Melatonin action on thyroid activity in the soft-shelled turtle, Lissemys punctata punctata

Adult female turtles (Lissemys punctata punctata) were treated with pineal indoleamine melatonin (100 micrograms/100 g) or the antithyroid agent, methylthiouracil (100 micrograms/100 g) or melatonin together with methylthiouracil (100 micrograms of each drug/100 g) for 12 days. Melatonin alone inhibited thyroid activity as evidenced by reduction in the gland weight, follicular epithelial cell height, thyroid peroxidase, and plasma thyroxine levels. Methylthiouracil caused hyperplasia of the gland, although it inhibited thyroid activity and reduced thyroid peroxidase and plasma thyroxine levels. Melatonin together with methylthiouracil produced changes similar to those obtained with melatonin alone. The results indicate that melatonin probably exerts inhibitory effects influences on both thyrotropin release from the pituitary and the activity of the thyroid itself in turtles.     

Lack of effects of growth hormone administration on ovarian function of lactating goats

During method development in support of non-clinical studies in animal models, BMS-186716 was found to be extremely unstable in blood and plasma. Stabilization of the compound was achieved by reacting the compound with methyl acrylate (MA) in blood, from which the plasma was then prepared. While the resulting BMS-186716-MA adduct was found to be stable in dog plasma, and hence it was used as the basis for the method developed for analysis of dog plasma samples, the BMS-186716-MA adduct was found to be unstable in rat plasma as it was readily hydrolyzed to BMS-186716-acrylic acid (AA) by native esterases found in rat plasma. Although the finding of the instability of BMS-186716-MA in rat plasma was not the result of prospective planning, we were able to successfully develop a quantitative bioanalytical method using BMS-186716-AA as the analyte instead of the originally planned BMS-186716-MA analyte. The standard and quality-control (QC) samples were prepared by spiking blank plasma with BMS-186716-MA, and then allowing them to stand at room temperature for 1 h to convert BMS-186716-MA to BMS-186716-AA. After adding the internal standard BMS-188035-AA, each sample was acidified with HCl and then extracted with methyl tert.-butyl ether. The reconstituted extract was injected into a HPLC-electrospray ionization mass spectrometric system for detection by positive ion electrospray ionization. A lower limit of quantitation (LLQ) of 5 ng/ml was achieved, using 0.1 ml plasma and a standard curve range of 5-5000 ng/ml.     

Sexual differentiation of gonads as a function of temperature in the turtle Emys orbicularis: endocrine function, intersexuality and growth

The aim of this study was to develop an inexpensive method for reducing the number of differentiated cells from granulocyte colony-stimulating factor-mobilized leukocytapheresis products (LPs) containing peripheral blood stem cells. Analysis of LPs showed the presence of significant numbers of monocytes and myeloid cells. The myeloid cells represented largely immature stages of the granulocyte lineage (myelocytes and metamyelocytes). We investigated whether these cells could be selectively depleted by filtration over nylon wool. Filtration of LP samples over nylon wool in a medium containing fetal calf serum resulted in variable but on average low yields of CD34+ cells (48 +/- 30%; n=13) and strongly variable depletions of myeloid cells. The adherence of CD34+ cells to the polyamide fiber was partially mediated by activated platelets that were present in the LPs. Removal of platelets by counterflow centrifugal elutriation before filtration resulted in increased yields of CD34+ cells in the filtrates (65 +/- 13%; n=10). The yield of progenitor cells was similarly enhanced when trisodium citrate, a chelating substance, was added to the filtration medium. Adherence of the myeloid cells to the nylon fiber was promoted by preincubation of the columns with human immunoglobulin (Ig) (2 mg/mL). Small-scale filtrations of LP samples in the presence of trisodium citrate over columns with Ig-coated nylon wool resulted in removal of 96 +/- 4% of the monocytes and 74 +/- 18% of the myeloid cells, with a yield of 71 +/- 15% CD34+ cells and 67 +/- 10% granulocyte-monocyte colony-forming units (CFU-GM) (n=23). There was no loss of primitive stem cells during the procedure: the yield of late-appearing cobblestone area-forming cells (CAFCs, week 6) was 110 +/- 30% (n=4). CFU-GM production per CAFC-derived clone was unchanged upon filtration, indicating that the quality of stem cells was not affected. Moreover, the proportions of CD34+ cells expressing a primitive immunophenotype (CD38low or Thy-1+) were unchanged after filtration. Further enrichment of progenitor cells was obtained by separation of LP samples by elutriation before filtration. The combination of these two techniques resulted in complete removal of platelets, 89 +/- 7% depletion of erythrocytes, and 91 +/- 6% reduction of leukocytes, with a 50% yield of CD34+ cells (n=14). In conclusion, we have developed a rapid filtration technique by which monocytes and myeloid cells can be depleted from LP samples, with only minor loss of colony-forming cells and complete recovery of primitive stem cells.     

Apoptosis and ovarian function

For decades, the mechanisms responsible for germ cell depletion from the ovary, either directly during the perinatal period or indirectly via follicular atresia during postnatal life, have remained relatively obscure. The recent application of sensitive biochemical techniques for the study of cell death to the analysis of ovarian function has revealed that these two events, as well as a third instance of ovarian cell degeneration (luteolysis), are dependent upon the activation of physiological cell death mechanisms. It is therefore hypothesized that the controlled deletion of ovarian cell populations is accomplished via activation of a 'universal' pathway of cellular suicide involving altered expression of a conserved cohort of genes. The identity of the hormonal and intracellular effectors responsible for the coordination of life and death decisions made by ovarian cells during development as well as the biological and clinical implications of gene-directed cell death in the ovary are explored in this review.     

Follicular fluid insulin-like growth factor-I and insulin-like growth factor-binding protein-1 and -3 vary as a function of ovarian reserve and ovarian stimulation

PURPOSE: Follicular fluid concentrations of insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein (BP)-1, and IGFBP-3 in 57 women undergoing in vitro fertilization and embryo transfer were examined to determine whether levels reflected differences in patients' exposure to gonadotropin stimulation and a diminished ovarian reserve. METHODS: Preovulatory follicular fluid was obtained from both gonadotropin-stimulated and unstimulated cycles. Subjects were grouped according to normal or decreased ovarian reserve and whether or not they received gonadotropin stimulation. RESULTS: The mean follicular fluid concentrations of IGF-I and IGFBP-1 were significantly lower in the "decreased" ovarian reserve group compared with the "normal" ovarian reserve group, with no change in estradiol or IGF-II levels. This resulted in a decreased molar IGF-I: BP ratio and an increased molar IGF-II:IGFBP-1 ratio. In unstimulated cycles, mean follicular fluid concentrations of IGFs did not differ significantly compared with those in stimulated cycles, whereas concentrations of IGFBP-1 and IGFBP-3 were significantly lower, leading to higher molar ratios of the IGFs to the binding proteins. CONCLUSIONS: Follicular fluid IGF and binding proteins vary as a function of ovarian reserve and gonadotropin stimulation. This may reflect either differences in oocyte quality or a suboptimal follicular fluid environment.     

Inhibitory effects of ginsenoside Rh2 on tumor growth in nude mice bearing human ovarian cancer cells

Ginsenoside Rh2 (Rh2), isolated from an ethanol extract of the processed root of Panax ginseng CA Meyer, inhibits the growth of B16 melanoma cells. This study was designed to evaluate the ability of Rh2 to inhibit growth of human ovarian cancer cells (HRA) in vitro and in nude mouse. Rh2 inhibited proliferations of various established human ovarian cancer cell lines in a dose-dependent manner between 10 and 60 microM in vitro and induced apoptosis at around the IC50 dose. When HRA cells were inoculated s.c. into the right flank of nude mice, all mice formed a palpable tumor within 14 days. Although i.p. administration of Rh2 alone hardly inhibited the tumor growth, when Rh2 was combined with cis-diamminedichloroplatinum(II) (CDDP) the tumor growth was significantly inhibited, compared to treatment with CDDP alone. When mice were treated p.o. with Rh2 daily (but not weekly), the tumor growth was significantly (P<0.01) inhibited, compared to CDDP treatment alone. When Rh2 was combined with CDDP, the degree of tumor growth retardation was not potentiated. The survival time was significantly (P<0.05) longer than that of medium alone-treated controls or the group treated with CDDP alone. Then, we examined whether p.o. administration of Rh2 has a dose-dependent inhibitory effect on the tumor growth. I.p. and weekly administration of CDDP had more potent antitumor activity in the order of 1 mg/kg, 2 mg/kg and 4 mg/kg, whereas p.o. and daily administration of Rh, (0.4 to 1.6 mg/kg) not only had antitumor activity comparable to that of 4 mg/kg CDDP, but also resulted in a significant increase of the survival. Doses of Rh2 used in this study did not result in any adverse side-effects as confirmed by monitoring hematocrit values and body weight, unlike 4 mg/kg CDDP, which had severe side-effects. It is noteworthy that p.o. but not i.p. treatment with Rh2 resulted in induction of apoptotic cells in the tumor in addition to augmentation of the natural killer activity in spleen cells from tumor-hearing nude mice. Thus, particularly in view of the toxicity of CDDP, Rh2 alone would seem to warrant further evaluation for treatment of recurrent or refractory ovarian tumor.     

Identification and initial characterization of serum growth hormone binding protein in the turtle Chrysemys dorbigni

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential early during eye development to inhibit the determination of excess photoreceptors. Ubiquitin is a small polypeptide that tags proteins for degradation by a multisubunit proteolytic complex called the proteasome. The FAT FACETS protein is thought to be required to remove ubiquitin from a particular protein, thereby rescuing if from proteolysis. In order to identify the genes encoding the substrate of FAT FACETS and other components of the neural inhibition pathway, a mutagenesis screen for dominant enhancers of the fat facets mutant eye phenotype was performed. Several genes were identified, one of which is an excellent candidate for encoding a component of the pathway regulated by FAT FACETS. Three different eye phenotypes were observed when the fat facets mutants were dominantly enhanced by different mutations, suggesting that fat facets has other functions in addition to its critical role early in eye development.     

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.     

Insulin-like growth factors and ovarian physiology

OBJECTIVE: To review the available information regarding the roles of insulin-like growth factor (IGF)-IGF binding protein (IGFBP) system in ovarian physiology. DESIGN: Studies that specifically relate to the roles of ovarian folliculogenesis, oocyte maturation, and ovulation were identified through the literature and Medline searches. RESULTS: Numerous actions of the IGFs have been demonstrated in the ovary, including an enhancement of cell proliferation, aromatase activity, and progesterone biosynthesis. The ovarian IGF system, comprised of IGF-I and IGF-II peptides, IGFBPs and IGF receptors, plays a significant role in the process of follicular development. In addition, IGF-I stimulates the meiotic maturation of follicle-enclosed oocytes in vitro via the IGF-I receptors. IGFBP-3 significantly inhibit gonadotropin-induced ovulation and oocyte maturation by neutralizing endogenously produced IGF-I. Thus, the intraovarian IGF-IGFBP system play a significant role in the processes of follicular development, oocyte maturation, and ovulation. CONCLUSION: IGF-IGFBP systems have autocrine/paracrine regulatory actions in ovarian physiology. The disturbance of the IGF-IGFBP system in human ovaries may lead to an ovulation, disorders of androgen excess, and infertility.     

Interrelation of the therapeutic effects of growth hormone and testosterone on growth in hypopituitarism

The present study evaluates the modifying effect of growth hormone on the growth-promoting action of testosterone in boys at pubertal bone age. Growth and bone maturation were analyzed in 42 boys with primary or secondary Leydig cell insufficiency who had been treated with testosterone in an attempt to induce puberty and the accompanying growth spurt. The dosage given was considered normal or high for physiologic replacement therapy at puberty. Sixteen boys had normal GH secretion (seven had isolated gonadotropin deficiency, nine had congenital anorchia); 26 were GH and Gn deficient (20 idiopathic, six craniopharyngiomas). Of the GH-deficient patients, 12 received hGH simultaneously, while 14 received only testosterone. Results from each group were compared with the normal pubertal growth spurt in 15 untreated healthy boys. In isolated Gn deficiency and in congenital anorchia, the growth rates increased to above normal during the first six months of treatment, indicating that the testosterone dosage was probably too high for the beginning of puberty. During two subsequent six-month treatment periods, the rates leveled off close to normal. The same was true in the GH- and Gn-deficient patients on adequate hGH replacement. For contrast, there was minimal or no stimulation of growth when an even higher testosterone dose was given to GH- and Gn-deficient boys without hGH therapy. Bone maturation was normal in the boys with normal GH secretion or with hGH replacement, but was subnormal in the GH-deficient boys not treated with hGH. We conclude that testosterone exerts its full growth-promoting action only in the presence of normal endogenous GH secretion or with sufficient hGH replacement and that both hormones should be continued simultaneously until final adult height is achieved.     

The influence of norethisterone oenanthate on ovarian function

The experiences acquired using a new dilutor in the serological field are dealt with here. The versatile dilutor is particularly adapted to the execution of two-fold dilutions. Among the many possible techniques, the relatives to ASLO, THPA and serum-agglutinins are thoroughly described.     

Vascular endothelial growth factor localization in human ovary and fallopian tubes: possible role in reproductive function and ovarian cyst formation

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that also increases vascular permeability. We hypothesized that VEGF plays a role in the regulation of cyclic ovarian angiogenesis in women, and that its ability to increase vascular permeability may be an important factor in the production of fallopian tube effluent and fluid formation in ovarian cysts. To examine these hypotheses, we assessed VEGF expression in ovaries and fallopian tubes from premenopausal (n = 10) and postmenopausal (n = 4) women. Immunohistochemical analysis for VEGF was performed using a rabbit polyclonal antibody directed against human VEGF. In normal ovaries from premenopausal women, VEGF within healthy follicles was localized to the thecal cell layer, with minimal VEGF peptide detected in the granulosa cell layer. VEGF was not expressed in atretic follicles or a degenerating corpus luteum. However, intense VEGF immunostaining was observed within the highly vascularized corpora lutea in all specimens examined. In normal ovaries from postmenopausal women, VEGF was detected only in epithelial inclusion cysts and a serous cystadenoma. In specimens from both pre- and postmenopausal women, the luminal epithelium of the fallopian tube as well as smooth muscle cells and pericytes lining small and large blood vessels within the tube and hilum of the ovary exhibited specific staining for VEGF. Based on these data, we suggest that during reproductive life, VEGF plays a role in the growth and maintenance of the ovarian follicle and corpus luteum by mediating angiogenesis. In addition, VEGF within the fallopian tube luminal epithelium may increase vascular permeability and modulate tubal luminal secretions. Similarly, VEGF in the epithelial lining of benign ovarian neoplasms may contribute to fluid formation in ovarian cysts.     

The effects of sodium on the growth velocity and growth morphology of silicon in Al-Si alloys

The effects of sodium additions to Al-Si alloys have been studied by determining the coupled zone for normal and "modified" alloys. Sodium slows the growth rate of silicon with respect to aluminum, and changes the morphology of the silicon. These effects were observed to be independent of growth temperature. A hypothesis that sodium adsorbs on the fast-growing faces of silicon can explain all observations.     

Patterns of ovarian growth and development in cattle with a growth hormone receptor deficiency

Nutritionally induced changes in growth hormone (GH) and IGF-I are associated with decreased ovarian function and may partially explain infertility and anestrus in undernourished cattle. The reproductive importance of GH and IGF-I was tested in cattle with a GH receptor deficiency (GHRD) that have reduced blood IGF-I. Blood was collected daily for plasma, and ovaries were examined daily by ultrasonography for 3 wk during an estrous cycle (estrus = d 0) in GHRD (n = 8) and control (n = 8) cattle. On d 18, blood samples were collected every 10 min for 6 h to measure LH. The GHRD cattle had fewer small antral ovarian follicles (2 to 5 mm, P < .01). After estrous cycle d 5, the first-wave dominant follicle stopped growing in GHRD but continued growing in controls (P < .001). Size of the CL was equivalent for GHRD and controls until d 5, after which CL development slowed in GHRD (P < .01). Likewise, plasma progesterone concentrations were less in GHRD (P < .001). During the luteal phase, GHRD cattle failed to develop follicles greater than 10 mm in diameter (endocrine status x day, P < .05). Size and rate of growth of preovulatory follicles, plasma estradiol, plasma FSH, and plasma LH (d 18 bleed) were similar in GHRD and controls. In conclusion, an important role for GH, GH receptor, and IGF-I in ovarian function was supported because GHRD cattle had distinctly different patterns of ovarian development compared with control cattle.     

The muscular contractions of the midgut of the cockroach, Diploptera punctata: effects of the insect neuropeptides proctolin and leucomyosuppressin

We have previously shown differential expression of leucomyosuppressin (LMS) mRNA in apparent endocrine cells in the anterior region of midguts of the cockroach Diploptera punctata, using in situ hybridization. In contrast, other FMRFamide-related peptides, as revealed by immunohistochemistry, have been found most abundantly in the posterior region in both apparent endocrine cells and nerve tracts. Here, we partially purified extracts of anterior and posterior cockroach midguts, using HPLC coupled with radioimmunoassay, and found, among multiple FMRFamide-like immunoreactive fractions, one fraction co-eluting with LMS in both regions. The presence of a co-eluting fraction in the posterior region, in the absence of LMS mRNA positive endocrine cells suggests that LMS might therefore be present in nerve tracts running along the length of the midgut. Using a circular muscle contraction assay from different portions of midgut, we determined the effects of LMS, proctolin and a variety of other midgut peptides on contractions of the midgut of Diploptera. Proctolin caused a sustained tonic contraction in the anterior midgut, the amplitude of which was dose-dependent. In contrast, LMS, and its relative SchistoFLRFamide, reduced the amplitude of these contractions. LMS and SchistoFLRFamide also inhibited spontaneous phasic contractions, which were elicited by proctolin application in only a few preparations. Other postulated midgut peptides did not induce or inhibit contractions, nor augment the proctolin-induced contractions. The C-terminal truncated sequences of LMS, HVFLRFamide and VFLRFamide, were sufficient to reduce the amplitude of the proctolin-induced contractions. This work illustrates a possible physiological role for LMS in Diploptera midguts, in the passage of food along the alimentary canal.     

Aging effects on growth hormone receptor binding in the brain

Recent years have seen an increasing interest in research focused on the role that growth hormone (GH) may have in the central nervous system. The psychological improvements seen in adults following GH therapy combined with the observation that the hormone may affect the cerebrospinal fluid levels of several brain transmitters have received a great deal of attention. Studies have also revealed the presence of specific GH receptors in distinct areas of the brain of many mammals. This article will review our recent studies on the aging effects on GH binding in these regions. It also includes some data on the age-related effects on the expression of the GH-receptor messenger ribonucleic acid (mRNA) in certain brain areas.     

Comparison of the effects of growth hormone and insulin-like growth factor I on substrate oxidation and on insulin sensitivity in growth hormone-deficient humans

Insulin-like growth factor-I (IGF-I) is considered to be the mediator of the growth-promoting effects of growth hormone (GH). The metabolic effects of these two hormones, however, are different. Whereas GH treatment leads to elevated insulin and glucose levels, reduced insulin sensitivity, and impaired glucose tolerance, IGF-I treatment leads to reduced insulin and GH levels and enhanced insulin sensitivity. IGF-I may, therefore, not only be the mediator of the growth-promoting effects of GH but also a modulator of the effects of GH on insulin action and glucose metabolism. To study the influence of GH and IGF-I on substrate metabolism and insulin sensitivity (assessed by euglycemic, hyperinsulinemic clamping combined with indirect calorimetry and glucose tracer infusion), we have treated eight GH-deficient adults with GH (2 IU/m2 daily subcutaneously [s.c.]), IGF-I (10 micrograms/kg.h s.c.), or both hormones together for 7 d, respectively, and compared the effects of these treatment regimens with a control phase. Our findings suggest that (a) both GH and IGF-I promote lipolysis and lipid oxidation, albeit by different mechanisms; (b) treatment with either hormone is followed by enhanced energy expenditure and reduced protein oxidation; and (c) IGF-I reverses the insulin resistance induced by GH.