The push-pull perfusion technique was used in combination with a sequential bleeding schedule to estimate simultaneously the release patterns of LHRH and LH in unanesthetized ovariectomized sheep and to determine the temporal relationship between the release of these two hormones. Ovariectomized (greater than 30 days) ewes received unilateral push-pull cannula (PPC) implants (od, 0.85 mm) into the median eminence. After at least 6 days of recovery, each ewe was fitted with an indwelling jugular catheter. For push-pull perfusion, a stylette was removed from the outer PPC, and an inner cannula assembly (od, 0.40 mm) was inserted. Artificial cerebrospinal fluid was pushed through the inner cannula and pulled up between the cannulae at 20 microliters/min. Continuous 10-min perfusate fractions were collected, acidified, and stored at -20 C for LHRH RIA. Blood samples were obtained every 10 min via the jugular catheter, each being drawn 5 min after the start of a perfusate collection interval. Plasma LH levels were determined by RIA. The duration of the sampling was 3-7 h. LHRH output was distinctly pulsatile, occurring at a frequency of approximately one pulse every 30-40 min (n = 5 sheep). LHRH pulse amplitude and frequency remained relatively constant throughout each perfusion. Plasma LH values also were pulsatile, and all LH peaks occurred either during the same interval or during the interval after a LHRH pulse. LH pulses always were accompanied or directly preceded by LHRH pulses, but LHRH pulses were not always followed by LH pulses. The amplitudes of LH pulses and corresponding LHRH pulses were highly correlated (r = 0.81; P less than 0.01). Histological examination revealed that detection of LHRH in perfusates depended upon the placement of the PPC tip into either the zona externa of the central median eminence or adjacent areas. These experiments demonstrate that 1) hypothalamic LHRH release in the Ovx ewe occurs in discrete pulses, with a mean interpulse interval of 38.7 +/- 1.5 min, 2) LH pulses invariably are preceded or accompanied by LHRH pulses, and 3) LH pulse amplitude is highly correlated with LHRH pulse amplitude.

The roles of FSH and androgen in the postnatal development of Sertoli cell number and function have been investigated using mice that lack FSH (FSHbetaKO), FSH-receptors (FSHRKO), or androgen receptors (Tfm). At birth and d 5, Sertoli cell number was normal in FSHRKO and FSHbetaKO mice, but was significantly reduced on d 20 and in adulthood. In contrast, Sertoli cell number was reduced at birth in Tfm mice and remained significantly less than normal up to adulthood. Sertoli cell activity was determined through measurement of 11 different mRNA transcript levels. From birth to adulthood, the expression of most transcripts increased, with a significant rise occurring between d 5 and 10. In animals lacking FSH stimulation, mRNA expression (measured per Sertoli cell) was largely normal on d 5, but was reduced in seven transcripts on d 20 and in five transcripts at adulthood. In Tfm mice two transcripts showed reduced expression on d 5, and four were reduced on d 20, although expression in adult Tfm mice did not differ from that in normal cryptorchid controls. The results show that 1) testosterone, but not FSH, is required for Sertoli cell proliferation during fetal and early neonatal life; 2) FSH and testosterone both regulate the late stages of Sertoli cell proliferation; 3) FSH has a general trophic effect on Sertoli cell activity in the pubertal and adult mouse; and 4) androgens are required for specific transcript expression during prepubertal development. Specific effects of androgens were not seen in the adult, although these may be masked by the effects of cryptorchidism.

Adult Leydig cell steroidogenesis is dependent on LH but fetal Leydig cells can function independently of gonadotropin stimulation. To identify factors that may be involved in regulation of fetal Leydig cells expressed sequence tag libraries from fetal and adult testes were compared, and fetal-specific genes identified. The ACTH receptor [melanocortin type 2 receptor (Mc2r)] was identified within this fetal-specific group. Subsequent real-time PCR studies confirmed that Mc2r was expressed in the fetal testis at 100-fold higher levels than in the adult testis. Incubation of fetal or neonatal testes with ACTH in vitro stimulated testosterone production more than 10-fold, although ACTH had no effect on testes from animals aged 20 d or older. The steroidogenic response of fetal and neonatal testes to a maximally stimulating dose of human chorionic gonadotropin was similar to the response shown to ACTH. The ED(50) for ACTH, measured in isolated fetal and neonatal testicular cells, was 5 x 10(-10) M and the lowest dose of ACTH eliciting a response was 2 x 10(-11) M. Circulating ACTH levels in fetal mice were around 8 x 10(-11) M. Neither alpha-MSH nor gamma-MSH had any effect on androgen production in vitro at any age. Fetal testosterone levels were normal in mice that lack circulating ACTH (proopiomelanocortin-null) indicating that ACTH is not essential for fetal Leydig cell function. Results show that both LH and ACTH can regulate testicular steroidogenesis during fetal development in the mouse and suggest that fetal Leydig cells, but not adult Leydig cells, are sensitive to ACTH stimulation.

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