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Intracerebroventricular (icv) infusion of human interleukin 1 beta (IL-1) into intact and adrenalectomized rats impairs immune function. Using antibody to IL-1 as well as an inhibitor of IL-1 action, we sought to determine if endogenous IL-1 in the central nervous system has a physiological role in mediating the immunosuppressive effects of stress. Compared with freely moving controls, rats given intermittent electric shock to the tail for 40 min exhibited a fall in T lymphocyte proliferation and natural killer (NK) cell cytotoxicity of 33% and 38%, respectively; however, when pretreated with icv human IL-1 monoclonal antibody, which significantly crossreacts with rat IL-1, the decrement was attenuated to 14.6% and 15%, respectively. When rats were pretreated with icv alpha-MSH, which blocks many IL-1 effects, shock-induced suppression of 42% in both T lymphocyte proliferation and NK cytotoxicity were blunted to 33% and 31%, respectively. Similar results were found in adrenalectomized rats. These findings suggest that endogenous IL-1 is a physiologically relevant mediator of the immune response to stress. As IL-1 has been reported to release CRF, which we have shown always plays a significant role in stress-induced immunomodulation, we then assessed the relationship of IL-1 and CRF in immunosuppression. Infusion of icv IL-1 caused a decrease of 35% in T lymphocyte proliferation and 34% in NK activity, but pretreatment with CRF antibody icv attenuated IL-1 suppression of T lymphocyte proliferation and NK activity to 10% and 8%, respectively. Comparable results were observed in adrenalectomized rats. These findings suggest that CRF antibody is able to block the immunosuppressive effects of IL-1. To further examine the interaction of CRF in mediating stress-induced immunosuppression, we found that animals pretreated with icv CRF antibody, shocked and then given icv IL-1, had a decrement in T lymphocyte proliferation and NK cytotoxicity of 24% and 21%, respectively, demonstrating that the immunosuppressive effect of icv IL-1 is blocked when central CRF has been neutralized by prior administration of icv CRF antibody. In contrast, animals pretreated with icv IL-1 antibody, shocked and then given icv CRF, had decrements of 38% and 40%, respectively, showing that icv CRF does act even when central IL-1 has been neutralized by prior administration of icv IL-1 antibody. Thus, we conclude there is a sequential relationship between two of the known mediators of stress-induced immunosuppression, with release of central IL-1 followed by that of CRF.

We examined the role of CRF, a key mediator of the endocrine response to stress, in modulating immunosuppression during the subacute stress of intermittent electrical shock over 1 h. Administration of shock to intact rats resulted in a 74% decrement in T-lymphocyte proliferation and a 59% decrease in natural killer cytotoxicity. Similar suppression of these two parameters of immune function in response to shock was noted in adrenalectomized rats as well. The immunosuppressive effects of this shock were significantly and comparably blunted when both intact and adrenalectomized animals were pretreated 1) iv with either a highly potent polyclonal CRF antibody or a specific CRF antagonist or 2) intracerebroventricularly with either a high affinity monoclonal antibody to CRF or a specific CRF antagonist. An immunomodulatory role for CRF is further supported by the findings that administration of exogenous CRF, either iv (10 micrograms/animal) or intracerebroventricularly (1 microgram/animal), resulted in significant decrements in lymphocyte proliferation and natural killer cytotoxicity, similar to those seen with the stress paradigm. Our observations indicate that CRF plays a significant role in modulating the immune response to subacute stress, largely by adrenal-independent mechanisms.

A correlative radioimmunological-biochemical-ultrastructural study of the rat pineal gland was undertaken during the summer months when pineal arginine vasotocin (AVT) immunoactivity increases up to 200-fold. RIA confirmed a rapid rise in AVT activity during mid-August regardless of the time of day sampled. Pineal indoles were separated by HPLC and measured using electrochemical detection. Serotonin (5-HT) and 5-hydroxyindoleacetic acid levels were consistently elevated in daytime samples, and there was a significant trend for increased day and nighttime levels of 5-HT from July to September. Mid-dark levels of melatonin also exhibited a significant increase over the sample period. Nighttime levels of N-acetylserotonin mirrored fluctuations in 5-HT in the preceding photoperiod. Ultrastructural components implicated in peptide/protein and/or indole biosynthesis were quantified by stereological morphometry. The greatest amounts of rough endoplasmic reticulum stacks, lipid droplets, and annulate lamellae-like bodies coincided with peak AVT activity. Dense-cored vesicles and synaptic ribbons were consistently more frequent during the dark period. The number of dense-cored vesicles and nucleolar size tended to be greatest before and after the peak in AVT immunoactivity. These observations are consistent with the hypotheses that endoplasmic reticulum and lipid are functionally related to the synthesis and/or storage of peptide/protein factors and that numerical changes in synaptic ribbons and dense-cored vesicles are more closely related to day/night differences in indole metabolism.

A high molecular weight antifertility factor (AF-1) was obtained in a high degree of purity from human seminal plasma by ultracentrifugation, CM-cellulose and concanavalin A chromatography, and Sepharose 6B gel filtration. A final purification step involving preparative disc electrophoresis was occasionally required. AF-1 showed a dose-dependent inhibition of the in vitro fertilizing ability of capacitated mouse spermatozoa, causing 50% inhibition of fertilization at approximately 27.5 micrograms/10(6) spermatozoa. Removal of the follicle cell layer of the oocyte did not decrease the antifertility effect of AF-1 but inhibition of fertilization was no longer observed after dispersal of the zona pellucida. The effect of AF-1 was on the spermatozoa and not on the oocytes. These results show that AF-1 treatment prevents capacitated spermatozoa from penetrating the zona pellucida and possibly the follicle cell layer. The mechanism by which AF-1 does so is not known because AF-1 did not prevent the in vitro acrosome reaction of guinea pig spermatozoa, nor did it inhibit the activity of human acrosin or bovine testicular hyaluronidase. The antifertility activity of AF-1 is reversed after recapacitation of the AF-1 treated spermatozoa and it can be assumed that AF-1 is either dissipated or loses activity during the transport of the spermatozoa through the female genital tract when capacitation takes place. AF-1 is heat labile. The properties of AF-1 are the same as those found previously for the pellet obtained by high-speed centrifugation of human seminal plasma, indicating that this is the primary, nonparticulate, high molecular weight factor with antifertility activity in human semen.

A monoclonal antibody to ovine corticotropin releasing factor (CRF) has been produced by fusion of a non-producing plasmacytoma cell line P3U1 with spleen cells of Balb/c mice immunized with the synthetic 41 amino acid peptide coupled covalently with rabbit myosin by a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. A total immunizing dose of 500 micrograms resulted in a highly specific, high-affinity antibody with a Ka of 0.15 x 10(12) M-1, which was used to establish a specific RIA with a sensitivity of 10 pg/tube. Levels of corticotropin releasing factor-like immunoreactivity (CRF-LI) in a pg/mg of hypothalamic tissue ranged from 4-10 in ovine, 2.5-8 in bovine, 47.5-67.5 in mouse and 2.3-20 in human tissue. Moreover, CRF-LI was widely distributed in extrahypothalamic mouse brain at concentrations approximately one half those seen in hypothalamus.

Groups of male and female laboratory rats, 28-30 days of age, were killed each week from July 1980 to September 1981. Pineal glands were collected, pooled, and extracted. Arginine vasotocin (AVT) activity in the extracts was measured by RIA. For most of the calendar year, pineal AVT immunoactivity ranged between 1.8-7.7 pg/gland. The average (+/- SE) basal AVT activity level was 4.1 +/- 0.3 pg/gland (n = 48). Both years in early August, pineal AVT activity increased several hundred fold. Values of 1720 and 1170 pg/gland were measured in mid-August of 2 successive years. The signal for this dramatic yearly rhythm, and its physiological consequences, are as yet unknown.

Impressive evidence has emerged indicating that immunoassayable and bioassayable CRF, which is immunoneutralizable, is present not only in the hypothalamus but in many peripheral tissues as well. Using highly specific and sensitive RIAs and immunoaffinity chromatography to investigate whether this extrabrain CRF circulates in the rat, we found low but clearly measurable levels in peripheral plasma (mean, 11.4 +/- 0.8 pg/ml). Immunological findings were corroborated by fast protein liquid chromatography, which resolves peptides by both hydrophobicity and ionic charge. With this approach the major immunoreactive peak was eluted at the position of synthetic rat CRF standard. To assess whether levels of peripheral plasma CRF-like immunoreactivity (CRF-LI) vary in parallel with those of hypothalamic CRF-LI, we performed studies with low and high dose dexamethasone administration and withdrawal, adrenalectomy, and hypophysectomy. Seven days after oral administration of dexamethasone, there was a decrement in the levels of peripheral plasma and hypothalamic CRF-LI. Depending on the dose, recovery was also found 7 days after cessation of the treatment. After either adrenalectomy or hypophysectomy, there were increments in the levels of CRF-LI in both peripheral plasma and hypothalamus. Thus, concentrations of CRF-LI in the peripheral plasma and in the hypothalamus vary in parallel in response to alterations in the pituitary-adrenal axis.

The preparation of a highly purified antifertility factor from human seminal plasma is described, and some of its biochemical properties have been determined. Purification was achieved by ultracentrifugation of particle-free human seminal plasma, followed by chromatography of the precipitate using carboxymethyl cellulose, concanavalin A, and Sepharose 6B columns. An occasional contaminant was further removed by preparative isoelectric focusing. During the purification procedures, the activity of the fractions was monitored by mixing them with capacitated mouse spermatozoa for 20 min before adding an aliquot to intact mouse oocytes, and determining fertilization after 24 h. The I50 (amount causing a 50% reduction in fertilization as compared to the control) of the final purified factor was 45 micrograms protein. Purity was established by standard and sodium dodecyl sulfate disc gel electrophoresis, isoelectric focusing, high-pressure liquid chromatography, and sedimentation analysis. These methods, as well as Sepharose gel filtration, were also used for the molecular weight estimations; good agreement was obtained between the various techniques. The factor appears to be a glycoprotein with a molecular weight of about 200,000. It consists of two subunits with molecular weights of about 125,000 and 72,000 and s-20,w of 6.2 s and 4.3 s. The factor contains relatively high amounts of aspartic acid and glutamic acid residues as well as leucine and serine, but only small amounts of tryptophan and no methionine was detected. The carbohydrate fraction is particularly rich in galactose and N-acetylgalactosamine but also contains mannose and N-acetylglucosamine and small amounts of fucose and sialic acid.

Neurophysin (Np) is generally found in close association with vasopressin and oxytocin in the hypothalamo-neurohypophyseal complex. Dog neurophysin I and II have been isolated from fresh and frozen posterior pituitaries. The proteins were characterized on the basis of disc electrophoresis, immunological properties, amino acid composition and partial sequence determination. The amino terminal sequence of dog Np I is Ala-Ala-Leu-Asp-Leu-Asp-Val-Arg-Gln-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Gln-Gly-while that of dog Np-II is Ala-Met-Ser-Asp-Leu-Glu-Leu. The dog Np I appears to be metabolically less stable than Np II. Isotope experiments with [35S]cystine or 3H-labeled amino acids using a design of "in vitro pulse and in vitro chase" as well as "in vivo pulse and in vivo chase," added further confirmation of the capability of the hypothalamic neurosecretory cells to synthesize concomitantly precursors of Np and vasopressin. The radioactively labeled precursors were converted to Np-like protein and vasopressin, both of which were isolated.