Thomas J. Collins

Characterization of a potent biotin-conjugated CRF analog and the response of anterior pituitary corticotropes

A biotin-conjugated synthetic corticotropin releasing factor (B-CRF) was prepared and characterized. Its biological activity and binding affinity were compared with that of unlabeled synthetic CRF. Both forms of the releasing factor were equipotent in in vitro studies measuring the release of corticotropin (ACTH) (ED50 = 1 nM). The IC50 in the binding assays was 1.5 nM for CRF and 4 nM for B-CRF. Dual avidin-biotin peroxidase complex stains were then used in pituitary monolayer cultures to visualize receptivity to the releasing factor and to confirm opiocortin storage in the target cells. All corticotropes showed stain for B-CRF. The percentage of cells that were double-labeled for ACTH and CRF increased with the dose of B-CRF during a four hour incubation period. The CRF stain was abolished, however, when an excess of unlabeled CRF was added to compete with B-CRF. The distribution of the B-CRF and ACTH stains varied in the cells with the time of exposure to the analog. These studies show that biotin-conjugate CRF is a potent analog that can be demonstrated cytochemically on cells identified immunocytochemically as corticotropes. It can be used to follow important events associated with CRF stimulation including the rapid internalization of CRF coupled with the mobilization of corticotropin stores and the formation of cellular processes.

Plasma and pituitary concentrations of LH, FSH, and Prolactin in aging C57BL/6 mice at various times of the estrous cycle ☆

Plasma and pituitary concentrations of LH, FSH, and prolactin (Prl) were measured by RIA in 2-4, 7-8, 12-13 and 16-20 month-old female C57BL/6 mice during various stages of the estrous cycle. In general, gonadotropin concentrations tended to rise with increasing age and Prl concentrations tended to decline. Pronounced differences existed, however, between the four age groups around the time of the LH surge. LH secretion declined progressively with increasing age at 21.00 hr of proestrus. Aged mice, 16-20 months old, had significantly lower plasma concentrations of LH than did other age groups. It is not known whether age-related changes in the ovary, pituitary, or hypothalamus are largely responsible for differences in the secretion of LH, FSH and Prl in aging C57BL/6 mice.

Peptide Biotinylation with Amine-Reactive Esters: Differential Side Chain Reactivity

N-hydroxysuccinimide (NHS) esters of biotin are reported to react specifically with amino groups of peptides and proteins. However, we have found that these reagents can readily acylate other functional groups in specific peptide sequences under relatively mild conditions. We have extended our inquiry of sequence-dependent acylation by evaluating the reactivity of a variety of commonly employed biotinylation reagents typically used for amino group modification. These included the p-nitrophenyl ester of biotin, NHS-esters of biotin containing aminohexanoic acid spacer arms, and a sulfonated NHS-biotin ester that contained a disulfide bond within its spacer. The decapeptide [D-Lys6]gonadotropin releasing hormone was employed as a model peptide. Reaction products were characterized by high-performance liquid chromatography, amino acid compositional analysis, reaction with hydroxylamine, and mass spectrometry. In addition to the O-acylation of Ser4 and Tyr5 in this peptide, we have also identified a novel biotinylation of the Arg8 side chain.

Inhibition of nitric oxide facilitates LH release from rat pituitaries.

We examined the effects of nitric oxide (NO) modulators on rat pituitary LH content in vivo and studied their response to LHRH-stimulated LH secretion in vitro in ovariectomized adult female Sprague Dawley rats. Alzet mini pumps (flow rate 10 microl/h) delivering either normal saline (Group I, 1.2 mg nitroglycerin, a donor of NO (Group II) or 50 mg of nitro-L-Arginine methyl ester, a NO synthase (NOS) inhibitor (Group III), were subcutaneously implanted into experimental animals. Following 36 h infusion, pituitaries were removed and either frozen for LH quantitation, or fragmented and challenged in the superfusion system with 10 min pulses of LHRH (1 ng/ml) at 90 min intervals for 10 hours. LH was assayed by radio-immunoassay (RIA) in the homogenates of pituitaries and in aliquots of the superfusate collected every 10 mins. Significantly lower pituitary LH levels were noted in Group III (150.3 +/- 18.6 ng) in comparison to Groups I (215.6 +/- 5.5 ng; p<0.04) or II (221.2 +/- 14.9 ng; p<0.01), suggesting that low levels of NO stimulate LH secretion in vivo. The pituitary LH contents were not significantly different in Groups I and II. In vitro studies reveal that exogenous LHRH stimulated response, measured as average pulse response (90 minute period after LHRH), and total LH released during the 10 hour perfusion, was 290 +/- 23.6 ng and 1646.7 +/- 270.8 ng, respectively, in Group III; 57.9 +/- 3.1, and 344.7 +/- 24.3 ng in Group I, and 105.3 +/- 6.3, and 633.7 +/- 77.1 mg in Group II. Thus, our in vitro studies demonstrate significantly enhanced (p<0.05) LHRH- stimulated LH secretion in Group III in comparison to Groups I and II, while Group II shows higher responsiveness than Group I (p<0.05). The results of the current studies provide evidence that NOS inhibition facilitates pituitary LH secretion. The differential responses to LHRH-stimulated LH secretion in vitro in the 3 groups suggest a possible role of NO in modulating pituitary LHRH receptor concentrations. However, this will have to be tested by further studies.

Lithium: Short-term and chronic effects on plasma testosterone and luteinizing hormone concentrations in mice

The effects of short-term and chronic lithium administration on the concentrations of plasma testosterone (T) and luteinizing hormone (LH) were evaluated in C57BL/6 mice, maintained on a fixed photoperiod of LD 14:10 (white lights on at 06:00 h, CST). Lithium chloride was injected intraperitoneally twice daily (at 09:00 and 16:00 h) in groups of adult male mice at a dosage of 2.5 meq/kg for 7 days, and 1.25 meg/kg for 21 days. Circulating levels of T and LH were measured by standard radioimmunoassay (RIA) methods. Plasma T levels showed a significant increase in mice treated with lithium for 7 days as compared to those in saline-injected control animals. However, there was no significant difference in the concentrations of plasma T between chronic (21 days) lithium-treated mice and the matched control. Plasma LH levels remained unchanged following both short-term and chronic lithium treatment.

Neuroendocrine Function in Transgenic Male Mice with Human Growth Hormone Expression

The neuroendocrine effects of human growth hormone (hGH) secretion were studied in adult male mice into which an hGH gene fused with mouse metallothionein 1 (mMT-1) promoter had been introduced. Intact transgenic mice had significantly greater plasma luteinizing hormone (LH) levels than did normal littermate controls. Castration increased LH levels in normal mice but was without effect on plasma LH levels in the transgenic mice. In vitro LH secretion and pituitary LH content were higher in the intact transgenic mice than in intact controls, while there was no significant difference in pituitary LH levels and in vitro LH secretion between the 2 groups of castrate animals. Intact transgenic mice exhibited a greater median eminence (ME) norepinephrine (NE) turnover than control animals, but ME NE turnover did not increase after castration in the transgenic animals as was the case in control mice. Castrate mice expressing the hGH gene had plasma levels of prolactin (PRL) similar to those seen in castrate controls, which was unexpected based on a previous study showing greatly attenuated PRL levels in intact hGH mice when compared to intact controls from the same line. Dopamine (DA) turnover in the ME was not significantly affected by the presence of the hGH gene, suggesting that the difference in plasma PRL levels between normal and transgenic mice is mediated through changes in PRL-regulating factors other than DA. In conclusion, the expression of the mMT-1/hGH hybrid gene in male mice leads to major alterations in LH secretion and lesser changes in PRL secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Lithium-induced changes in the plasma and pituitary levels of luteinizing hormone, follicle stimulating hormone and prolactin in rats

Adult male Sprague-Dawley rats, maintained under a controlled photoperiod of LD 14:10 (white lights on at 06:00 h, CST), were injected with lithium chloride and changes in the levels of plasma and pituitary homogenates of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) were examined to evaluate the effects of this anti-manic drug on reproductive function. Two groups of rats were injected with lithium chloride intraperitoneally, twice daily at 09:00 and 16:00 h, for 2 and 7 days at a dosage of 2.5 meq/Kg body weight. Plasma and pituitary levels of LH, FSH and PRL were measured by radioimmunoassay. Plasma levels of LH were significantly (P less than 0.05) increased after 2 days of lithium treatment. In contrast, a significant (P less than 0.005) reduction in plasma levels of LH was evident when lithium injections were continued for 7 days. The plasma levels of FSH remained unaffected by lithium treatment by either time period. Lithium administered for 2 days did not bring about any significant alteration in the plasma levels of PRL, although there was a significant (P less than 0.002) reduction in plasma PRL levels after 7 days treatment. The concentrations of pituitary LH, FSH and PRL remained unchanged after 2 and 7 days of lithium treatment.

Effects of Aging on the Activity of Hypothalamic Dopamine-Beta-Hydroxylase during Various Stages of the Estrous Cycle in C57BL/6 Mice

Hypothalamic dopamine-β-hydroxylase (DBH) activity and plasma luteinizing hormone (LH) concentrations were analyzed in female C57BL/6 mice aged 2–4, 7–8, 12–13, and 16–20 months at various times of th

Plasma and pituitary concentrations of luteinizing hormone, follicle-stimulating hormone and prolactin in aged, ovariectomized CD-1 and C57BL/6 mice

Plasma and pituitary concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (Prl) were determined by radioimmunoassay in young (2-4 months-old) and aged CD-1 (14-18 months-old) and C57BL/6 (16-22 months-old) mice one month after ovariectomy. In young, ovariectomized mice, plasma and pituitary concentrations of LH and FSH were significantly higher, whereas concentrations of Prl were significantly lower than those in control mice (sham-operated). In contrast, plasma concentrations of LH, FSH and Prl were not statistically different in aged, ovariectomized mice and aged, control mice. There were also no differences in pituitary concentrations of the three hormones when comparing the same aged C57BL/6 mice, although the aged, ovariectomized CD-1 mice exhibited higher pituitary levels of each hormone than those of their controls. The pituitary of the aged mouse responds differently to ovariectomy than that of the young mouse because of age-related changes in the ovary and/or hypothalamic-hypophyseal complex.

Ovarian dysfunction in peripubertal hyperinsulinemia.

Objective: Increasing evidence suggests the hyperinsulinemia plays an important role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the timing for the onset of hyperinsulinemia is not clear. The objective of this study was to examine the effect of peripubertal hyperinsulinemia on the maturing female reproductive axis. Methods: Hyperinsulinemia was induced in 28-day-old peripubertal female rats by infusing insulin (0.14 IU/d) via subcutaneously implanted Alzet minipumps (Model #2004; Durect Corp, Cupertino, CA; constant flow rate 0.25 μL/h) for 4 weeks. Control animals were administered normal saline. Estrus cyclicity was monitored regularly. Upon termination of the experimental period, the animals were killed, trunk blood and pituitaries were collected for hormone assays, and ovaries were collected for histological and immunocytochemical studies. Results: In contrast to the control animals, hyperinsulinemic animals had (1) erratic estrus cycles, with prolonged (2 to 3 days) metestres-diestrus-diestrus-proestrus stages; (2) significantly (P <.05) decreased levels of serum progesterone, and signiicantly (P <.05) increased levels of serum testosterone and dehydroepiandrostene sulfate; (3) prematurely luteinized ovarian follicles with prominent thecal and interfollicular stromal proliferation; and (4) markedly reduced expression of growth differentiation factor-9 (GDF-9) and activin receptors (ActR) I and IB in the ovaries. Conclusion: Peripubertal hyperinsulinemia in rats causes hormonal and ovarian changes similar to those in women with PCOS. Based on these novel findings, we speculate that peripubertal hypeinsulinemia may be a risk factor for the development of PCOS later in life.