Initiation of periovulatory events in gonadotrophin-stimulated macaques with varying doses of recombinant human chorionic gonadotrophin

During in-vitro fertilization (IVF) cycles, a large bolus of human chorionic gonadotrophin (HCG) is used to induce periovulatory events, but the efficacy of lower doses is undefined. Following follicular stimulation in rhesus monkeys, oocyte nuclear maturation, IVF, granulosa cell luteinization and corpus luteum function were compared after injection of 100, 300 or 1000 IU recombinant HCG or 1000 IU urinary HCG. Bioactive HCG rose to peak concentrations within 2 h that were proportional to the dose administered (100 < 300 < 1000 IU, recombinant HCG = urinary HCG). The duration of surge values (>100 ng/ml) was also dose-dependent (0 h, 100 IU; 24 h, 300 IU; >48 h, 1000 IU, recombinant and urinary HCG). While the proportions of oocytes resuming meiosis and undergoing IVF were similar among groups, fewer animals yielded fertilizable oocytes following 100 and 300 IU (five of nine) compared to 1000 IU recombinant and urinary HCG (nine of 10). Peak values of serum progesterone in the luteal phase were similar, but declined 2 days earlier after 100 and 300 IU relative to 1000 IU recombinant and urinary HCG. Thus, 3-10 fold lower doses of HCG elicit low amplitude surges of short duration that induce periovulatory events such as re-initiation of oocyte meiosis and granulosa cell luteinization. However, oocyte fertilization and luteal function may optimally require surges of higher amplitude and longer duration similar to those produced by standard doses of 1000 IU recombinant or urinary HCG.     

The effects of phenobarbital and gonadal steroids on periovulatory serum levels of luteinizing hormone and follicle-stimulating hormone in the hamster

The occurrence of ovulation and serum levels of LH and FSH (measured by radioimmunoassay) were determined in periovulatory hamsters injected with an ovulation-blocking dose of phenobarbital (Phen) combined with progesterone (P), estradiol-17beta (E2), or testosterone (T). Proestrous hamsters were treated at 1300 h with Phen plus oil, P, P plus E2, E2, T, or a second injection of Phen at 2000 h. Each treatment group was divided into 3 subgroups, each of which was serially bled 4 times at 6 h intervals beginning at 1200, 1400, and 1600 h on proestrus. Phen blocked ovulation on the next morning in all animals, while treatments that included P (1 mg) restored the normal complement of ova in 65-75% of the animals. Neither E2 (1, 10 or 50 mug) nor T (0.1 or 1 mg) overcame the Phen block of ovulation. Control hamsters had peak levels of LH between 1400 and 1800 h and a biphasic release of FSH consisting of a peak at 1600 h on proestrus, a return to basal levels at 2200 h, and a second more sustained surge between 2400 and 0800 h on the morning of estrus. Phen completely depressed the proestrous surge of both gonadotropins but only partially inhibited the second FSH elevation on the morning of estrus. In ovulatory animals, P alone or combined with 1 or 10 mug E2 restored peak LH levels at 1600 h. FSH levels on proestrus in hamsters treated with Phen plus P peaked at 1800 h, while the addition of 1 mug E2 resulted in increased FSH levels at 1600 h; peak levels in both groups were about half of control values. No proestrous increase was detected in ovulatory animals treated with P and 10 mug E2. FSH levels on estrus in hamsters injected with P alone or in combination with E2 were intermediate between those of controls and animals given Phen only. Levels of LH and FSH in animals treated with a single or double dose of Phen or Phen plus E2 or T were not different during the periovulatory period.     

A bolus of recombinant human follicle stimulating hormone at midcycle induces periovulatory events following multiple follicular development in macaques

The efficacy of follicle stimulating hormone (FSH) as an alternative to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) for the initiation of periovulatory events in primate follicles is unknown. A single bolus of 2500 IU recombinant (r)-hFSH was compared to 1000 IU r-HCG for its ability to promote oocyte nuclear maturation and fertilization, granulosa cell luteinization and corpus luteum function following r-hFSH (60 IU/day) induction of multiple follicular development in rhesus monkeys. Following the r-hFSH bolus, bioactive luteinizing hormone concentrations were <3 ng/ml. Peak concentrations of serum FSH (1455+/-314 mIU/ml; mean+/-SEM) were attained 2-8 h after r-hFSH, and declined by 96 h. Bioactive HCG concentrations peaked between 2-8 h after r-HCG and remained > or = 100 ng/ml for >48 h, while immunoreactive FSH concentrations were at baseline. The proportion of oocytes resuming meiosis and undergoing in-vitro fertilization (IVF) were comparable for r-hFSH (89%; 47+/-19%) and r-HCG (88%; 50+/-17%). In-vitro progesterone production and expression of progesterone receptors in granulosa cells did not differ between groups. Peak concentrations of serum progesterone in the luteal phase were similar, but were lower 6-9 days post-FSH relative to HCG. Thus, a bolus of r-hFSH was equivalent to r-HCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function in primates.     

D4 dopamine receptor-mediated signaling events determined in transfected Chinese hamster ovary cells

A Chinese hamster ovary (CHO) cell line stably expressing a recombinant human D4 dopamine receptor made from a synthetic gene has been used to determine potential D4-mediated signaling events. We designed and synthesized a modified gene coding for a human D4 receptor with reduced G + C content but unaltered encoded amino acids. Stable expression of this gene was obtained in two cell lines, inducible expression in CHO lacI cells and constitutive expression in HEK293 cells. In CHO lacI cells induced to express D4 receptors but not in uninduced cells, dopamine and quinpirole inhibit forskolin-stimulated cAMP accumulation and potentiate ATP-stimulated [3H]arachidonic acid release through a mechanism that requires protein kinase C but is unaffected by membrane-soluble cAMP analogs. In addition, D4 receptor activation causes an increase in the rate of extracellular acidification measured by microphysiometry. This response is unaffected by protein kinase C down-regulation but is inhibited by removal of extracellular sodium and inhibitors of NaH-1 exchange, suggesting the involvement of a Na+/H+ exchanger. All responses are blocked by clozapine and are sensitive to pertussis toxin. D4 receptors, like other G(i)/G(o)-linked receptors, mediate multiple signaling events, and the pathways activated are similar to those used by D2 and D3 receptors expressed in similar cells.     

The role of telomerase for pancreatic carcinogenesis in human and hamster

Telomerase activity and terminal restriction flagment (TRF) length were investigated in human and hamster pancreatic duct adenocarcinomas. In the hamster primary and transplantable pancreatic carcinomas and cell lines, telomerase activity increased 86 to 215.7 times relative to the levels in normal spleen and pancreas, and reduction of TRF length was observed. In 38 human pancreatic ductal carcinomas, 32 (84%) exhibited increased telomerase activities with no apparent relation to the histological type of tumor, tumor size, regional lymph node involvement and distant metastasis. These results suggest that telomerase play an important role for pancreatic duct carcinogenesis.     

Examination of the role of DNA polymerase proofreading in the mutator effect of miscoding tRNAs

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, epsilon, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered epsilon subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutD alleles. In the present work, we extended the study of specific mutD alleles and constructed 16 altered mutD genes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes epsilon. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the epsilon subunit playing a major role in the mutator effect of misreading tRNAs.     

Predicting dementia from the Mini-Mental State Examination in an elderly population: the role of education

Steroid hormone regulation and cell-type specific expression of the jun-D protooncogene in rat uterus was examined. Adult, ovariectomized rats were injected with progesterone, testosterone, 17beta-estradiol (E2-17beta), 16alpha-estradiol (E2-16alpha), dexamethasone or cycloheximide. Uteri were collected between 0 and 6 h post-treatment. Northern blot analysis of uterine RNA revealed that induction of jun-D was specific for estrogenic steroids, as progesterone and testosterone had no effect on expression of this member of the jun gene family. Treatment with E2-17beta increased jun-D mRNA levels by approx. 5-fold, with expression reaching peak levels at 3 h after treatment and declining thereafter. Administration of E2-16alpha, a short-acting estrogen that does not cause uterine cell proliferation, increased expression of jun-D but with different kinetics compared to the long-acting E2-17beta. The mRNA levels of jun-D increased by 3-fold 1 h after administration of E2-16alpha but declined soon after. Slight induction of jun-D mRNA by dexamethasone was apparent, but to a much lesser extent compared to estrogen. The protein synthesis inhibitor, cycloheximide, did not block jun-D induction, indicating that this is an "immediate early" response. Expression of Jun-D protein was examined by immunohistochemical methods. E2-17beta treatment activated jun-D primarily in the nuclei of luminal and glandular epithelial cells of the endometrium. These results demonstrate that hormonal induction of jun-D is specific for estrogens and that uterine expression of this protooncogene occurs in a cell-type specific manner.     

Examination of the role of the proteolytically-activated form of protein kinase C in the differentiation of human haemopoietic cells

In neutrophils, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the translocation of the Ca(++)- and phospholipid-dependent protein kinase, protein kinase C (PK-C) from the soluble to the particulate fraction. At the same time there was a corresponding increase in the amount of Ca(++)- and phospholipid-independent protein kinase activity recovered in the soluble fraction. This soluble Ca(++)- and phospholipid-independent protein kinase presumably reflects proteolytic activation of the particulate associated PK-C. Bone marrow and undifferentiated HL-60 cells also translocated PK-C to the particulate fraction in response to TPA but did not accumulate the soluble Ca(++)- and phospholipid-independent form of the enzyme. Similar results were obtained using HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO), recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) or 1 alpha,25-dihydroxyvitamin D3. There was also no significant change in either the number or time of expression of differentiation-specific cell surface antigens observed on HL-60 cells induced to differentiate with either DMSO, 1 alpha,25-dihydroxyvitamin D3 or TPA in the presence of cyclosporin A, an agent reported to inhibit the proteolytic breakdown of PK-C to the Ca(++)- and phospholipid-independent form. Likewise, cyclosporin A did not affect the rate of extent of differentiation of primary bone marrow cell cultures. These results suggest that the proteolytically activated and phospholipid-independent form of PK-C is probably not involved in haemopoietic cell differentiation.     

The role of childhood abuse and neglect in the sensitization to stressful life events in adolescent depression.

This study examined the role of childhood abuse and neglect in sensitizing adolescents to the effects of proximal stressful life events in a cross-sectional sample of 103 depressed and nondepressed adolescents. Consistent with hypotheses, adolescents with a history of childhood abuse and/or neglect reported a lower level of threat of stressful life events prior to episode onset than that reported by those without. This effect was specific to those on their 1st episode of depression and was specific to independent events (i.e., stressors outside of adolescents' control). Further, this effect was robust when controlling for level of chronic difficulties, which was higher in those with childhood abuse and/or neglect. The authors suggest that childhood abuse and/or neglect may be an important risk factor that sensitizes individuals to the effects of acute independent life events. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Follicle-stimulating hormone (FSH) regulates the expression of FSH receptor messenger ribonucleic acid in cultured Sertoli cells and in hypophysectomized rat testis

FSH acts on Sertoli cells via interaction with a transmembrane receptor (FSHr). Control of expression of the receptor is surely a factor in the regulation of the action of FSH. The regulation of FSHr by FSH and testosterone was studied both in culture and in vivo. Sertoli cells from 18- to 20-day-old male rats were cultured in the presence of 25 ng/ml ovine (o) FSH. At 8 h after addition of FSH, expression of FSHr mRNA decreased significantly. Addition of FSH and actinomycin D to cells did not result in a further decrease in FSHr mRNA levels, suggesting that FSH does not alter turnover of FSHr mRNA. Treatment of cells with 40 ng/ml testosterone did not have any significant effect on the expression of FSHr mRNA. Hypophysectomy of 20-day-old male rats resulted in an increase in expression of FSHr mRNA as compared to that in sham-hypophysectomized animals. This increase was measured at 24 h posthypophysectomy and was maintained at 72 h after surgery. Injection of rats with 0.2 U oFSH at 48 h posthypophysectomy resulted in a reduction in FSHr mRNA when compared to the levels in hypophysectomized rats. Treatment with 2 mg testosterone propionate had no effect on FSHr mRNA levels. The findings confirm that FSH plays an important role in regulating mRNA expression of the FSHr in Sertoli cells in culture and show for the first time that FSHr mRNA is regulated in vivo by FSH in the immature rat testis.     

Aging and memory for action events: The role of familiarity.

Two experiments with young and elderly adults explored age-related memory differences for performed action events varying in familiarity. Memory for similar items encoded verbally was also assessed. The findings demonstrated that type of encoding and item-familiarity influenced immediate as well as delayed free recall in both age groups. Highest recall performances were found for familiar performed items. Both factors affected memory performance separately and did not compensate for each other, either in immediate or in delayed free recall. These findings held true regardless of age. Performed actions were especially resistant against forgetting, indicating that, besides the amount of items encoded, performing while encoding especially enhances the retention of knowledge. Recognition memory also varied with type of encoding. Age-related memory differences were found in all free recall tests irrespective of item familiarity and type of encoding, favoring young adults. No age-related memory differences were found in the recognition test. Because of possible ceiling effects, this finding must be treated with care. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Examination of the role of ADP-ribosylation factor and phospholipase D activation in regulated exocytosis in chromaffin and PC12 cells

The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.     

Hypothalamic regulation of pituitary FSH secretion

Temporal changes in plasma LH and FSH concentrations were monitored during the afternoon of proestrus in controls and in rats in which the spontaneous LH/FSH surges were blocked with Nembutal. These values were compared with those obtained following electrochemical stimulation (ECS) of either the medial preoptic area (MPOA) or the dorsal anterior hypothalamic area (DAHA) in similar Nembutal-blocked animals. Whereas MPOA-ECS (60 muA/60 sec) elicited a release of both FSH and LH, similar unilateral stimulation of the DAHA resulted in a pronounced increase in plasma FSH and only a slight elevation in plasma LH. Increasing the amount of DAHA tissue stimulated (100 muA/60 sec) caused a significantly greater release of FSH but not LH. Bilateral DAHA-ECS (60 muA/60 sec) failed to produce a greater release of FSH than that observed after unilateral 100 muA/60 sec ECS but resulted in increased concentrations of LH in plasma. Surgical separation of the MPOA from the DAHA, leaving the preopticotuberal fibers intact, did not alter the spontaneous temporal patterns of discharge of FSH or LH 19-21 days post-operatively, although peak LH concentrations were reduced. Further, unilateral ECS (60 muA/60 sec) of the MPOA in such preparations elicited a release of FSH and LH similar to that observed in intact MPOA-ECS rats. In contrast, unilateral DAHA ECS (60 muA/60 sec) in rats with transected hypothalami, caused no release of LH and an attenuated FSH discharge when compared with intact DAHA-ECS rats (peak valued 189 +/- 8 ng/ml vs 274 +/- 11 ng/ml). These studies suggest the existence of specific cell bodies in the DAHA which can cause selective release of FSH when activated. Coexisting with this system is that level of control which is believed to be responsible for the cyclic discharge of both FSH and LH of which the MPOA is a component part.     

Prognostic value of high FSH concentrations

Ovarian age is a major determinant of success in assisted reproductive technologies. The number and quality of oocytes retrieved directly depend upon the ovarian reserve. The evaluation of FSH concentration during the first days of the menstrual cycle provides a simple and accurate means of assessing ovarian reserve. Plasma FSH value is correlated with cancellation rate, peak estradiol, number of oocytes retrieved and of the resulting embryos, pregnancy rate, outcome of cryopreservation. However, a poor response to the stimulation treatment is unpredictable in a subgroup of women whose FSH concentration remain within the normal range. Ovarian aging begins several years before any clinical or endocrinological modification. Recurrent FSH determinations may help in predicting a poor response, as a large intercycle FSH variability is correlated with ovarian failure. The value of ovarian reserve tests is presently not fully established.     

Effects of smokeless tobacco on chemically transformed hamster oral keratinocytes: role of angiotensin I-converting enzyme

The purpose of this study was to determine whether exposure of chemically transformed golden Syrian hamster oral epidermoid carcinoma cell (HCPC-1) cultures to smokeless tobacco extract (STE) is associated with a decrease in specific angiotensin I-converting enzyme (ACE) activity and whether this decrease potentiates bradykinin-induced cell growth. We found that STE induced a significant concentration- and time-dependent decrease in ACE activity in cultured HCPC-1 cells (P < 0.05). STE alone had no significant effect on cell number. Bradykinin alone induced a slight, but significant, increase in cell number (P < 0.05). These effects were significantly potentiated by STE (P < 0.01). We conclude that STE potentiates bradykinin-induced HCPC-1 cell growth, in part by attenuating specific ACE activity in these cells.