character with a mean (+/- SEM) pulse interval of 11.3 +/- 0.8 min. The mean pulse amplitude was 49.7 +/- 6.3 pg, with a nadir to peak increment of 90.7 +/- 10.4%. The mean ACTH release rate was 87.2 +/- 13.3 pg/2 min. Addition of the calcium chelator EGTA (4 nM) to the perifusion medium induced a significant (P less than 0.01) decrease in both ACTH release rate (from 102.0 +/- 8.5 to 52.0 +/- 9.9 pg/2 min) and ACTH pulse amplitude (from 57.7 +/- 2.8 to 31.3 +/- 4.6 pg) (n = 3). Administration of either 2 nM corticotrophin releasing factor (CRF) or 56 mM KCl induced 10- and 2-fold increases in ACTH secretion, respectively (n = 2). Quarters of adult human pituitaries (n = 6) also secreted ACTH in a pulsatile fashion, with a pulse interval of 14.8 +/- 1.7 min, pulse amplitude of 86.7 +/- 10.0 pg, nadir to peak increment of 84.5 +/- 9.8%, and overall release rate of 167.2 +/- 8.8 pg/2 min. These studies demonstrate that ACTH release from the isolated human pituitary in vitro is characterized by high frequency/low amplitude pulses, independent of hypothalamic stimulation. Accordingly, this spontaneous calcium-dependent pulsatile ACTH release apparently reflects the activity of an intrinsic intrapituitary pulse-generating mechanism.

Human fetal hypothalamic GnRH neurosecretion: dopaminergic regulation in vitro.

An in-vitro perifusion system was used to investigate GnRH release from fetal (21-23 weeks gestation) human hypothalami in response to dopamine (DA) and the DA receptor antagonist haloperidol. Administration of 1 mumol/l DA during five perifusions in which 1 mumol/l haloperidol was added to the medium failed to alter basal GnRH release. In contrast DA evoked a rapid and sustained 95.8 +/- 20.3% increase (P less than 0.01) in GnRH release during five matching perifusions with medium containing the alpha-adrenergic antagonist phentolamine. While exposure to 0.01 mumol/l DA failed to alter basal GnRH release during three perifusions, 0.1 mumol/l DA elicited a 145.7 +/- 65.2% increase (P less than 0.05) in GnRH release in three matching perifusions, indicating a dose-dependent effect. These studies demonstrate that DA can stimulate in-vitro release of GnRH from the mid-gestation fetal human hypothalamus by a DA receptor mediated mechanism.