Gary T. Campbell

Estrogen can protect splenocytes from the toxic effects of the environmental pollutant 4-tert-octylphenol

Four-tert-octylphenol (OP), an environmental pollutant, exerts apoptotic effects on cultured mouse splenocytes. Although OP binds to estrogen receptors, these apoptotic effects are not exerted by 17β-estradiol (E). It remained possible that OP might bind to estrogen receptors and subsequently exert apoptotic effects not exerted by E after it binds to the same receptors. It also remained possible that E-primed splenocytes might respond to OP differently than splenocytes not exposed to E. Thus, we investigated OP and E interactions on the viability of mouse splenocytes in culture. The total number of splenocytes (cells stained and not stained with trypan blue) was not altered or altered slightly after incubation with any agent for 24 h. Incubation of splenocytes in medium containing 5×10−5 or 5×10−7M OP decreased the percentage of viable cells by approx 47% and 25%, respectively. The addition of 0.8×10−5 to 0.8×10−9M E to cultures was without effect or decreased the percentage of viable cells by only approx 5%. The addition of these concentrations of E simultaneously with or at 2 h after the addition of 5×10−5M or 5×10−7M OP to cultures did not interfere with the OP-induced decreases in cell viability. By contrast, incubation of splenocytes in medium containing E for 2 h prior to the subsequent addition of either dose of OP blocked the OP-induced decreases in cell viability in a dose-response manner. There was a marked reduction in the percentage of viable cells (70%) when splenocytes were incubated with 0.5×10−5M dexamethasone. The addition of 0.8×10−5M E at 2 h prior to the addition of dexamethasone did not prevent the decreased cell viability. Incubation of cells in medium with 0.8×10−5M testosterone caused a small decrease in splenocyte viability similar to that observed with E. However, unlike E, the addition of testosterone at 2 h prior to the addition of 5×10−5M OP did not prevent the OP-induced decrease in cell viability. These data suggest the presence of estrogen receptors in some splenocytes. They also suggest that if OP binds to these estrogen receptors or other receptors in the absence or initial presence of E, the resulting effect is toxic to the cells. By contrast, exposure of splenocytes to E prior to their exposure to OP can prevent the toxicity of OP.

Intraperitoneal injection of chloral hydrate causes intra-abdominal adhesions and unilateral testicular atrophy in golden syrian hamsters

We investigated the reason for the high mortality we had observed in hypophysectomized-orchidectomized Golden Syrian hamsters that were anesthetized with intraperitoneal (i.p.) injections of chloral hydrate (CH). Intact male Golden Syrian hamsters were injected intraperitoneally with 0.1cc/100g BW of a 35% solution of CH, a 35% solution of sodium chloride, or double-distilled water. Equal numbers of hamsters in each group were injected on the right or left side of the abdomen. Within 10 days, 35% of the CH-injected hamsters were dead or had to be euthanized. Autopsy revealed severe peritonitis and adynamic ileus. CH-injected hamsters that survived gained weight at a rate similar to that of the controls. All surviving hamsters were killed 18 days after the injections. Among the surviving CH-injected hamsters, 84.6% had intra-abdominal adhesions, 61.5% had unilateral testicular atrophy, and 53.8% had a yellowish necrotic mass in the epididymal fat pad (EFP). All the lesions occurred on the side that was injected. The atrophied testes had been rendered cryptorchid due to involvement with intra-abdominal adhesions. In the water-treated controls, there were no abnormalities; whereas, in the saline controls, 75% had a mass in the EFP. Histology of the EFP mass was similar in hamsters injected with CH or hypertonic saline and suggested a diagnosis of fat necrosis. The results suggest that the mortality, the intra-abdominal adhesions, and the unilateral cryptorchidism were caused by a single i.p. injection of CH, but the fat necrosis in the EFP was probably caused by high concentrations of salt. The results further suggest that high concentrations of CH should not be injected intraperitoneally for anesthesia in chronic studies, particularly of the male reproductive system.

Effects of corticotrophin-releasing hormone on corticotrophs in anterior pituitary gland allografts in hypophysectomized, orchidectomized hamsters

SummaryWe investigated the effects of corticotrophin-releasing hormone (CRH) on the percentage of anterior pituitary gland (APG) cells which are corticotrophs as well as the size and shape of corticotrophs. Pituitary glands were removed from 7-week-old male hamsters and placed beneath the renal capsules of hamsters that had been hypophysectomized and orchidectomized 3 weeks previously. Beginning 6 days after each host had received a single allograft, each was injected subcutaneously twice daily with 4 ⧎g CRH or vehicle for 16 days. Six hosts in each group were decapitated 16 h after the last injection. Sections of anterior pituitary tissue were stained for ACTH and with hematoxylin. The percentage of corticotrophs among APG cells was greater in allografts exposed to exogenous CRH (∼20%) than in allografts exposed to vehicle (∼15%). Exposure to exogenous CRH increased the cross-sectional area of corticotroph cells in allografts to values greater than those measured for corticotrophs in allografts exposed to vehicle, without altering the shape of cells. Results of subsequent studies suggested that hamsters with allografts injected with vehicle do not release ACTH and that exogenous CRH causes an abrupt release of ACTH from allografts. These results indicate that CRH releases ACTH from ectopic corticotrophs and that administration of CRH can increase corticotroph size and the percentage of APG cells that are corticotrophs.

Neuropeptide Y and luteinizing hormone releasing hormone synergize to stimulate the development of cellular follicle-stimulating hormone in the hamster adenohypophysis.

Luteinizing hormone releasing hormone (LHRH) stimulates the development of cellular FSH immunoreactivity in the perinatal hamster adenohypophysis. Because neuropeptide Y (NPY) can act directly on rat adenohypophysial cells to stimulate FSH and LH release and potentiate the stimulatory effect of LHRH on FSH and LH release, we investigated the effects of NPY alone and in combination with a low, ineffective dose of LHRH on inducing cellular FSH immunoreactivity in the neonatal hamster adenohypophysis. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized‐ovariectomized adult hamster hosts with a catheter implanted in the external jugular vein. After treatment, hosts were decapitated and graft tissue was stained for FSH and LH immunoreactivity. The mean percentage of adenohypophysial cells that stained for FSH was low (2.8%) in grafts in hosts infused continuously with heparinized saline vehicle for 7 days. In other hosts, peptides were pulsed through the catheter every 12 h for 7 days. The mean percentage of FSH cells also was low after pulsing 6 ng LHRH or 2 μg NPY but increased substantially when the two peptides were pulsed simultaneously. No differences in the mean percentage of LH cells existed between any of the groups. The results demonstrate that NPY and LHRH can synergize to induce cellular FSH immunoreactivity in the neonatal female hamster.

Effects of growth hormone-releasing hormone on somatotrophs in anterior pituitary gland allografts in hypophysectomized, orchidectomized hamsters.

SummaryWe investigated the influences of growth hormone-releasing hormone (GHRH) on the percentage, size, and shape of somatotrophs in ectopic anterior pituitary tissue. Entire pituitary glands removed from 7-week-old male hamsters were placed beneath the renal capsules of 12-week-old hamsters that had been hypophysectomized and castrated 3 weeks previously. Beginning 6 days after each host had received a single allograft, each was injected subcutaneously twice daily with 4 μg GHRH in 100 μl of vehicle or 100 μl of vehicle for 16 days. Six hosts in each group were killed by decapitation on day 17, 16 h after the last injection. Nine normal male hamsters were also decapitated and their pituitary glands were removed. Sections of anterior pituitary tissue were stained for GH and with hematoxylin. The percentage of anterior pituitary cells that stained for growth hormone was similar in the 3 groups. In contrast, somatotrophs in grafts had a smaller mean cross-sectional area than those observed in glands in situ. This effect was reversed by GHRH. Analysis of the shape of somatotrophs in both groups of grafts disclosed that they were less circular in cross-section than those in glands in situ. The results suggest that GHRH may not play a role in maintaining the percentage of somatotrophs among anterior pituitary cells, but that it does play a role in maintaining their size.

Adenohypophysial Allografts Releasing Prolactin Decrease Prolactin mRNA Concentration in the Host Hamster’s Adenohypophysis in situ

The inhibitory effects of pituitary allografts on the prolactin (PRL)-secretory system are presumed to be consequences of the unabated release of PRL by the allografts. In the present studies we used pituitary allografts in the Golden Syrian hamster to address the following questions: (a) Do allografts of adult adenohypophysial tissue which elevate serum PRL levels decrease the concentration of PRL mRNA in the hosts adenohypophysis? (b) Is this effect shared by allografts of neonatal hypophysial tissue or neonatal muscle tissue which do not elevate serum PRL levels? (c) Do any of these types of allograft alter growth hormone mRNA in the hosts adenohypophysis? Prolactin mRNA concentration, but not growth hormone mRNA concentration, was decreased in the adenohypophyses in situ in the hosts bearing adult adenohypophysial allografts in which serum PRL levels were elevated. In contrast, serum PRL in hosts with neonatal hypophysial or muscle allografts were not elevated and PRL mRNA levels in the adenohypophysis in situ were not decreased when compared to the levels measured in hamsters with sham transplants. Prolactin mRNA levels in hosts with neonatal muscle allografts were not different from levels in hosts with neonatal hypophysial allografts but were increased when compared to the levels measured in hamsters with sham transplants. There were no differences in PRL concentration in the adenohypophyses in situ between any of the groups. Also, PRL concentrations in neonatal hypophysial allografts were similar to those in adult adenohypophysial allografts. To our knowledge these observations are the first demonstrating that short-loop feed-back of PRL includes a decrease in PRL mRNA concentration. The observations also support the working hypothesis that PRL and not another pituitary factor exerts the negative feedback.

Effects of Hypothalamic Neurohormones on Prolactin Release from Pituitary Allografts in the Hamster

Abstract Recent reports indicate that luteinizing hormone-releasing hormone (LHRH) releases prolactin (PRL) under some circumstances. We examined the chronic effects of LHRH, growth hormone-releasing hormone (GHRH), and corticotrophin-releasing hormone (CRH) on the release of PRL, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by pituitary allografts in hypophysectomized, orchidectomized hamsters. Entire pituitary glands removed from 7-week-old-male Golden Syrian hamsters were placed under the renal capsule of hypophysectomized, orchidectomized 12-week-old hamsters. Beginning 6 days postgrafting, hamsters were injected subcutaneously twice daily with 1 μg LHRH, 4 μg GHRH, or 4 μg CRH in 100 μl of vehicle for 16 days. Six hosts from each of the four groups were decapitated on Day 17, 16 hr after the last injection. Prolactin, LH, and FSH were measured in serum collected from the trunk blood. Treatment with LHRH significantly elevated serum PRL levels above those measured in the other three groups, which were all similar to one another. Serum LH levels in hosts treated with vehicle were elevated above those measured in the other three groups. Serum FSH levels in hosts treated with LHRH were greater than FSH levels in any of the other three groups. These results indicate that chronic treatment with LHRH can stimulate PRL and FSH release by ectopic pituitary cells in the hamster.

4 – Hypophysial Grafts beneath the Renal Capsule: A Model to Study Endocrine Control Systems

Publisher Summary This chapter provides an overview of the preparation of allografts of pituitary tissue beneath the renal capsule of the Golden Syrian hamster, and a synopsis of the utility of the model for studying endocrine control systems. Grafting pituitary tissue into ectopic sites in a variety of hosts is used as a model to study diverse physiological control systems and the genesis of pathological states. The use of pituitary allografts also contributes to the understanding of the development of the adenohypophysis. The results contributed to the understanding of the development of other pituitary cell types. Donor hypophyses removed from neonatal hamsters contain abundant somatotrophs, but are virtually devoid of lactotrophs. Pituitary allografts are used to provide prolactin (PRL) replacement with accompanying minimal amounts of other pituitary hormones in hypophysectomized hosts or to produce hyperprolactinernia in almost any type of host. The majority of studies using pituitary allografts in any species utilize the ability to manipulate circulating PRL levels to study the physiological and pathological roles of PRL. The preponderance of studies dealt with PRL and reproduction. This chapter has culminated in the acceptance of PRLs role in the maintenance of pregnancy, regulation of the mammary gland, regulation of gonadal receptors for gonadotrophins, and controlling gonadotrophin secretion. Thus, the pituitary-grafted animal proves to be quite valuable in studying the many actions of PRL.