Mark J. Horacek

The extracellular matrix components laminin, fibronectin and collagen IV are present among the epithelial cells forming Rathke's pouch

Cell-matrix interactions probably play a cooperative role with cell-hormone interactions to ensure normal differentiation of the adenohypophysis. The extracellular matrix (ECM) surrounding adult adenohypophysial cells contains laminin but its embryonic development has not been described. This study was carried out to test the hypothesis that adenohypophysial cells are associated with components in the ECM prior to cellular differentiation in the adenohypophysis. Fetuses were removed from Golden Syrian hamsters every 12 h from embryonic days 8.5-14. Coronal and sagittal 5-microns-thick sections of paraplast-embedded embryos were stained for fibronectin, laminin, or collagen IV using avidin-biotin immunoperoxidase staining. The adenohypophysial anlage was observed initially as a group of epithelial cells (Rathkes pouch) in the roof of the stomatodeum in contact with a basement membrane. The basement membrane stained positively for laminin, collagen IV and fibronectin. Lighter staining for laminin, fibronectin and collagen IV was observed between the developing adenohypophysial cells in Rathkes pouch. Proliferative activity was apparent in the antero-inferior region of Rathkes pouch and resulted in the formation of the bulk of the adenohypophysis. Mesenchyme infiltrates the region between the base of Rathkes pouch and the oral epithelium, thus separating the two. The basement membrane surrounding the pouch appears to become discontinuous in the regions of high proliferative activity. These results show that ECM components appear early during the development of adenohypophysial cells prior to their cellular differentiation into hormone-containing cells. This association between ECM components and developing adenohypophysial cells provides the anatomical basis for cell-ECM interactions to influence adenohypophysial development and differentiation.

Adult adenohypophysial cells express β1 integrins and prefer laminin during cell-substratum adhesion

Summaryβ1 Integrins are a family of structurally related heterodimeric cell surface receptors that are involved in adhesion to molecules in the extracellular matrix (ECM) such as laminin (LN), fibronectin (FN), and collagen. These receptors are expressed by many cell types and mediate a variety of processes such as cell-matrix and cell-to-cell adhesion, cell migration, growth, and differentiation. The purpose of these studies was to identify and partially characterize β1 integrins on adenohypophyseal cells and to begin to elucidate their functional importance. Adenohypophyses were removed from adult male rats, dispersed using 0.25% trypsin, rinsed, and resuspended in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and F12 medium containing 10% fetal bovine serum and antibiotics. Ten million cells were allowed to attach to each of five plastic culture dishes overnight. The next day, the adenohypophyseal cells were surface-labeled with125I. The labeled cells were lysed and centrifuged. The supernatant was immunoprecipitated using preimmune IgGs (100 µg/ml) and was then incubated with a polyclonal antibody against the rat β1 family of integrins or with a variety of immune IgGs directed against the α subunit of the receptor (anti α1, anti α2, anti α3, and anti α5 antibodies). The receptors were then immunoprecipitated by addition of protein A-Sepharose or IgG1 Sepharose. After washing, the immunoprecipitates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Cultured adenohypophyseal cells expressed the β1 integrin subunit, which was associated with the α1, α2, α3, and α5 integrin subunits. These integrins are known to have binding specificities for LN, FN, epiligrin, and several collagens. Immunocytochemical staining and confocal microscopy verified that these receptors were present on the cell surface in vitro. The addition of anti rat β1 integrin antibodies to dispersed adenohypophyseal cells partially blocked their attachment to ECM ligands in cell adhesion assays. In addition, peptides containing Agr-Gly-Asp-Ser (RGDS) partially blocked adenohypophyseal cell attachment to FN and to a lesser extent to LN. These studies show for the first time that adult adenohypophyseal cells express several β1 integrin dimers and attach to ECM ligands corresponding to their binding specificities. The fact that these interactions are only partially blocked by RGDS peptides and antibodies against the β1 family of integrins may indicate that other cell-matrix receptors are also present. Additional studies are necessary to determine whether these interactions have a functional significance (such as an effect on hormone secretion) beyond their role in cell-matrix adhesion.

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.

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.

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.