Growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) are members of the PACAP/Glucagon superfamily. The family is proposed to have developed from an ancestral PACAP-like molecule in invertebrates. Through successive exon, gene and genome duplications the family has grown to include seven other members. In mammals GRF and PACAP are located on different genes, but in fish, amphibians and birds they are located on the same gene. The main function of GRF is the release of growth hormone (GH) from the pituitary. Also, during development GRF influences the fetal pituitary and stimulates GH release during late gestation. In contrast, the functions of PACAP are extremely varied. PACAP is the newest member of the superfamily and there is still much work to be done before its actions are well understood. Like GRF, PACAP is a releasing hormone acting on the pituitary and in addition, the adrenal gland, pancreas and heart, as well as other organs. Also, PACAP regulates smooth muscle in the vascular system, gut, respiratory tract and reproductive tract During development PACAP affects proliferation, differentiation and apoptosis. GRF and PACAP are expressed throughout development in fish, beginning during the blastula period in rainbow trout and at the end of gastrulation in zebrafish (earliest stage examined). In rainbow trout the grf/pacap gene is expressed as two transcripts, a short and a long transcript. The short transcript is produced by alternative splicing of the gene and does not include the fourth exon which codes for GRF. The long transcript includes the coding regions for both GRF and PACAP. By this means PACAP can be regulated separately from GRF. With the extensive role PACAP appears to play in development, separate regulation of the hormone may be necessary. Expression of the grf/pacap gene in zebrafish is widespread early in development and gradually becomes localized. Of particular interest is the expression of the grf/pacap transcript in regions associated with the prechordal plate, an important organizing center in development. Although it is not yet confirmed, there is evidence to suggest GRF and PACAP are expressed in the prechordal plate and its derivatives in the gut and hatching gland. In addition, expression of the grf/pacap transcript is observed in the neuroectoderm (eye, brain and spinal cord) and the developing heart. Considering the expression pattern of GRF and PACAP, I propose that one of both of these hormones may be involved in patterning during vertebrate embryogenesis. The evolution of gene families is thought to occur through successive exon, gene and genome duplications. Duplicate exons or genes become differentiated and eventually gain new functions or become functionless. During evolution of the grf/pacap lineage, several duplication events have occurred. Analysis of rainbow trout leads me to think that this fish and other salmonids possess two copies of the grf/pacap gene. This is not unexpected considering the tetraploid nature of salmonids. Present day mammals encode GRF and PACAP on separate genes. At some point during the evolution of this lineage a duplication event has occurred, possibly in early mammals or prior to the divergence of birds. The study of multigene families is a useful way to understand evolutionary processes. To this end I examined three members of multigene families from sockeye salmon. Therefore, in addition to the evolutionary mechanisms and pathways that directed grf/pacap gene evolution, I examined the ferritin-H subunit, the alpha-tubulin subunit and the beta-globin subunit. These cDNA sequences are similar to their counterparts in other teleost. The evolution of the ferritin gene family is particularly interesting because it involves the addition or deletion of DNA sequences that affect regulation and cytosolic location. / Graduate

Call number: LD2668 .T4 1984 S585 / Master of Science

A series of experiments were conducted to investigate the regulatory mechanisms governing the absence of and the initiation of pulsatile pituitary LH secretion during the infantile and prepubertal periods of development in the bull calf. In the first experiment, patterns of hypothalamic GnRH secretion into hypophyseal portal vessels were measured in sixteen Holstein bull calves at 2, 5, 8 and 12 weeks of age. The results of this study correlated the attainment of an hourly rate of pulsatile GnRH release to the onset of prepubertal LH secretion. In the second experiment, nine Holstein bull calves were infused with exogenous GnRH (200 ng) on an hourly basis from 1 to 6 weeks of age. From this experiment, we were able to demonstrate that an hourly rate of pulsatile GnRH release stimulates the age-associated changes in the hypothalamic-pituitary axis necessary for initiating pituitary LH secretion during the transition from the infantile to prepubertal period of development.

Pineal-mediated depressions in prolactin cell activity after light deprivation were studied in the male and female Golden Syrian hamster. Prolactin cell activity was determined by measuring radioimmunoassayable prolactin, newly synthesized prolactin and prolactin mRNA levels in the pituitary. Serum prolactin was also measured by radioimmunoassay. Use of the recombinant DNA plasmid, pPRL-1, which contains the rat prolactin complimentary DNA sequence, was validated in this dissertation for measuring prolactin mRNA in the hamster. Male hamsters blinded for 11, 21, or 42 days showed significant and progressively greater declines in prolactin mRNA levels which were completely prevented by pinealectomy. The decline seen after 11 days is the earliest depression in prolactin cell activity reported after light deprivation in the hamster. Female hamsters blinded for 28 days, however, showed no such decreases in prolactin cell activity if they continued to display estrous cyclicity. This supports the hypothesis that, unlike the male, there is not a gradual decline in prolactin cell activity after blinding in the female hamster and that loss of estrous cyclicity may precede or possibly accompany declines in prolactin cell activity. After 12 weeks of blinding, females were acyclic and had dramatically depressed levels of prolactin cell activity. However, pinealectomy did not completely prevent this decline due to blinding unless the females continue to display estrous cyclicity. Thus, when pinealectomy was ineffective in preventing the loss of estrous cyclicity due to blinding, it was also ineffective in preventing declines in prolactin cell activity. In ovariectomized females, blinding caused a decline in prolactin cell activity. Pinealectomy was not consistently effective in preventing this decline after 12 weeks of treatment, although, in females blinded for 4 weeks (at which time all animals were cycling) and then ovariectomized for an additional 4 weeks, pinealectomy completely prevented this decline in prolactin cell activity. In a separate study, significant changes in prolactin cell activity during the estrous cycle were seen in untreated normally cycling female hamsters. These changes in prolactin mRNA, prolactin synthesis, and radioimmunoassayable prolactin in the pituitary were measured in the morning, when, consistent with other reports, no differences in serum prolactin were observed.

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

The ovarian steroids, progesterone and estradiol, regulate luteinizing hormone synthesis and secretion during the estrous cycle of mature ewes. During the luteal phase of the cycle the ovarian steroids inhibit luteinizing hormone secretion. Luteinizing hormone is secreted from the pituitary when stimulated by the hypothalamic neuropeptide, gonadotropin-releasing hormone. Ovarian steroids can inhibit luteinizing hormone secretion indirectly, by decreasing the secretion of gonadotropin-releasing hormone or directly, by modulating the response of the pituitary to gonadotropin-releasing hormone. These studies have examined the direct control of pituitary luteinizing hormone secretion by using an in vivo model in which endogenous gonadotropin-releasing hormone (GnRH) release has been ablated and replaced with exogenous GnRH release at a constant frequency. Progesterone directly inhibited pituitary LH secretion in an estradiol-dependent manner and this may not require inhibition of pituitary LH synthesis. Progesterone inhibition of pituitary luteinizing hormone secretion is associated with enhanced progesterone binding by the pituitary.

To test the hypothesis that the hypothalamic tuberoinfundibular dopaminergic neuronal system plays a role in the dynamic regulation of pituitary prolactin secretion, its activity was correlated with experimentally-induced prolactin secretory episodes in the male rat. Direct estimates of tuberoinfundibular neuronal activity were made by measuring its rates of dopamine and norepinephrine synthesis or release. Prolactin secretion was assessed in vivo by measuring radioimmunoassayable prolactin levels in peripheral blood and the pituitary and in vitro by measuring prolactin concentrations released into incubation media. The anesthetic urethane and a substance isolated from the pineal gland were both demonstrated to inhibit prolactin secretion. Significant elevations of newly synthesized tuberoinfundibular dopamine were observed concomitant with this decreased prolactin secretion suggesting that acute increases in tuberoinfundibular dopaminergic neuronal activity were perhaps causally related to acute decreases in prolactin secretion since these substances were without a direct effect on the pituitary in vitro. Conversely, acute decreases in tuberoinfundibular neuronal activity induced by dopamine biosynthesis inhibition or mimicked by pituitary receptor blockade induced acute increases in prolactin secretion. As another prerequisite for its involvement in the dynamic regulation of prolactin secretion, the tuberoinfundibular neuronal system was demonstrated to be involved in the negative feedback control of prolactin over its own secretion. Elevated circulating prolactin levels produced by pituitary homografts transplanted beneath the kidney capsule accelerated tuberoinfundibular dopaminergic neuronal activity. In two unrelated experimental conditions, rats rendered blind and anosmic or hyperprolactinemic, the chronic inhibition of prolactin secretion was not associated with the maintenance of an increased tuberoinfundibular neuronal activity, but rather with a supersensitivity of the anterior pituitary to the prolactin-release-inhibitory action of dopamine. Long-lasting alterations in tuberoinfundibular dopaminergic neuronal activity appeared to induce this pituitary supersensitivity to dopamine. The tuberoinfundibular neuronal system appears to have the capacity to modulate prolactin secretory episodes via the alteration of its dopaminergic activity. Long-lasting alterations in this activity may induce changes in anterior pituitary sensitivity to dopamine essential for the chronic inhibition of pituitary prolactin secretion.

Call number: LD2668 .T4 1950 S666 / Master of Science