Evidence for the involvement of non-androgenic testicular factors in the regulation of hypothalamic somatostatin and GHRH mRNA levels

Sequential blood samples were obtained from undisturbed freely-behaving male rats bearing chronic intracardiac venous cannulae. Blood was withdrawn every 15 min for periods of 4-24 h; plasma was separated, and saline-resuspended red cells were reinjected. Plasma GH was determined by radioimmunoassay. Pulsatile GH secretion was evident in each animal with most peak values greater than 200 ng/ml and most trough values less than ng/ml. The GH secretory episodes occurred at approximately 3 h intervals, and this rhythmic pattern of GH secretion persisted unchanged across all phases of a 12-h light-dark (L-D) cycle. Seven major episodes of GH secretion were observed during a single 24-h period. The mean period, or time interval between episodes, in 24 animals was 3.32 +/- 0.07 (SEM)h. The timing of the pulses with respect to the L-D cycle was similar in most animals, indicating that the rhythm may be entrained to the L-D cycle. The role of environmental lighting was further assessed in 14 animals exposed to constant light for 7 weeks. The results show that the basic rhythm was unchanged (mean period 3.18 +/- 0.06 h, peaks greater than 200 ng/ml, troughs less than 1 ng/ml), although entrainment to time of day was not evident. Subsequent exposure to the 12-h L-D cycle resulted in reversion to an entrained rhythm. These results suggest 1) that GH secretion in the rat is governed by an endogenous ultradian rhythm, with a periodicity of approximately 3.3 h, and 2) that the alternation of light and darkness probably serves as a Zeitgeber which sets the biological "clock" for GH secretion, but is not necessary for maintenance of the basic rhythm.

Ultrastructural changes in the interstitial cells of the adult rat testis were studied up to 45 days after administration of a single dose (100 mg/kg) of the antifertility compound ethylene dimethanesulfonate (EDS). Most Leydig cells showed degenerative changes 12 h after treatment. Twenty-four and 48 h after injection, all Leydig cells observed showed gross degenerative changes. At 4 and 14 days, intact Leydig cells could not be identified in the interstitial spaces. Twenty-one days after treatment with EDS, small Leydig cells were visible, and at 45 days, Leydig cells appeared normal. The seminiferous epithelium appeared morphologically normal until 4 days after injection of EDS, when slight abnormalities were observed. At 14 and 21 days, the seminiferous epithelium was grossly abnormal, but at 48 days, spermatogenesis appeared normal. Twelve, 24, and 48 h after treatment, large quantities of material, presumably from dead Leydig cells, were observed within the macrophage cytoplasm. The predominant cell in the interstitial space 4 and 14 days after EDS was the macrophage. Inclusions from the dead Leydig cells within the cytoplasm of the macrophages had almost disappeared. LH receptors (hCG binding) in testicular homogenates were consistent with the cytological changes in Leydig cells. Receptor concentration was low at 24 h and was almost zero at 4 days. This change was accompanied by a decrease in serum testosterone to castrate levels by 2 days. The responses of the endocrine system to destruction of the Leydig cell by EDS, as monitored by serum FSH, LH, and testosterone, were slower than those after castration, indicating that the response to EDS reflects the time required to kill the Leydig cell rather than direct impairment of the steroidogenic pathway. These experiments demonstrate that Leydig cells can be specifically destroyed by a cytotoxic drug. The availability of a specific cytotoxic agent for Leydig cells offers further opportunities to study the interrelationships between the Leydig cell and the seminiferous tubule.

LHRH and somatostatin or somatotropin-release inhibiting factor (SRIF) are produced by neurons whose cell bodies are located in telencephalic and diencephalic regions in the rat. Many, but not all, of these neurons project to the external zone of the median eminence (ME), where the peptides are released from the nerve terminals into hypophysial portal vessels. In the present study, we identified these neurons by in vivo injection of a retrograde tracer, the lectin wheat germ agglutinin (WGA), into the external zone of the ME. Subsequently, colchicine was given into the lateral ventricle 10-24 h after the WGA injection. The animals were killed 24-48 h after the WGA injection. Vibratome sections of the brains were stained for both WGA and LHRH or SRIF with a dual immunocytochemical technique. Approximately 70% of the LHRH neurons in the septum and the anterior hypothalamus and about 70% of the SRIF neurons in the medial preoptic area, the anterior periventricular area, and the paraventricular nucleus were double labeled, indicating that they projected to the ME. None of the SRIF neurons in the ventromedial and arcuate nuclei were labeled with WGA. Double labeled LHRH cells were either smooth and fusiform or spiny. WGA-accumulating LHRH or SRIF perikarya were intermixed with single labeled LHRH or SRIF cells, which apparently did not project to the ME. The results indicate that there are at least two populations of LHRH neurons in the preoptic-septal region and two populations of SRIF neurons in the medial preoptic and anterior periventricular areas and the paraventricular nucleus of the rat brain: one with access to the portal capillaries of the ME and, therefore, functionally related to the regulation of the pituitary, and another without access to portal capillaries, perhaps functionally related to intracerebral neurotransmission or modulation. Moreover, some hypophysiotropic LHRH and SRIF neurons may have axon collaterals reaching multiple targets within the central nervous system.