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M. B. Kerr, K. Marshall and J. Senior

ABSTRACT

The uterotrophic response in the normotensive (CD) and the hypertensive (SHR) rat was compared in intact cyclic rats and in ovariectomized rats given oestradiol. The parameters measured were blood flow, and uterine wet and dry weights. In the cyclic animals blood flow to the oestogen target tissue varied throughout the oestrous cycle, peak flows being achieved at pro-oestrus; in the SHR rat, however, the pro-oestrous maximum was significantly attenuated compared with the CD rat. Uterine wet and dry weights were similar.

The temporal response to oestradiol in ovariectomized rats showed that in the CD rat the hyperaemic response peaked earlier than in the SHR rat, significant changes in terms of increased water imbibition also occurred more quickly in the CD strain. In both strains, uterine dry weight was the last parameter to be significantly increased, the maximum weight being attained more quickly in the SHR rat.

The results of this study indicated that it is the blood flow to the oestrogen target tissues of the uterus and vagina that is most susceptible to change with strain of rat.

Journal of Endocrinology (1992) 135, 263–269

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M. B. Kerr, K. Marshall and J. Senior

ABSTRACT

The role of thromboxane in the gravid normotensive (CD) and hypertensive (SHR) rat was investigated (by utilizing two thromboxane receptor-blocking drugs, EP092 and AH23848) both at mid-gestation and at term. The parameters examined were uterine blood flow (blood flows were measured by the microsphere technique) and uterine weight and placental blood flow at term, fetal mass and number.

At mid-gestation EP092 significantly (P< 0·005) increased uterine blood flow in both strains whilst the increases seen with AH23848 were not statistically significant. At term (day 22 in the CD and day 23 in the SHR rat) the antagonists increased uterine blood flow in the CD rats alone. However, at this time the antagonists caused an increase in placental blood flow in both strains.

Thromboxane appears to be involved in the regulation of uteroplacental blood flow. The observation that the antagonists were able to potentiate blood flow by mid-gestation may provide a clinical indication with respect to potential prophylactic use of this class of compounds in cases of pregnancy-induced hypertension in women.

Journal of Endocrinology (1992) 135, 257–261

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J. B. Kerr and R. M. Sharpe

ABSTRACT

The objective of this study was to examine the role of intertubular macrophages in modifying the response of the rat testis to stimulation by human chorionic gonadotrophin (hCG). The phagocytic activity of macrophages was stimulated by a unilateral intratesticular injection of polystyrene latex beads. Latex beads were engulfed by the resident macrophages and retained within their cytoplasm. Contralateral testes received injection of vehicle alone. A group of control rats was killed 3 days later; other groups received 100 IU hCG s.c. and the morphological and functional responses of the testes were examined 12, 24 and 48 h later. Spermatogenesis was unaffected in control rats, whereas in the testes of all hCG-treated rats leukocytes infiltrated into the intertubular tissue and the seminiferous tubules exhibited focal disruptions of spermatogenesis which were more severe in testes containing activated macrophages. Spermatogenic disruption was dependent upon the stage of the spermatogenic cycle, with the maximum tubule degeneration occurring at or near stages III and IX–XI. However these changes were not a consequence of androgen deprivation, since no consistent correlation was demonstrated between alterations in testosterone levels in testicular interstitial fluid and the accompanying damage to germ cells. It is concluded that hCG alone or in combination with activated macrophages induces an inflammatory-type response of the intertubular tissue and localized degeneration of the seminiferous epithelium. The antispermatogenic effects of hCG may have important implications for in-vivo investigations of Leydig cell function in laboratory animals and for the efficacy of hCG administration used in the clinical treatment of male hypogonadism.

Journal of Endocrinology (1989) 121, 285–292

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D. E. Kerr, B. Laarveld and J. G. Manns

ABSTRACT

The physiological importance of circulating as opposed to locally produced insulin-like growth factor-I (IGF-I) has not been determined. By using a passive immunoneutralization technique, our objectives were to evaluate the role of circulating IGF-I in the regulation of animal growth and pituitary GH content.

A monoclonal antibody (MAb) to IGF-I, generated in our laboratory, has an affinity (K a) of 0·13 litres/pmol for recombinant human IGF-I (rhIGF-I). Cross-reactivities of recombinant des-tripeptide IGF-I and recombinant bovine IGF-II were approximately 40 and 8% respectively. This MAb inhibited binding of purified hIGF-I to human placental membranes. In a radioimmunoassay based on displacement of 125I-labelled rhIGF-I from the MAb, displacement curves generated with dilutions of acid–gel chromatography extracts of guinea-pig serum and rhIGF-I standards were parallel.

Twenty-four, 3-week-old male guinea-pigs were treated with the IGF-I MAb, a bovine herpes virus-I (BHV-I) MAb (control MAb) or vehicle (phosphate-buffered saline) (n = 8 per group). Treatments were administered i.p. every 3 days for 24 days at a dose of 20 mg/kg body weight. Blood was obtained on day 23 (48 h after treatment) and on day 25 (24 h after treatment). In a liquid-phase assay, serum from the IGF-I MAb-treated group bound 38 ± 8% (mean ± s.e.m.) (day 23) and 56 ± 7% (day 25) of an 125I-labelled rhIGF-I trace at a final dilution of 1:10 000. Because of the development of an anti-mouse immune response in the guinea-pigs, these parameters would probably have been much greater during the first 2 weeks of the trial. Of the total IGF-I in serum, 50 ± 5% and 61±4% could be immunoprecipitated with an excess of rabbit anti-mouse immunoglobulin in samples from days 23 and 25 respectively. Comparisons between the groups treated with IGF-I MAb and BHV-I MAb revealed no significant differences in whole animal growth rate, growth of individual tissues, or pituitary GH content. Mean serum concentrations of IGF-I were 69 and 99% greater in IGF-I MAb-treated group than in the BHV-I MAb-treated group on days 23 and 25 respectively. These differences probably resulted from an extension of the half-life of IGF-I in serum of animals treated with the IGF-I MAb.

The lack of effect of treatment with the IGF-I MAb suggests that local production of IGF-I is generally sufficient to maintain normal growth or that local production or activity of IGF-I is increased in a compensatory fashion.

Journal of Endocrinology (1990) 124, 403–415

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S. Maddocks, J. B. Kerr, G. Allenby and R. M. Sharpe

ABSTRACT

During normal sexual maturation of the male rat there is a progressive change in the route of secretion of inhibin by the Sertoli cell, from a predominantly basal route of secretion in prepuberty to a predominantly apical route of secretion in adulthood. This change may be monitored by comparing the levels of inhibin in testicular (TV), spermatic and peripheral (PV) venous blood and the levels in testicular interstitial fluid (IF). This study has assessed the role of germ cells in effecting this change by assessing (a) the effect of total germ cell depletion by X-irradiation of the males in utero, and (b) the effect of selective germ cell depletion in adulthood using the testicular toxicant, methoxyacetic acid (MAA).

Female rats were X-irradiated on day 20 of gestation to produce male offspring whose testes were germ-cell deficient. Blood and IF samples were collected from groups of these offspring and age-matched controls at 35 and 100 days of age. In blood and IF samples, inhibin concentrations were significantly higher at 35 days of age than at 100 days. The absence of germ cells in X-irradiated animals did not affect the age-related fall in inhibin levels, nor the change in the predominant route of secretion of inhibin from the testis into blood. Testosterone was almost undetectable in 35-day-old controls, but was raised significantly by 100 days of age. In X-irradiated animals, testosterone levels were increased significantly at 35 days of age, and the levels in most samples were increased even more substantially by 100 days of age. However, PV levels of testosterone in 100-day-old X-irradiated animals were significantly lower than in controls. LH and FSH levels were raised in X-irradiated animals compared with their age-matched controls, but FSH levels in X-irradiated animals still fell with age, as in the controls.

The role of specific germ cell types in regulating the route of secretion of inhibin from the normal adult testis was studied after depletion (80–100%) of pachytene and later spermatocytes by a single oral administration of MAA (650 mg/kg) to adult rats. At 3 days after MAA treatment, coincident with the loss of pachytene spermatocytes, plasma inhibin levels were increased significantly in blood and IF samples, and this was associated with a dramatic change in the route of secretion of inhibin from the testis, with increased secretion of this peptide via the base of the Sertoli cell into IF and TV blood. However, previous studies suggest that this may be a consequence of direct stimulation by MAA, rather than the absence of pachytene spermatocytes. By 21 days after MAA treatment, when late-stage spermatids are absent, plasma inhibin levels were reduced significantly compared with controls, although the route of secretion of inhibin from the testis was comparable with that of controls. By 42 days, when a normal germ cell complement has been restored, plasma concentrations and the route of secretion of inhibin from the testis were similar to controls.

It is concluded that: (1) the presence of germ cells is not necessary for the maturational changes in the rate and route of secretion of inhibin by the Sertoli cell; these changes are most likely a consequence of formation of the blood–testis barrier, (2) in the normal adult testis, MAA-induced depletion of the most mature germ cell types affects the rate, but not the route, of inhibin secretion, whilst depletion of pachytene spermatocytes affects both parameters; the latter may indicate an early effect of MAA on the functional competence of the blood–testis barrier.

Journal of Endocrinology (1992) 132, 439–448

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G. P. Risbridger, J. B. Kerr and D. M. de Kretser

ABSTRACT

Leydig cells were selectively eliminated from the testis by treatment with the cytotoxin, ethane dimethane sulphonate. Rats were then made unilaterally cryptorchid and the morphological and functional response of the interstitial tissue in abdominal compared with scrotal testes was examined for up to 8 weeks. Regeneration of new Leydig cells occurred more rapidly in the interstitial tissue of abdominally cryptorchid testes compared with the interstitial tissue of contralateral scrotal testes. More rapid recovery of Leydig cells was coincident with significant increases in the bioactivity of a local factor(s), collected from interstitial fluid of cryptorchid testes, which stimulated testosterone production by isolated Leydig cells in vitro. These results support the theory that the production of a local factor(s) plays a role in the regeneration of Leydig cells and is affected by damage to the seminiferous epithelium.

J. Endocr. (1987) 112, 197–204

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J M Fleming, B J Leibowitz, D E Kerr and W S Cohick

Elucidating how mitogens facilitate epithelial/stromal interactions is critical given that mitogens regulate mammary gland development and function. IGF-I is a potent mammary cell mitogen that is locally produced in the mammary gland. Since IGF-binding proteins (IGFBPs) regulate IGF-I bioavailability, we characterized the cell-type-specific production of IGFBP in primary bovine mammary epithelial (BME) and fibroblast (BMF) cells. Cells were treated with IGF-I and mRNA levels were analyzed via quantitative real-time (qRT)-PCR and Northern blot analysis. Media conditioned by cells treated with IGF-I for 48 h were analyzed via ligand blotting with 125I-labeled IGF-I and -II and immunoblotting with specific IGFBP antibodies. A reciprocal regulation of IGFBP-3 and -5 by IGF-I was observed between the two cell types. IGF-I induced large dose-dependent increases in IGFBP-3 mRNA and protein levels in BME cells, while IGFBP-5 protein was barely detectable and mRNA levels were detectable only by qRT-PCR. In BMFs, IGF-I induced large increases in IGFBP-5 mRNA and protein while IGFBP-3 mRNA was only slightly increased by IGF-I treatment and the protein was difficult to detect. IGFBP-6 mRNA was detected by Northern blot analysis in both cell types but was not regulated by IGF-I. In BME cells, IGFBP-6 protein levels were readily detectable under basal conditions and were increased by IGF-I. Interestingly, IGFBP-6 protein could not be detected in media conditioned by BMFs. IGFBP-4 mRNA was readily seen by Northern blot analysis in BMFs, however qRT-PCR was required to detect IGFBP-4 mRNA in BME cells. IGF-I increased IGFBP-4 mRNA levels by 2-fold in both cell types. IGFBP-4 protein was only detectable in media conditioned by BME cells when stimulated by IGF-I. In contrast, IGFBP-4 was present in media conditioned by untreated BMFs but was not consistently increased by IGF-I treatment. This was explained by the finding that IGF-I stimulated proteolysis of IGFBP-4, as evidenced by the appearance of two immuno-responsive fragments of 18 and 14 kDa. This proteolysis was specific to IGFBP-4, and was not observed in BME cells. We confirmed the protease to be pregnancy-associated plasma protein A (PAPP-A) by immunoblotting with an antibody against human PAPP-A/proMBP (pro form of eosinophil major basic protein) complex. In vitro immuno-neutralization experiments showed that blocking PAPP-A prevented the ability of IGF-I to stimulate IGFBP-4 proteolysis. IGFBP-2 mRNA and protein levels were observed under basal conditions in both cell types, with no significant regulation by IGF-I. The analysis of cell-type-specific regulation of the IGF system in both primary mammary epithelial cells and stromal cells will assist in the characterization of the mechanisms behind the role of the IGF system in normal mammary physiology and ultimately breast cancer.

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R Riachy, B Vandewalle, S Belaich, J Kerr-Conte, V Gmyr, F Zerimech, M d'Herbomez, J Lefebvre and F Pattou

We examined whether 1,25 dihydroxyvitamin D(3) (1,25 D(3)), the active form of vitamin D involved in the regulation of the immune system, may also protect human pancreatic islet cells from destruction induced by cytokines. In this study, we specifically investigated the effect of 1,25 D(3) on oxidative stress and major histocompatibility complex (MHC) induction, both implicated in cytokine-induced islet cell dysfunction and destruction. We also investigated the effects of 1,25 D(3) on interleukin (IL)-6, a pleiotropic cytokine implicated in the pathogenesis of immunoinflammatory disorders. Human pancreatic islets, isolated from heart-beating donors, were treated with a combination of three cytokines, IL-1beta+tumor necrosis factor alpha+interferon gamma, in the presence or absence of vitamin D, and compared with with untreated control cells. Metabolic activity was assessed by cell viability and insulin content. Oxidative stress was estimated by heat shock protein 70 (hsp70) expression, cell manganese superoxide dismutase (MnSOD) activity and nitrite release, a reflexion of nitric oxide (NO) synthesis. Variation of immunogenicity of islet preparations was determined by analysis of the MHC class I and class II transcripts. Inflammatory status was evaluated by IL-6 production. After 48 h of contact with cytokines, insulin content was significantly decreased by 40% but cell viability was not altered. MHC expression significantly increased six- to sevenfold as well as NO and IL-6 release (two- to threefold enhancement). MnSOD activity was not significantly induced and hsp70 expression was not affected by the combination of cytokines. The addition of 1,25 D(3) significantly reduced nitrite release, IL-6 production and MHC class I expression which then became not significantly different from controls. These results suggest that the effect of 1,25 D(3) in human pancreatic islets cells may be a reduction of the vulnerability of cells to cytotoxic T lymphocytes and a reduction of cytotoxic challenge. Hence, 1,25 D(3) might play a role in the prevention of type 1 diabetes and islet allograft rejection.

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G Tachas, S Lofthouse, C J Wraight, B F Baker, N B Sioufi, R A Jarres, A Berdeja, A M Rao, L M Kerr, E M d’Aniello and M J Waters

Diabetic retinopathy and acromegaly are diseases associated with excess action of GH and its effector IGF-I, and there is a need for improved therapies. We have designed an optimised 2′-O-(2-methoxyethyl)-modified phosphorothioate oligodeoxynucleotide, ATL 227446, and demonstrated its ability to suppress GH receptor mRNA in vitro. Subcutaneous injections of ATL 227446 reduced GH receptor mRNA levels, GH binding activity and serum IGF-I levels in mice after seven days of dosing. The reduction in serum IGF-I could be sustained for over ten weeks of dosing at therapeutically relevant levels, during which there was also a significant decrease in body weight gain in antisense-treated mice relative to saline and mismatch control-treated mice. The findings indicate that administration of an antisense oligonucleotide to the GH receptor may be applicable to human diseases in which suppression of GH action provides therapeutic benefit.