SUMMARY

Incidence and patterns of mitoses and histochemical localization of alkaline phosphatase (APase) and adenosine triphosphatase (ATPase) were studied from days 5 to 20 in pseudopregnant mice in which deciduomata had been induced in the left uterine horns by intrauterine injection of oil on day 4, the right horns serving as controls.

In stromal cells, mitoses were very numerous throughout the endometrium of the left (but not the right) horn on days 5 and 6, in the basal, non-decidualized stroma until day 8 or day 9, and were not seen in stromal cells of either horn thereafter. In glandular epithelium, mitoses were absent from days 5 to 10, and numerous from day 11 or 12 until at least day 17 in both horns. Mitoses were present in capillaries within developing deciduomata on days 5 and 6, then seldom seen until day 12 and during the next 3 days were numerous in endothelial and pericapillary cells in the mesometrial quadrant of the left horn and around glands in both horns.

The deciduoma cells reacted strongly with AP and ATP substrates from days 5 to 10, after which the intensity of the reaction weakened and had usually disappeared by day 13. ATPase activity disappeared from vascular endothelium within the deciduoma a few hours after APase had appeared within the deciduoma cells on day 5. It reappeared in the vessel walls on day 9 and thereafter was usually present until the deciduoma was shed. In the basal, non-decidualized stroma, APase was absent until about day 10, then appeared in the stroma cells nearest to the myometrium, extending gradually into the densely packed cells nearest to the regressing deciduoma. The possible role of this enzyme in reparative growth of the endometrium after regression of the deciduoma is discussed.

SUMMARY

The effect of s.c. injections of cadmium chloride (3 μmole/100 g. body wt.) on the blood flow and vascular permeability of the testis, epididymis and accessory reproductive organs of rats has been examined. Sapirstein's indicator fractionation technique was used to measure blood flow with [¹³¹I]iodoantipyrine and [⁸⁶Rb]-rubidium chloride. A rubidium-rejecting compartment was found in the testis similar to, but smaller than, that in the brain and pineal body.

Testicular blood flow started to decrease within 3 hr. of giving cadmium (Cd) and by 12 hr. was only 2–9 % of the control values; it then started to recover but after 14 days it was still only 31 % of the control values.

Apart from a small reduction of blood flow to the first part of the head of the epididymis 6 hr. after Cd administration, blood flow was not strikingly reduced in any part of the epididymis by Cd treatment. However, blood flow to all parts of the epididymis had increased markedly when examined 7 days after giving Cd; this was most evident in the first part of the head of the epididymis.

Blood flow through the accessory reproductive organs was reduced within 6 hr. of Cd injection. Blood flow in the seminal vesicles returned to normal between 1 and 14 days after treatment but blood flow in the prostate gland did not recover.

The movement of albumin from intravascular to extravascular compartments was used as an index of vascular permeability. This index increased in the testis in the period 1–6 hr. after Cd administration and the change occasionally occurred before blood flow decreased. A similar increase was seen in the first part of the head of the epididymis 3–6 hr. after Cd, but no change was seen in the rest of the epididymis.

The evidence suggests that Cd acts by damaging the endothelium of the capillaries in the testis leading to prolonged stoppage of blood flow which would lead to hypoxia in the spermiogenic epithelium.

Plasmalemma vesicle protein-1 (PV-1) is an integral membrane protein associated with endothelial cell caveolae and fenestrae. Since endocrine glands are enriched with fenestrated endothelium, we examined the distribution of PV-1 mRNA and protein in endocrine glands and determined its cellular localization. A single transcript was detected by RT-PCR in all endocrine glands examined. A synthetic peptide was used to generate antibodies for Western blotting and immunohistochemistry (IHC). Western blotting of membrane fractions from lung, pituitary, adrenal, testis and PV-1-transfected Cos-1 cells revealed a major 65 kDa protein. This protein binds to heparin with high affinity. Using IHC, PV-1 was localized to both endothelial cells of the adrenal zona reticularis and chromaffin cells of the medulla. In the pancreas, PV-1 expression was restricted to a few cells in the islets of Langerhans that partially overlap with somatostatin-positive delta-cells. In both neonatal and adult pituitaries, strong PV-1 immunoreactivity was detected in neural lobe pituicytes in a pattern similar to that of glial fibrillary acidic protein (GFAP). PV-1 and GFAP expression was seen in the adult, but not neonatal, intermediate lobe. Endothelial cells throughout the neonatal anterior lobe were PV-1 positive, but PV-1 in the adult was restricted to some endothelial and endocrine cells localized near the margins of lobe. In the adult testis, strong PV-1 expression was seen in germ cells within the seminiferous tubules that varied with the stage of spermatogenesis. In contrast, PV-1 in the neonatal testis was localized to the interstitial cells but not seminiferous tubules. In the ovary, PV-1 was expressed in stromal endothelial cells as well as the thecal layer of developing follicles. Over half the corpus luteal cells were positive for PV-1. Our data have shown that PV-1 is not restricted to endothelial cells but is localized in many types of endocrine and non-endocrine cells. Furthermore, PV-1 expression in the pituitary and testis is developmentally regulated.

Parathyroid hormone (PTH)-related protein (PTHrP) is produced in smooth muscles and endothelial cells and is believed to participate in the local regulation of vascular tone. No direct evidence for the activation of endothelium-derived nitric oxide (NO) signaling pathway by PTHrP has been found despite attempts to identify it. Based on direct in situ measurements, it is reported here for the first time that the human PTH/PTHrP receptor analogs, hPTH(1--34) and hPTHrP(1--34), stimulate NO release from a single endothelial cell. A highly sensitive porphyrinic microsensor with a response time of 0.1 ms and a detection limit of 1 nmol/l was used for the measurement of NO. Both hPTH(1--34) and hPTHrP(1--34) stimulated NO release at nanomolar concentrations. The peak concentration of 0.1 micromol/l hPTH(1--34)- and 0.1 micromol/l hPTHrP(1--34)-stimulated NO release was 175+/-9 and 248+/-13 nmol/l respectively. This represents about 30%--40% of maximum NO concentration recorded in the presence of (0.1 micromol/l) calcium ionophore. Two competitive PTH/PTHrP receptor antagonists, 10 micromol/l [Leu(11),d -Trp(12)]-hPTHrP(7--34)amide and 10 micromol/l [Nle(8,18),Tyr(34)]-bPTH(3--34)amide, were equipotent in antagonizing hPTH(1--34)-stimulated NO release; [Leu(11),d -Trp(12)]-hPTHrP(7--34)amide was more potent than [Nle(8,18),Tyr(34)]-bPTH(3--34)amide in inhibiting hPTHrP(1--34)-stimulated NO release. The PKC inhibitor, H-7 (50 micromol/l), did not change hPTH(1--34)- and hPTHrP(1--34)-stimulated NO release, whereas the combined effect of 10 micromol/l of the cAMP antagonist, Rp-cAMPS, and 50 micromol/l of the calmodulin inhibitor, W-7, was additive. The present studies show that both hPTH(1--34) and hPTHrP(1--34) activate NO production in endothelial cells. The activation of NO release is through PTH/PTHrP receptors and is mediated via the calcium/calmodulin pathway.

ABSTRACT

We report the light microscopic, transmission and scanning electron microscopic features of the adrenal cortices in rats bearing a medullary thyroid carcinoma cell line transfected with a corticotrophin-releasing hormone (CRH) cDNA expression vector. The animals had elevated CRH, ACTH and corticosterone blood levels, involuted thymuses and markedly enlarged adrenal glands with prominent lipid-depleted cortices and dilated congested capillaries, similar to those of animals treated with ACTH. Using electron microscopy it was found that the enlarged fasciculata and reticularis zones were composed of large, compact cells with abundant smooth endoplasmic reticulum, prominent Golgi complexes, increased number of large mitochondria with focal loss of cristae and cavitation of the internal compartments, numerous lysosomes and prominent elongated microvilli. In addition, small cytoplasmic fragments were seen within the capillary lumina; these structures resembled microvilli that were apparently detached from adrenocortical cells and entered the blood stream via discontinuous endothelium of dilated capillaries. By scanning electron microscopy it was found that the cells had bulging surfaces with scattered pits and numerous long microvilli pointing in different directions.

This animal model allows analysis of the effects of protracted CRH excess resembling tumoural CRH-dependent Cushing's syndrome in human patients. Our findings call attention to the role of microvilli in adrenocortical secretion. The increased number and size of microvilli has been thought to lead to an increase in the surface area of adrenocortical cells, thereby facilitating hormone discharge. The detachment of microvilli from adrenocortical cells may represent a form of apocrine secretion and may contribute to hypercorticosteronaemia in CRH excess.

Journal of Endocrinology (1992) 135, 271–277

Abstract

Hyperthyroidism is associated with elevated plasma levels of endothelium-derived proteins such as von Willebrand factor (vWF), fibronectin (FN) and endothelin-1 (ET-1). This study was designed to characterize the mechanisms involved in this phenomenon at the cellular level. vWF, FN and ET-1 secretion and mRNA expression were measured in human umbilical vein endothelial cells (HUVECs) exposed to tri-iodothyronine (T₃) for 13 ± 1 days, using ELISA, Western blot, RIA and Northern blot analysis respectively. Exposure of HUVECs to T₃ significantly increased vWF secretion (50 ng T₃/ml: 117 ± 5%, P<0·01; 100 ng T₃/ml: 127 ± 26%, P<0·01) as well as vWF mRNA expression (50 ng/ml: 116 ± 13%, P<0·001; 100 ng/ml: 136 ± 30%, P<0·002) (results are means ± s.d. analysed by the Wilcoxon signed rank test). FN secretion was significantly affected by 50 (145 ± 42% of control, P<0·05) and 100 (116·8 ± 16% of control, P<0·05) ng T₃/ml, and FN mRNA expression by 50 ng T₃/ml (123 ± 20%, P<0·05). Long-term incubation with T₃ increased both ET-1 secretion (25 ng/ml: 124 ± 25%, P<0·001; 50 ng/ml: 165 ± 53%, P<0·05; 100 ng/ml: 116 ± 17%, P<0·05) and prepro-ET-1 mRNA expression (25 ng/ml: 112 ± 16%, P<0·05; 50 ng/ml: 134 ± 43%, P<0·02; 100 ng/ml: 120 ± 20%, P<0·02). Protein kinase C (PKC) isoforms ε and βII were not significantly affected by T₃, whereas PKC α was increased in whole cell lysates and in membrane fractions of cells incubated with 100 but not 50 ng T₃/ml. Prepro-ET-1 mRNA stability, cell numbers and proliferation, measured by [³H]thymidine assays, remained unaffected in HUVECs after exposure to T₃. These data indicate thyroid hormone-induced upregulation of mRNA expression and protein synthesis of vWF, FN and ET-1, by PKC α-, βII- and ε-independent pathways, explaining, at least in part, increased plasma concentrations of endothelial proteins and peptides in the hyperthyroid state.

Journal of Endocrinology (1997) 154, 231–239

Abstract

Plasma clearance of insulin-like growth factors-I and -II (IGF-I and -II) and insulin-like growth factor-binding protein-2 (IGFBP-2) from lactating goats (n=4) was determined following a single intravenous injection of the corresponding ¹²⁵I-labelled human protein. Transfer of these proteins out of the vascular space was monitored by their subsequent appearance in mammary-derived lymph and milk. Clearance of ¹²⁵I-IGFBP-2 from circulation was 0·37 ± 0·06 ml/min/kg, which is markedly greater than that of ¹²⁵I-IGF-I or -II (0·11 ± and 0·12 ± 0·01 ml/min/kg respectively). This was also reflected in longer elimination half-lives for IGF-I (353 ± 6 min) and -II (254 ± 8 min) compared with IGFBP-2 (110 ± 9 min). Three hours after injection of the ¹²⁵I-labelled protein, the plasma:lymph ratio of trichloroacetic acid-precipitable radioactivity was 1·54 ±0·04, 3·3 ±0·6 and 4·1 ±0·4 for IGFBP-2, IGF-I and -II respectively. The form of ¹²⁵I-IGFBP-2 in lymph was not different from that of plasma. Elevation of plasma concentrations of IGFBP-2 by its intravenous infusion significantly decreased plasma half-life of both IGF-I and -II (251 ± 8 and 198 ±7 min respectively). Although the amount and rate of transfer of IGF into mammary-derived lymph was decreased slightly by IGFBP-2, concentrations eventually obtained were not different from control. However, secretion of IGFs into milk was significantly reduced by IGFBP-2, particularly in the case of IGF-I. These results are consistent with the ability of all three compounds to cross the vascular endothelium intact and of IGFBP-2 to decrease the uptake of IGF by mammary epithelium and subsequent secretion into milk. IGFBP-2 may well have acted to target plasma IGF towards non-mammary tissues, thus explaining the more rapid plasma clearance of IGFs in the presence of elevated IGFBP-2.

Journal of Endocrinology (1996) 150, 121–127

Abstract

We studied the expression of epidermal growth factor (EGF) receptor protein and messenger RNA (mRNA) in human fallopian tubes at three stages of the menstrual cycle: early follicular (n=3), late follicular (n=3) and luteal (n=3). Immunohistochemical studies in the ampullary portion of the tubes showed that specific staining was localized to the epithelium and the vascular endothelium. Staining of the epithelium was intense at the late follicular and luteal stages, while it was weak at the early follicular stage. ¹²⁵I-EGF binding study in the tubal plasma membranes revealed a class of high-affinity EGF receptors. Although dissociation constants were similar between the stages, numbers of binding sites at the late follicular and luteal stages were significantly (^P<0·01) greater than those at the early follicular stage. Western blotting showed that tubal plasma membranes contain M _r 170 000 EGF receptor protein. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage. Reverse transcription and polymerase chain reaction (RT-PCR) revealed that EGF receptor mRNA was expressed in all the 9 RNA samples (n=3 for each stage) from the tubal ampullary portion. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage (by a competitive PCR). Increase in the amounts of EGF receptor protein and mRNA occurred in association with an increase in serum oestradiol but not progesterone levels. Next we examined whether EGF receptor and its ligands (EGF and transforming growth factor a) are directly induced by oestrogen. We found that specific staining for EGF receptor and its ligands in the tubal epithelium was detected (by immunohistochemistry) in postmenopausal women with oestrogen replacement (n = 3), but not in subjects without oestrogen replacement (n=3). These results suggested that EGF receptors in the human tubal epithelium are expressed in relation to specific stages of the menstrual cycle and that the expression may be induced by oestrogen.

Journal of Endocrinology (1995) 147, 553–563

Angiogenesis is an important component in the development of thyroid goitre. Vascular endothelial growth factor (VEGF) represents a family of specific endothelial cell mitogens involved in normal angiogenesis and in tumour development. The purpose of this study was to determine the distribution of VEGF in thyroid tissues during goitre formation, and to study the actions of VEGF on the regulation of thymidine incorporation and iodine uptake by thyroid follicular cells. Goitre was induced in adult rats by administration of methimazole together with a low iodine diet. Thyroid from normal or goitrous rats was removed, fixed and sectioned. Immunocytochemistry performed for VEGF using the avidin-biotin system showed that VEGF is present in normal thyroid and is located mainly in the vascular endothelium and interfollicular stromal tissue. After administration of goitrogen for 2 weeks, which caused a two- to threefold increase in thyroid weight, staining of VEGF was less apparent within the interfollicular stroma, but strongly increased throughout the thyroid follicular and endothelial cells. Uptake of [125I] and incorporation of [3H]thymidine by Fisher rat thyroid cells (FRTL-5) were measured after 72 h culture with or without TSH or VEGF, or both. In the absence of TSH, incubation with VEGF caused a significant reduction in [3H]thymidine incorporation, but did not significantly alter [125I] uptake. Incubation with TSH (1 mU/ml) caused a fourfold increase in [3H]thymidine incorporation that was diminished by co-incubation with 10 ng/ml or greater VEGF. Similarly, 10 ng/ml or greater VEGF significantly reduced the ability of TSH to increase [125I] uptake. The antagonistic effects of VEGF on TSH-stimulated [3H]thymidine incorporation or [125I] uptake were significantly reduced in the presence of an anti-VEGF antiserum. A DNA fragment representing mRNA encoding the VEGF receptor, flt-1, was identified in FRTL-5 cells by reverse transcription PCR analysis, and the abundance of this fragment was increased in FRTL-5 cells cultured in the medium containing TSH (1 mU/ml) or fibroblast growth factor (FGF)-2 (25 ng/ml). These results indicated that VEGF and one of its receptors, Flt-1, are present in epithelial cells of the thyroid, and that VEGF could contribute to the regulation of development and function of thyroid epithelial cells.