bluuebunny/crossref_metadata_2025 huggingface dataset:
A possible role for D8/PSF-A-like sequences in lactotroph versus somatotroph expression of the human prolactin gene
Authors: Leite V , Cardoso E A , Bock M E , Sobrinho L G , Cattini P A
Published: December 2004

The transcription factor GHF-1/Pit-1 is essential for the expression of GH and prolactin (PRL) by somatotrophs and lactotrophs respectively. However, PRL is not expressed in mature somatotrophs despite the presence of GHF-1/Pit-1. A possible mechanism is the presence of a somatotroph-specific repressor in the 5′-flanking sequences of the PRL gene. The region −3500/−1750 of the human (h) PRL gene is associated with negative regulatory activity and contains an element, designated D8, that resembles repressor PSF-A sequences which are located in the distal upstream region of placental members of the human GH family. An internal deletion of D8 sequences resulted in a significant stimulation of promoter activity in somatotroph GC (P<0·005) and somatolactotroph-like GH3 and GH4C1 cells (P<0·05), but not lactotroph-like 235–1 cells after gene transfer. However, D8 binding was observed by nuclease protection with lactotroph- as well as somatotroph-like cell nuclear protein. Although proteins that bind to the D8 element appear ubiquitous, this element does yield tissuespecific complexes in mobility shift assays. Further, competition studies do not suggest an interaction between GHF-1/Pit-1 and D8 proteins. The hPRL D8 element was inserted upstream of a thymidine kinase promoter and used to transfect pituitary and non-pituitary HeLa cells, to assess intrinsic repressor activity and/or promoter specificity. Although no repression was observed, a significant ninefold increase in expression was observed in HeLa cells (P<0·001) which was at least twofold greater than observed in any of the pituitary cell lines tested. These results implicate D8 in the somatotroph-specific repression of hPRL; however, they also suggest that D8 can act as a stimulator as well as a repressor, depending on the interaction of a ubiquitous D8 factor forming promoter and cell-specific complexes with other elements/factors.

Journal of Endocrinology (1996) 149, 473–483
New insights into zonal differentiation of adrenal autotransplants in the rat: an immunohistochemical study
Authors: Vendeira P , Pignatelli D , Neves D , Magalhães M M , Magalhães M C , Ho M M , Vinson G P
Published: December 2004

Adrenal gland autotransplantation, an interesting model of adrenal regeneration, provides the reconstruction of distinct functional and morphological zonae. An immunohistochemical study of the adrenal gland of adult male rats after autotransplantation and endothelin-1 (ET-1) stimulation was carried out. The technique involved total adrenalectomy and immediate autotransplantation of small adrenal pieces under the skin of the dorsal region. The animals were killed 90 days after the autotransplantation and 1 h after intravenous ET-1 administration. Sections of recovered adrenal grafts were incubated with IZAb, a monoclonal antibody which interacts with an antigen (IZAg) predominantly found in rat adrenal inner zones. Saline-treated control autotransplanted animals showed IZAb immunostaining in almost all adrenocortical tissue, with the exception of small clusters of cells beneath the capsule. ET-1-treated animals exhibited an extended zone devoid of immunostaining and located in the subcapsular area. In addition, ET-1-stimulated animals showed significant increases in aldosterone as well as corticosterone concentrations in plasma. These results revealed that ET-1 stimulated the development of an extended subcapsular zone lacking IZAg expression, an effect that suggests its role in zona glomerulosa induction in these animals.

Journal of Endocrinology (1996) 149, 497–502
Molecular forms of GnRH in three model fishes: rockfish, medaka and zebrafish
Authors: Powell J F F , Krueckl S L , Collins P M , Sherwood N M
Published: December 2004

Three species of fish have become important in the study of reproduction and development. Rockfish are a model for developmental studies of live-bearing perch-like fish, whereas medaka and zebrafish are models for developmental and genetic studies. The forms of GnRH are identified in the brains of each of these fish and in the pituitary of the rockfish to investigate the role of GnRH in reproduction. Here, we report that grass rockfish (Sebastes rastrelliger) have three forms of GnRH in brain extracts as determined by HPLC elution position and RIA. These forms are identified as sea bream GnRH, chicken GnRH-II and salmon GnRH. In contrast, only two forms of GnRH were detected in brain extracts of medaka (Oryzias latipes) and zebrafish (Brachydanio rerio): salmon GnRH and chicken GnRH-II. Rockfish is distinct from medaka and zebrafish in that the most abundant form of GnRH in the rockfish pituitary is sea bream GnRH, whereas this form is absent in the other two fishes. The identification of sea bream GnRH in the rockfish brain and pituitary extracts indicates that the phylogenetic emergence of sea bream GnRH is earlier than the order Perciformes.

Journal of Endocrinology (1996) 150, 17–23
Trophoblastic giant cells of the mouse placenta contain calbindin-D9k but not the vitamin D receptor
Authors: Shamley D R , Veale G , Pettifor J M , Buffenstein R
Published: December 2004

The effects of vitamin D deficiency on the ontogeny of calcium-binding proteins (CaBPs) and the vitamin D receptor (VDR) in the placenta and yolk sac of the mouse were examined. Maternal vitamin D status did not affect the time of appearance of CaBP-D9k (9 kDa) in the yolk sac endoderm or trophoblastic giant cells (TGCs) of the placenta. VDRs were undetectable in TGCs and yolk sac endoderm, but were present in the intraplacental yolk sac. Since yolk sac endoderm and TGCs contain CaBP but not VDR, it is unlikely that CaBP synthesis and/or activity in these cells is controlled by vitamin D. The TGCs, therefore, may be involved in vitamin D-independent transplacental transfer of calcium.

Journal of Endocrinology (1996) 150, 25–32
Recombinant FSH-induced follicle development in immature rats treated with an LHRH antagonist: a direct effect of RU486 on follicular atresia
Authors: Uilenbroek J Th J , Kramer P , van Leeuwen E C M , Karels B , Timmerman M A , de Jong F H , de Leeuw R
Published: December 2004

To investigate whether the progesterone antagonist RU486 has a direct effect on ovarian function, it was administered to immature female rats rendered hypogonadotrophic by administration of an LHRH antagonist and in which follicle development was stimulated by recombinant human FSH (recFSH).

In the first experiments the effects of LHRH antagonist and recFSH on follicle growth were evaluated. Female rats of 22 days of age were injected with an LHRH antagonist (Org 30276; 500 μg/100 g body weight) every other day. This treatment resulted in a tenfold decrease in serum LH concentrations and a twofold decrease in serum FSH concentrations at day 30 and caused a reduction in the number and size of antral follicles. Treatment with recFSH (Org 32489) twice daily from day 26 for 4 days in a total dose ranging from 5 to 20 IU/animal increased the number and size of antral follicles in a dose-related manner and resulted after 20 IU recFSH in a tenfold increase in the concentration of inhibin in serum and ovaries at day 30. Once it was established that LHRH antagonist treatment in immature rats could be used to study the effects of gonadotrophins or steroids on follicle function, this animal model was used to study the effects of RU486 on the ovary. RU486 was administered (twice daily for 4 days, 1 mg/injection) to LHRH antagonist-treated rats in which follicular growth and differentation were stimulated by 10 IU recFSH or by 10 IU recFSH plus 0·5 IU human chorionic gonadotrophin (hCG). RU486 had no effect on circulating levels of LH and FSH, but stimulated follicular atresia both in rats treated with recFSH alone and in rats treated with recFSH and hCG. Inhibin concentrations both in serum and ovaries were significantly increased after hCG treatment. RU486, however, did not increase inhibin in the rats treated with recFSH and in those treated with recFSH and hCG.

In summary, the present study has demonstrated that (1) immature rats treated with an LHRH antagonist can be used to study the effects of gonadotrophins and steroids on follicular function and (2) RU486 has a direct stimulatory effect on follicular atresia.

Journal of Endocrinology (1996) 150, 85–92
A study of the luteolytic mechanism of the antiprogesterone RU486 during the late-luteal phase in pseudopregnant rats
Authors: Nakamura Y , Tamura H , Ono M , Shimamura K , Sugino N , Numa F , Ueda K , Kato H
Published: December 2004

The purpose of this study was to examine the possible mechanism through which RU486 induces luteolysis during the late-luteal phase in pseudopregnant (PSP) rats. PSP rats received a subcutaneous injection of RU486 in sesame oil (5 mg/kg body weight) or sesame oil alone once a day between day 9 and day 11 of pseudopregnancy. Serial blood samples were collected on days 5, 9, 10, 11 and 12 and assayed for progesterone content. To examine the possible action of RU486 through a uterine and/or a pituitary (prolactin-dependent) mechanism, PSP rats and chronic hysterectomized PSP rats which had been hysterectomized before PSP induction received a subcutaneous injection of RU486 in sesame oil (5 mg/kg body weight), sesame oil alone, prolactin in 50% polyvinylpyrrolidone (15 IU/day), or RU486 and prolactin once a day between day 9 and day 11 of pseudopregnancy. Serial blood samples were collected on days 5, 9, 10 and 11 and assayed for progesterone content. Blood samples were also collected at 0400 h on day 12 and used for prolactin and progesterone determinations. To examine the direct effect of RU486 on corpus luteum and/or pituitary, hysterectomized rats underwent hypophysectomy and pituitary autotransplantation on dioestrus 1 and received a subcutaneous injection of RU486 in sesame oil or sesame oil alone for 3 days between day 21 and day 23 after surgery. Serial blood samples were collected on days 10, 21, 22, 23 and 24 and assayed for progesterone and prolactin contents.

In ordinary PSP rats, serum progesterone levels were significantly (P<0·01) lower in the RU486-treated group than in the control group (9 ± 1 vs 53 ± 7 ng/ml; mean ± s.e.m.) on day 11. Serum prolactin levels at 0400 h on day 12 of pseudopregnancy were significantly (P<0·05) lower in the RU486-treated group than in the control group (16 ±4 vs 154 ±44 ng/ml; mean ± s.e.m.). The concomitant prolactin treatment reversed the luteolytic effects of RU486 on day 11 of pseudopregnancy. In hysterectomized PSP rats, RU486 also suppressed serum prolactin levels, and the concomitant prolactin treatment again reversed the luteolytic effects of RU486. In hysterectomized rats which were hypophysectomized and pituitary autotransplanted, RU486 treatment did not induce any significant changes in serum progesterone and prolactin levels.

These results indicated that RU486 induced luteolysis during the late-luteal phase in PSP rats by suppressing prolactin secretion via a hypothalamic mechanism.

Journal of Endocrinology (1996) 150, 93–98
GH inhibition of lipogenesis and stimulation of lipolysis in sheep adipose tissue: involvement of protein serine phosphorylation and dephosphorylation and phospholipase C
Authors: Vernon R G
Published: December 2004

The intracellular signalling systems involved in the chronic insulin-antagonistic, anti-lipogenic effects and also the lipolytic effect of GH have been investigated in sheep adipose tissue in an in vitro tissue culture system. During culture, chronic exposure to GH decreased the rate of lipogenesis and prevented the increase in lipogenesis induced by insulin. GH also increased glycerol release into the culture medium. GH had no acute, insulin-like effect on lipogenesis in sheep adipose tissue.

Pretreatment with phorbol ester to down-regulate isoforms of protein kinase C or addition of the protein serine kinase inhibitor staurosporine decreased the anti-lipogenic effect of GH while the protein serine kinase inhibitor H7 eliminated it completely. Pretreatment with phorbol ester or addition of H7 also decreased the insulin-antagonistic effect of GH on lipogenesis. Addition of the protein serine phosphatase inhibitor okadaic acid or the phosphatidyl choline phospholipase C inhibitor D609 both diminished the anti-lipogenic and insulin-antagonistic effects of GH.

Chronic exposure of adipose tissue to GH had no effect on the total activity of acetyl CoA carboxylase or its activation status but it did diminish the increase in activation status induced by insulin. H7 and okadaic acid also diminished the increase in activation status of acetyl CoA carboxylase induced by insulin but did not alter the effect of GH on this variable. Okadaic acid decreased total acetyl CoA carboxylase activity.

Pretreatment with phorbol ester or the addition of H7, staurosporine or okadaic acid increased glycerol release into the culture medium to the same extent as GH itself; the effects of GH and these various agents were not additive.

These studies suggest that the anti-lipogenic, insulin-antagonistic effects of GH involve both protein serine kinases and phosphatases, possibly including one or more isoforms of protein kinase C, and a phosphatidyl choline-specific phospholipase C. Comparison with studies by others on the GH enhancement of preadipocyte differentiation and prolactin stimulation of lipogenesis in mammary tissue suggests involvement of protein kinase C at an early stage in all three systems. In contrast, effects of okadaic acid vary with the system, suggesting the involvement of protein serine phosphatase activity in a late stage of the action of GH. The effects of GH on lipogenesis and lipolysis do not occur via identical mechanisms.

Journal of Endocrinology (1996) 150, 129–140
Regulation of insulin-like growth factor-binding protein-4 protease activity by estrogen and parathyroid hormone in SaOS-2 cells: implications for the pathogenesis of postmenopausal osteoporosis
Authors: Kudo Y , Iwashita M , Itatsu S , Iguchi T , Takeda Y
Published: December 2004

The cellular mechanisms involved in the accelerated bone loss occurring in association with estrogen deprivation as seen following the menopause are not fully understood. Insulin-like growth factor-I (IGF-I) is the local regulator of osteoblasts and one of its binding proteins, insulin-like growth factor-binding protein-4 (IGFBP-4), binds to IGF-I and suppresses biological activity. Previous studies have shown that the binding activity of IGFBP-4 in the conditioned medium of parathyroid hormone (PTH)-treated SaOS-2 osteoblastic-like cells is enhanced twofold and that this PTH-enhanced IGFBP-4 binding activity is abolished by 17β-estradiol. Levels of IGFBP-4 in the conditioned medium have been reported to be regulated not only at the level of production but also at the level of degradation which is catalyzed by a protease that specifically cleaves IGFBP-4. We have, therefore, studied the effects of 17β-estradiol and PTH on IGFBP-4 protease activity using SaOS-2 cells. SaOS-2 cells produce a protease that specifically cleaves IGFBP-4 into two fragments of approximately 18 and 14 kilodaltons. IGFBP-4 protease activity in the conditioned medium from PTH-treated cells was suppressed, while this PTH-induced suppression of protease activity was reversed by the addition of 17β-estradiol to the cultures. IGFBP-4 proteolytic activity was stimulated by IGF-I or IGF-II added exogenously and was inhibited by EDTA or protease inhibitors. IGFBP-4 proteolyzed in the conditioned medium from cells treated with PTH and 17β-estradiol was less effective at inhibiting IGF-I-stimulated [3H]thymidine incorporation into DNA compared with that proteolyzed in the conditioned medium from PTH-treated cells. The simplest explanation is that 17β-estradiol suppressed the inhibitory effect of PTH on osteoblastic activity by inhibiting the PTH-induced suppression of IGFBP-4 protease activity.

Journal of Endocrinology (1996) 150, 223–229
Pituitary and ovarian expression of the endogenous follicle-stimulating hormone (FSH) subunit genes and an FSH β-subunit promoter-driven herpes simplex virus thymidine kinase gene in transgenic mice; specific partial ablation of FSH-producing cells by antiherpes treatment
Authors: Markkula M , Kananen K , Klemi P , Huhtaniemi I
Published: December 2004

The ovarian expression of the endogenous follicle-stimulating hormone β-subunit (FSHβ) and common α-subunit (Cα) genes, and a herpes simplex virus thymidine kinase (tk) transgene, driven by a 2·3 kb bovine FSHβ promoter, was studied in normal and transgenic (tg) mice. tk functions not only as a neutral reporter that enables the study of the promoter function but also as an exogenously inducible toxigene. Reverse transcriptionPCR followed by Southern blot hybridization with a nested probe was used to show the expression of the gene at the mRNA level. Common α-subunit mRNA was detected in the pituitary gland and ovaries of normal adult mice. We have previously detected endogenous FSHβ and tg tk mRNAs in the mouse pituitary, testis and ovary. In this study, the cellular localization of the corresponding proteins was visualized by immunocytochemistry. In normal mouse ovaries a positive reaction with FSHβ and Cα antisera was seen in some of the corpora lutea and most prominently in the interstitial cells. A positive reaction with the tk antiserum was seen in the same cell types of tg mouse ovaries, but not in those of non-tg mice. Cell-ablation-inducing treatment (gancyclovir, 20 mg/kg per day, for 14 days) of tg female mice reduced pituitary FSH concentrations by 52% (P<0·05) but did not affect pituitary LH or plasma gonadotropins compared with non-tg females treated in the same way. A longer period of cell ablation induction (acyclovir 400 mg/kg per day, for 21 days) reduced not only pituitary but also plasma FSH concentrations (55 and 57% respectively; P<0·05) without affecting LH. This treatment also reduced ovarian weight by 38% (P<0·01). In conclusion, our results show first that the endogenous FSHβ and Cα proteins are produced in the mouse ovary. Hence, endogenously synthesized FSH or its subunits may have a role in the paracrine regulation of ovarian function. Secondly, the FSHβ promoter directs the expression of tg tk in the pituitary gonadotrope cells, as shown by specific but partial ablation of FSH-producing cells after induction by gancyclovir and acyclovir. In the ovary, tk protein was localized to the same compartments as the endogenous gonadotropin subunit proteins. This further confirms our finding of ovarian expression of the FSH subunit genes.

Journal of Endocrinology (1996) 150, 265–273
Activation of protein kinase A and protein kinase C mediates the depolarising effect of LH in ovine cumulus-corona cells
Authors: Mattioli M , Barboni B , Gioia L
Published: December 2004

We have studied the changes in membrane potential induced by LH in cumulus and granulosa cells isolated from sheep antral follicles. The investigation was carried out by using a non-invasive technique based on the use of a membrane potential sensitive probe, bis-oxonol. The membrane potential of mural granulosa cells was totally unaffected by LH, while that of cumulus or corona cells showed a marked depolarisation, starting 2–3 min after the addition of the hormone and plateauing after 5–10 min. None of the cells tested reacted to FSH.

In the second part of the experiment the role of protein kinase A (PKA) and protein kinase C (PKC) in mediating the effect of LH was studied. The selective activation of PKA or PKC induced in cumulus-corona cells a rapid hyperpolarisation due to increased Cl and K conductance respectively. By contrast, the simultaneous activation of the two kinases induced a rapid membrane depolarisation due to the progressive decrease in K conductance. The activation of each kinase or their combined stimulation did not induce any change in the membrane potential of mural granulosa cells. These data demonstrated that LH has a depolarising effect regionally circumscribed to cumulus-corona cells and that this depolarisation depends on a reduction of K conductance caused by the activation of PKA and PKC.

Journal of Endocrinology (1996) 150, 445–456
Labour-associated increase in interleukin-1 α release in vitro by human gestational tissues
Authors: Laham N , Brennecke S P , Bendtzen K , Rice G E
Published: December 2004

The aims of this study were to investigate the concentration and release of interleukin-1α (IL-1α) at the time of human term labour, and to study the regulation of IL-1α release from human gestational tissue explants by bacterial endotoxin. Immunoreactive IL-1α concentrations in maternal plasma, amniotic fluid and conditioned media from human amniotic, choriodecidual and placental explants were quantified before and after spontaneous term labour-onset and delivery. Furthermore, the effects of a bacterial endotoxin, lipopolysaccharide (LPS), on the release of IL-1α from human gestational tissue explants over a time course of 24 h (n=3) and LPS concentrations ranging from 10–107 pg/ml (n=3) were investigated. IL-1α concentrations in maternal plasma and amniotic fluid did not change significantly with spontaneous term labour-onset. In contrast, IL-1α was released in detectable amounts from human amniotic and choriodecidual explants only in association with term labour-onset and delivery. Similarly, placental release of IL-1α was increased significantly in explant cultures in association with term labour-onset and delivery. LPS increased IL-1α release significantly only from human placental explants from both term not-in-labour and term after-labour tissues. The data demonstrate differential regulation of IL-1α release from human gestational tissues in association with labour and LPS treatment and the observations support the hypothesis that the labour-associated increase in IL-1α release from the fetal membranes is independent of exposure to bacterial endotoxin.

Journal of Endocrinology (1996) 150, 515–522
The localisation and characterisation of insulin-like growth factor-I receptors and the investigation of melatonin receptors on the hair follicles of seasonal and non-seasonal fibre-producing goats
Authors: Dicks P , Morgan C J , Morgan P J , Kelly D , Williams L M
Published: December 2004

To define the hormonal influences that are directly involved in the hair follicle cycles of animals with differing patterns of fibre growth and moulting, we have investigated the possible presence of IGF-I and melatonin receptors on the dermis and hair follicles of cashmere and Angora goats, sampled in February, March and June, using quantitative in vitro autoradiography. The presence of IGF-I receptors in the dermis of both breeds of goat was determined using cryostat sections incubated with 50 pm 125I-labelled IGF-I in the presence or absence of 50 nm IGF-I. Sections of the growing tip of deer antlers containing the cartilaginous zone, a tissue known to contain high concentrations of specific IGF-I receptors, were used as a positive control. As the production of antler velvet uniquely involves the generation of hair follicles de novo, the presence of IGF-I receptors in the velvet-producing region was also investigated. In both breeds of goat, specific 125I-IGF-I binding was localised over the inner and outer root sheath, the matrix, the germinal matrix, the dermal papilla and the sebaceous glands and satisfied the basic kinetic criteria considered to be representative of a specific IGF-I receptor. Analysis of saturation isotherms using a one-site binding model revealed dissociation constants (Kd) in the range 0·1–0·9 nm and theoretical maximal numbers of binding sites (Bmax) between 21·4 and 45·6 fmol/mg tissue. Kd and Bmax values derived from cashmere and Angora goats sampled at different times of the year did not differ significantly between breeds or sampling times. Specific 125I-IGF-I binding was also localised to the developing follicles on the deer antler dermis. The presence of melatonin receptors within the goat dermis was also investigated. Sections were incubated with 100 pm 2-[125I]iodomelatonin with or without 0·1 μm melatonin, along with sections of sheep pars tuberalis which are known to contain high levels of high-affinity melatonin receptors. No displaceable 2-[125I]iodomelatonin binding was found on any sections of the cashmere or Angora skin analysed. It is therefore concluded that melatonin receptors are not present on the hair follicles or associated structures. IGF-I receptors are present on the hair follicle and sebaceous gland and may be involved in the growth of both seasonally and non-seasonally produced fibre and in the development of antler velvet.

Journal of Endocrinology (1996) 151, 55–63
In vitro activation of fish phagocytic cells by GH, prolactin and somatolactin
Authors: Sakai M , Kobayashi M , Kawauchi H
Published: December 2004

The activation of rainbow trout (Oncorhynchus mykiss) phagocytic cells by chum salmon GH, prolactin (PRL) and somatolactin (SL) was investigated in vitro. Rainbow trout kidney leucocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng of each hormone/ml and the production of superoxide anion was measured using reduction of nitroblue tetrazolium (NBT) and ferricytochrome C. Macrophages incubated with 10–100 ng GH or PRL/ml showed significantly enhanced production of superoxide anion in both the NBT and ferricytochrome C tests compared with control macrophages (without hormone). SL did not induce enhanced production of superoxide anion in macrophages. The phagocytic cells treated with GH or PRL also showed increased phagocytic activity and phagocytic index. However, the cells treated with SL showed no enhancement of phagocytic activity or index. These results indicate that GH and PRL stimulate macrophages in vitro.

Journal of Endocrinology (1996) 151, 113–118
Expression of prostaglandin G/H synthase-1 and -2 in ovine amnion and placenta following glucocorticoid-induced labour onset
Authors: McLaren W J , Young I R , Wong M H , Rice G E
Published: December 2004

Parturition in the sheep is preceded by an increase in the synthesis of prostaglandins by intrauterine tissues. Prostaglandin G/H synthase (PGHS) is the central enzyme involved in prostanoid production. Its expression is enhanced during late gestation in the ewe. Recent studies have identified two PGHS isozymes, termed PGHS-1 and PGHS-2. The labour-associated expression of the two isozymes of PGHS in the sheep has not been characterized.

This study investigated the changes in expression of immunoreactive PGHS-1 and PGHS-2 in ovine amnion and placenta following glucocorticoid-induced labour. Ewes underwent surgery to implant fetal and maternal vascular cannulae and uterine electromyogram electrodes between 118 and 125 days of gestation. Fetal sheep were administered either the glucocorticoid betamethasone (n=5) or saline (control n=6) by direct transabdominal intrafetal injection. Ewes from the betamethasone-injected group were killed in the first stage of labour as indicated by uterine electromyographic activity. Ewes from the saline-injected group were killed at the same time to obtain age-matched control tissue. The time taken to euthanasia following induced-labour onset in the glucocorticoid-injected animals was 56·6 ± 0·8 h post-injection.

Plasma endocrine profiles in the maternal and fetal circulation following glucocorticoid injection were comparable to those observed following normal spontaneous delivery. At post-mortem, amnion and cotyledons were collected in liquid N2 and stored at −70 °C. Solubilized tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PGHS-1 and PGHS-2 isozymes. Fetal amnion contained PGHS-1 isozyme at day 133 of gestation, as demonstrated in the saline-injected animals. Slightly higher PGHS-1 immunoreactivity was observed following induced-labour onset, although this did not reach statistical significance (P>0·05). PGHS-2 enzyme was not detectable in amnion. PGHS-2 expression was also not induced following labour onset.

In contrast, PGHS-2 demonstrated enhanced expression following glucocorticoid-induced labour in ovine cotyledon. This tissue contained PGHS-1 enzyme, but immunoreactive levels were minimal and demonstrated limited regulation at labour.

These data suggest that the previously reported rise in placental PG production at term in the sheep is predominantly due to increased expression of the PGHS-2 isozyme. This suggests that PGHS-2 contributes to PG production at term labour in sheep or is induced by the mechanisms controlling ovine parturition. PGHS-1 isozyme is produced constitutively in ovine amnion and may contribute to the gestational increase in PG formation by intrauterine tissues.

Journal of Endocrinology (1996) 151, 125–135
Expression of tissue inhibitor of metalloproteinases-1 in the primate ovary during induced luteal regression
Authors: Duncan W C , Illingworth P J , Fraser H M
Published: December 2004

Although tissue inhibitor of metalloproteinases-1 (TIMP-1) is one of the major secretory products of the corpus luteum, the functional significance of this is not clear. In addition to its role as a specific inhibitor of the matrix metalloproteinase enzymes involved in tissue remodelling, it has recently been suggested that TIMP-1 is also a potent stimulator of steroidogenesis in vitro. However, in the ruminant, TIMP-1 expression increases during luteal regression. This study sought to determine (i) the effect of induced luteal regression on ovarian TIMP-1 expression in the primate and (ii) the expression of TIMP-1 in other steroidogenic and non-steroidogenic tissues.

Marmoset ovaries were studied on day 10 of the normal luteal phase and 12 and 24 h after induced luteolysis, with either gonadotrophin-releasing hormone (GnRH) antagonist or prostaglandin F2α analogue. Ovaries from different stages of the normal ovarian cycle were also studied. Expression of TIMP-1 was investigated by isotopic in situ hybridisation. TIMP-1 expression was also examined in a wide range of other marmoset tissues by Northern blotting and in situ hybridisation. TIMP-1 was found to be highly expressed in the marmoset corpus luteum. Luteolysis induced with either prostaglandin F2α or GnRH antagonist was associated with a significant fall in TIMP-1 expression in luteal tissue. TIMP-1 mRNA was also localised to ovarian follicles throughout the ovarian cycle. Expression occurred in the thecal layer of smaller follicles (<1·5 mm) and the granulosal layer of larger pre-ovulatory follicles. In atretic follicles, TIMP-1 was highly expressed at the interface between the thecal and granulosal cells. TIMP-1 was found to be predominantly expressed in steroidogenic tissues, particularly the ovary, adrenal and placenta.

These data support a role for changes in TIMP-1 expression in tissue remodelling in the ovary and are consistent with an additional function of TIMP-1 as a facilitator of steroidogenesis.

Journal of Endocrinology (1996) 151, 203–213
Introducing a point mutation identified in a patient with pituitary resistance to thyroid hormone (Arg 338 to Trp) into other mutant thyroid hormone receptors weakens their dominant negative activities
Authors: Ando S , Nakamura H , Sasaki S , Nishiyama K , Kitahara A , Nagasawa S , Mikami T , Natsume H , Genma R , Yoshimi T
Published: December 2004

Clinical resistance to thyroid hormone (RTH) has been classified into generalized resistance to thyroid hormone (GRTH) and pituitary resistance to thyroid hormone (PRTH) types. Since similar mutations have been identified in tri-iodothyronine (T3) receptor (TR) β gene in GRTH and PRTH, and since considerable overlap has been seen in the clinical manifestations in patients with GRTH and PRTH, two subtypes of RTH are now considered to be a continuous spectrum with the same genetic defect. A point mutation at amino acid Arg 338 to Trp (R338W) which we identified in a patient with PRTH is very interesting, since R338W has been found in several other patients with PRTH, raising the possibility that this mutation may tend to associate with a phenotype of PRTH.

In our previous study, we found that R338W had relatively less impaired transcriptional potency, weaker dominant negative activity on various T3 response elements and poor homodimer formation, as compared with another GRTH mutant. In this study, to investigate the functional properties of R338W further, especially in terms of the relation between transcriptional activity and dimer formations, we introduced the R338W mutation into the mutant receptors, K443E and F451X, constructing the double mutants, R338W/K443E and R338W/F451X. Both R338W/K443E and R338W/F451X showed negligible T3 binding and transcriptional activities. The dominant negative activities of K443E and F451X were, however, significantly weakened by introducing the R338W mutation. As a control, a double mutant G345R/K443E was constructed by introducing a point mutation, G345R, located in the same exon 9 as R338W, into the K443E mutant. Dominant negative activity did not differ between G345R/K443E and K443E. Homodimer formation was significantly reduced in the double mutants containing R338W, but not G345R.

In summary, introducing the R338W mutation, but not G345R, into the mutant TR significantly weakened the dominant negative activity, despite further impairment of the T3 binding and transcriptional activities.

Journal of Endocrinology (1996) 151, 293–300
Glyceryl trinitrate enhances nitric oxide mediated perfusion within the equine hoof
Authors: HINCKLEY K. A. , FEARN S. , HOWARD B.R. , HENDERSON I.W.
Published: December 2004

Laminitis, a microvascular disease of the equine hoof leads to severe lameness. Exogenous iv 1-arginine and transdermal nitric oxide donors, such as GTN, applied to the pasterns improve lameness during acute laminitis. Near Infrared spectroscopy in an earlier study showed haemostasis and ischaemia in the hoof during acute laminitis, both were alleviated by 1-arginine. Quantitative NIRS in the present study shows that transdermal GTN increases blood flow in the equine hoof. It is concluded that glyceryl trinitrate enhances nitric oxide mediated perfusion within the equine hoof in normal and chronically laminitic horses and ponies.
Localisation of mRNA for interleukin-1 receptor and interleukin-1 receptor antagonist in the rat ovary
Authors: Wang L-J , Brännström M , Cui K-H , Simula A P , Hart R P , Maddocks S , Norman R J
Published: December 2004

Interleukin-1 (IL-1) is a multifunctional cytokine with profound effects on ovarian function. The effects of IL-1 on ovarian steroidogenesis have been demonstrated in several species. IL-1 mRNA levels are increased in the thecal layer of the ovulating follicle and IL-1β has been shown to induce ovulations in vitro. In this study we have investigated the presence and distribution of the mRNAs for type I IL-1 receptor (IL-1RtI) and for the naturally occurring IL-1 receptor antagonist (IL-1ra) in ovaries of adult cycling rats, to elucidate the target cells for IL-1 action. We have demonstrated the presence of mRNA for both substances by in situ hybridisation and reverse transcription PCR. mRNA for IL-1RtI was not found in primordial follicles but was abundant in the granulosa and thecal layer in developing follicles with stronger signals in the granulosa layer. In the preovulatory and ovulatory follicles, there was a further increase in the signal for IL-1RtI mRNA in the thecal layer compared with the granulosa layer. Corpora lutea were weakly positive at all stages and atretic follicles were largely negative. No mRNA was detected in oocytes of any stage. mRNA for IL-1ra showed a similar distribution to that of IL-1RtI. The changes in distribution suggest an action of IL-1 on rat granulosa cells during follicular development and on thecal cells during ovulation.

Journal of Endocrinology (1997) 152, 11–17
GH, GH-releasing factor and somatostatin in the growing lamb: sex differences and mechanisms for sex differences
Authors: Gatford K L , Fletcher T P , Rao A , Egan A R , Hosking B J , Clarke I J
Published: December 2004

Factors contributing to sex differences in the somatotrophic axis were investigated in growing lambs. In the first experiment, circulating patterns of GH in venous blood, pituitary content of GH and GH mRNA, and median eminence (ME) contents of GH-releasing factor (GRF) and somatostatin (SRIF) were characterized in prepubertal ram and ewe lambs which were pair-fed to remove sex differences in feed intake. Mean and baseline plasma GH concentrations, GH pulse amplitude, and integrated plasma GH were greater in ram lambs than in ewe lambs, but GH interpulse interval did not differ between sexes. The pituitary GH content and ME contents of GRF and SRIF were greater in rams than in ewes, but steady-state levels of mRNA for GH in the pituitary gland did not differ between sexes. A second experiment investigated sex effects on the levels of SRIF in hypophysial portal blood, and found that these did not differ between sexes. We concluded that the presence of sexually dimorphic patterns of GH secretion in the growing lamb is independent of feed-intake differences between sexes. The lack of sex differences in circulating patterns of SRIF in portal plasma implies that there may be a difference in GRF secretion which may produce sexually dimorphic patterns of GH secretion in lambs.

Journal of Endocrinology (1997) 152, 19–27
Pulsatile secretion of α-MSH and the differential effects of dexamethasone and haloperidol on the secretion of α-MSH and ACTH in dogs
Authors: Kooistra H S , Greven S H , Mol J A , Rijnberk A
Published: December 2004

This study was performed to determine whether, in the dog, there is at any time pulsatile release of α-MSH and whether secretion of ACTH from the pars intermedia (PI) contributes to the circulating concentrations of ACTH.

The 24-h secretory profiles of α-MSH, ACTH, and cortisol were determined in eight dogs. Plasma samples were obtained at 10-min intervals via an indwelling jugular catheter during two 12-h periods. Pulsatile secretion of α-MSH was found in all dogs, with wide variations in peak height. Plasma α-MSH levels were usually low (mean 15 pmol/l), but brief, distinct periods of increased plasma α-MSH concentrations as high as 489 pmol/l were found. Analysis of pulse frequency revealed a mean of 4·75 significant α-MSH peaks/24 h. The highest α-MSH peaks were associated with definite changes in the plasma concentrations of ACTH.

In separate studies, the influence of dexamethasone on the 6-h secretory profiles and on the haloperidol-stimulated secretion of α-MSH, ACTH, and cortisol was investigated. In these two studies, plasma ACTH was measured by a highly sensitive immunoradiometric assay. Dexamethasone pretreatment significantly suppressed the plasma concentrations of ACTH, cortisol, and α-MSH to 10·3%, 3·9%, and 74·6% respectively. Dexamethasone pretreatment also significantly reduced the haloperidol-stimulated secretion of ACTH and cortisol, but had no influence on the haloperidol-stimulated secretion of α-MSH. After the administration of haloperidol to the dexamethasone-pretreated dogs, there were small increases in the plasma concentrations of ACTH and cortisol, the latter being significant.

These data demonstrate that α-MSH is secreted spontaneously in a pulsatile manner in the dog and suggest that the canine PI contributes to circulating ACTH concentrations.

Journal of Endocrinology (1997) 152, 113–121
Adrenal and gonadal function in hypothyroid adult male rats
Authors: Tohei A , Akai M , Tomabechi T , Mamada M , Taya K
Published: December 2004

The functional relationship between thyroid, adrenal and gonadal hormones was investigated using adult male rats. Hypothyroidism was produced by the administration of 4-methyl-2-thiouracil (thiouracil) in the drinking water for 2 weeks. Plasma concentrations of TSH dramatically increased, whereas plasma concentrations of tri-iodothyronine and thyroxine decreased in thiouracil-treated rats as compared with euthyroid rats. Hypothyroidism increased basal levels of plasma ACTH and pituitary content of ACTH. The pituitary responsiveness to CRH for ACTH release markedly increased, whereas the adrenal responsiveness to ACTH for corticosterone release decreased. These results indicated that hypothyroidism causes adrenal dysfunction in adult male rats. Pituitary contents of LH and prolactin decreased in hypothyroid rats as compared with euthyroid rats. In addition, hypothyroidism lowered pituitary LH responsiveness to LHRH. Testicular responsiveness to human chorionic gonadotrophin for testosterone release, however, was not different between euthyroid and hypothyroid animals. These results indicated that hypothyroidism causes adrenal dysfunction and results in hypersecretion of ACTH from the pituitary gland. Adrenal dysfunction may contribute to the inhibition of LHRH secretion from the hypothalamus, possibly mediated by excess CRH.

Journal of Endocrinology (1997) 152, 147–154
Mouse anococcygeus muscle: sexual dimorphism and responsiveness to sex hormones
Authors: Fukazawa Y , Iguchi T , Bern H A
Published: December 2004

The anococcygeus muscle (AcM) is one of a pair of thin sheets of smooth muscle inserting on the rectum, having a tendinous origin largely on sacral vertebrae. The cross-sectional area of AcM in the juxtarectal region in 90-day-old male mice was significantly larger than that in females of three strains: BALB/cCrgl, ICR/Jcl and C57BL/Tw. The AcM area in female mice showed strain differences: BALB/c>ICR>C57BL. Five daily injections of testosterone into newborn ICR mice from the day of birth significantly increased the areas of AcM in both sexes at 30 days of age, but five daily injections of oestradiol-17β (OE) decreased them. The AcM area in 60-day-old ICR male mice castrated at 30 days of age was significantly smaller than in intact males, and that in ovariectomized females was significantly larger than in intact females. In both sexes, implantation of a testosterone pellet (12 mg) into gonadectomized mice on the day of gonadectomy stimulated the growth of AcM, and implantation of an OE pellet (12 mg) inhibited the growth of AcM. The AcM in both ICR and C57BL strains showed positive androgen receptor and oestrogen receptor immunostaining at 15 days. Female ICR mice exposed neonatally to diethylstilboestrol (DES) had significantly larger AcM than controls; ovariectomy at 30 days of age did not change the AcM area in 60-day-old DES-exposed mice. However, male mice exposed neonatally to DES had significantly smaller AcM than controls; castration at 30 days of age nullified this inhibition. These results suggest that both androgen and oestrogen play an important role in sexual dimorphism of the mouse AcM. Neonatal exposure to DES (but not to oestradiol) had an irreversible stimulatory effect on the AcM area in female mice.

Journal of Endocrinology (1997) 152, 229–237
Enhancement of FSH bioactivity in vivo using site-specific antisera
Authors: Ferasin L , Gabai G , Beattie J , Bono G , Holder A T
Published: December 2004

The ability of site-specific antipeptide antisera to enhance the biological activity of ovine FSH (oFSH) in vivo was investigated using hypopituitary Snell dwarf mice. These animals were shown to respond to increasing doses of oFSH (3·3–90 μg/day), administered in two daily injections over a 5-day treatment period, in a highly significant dose-dependent fashion. The responses measured were increases in uterine weight, ovarian weight and the index of keratinisation in vaginal smears. The dose-dependent response to oFSH confirmed the suitability of this animal model for these investigations and suggested the suboptimal dose of oFSH (20 μg/day) for use in enhancement studies.

Five peptides derived from the β subunit of bovine FSH (bFSH) (A, residues 33–47; B, 40–51; C, 69–80; D, 83–94; E, 27–39) were used to generate polyclonal antipeptide antisera. Of these peptides, only A and B produced an antiserum (raised in sheep) capable of recognising 125I-bFSH in a liquid phase RIA. Antisera prepared against peptide A or peptide B were found to significantly enhance the biological activity of 20 μg oFSH/day over a 5-day treatment period. The response to antipeptide antisera alone did not differ significantly from that observed in PBS-injected control animals, neither did the response to FSH alone differ from that observed in animals treated with FSH plus preimmune serum. Thus the enhanced responses are dependent upon the presence of FSH plus antipeptide antiserum. Peptides A and B are located in a region thought to be involved in receptor recognition, this may have implications for the mechanism underlying this phenomenon and/or the structure/function relationships of FSH.

That FSH-enhancing antisera can be generated by immunisation of animals with peptides A and B suggests that it may be possible to develop these peptides as vaccines capable of increasing reproductive performance, such as ovulation rate. The high degree of sequence homology between ovine, bovine and porcine (and to a lesser extent human and equine) FSH in the region covered by peptides A and B suggests that these peptides could also be used to promote and regulate ovarian function in all of these species.

Journal of Endocrinology (1997) 152, 355–363
The expression of c-myc during follicular growth and luteal formation in the rat ovary in vivo
Authors: Piontkewitz Y , Sundfeldt K , Hedin L
Published: December 2004

The processes of folliculogenesis and formation of corpora lutea involve proliferation and differentiation of the follicular cells. The expression of several oncogenes is associated with the proliferative phase in many cell types. The present study examined the expression and hormonal regulation of the c-myc proto-oncogene during follicular development and the luteal phase of pseudopregnancy. Follicular development was initiated by pregnant mare's serum gonadotropin (PMSG) in immature rats followed two days later by the injection of human chorionic gonadotropin (hCG) to induce ovulation and luteal formation. Ovaries were collected at different time points and the content and distribution of c-myc mRNA/protein were examined. C-myc increased rapidly after the administration of both PMSG and hCG, but the effect of PMSG was less pronounced. The increase after PMSG was transient and localized primarily to the granulosa cells of developing follicles. The ovulatory dose of hCG resulted in a rapid and substantial increase of c-myc mRNA and protein with maximal levels at 1 h and 2–4 h respectively. At this stage, the c-myc protein was localized to the follicular cells, the surface epithelium and, to some extent, to the interstitial tissue. There was a subsequent decrease prior to ovulation. The luteal phase was characterized by decreasing levels of c-myc with increasing luteal age.

In order to examine the involvement of specific hormones in the regulation of c-myc, hypophysectomized, immature rats were injected sequentially with estradiol (E2) and follicle-stimulating hormone (FSH). Hypophysectomy resulted in a decrease of c-myc compared with intact animals. The administration of E2 resulted in an increase of c-myc mRNA and protein. The subsequent treatment with FSH did not result in a further increase and the levels remained at the same level as with E2 only. However, an ovulatory dose of hCG to E2 and FSH primed animals resulted in an additional increase of c-myc mRNA and protein. The levels after E2 and FSH were considerably lower compared with those of untreated ovaries of intact, immature animals, suggesting the involvement of other endocrine and paracrine factors.

The presence of proliferating cell nuclear antigen in cell extracts indicated that the expression of c-myc was associated with phases of increased proliferation of follicular cells after hormonal stimulation.

The results demonstrate that c-myc is regulated by hormones (E2, gonadotropins) in the rat ovary during follicular development to the preovulatory stage. The pronounced increase prior to ovulation also suggests a role for c-myc in the regulation of proliferative events involved in luteal formation.

Journal of Endocrinology (1997) 152, 395–406