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Adiponectin is a hormone secreted from adipose tissue, and serum levels are decreased with obesity and insulin resistance. Because prolactin (PRL) and growth hormone (GH) can affect insulin sensitivity, we investigated the effects of these hormones on the regulation of adiponectin in human adipose tissue in vitro and in rodents in vivo. Adiponectin secretion was significantly suppressed by PRL and GH in in vitro cultured human adipose tissue. Furthermore, PRL increased adiponectin receptor 1 (AdipoR1) mRNA expression and GH decreased AdipoR2 expression in the cultured human adipose tissue. In transgenic mice expressing GH, and female mice expressing PRL, serum levels of adiponectin were decreased. In contrast, GH receptor deficient mice had elevated adiponectin levels, while PRL receptor deficient mice were unaffected. In conclusion, we demonstrate gene expression of AdipoR1 and AdipoR2 in human adipose tissue for the first time, and show that these are differentially regulated by PRL and GH. Both PRL and GH reduced adiponectin secretion in human adipose tissue in vitro and in mice in vivo. Decreased serum adiponectin levels have been associated with insulin resistance, and our data in human tissue and in transgenic mice suggest a role for adiponectin in PRL and GH induced insulin resistance.

Progesterone-receptor (PR) stimulation promotes survival in rat and human periovulatory granulosa cells. To investigate the mechanisms involved, periovulatory rat granulosa cells were incubated in vitro with or without the PR-antagonist Org 31710. Org 31710 caused the expected increase in apoptosis, and expression profiling using cDNA microarray analysis revealed regulation of several groups of genes with functional and/or metabolic connections. This regulation included decreased expression of genes involved in follicular rupture, increased stress responses, decreased angiogenesis, and decreased cholesterol synthesis. A decreased cholesterol synthesis was verified in experiments with both rat and human periovulatory granulosa cells treated with the PR-antagonists Org 31710 or RU 486 by measuring incorporation of [14C]acetate into cholesterol, cholesterol ester, and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory rat granulosa cells using 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (lovastatin, mevastatin, or simvastatin) increased apoptosis, measured as DNA fragmentation and caspase-3/7 activity. The increase in apoptosis caused by simvastatin was reversed by addition of the cholesterol synthesis-intermediary mevalonic acid. These results show that PR antagonists reduce cholesterol synthesis in periovulatory granulosa cells and that cholesterol synthesis is important for granulosa cell survival.

The small ubiquitin-related modifier-1 (SUMO-1) with broad cellular expression has been implicated in a range of cellular processes, such as cell proliferation, differentiation, and apoptosis. As shown recently, SUMO-1 is expressed and regulated by gonadotropins, in particular an ovulatory hCG stimulus in mouse granulosa cells in vivo. To test the hypothesis that modulation of granulosa cell apoptosis changes SUMO-1 expression during granulosa cell differentiation in the mouse ovary, we demonstrate that progesterone receptor (PR) proteins are absent in pre-ovulatory granulosa cell nuclei, whereas they are expressed in periovulatory granulosa cell nuclei in parallel with decreases in SUMO-1 expression, caspase-3 activation, and DNA fragmentation in vivo. Second, treatment with either PR antagonists or a cell permeable ceramide analog consistently increases SUMO-1 expression in parallel with an increase in apoptosis as well as a decrease in cell proliferation in periovulatory granulosa cells in vitro. However, we do not observe an increase in SUMO-1 expression in pre-ovulatory granulosa cells that have undergone the same treatment. Third, we have also demonstrated, in pre-ovulatory granulosa cells in vitro, neither induction of spontaneous apoptosis nor the protective effect of EGF against spontaneous apoptosis changes SUMO-1 protein expression. Fourth, we show that induction of apoptosis enhances SUMO-1 conjugation in periovulatory granulosa cells in vitro, pointing to the pivotal link between the SUMO-1 conjugation and cell death. Taken together, our observations suggest that SUMO-1 via sumoylation has an important role in the regulation of granulosa cell apoptosis during granulosa cell differentiation in the mouse ovary.

The small ubiquitin-related modifier-1 (SUMO-1) is a member of a family of ubiquitin-related proteins that have effects on several important physiological functions, including reproduction. However, the regulation of SUMO-1 expression and functional distribution of SUMO-1 in vivo remain poorly understood. In the present study, we show that SUMO-1 protein is widely expressed in various tissues. In the ovary, the expression of SUMO-1 protein is suppressed around ovulation, in both the whole ovary and the granulosa cells, after gonadotropin treatment. Additionally, when the ovulatory signal, the endogenous LH surge, is blocked in vivo by pentobarbitone sodium, the expression of SUMO-1 protein in granulosa cells is increased. This effect is reversed when the missing endogenous LH surge is substituted by human chorionic gonadotropin treatment. Our findings provide the first evidence that inhibition of SUMO-1 expression is regulated by LH receptor stimulation in granulosa cells concomitant with ovulation in the mouse ovary. Furthermore, the levels of SUMO-1 protein are increased in granulosa cells treated with progesterone receptor (PR) antagonists both in vivo and in vitro, demonstrating that SUMO-1 expression is regulated by functional PR. SUMO-1 interacts with nuclear receptors in vitro, and LH receptor-mediated induction of PR is crucial for ovulation. SUMO-1 and PR are coexpressed and can be coprecipitated, providing additional evidence for a direct interaction between SUMO-1 and PR in periovulatory granulosa cells in vivo. These results suggest that a functional link between SUMO-1 and PR is of physiological importance for the local modulation of PR-mediated events in the ovary.

Accumulation of lipids and cholesterol by macrophages and subsequent transformation into foam cells are key features in development of atherosclerosis. Serum copper concentrations have been shown to be associated with cardiovascular disease. However, the mechanism behind the proatherogenic effect of copper is not clear. We used DNA microarrays to define the changes in gene expression profile in response to copper exposure of human macrophages. Expression monitoring by DNA microarray revealed 91 genes that were regulated. Copper increased the expression of seven cholesterogenic genes (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, IPP isomerase, squalene synthase, squalene epoxidase, methyl sterol oxidase, H105e3 mRNA and sterol-C5-desaturase) and low-density lipoprotein receptor (LDL-R), and decreased the expression of CD36 and lipid binding proteins. The expression of LDL-R and HMG CoA reductase was also investigated using real time PCR. The expression of both of these genes was increased after copper treatment of macrophages (P

During lactation serum levels of prolactin (PRL) are elevated, and the activity of lipoprotein lipase (LPL) is decreased in the adipose tissue and increased in the mammary gland. However, PRL has been suggested to affect the adipose tissue in an indirect fashion during lactation. In the present study, we demonstrated expression of four PRL receptor (PRLR) mRNA isoforms (L, I, S1(a), and S1(b)) in human sc abdominal adipose tissue and breast adipose tissue using RT-PCR/Southern blot analysis. In addition, L-PRLR [relative molecular mass (M(r)) 90,000] and I-PRLR (M(r) 50,000) protein expression was detected in human sc abdominal adipose tissue and breast adipose tissue using immunoblot analysis. Two additional protein bands with the molecular weight M(r) 40-35,000 were also detected. The direct effect of PRL on the regulation of LPL activity in human abdominal adipose tissue cultured in vitro was investigated. PRL (500 ng/ml) reduced the LPL activity in human adipose tissue to 31 +/- 7.7%, compared with control. GH (100 ng/ml) also reduced the LPL activity, to 45 +/- 8.6%, compared with control. In agreement with previous studies, cortisol increased the LPL activity and GH inhibited cortisol-induced LPL activity. Furthermore, we found that PRL also inhibited the cortisol-induced LPL activity. Taken together, these results demonstrate a direct effect of PRL, via functional PRLRs, in reducing the LPL activity in human adipose tissue, and these results suggest that LPL might also be regulated in this fashion during lactation.

The intracellular progesterone receptor (PR) in the mammalian ovary is a part of the physiological pathway that facilitates ovulation. Two PR isoforms (A and B) exist, with different molecular and biological functions. Previous studies have revealed that the cellular ratio of the PR isoforms is important for progesterone-responsive tissues and is under developmental control in different species. However, the relative expression of PR isoforms in the ovary is unknown. In this study we have demonstrated first that the expression of both PR isoforms in mouse granulosa cells was rapidly upregulated by hCG treatment and dramatically down-regulated when the granulosa cells were undergoing luteinization. The relative level of protein expression of the A and B forms was 2:1 and the highest total PR protein expression was found after hCG stimulation. Second, we demonstrated that the expression of PR protein was specific to granulosa cells of periovulatory follicles and was absent in undifferentiated granulosa cells of growing follicles. It was not detected in other cell types (i.e., corpora lutea or any stage of follicles with features of apoptosis). Third, we demonstrated that treatment with the PR antagonist RU 486 in vivo resulted in down-regulation of both isoforms in parallel with increased activation of caspase-3, a decreased level of proliferating cell nuclear antigen, and a reduced rate of ovulation. Fourth, we demonstrated, in vitro, that the PR antagonists RU 486 and Org 31710 increased internucleosomal DNA fragmentation parallel with a decrease in DNA synthesis in granulosa cells, which express PR. These results indicate that PR and its isoforms participate in regulation of ovulation, along with suppression of granulosa cell apoptosis and promotion of cell survival in the mouse ovary. apoptosis, granulosa cells, progesterone receptor

Decreased estrogen production is associated with changes in the skeletal, cardiovascular and muscular systems. At the level of skeletal muscles, it has been shown that a reduction in force production occurs at menopause but the underlying mechanisms are still unknown. The aim of the study was to investigate the effects of ovariectomy on myosin heavy chain (MyHC) composition. Additionally, we studied the effects of physical activity and the combined effects of physical activity and estrogen treatment on MyHC content in ovariectomised (OX) animals. Twenty-five rats were randomly assigned to five different groups: controls, runners, OX, ovariectomised runners and ovariectomised runners receiving estrogen. Exercise consisted of voluntary running for 5 weeks. Two muscles were analysed: m. extensor digitorum longus, EDL, (fast muscle) and m. soleus (slow muscle). MyHC content was analysed on 8% gel electrophoresis. The level of running activity is reduced in OX animals and estrogen administration is associated with the normalisation of the level of physical activity. Ovariectomy induces a shift from fast to slow MyHC isoforms in both the soleus and EDL. When OX animals are allowed to run, alterations in MyHC isoforms are still observed in the EDL but not in the soleus. When physical activity is combined with estrogen treatment no alterations are observed in both muscles. In conclusion, this study shows that ovariectomy induces alterations in the contractile properties of skeletal muscles and that physical activity in combination with estrogen treatment are associated with the maintenance of slow and fast muscle characteristics.

Background The fallopian tube transports the gametes to the fertilization site and delivers the embryo to the uterus at the optimal time for implantation. Progesterone and the classical progesterone receptor are involved in regulating both tubal ciliary beating and muscular contractions, likely via both genomic and non-genomic actions. Methods To provide more details of the underlying mechanisms, we investigated the effect of progesterone on gene expression in mice fallopian tubes in vitro at 20 min, 2 h and 8 h post progesterone treatment using microarray and/or quantitative PCR. In parallel, oocyte cumulus complex transport was investigated in ovulating mice that were injected with one of the progesterone receptor antagonists, Org 31710 or CDB2194. Results Microarray analyses did not reveal any apparently regulated genes 20 min after progesterone treatment, consistent with the proposed non-genomic action of progesterone controlling ciliary beating. After 2 h, 11 genes were identified as up-regulated. Analyses using quantitative PCR at 2 h and 8 h showed a consistent up-regulation of endothelin1 and a down-regulation of its receptor Endothelin receptor A by progesterone. We also confirmed that treatment with progesterone receptor antagonists before ovulation accelerates the transport of the oocyte cumulus complex. Conclusions This is the first study showing that progesterone regulates the expression of endothelin1 and endothelin receptor A in the fallopian tube. Together with previous studies of the effects of endothelin on muscular contractions in the fallopian tube, the results from this study suggest that endothelin is a mediator of the progesterone-controlled effects on muscular contraction and eventually gamete transport in the fallopian tube. Electronic supplementary material The online version of this article (doi:10.1186/s12958-015-0038-8) contains supplementary material, which is available to authorized users.

Polycystic ovary syndrome (PCOS) is a state of altered steroid hormone production and activity. Chronic estrogen exposure or lack of progesterone due to ovarian dysfunction can result in endometrial hyperplasia and carcinoma. A key contributor to our understanding of progesterone as a critical regulator for normal uterine function has been the elucidation of progesterone receptor (PR) expression, regulation, and signaling pathways. Several human studies indicate that PR-mediated signaling pathways in the nucleus are associated with progesterone resistance in women with PCOS. The aim of this review is to provide an overview of endometrial progesterone resistance in women with PCOS; to present the PR structure, its different isoforms, and their expression in the endometrium; to illustrate the possible regulation of PR and PR-mediated signaling in progesterone resistance in women with PCOS; and to discuss current clinical treatments for atypical endometrial hyperplasia and endometrial carcinoma in women with PCOS and accompanying progesterone resistance.

Objective: To investigate if progesterone receptor (PR)-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated human luteinizing granulosa cells. Design: Laboratory study. Setting: Goteborg University and an in vitro fertilization laboratory of a university hospital. Patient(s): Women undergoing oocyte retrieval for in vitro fertilization after ovulation induction with gonadotropins. Intervention(s): Luteinizing granulosa cells were isolated from follicular aspirates after oocyte removal. The cells were treated with or without RU 486 (1 μM–100 μM), Org 31710 (1 μM–100 μM), progesterone (1 nM–10 μM), dexamethasone (0.5 μM–100 μM), dihydrotestosterone (1 nM–25 μM), RU 486 (10 μM–100 μM) + dexamethasone (50 μM), and picrotoxin (1 μM–100 μM) and were cultured under serum-free conditions. Main Outcome Measure(s): Measurement of caspase-3 activity; detection of internucleosomal DNA fragmentation using gel electrophoresis and fluorospectrophotometry; progesterone analysis of spent medium. Result(s): Addition of the PR antagonists RU 486 or Org 31710 in vitro to human luteinizing granulosa cells caused an increase in caspase-3 activity and a dose-dependent increase in internucleosomal DNA fragmentation. No effect on DNA fragmentation was seen after addition of dexamethasone, dihydrotestosterone, or picrotoxin . Conclusion(s): Nuclear PR-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated human luteinizing granulosa cells.

PRL has been reported to regulate fat metabolism in several species. We recently reported PRL receptor (PRLR) expression in mouse adipocytes and increased levels of PRLR expression in the adipose tissue of lactating and PRL-transgenic mice compared with controls. These results suggest PRLR-mediated effects in adipose tissue. However, to date most studies have been performed in vivo, and it is unclear whether PRL has direct effects on adipocytes. The PRLR belongs to the cytokine receptor family, and a family of suppressors of cytokine signaling (SOCS) was recently identified. The present study was performed to investigate whether PRL has direct effects on adipocytes. The expression of cytokine-inducible SH2-domain-containing protein (CIS), SOCS-3, and SOCS-2 mRNA and protein was analyzed using ribonuclease protection assay and immunoblotting, respectively. Ovine PRL induced CIS mRNA expression and a combination of oPRL and insulin induced SOCS-3 mRNA expression in adipocytes cultured in vitro for 0-240 min, demonstrating PRLR-mediated direct effects in these cells. Furthermore, CIS, SOCS-3, and SOCS-2 mRNA and protein were all transiently expressed in adipose tissue obtained from female mice stimulated with oPRL (1 microg/g BW) for 0-24 h. In adipose tissue of female mice with endogenously high PRL levels, PRL-transgenic mice, only SOCS-2 expression was increased. The level of SOCS-2 mRNA was also increased in adipose tissue during pregnancy and lactation compared with that in wild-type virgin female mice. A possible reason for increased SOCS-2 expression after prolonged PRL exposure during lactation and in the PRL transgenes could be to restore the sensitivity of adipose tissue to PRL. In addition, the direct effect of PRL on leptin production was investigated in adipocytes cultured in vitro for 6 h. PRL inhibited insulin-induced leptin production in vitro. However, PRL had no effect on leptin production in the absence of insulin. In contrast, serum leptin concentrations were increased in PRL-transgenic females compared with control mice. In conclusion, our results demonstrate functional PRLRs in mouse adipocytes and suggest a role for CIS, SOCS-3, and SOCS-2 in regulating PRL signal transduction in adipose tissue.

The majority of oocytes present in fetal ovaries are depleted before birth, and only about 400 will ovulate during the normal fertile life span. Studies on animals have shown that apoptosis is the mechanism behind oocyte depletion and follicular atresia. In the present study, we investigated the extent and localization of apoptosis in human fetal (aged 13–40 weeks) and adult ovaries. Furthermore, the expression of apoptosis-regulating proteins, bcl-2 and bax, and the relationship of transcription factor GATA-4 were studied. Apoptosis was found in ovarian follicles throughout fetal and adult life. During fetal development, apoptosis was localized mainly to primary oocytes and was highest between weeks 14–28, decreasing thereafter toward term. Expression of bcl-2 was observed only in the youngest fetal ovaries (weeks 13–14), and bax was present in the ovaries throughout the entire fetal period. In adult ovaries, apoptosis was detected in granulosa cells of secondary and antral follicles, and Bcl-2 and bax were expressed from primary follicles onwards. During fetal ovarian development, GATA-4 messenger RNA and protein were localized to the granulosa cells, with expression being highest in the youngest ovaries and decreasing somewhat toward term. The expression pattern of GATA-4 suggests that it may be involved in the mechanisms protecting granulosa cells from apoptosis from fetal to adult life. The results indicate that depletion of ovarian follicles in the human fetus occurs through intrinsic mechanisms of apoptosis in oocytes, and later in adult life the survival of growing follicles may be primarily determined by granulosa cell apoptosis.

Endometriosis is a complex and challenging disease that involves aberrant adhesion, growth, and progression of endometrial tissues outside of the uterine cavity, and there is evidence to suggest that estrogen plays a key role in its development and progression. Numerous in vivo clinical studies have described the ectopic expression and regulation of estrogen receptor (ER) and progesterone receptor (PR) in the different types of endometriosis compared to normal or eutopic endometrium. However, we have noticed that conflicting and contradictory results have been presented in terms of ER subtype (ERα and ERβ) and PR isoform (PRA and PRB) expression. Both ER and PR are transcription factors and ER/PR-mediated responses depend on the coordinated, opposing, and compensatory functions of ER subtypes and PR isoforms. Moreover, analysis of the uterine phenotypes of ERα/ERβ and PRA/PRB knockout mice indicates that different ER subtypes and PR isoforms mediate distinct responses to steroid hormones and play different roles in uterine function. In this review, we outline studies that have elucidated the molecules and signaling pathways that are linked to ER and/or PR signaling pathways in the development and progression of endometriosis.

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.

There are indications that PRL may exert important metabolic actions on adipose tissue in different species. However, with the exception of birds, the receptor has not been identified in white adipose tissue. The present study was designed to examine the possible expression and regulation of the PRL receptor (PRLR) in mouse adipose tissue. The long PRLR messenger RNA (mRNA) splice form (L-PRLR) and two short splice forms (S2- and S3-PRLR) were detected in mouse adipose tissue by RT-PCR. Furthermore, L-PRLR mRNA was detected by ribonuclease protection assay. Immunoreactive PRLR with a relative molecular mass of 95,000 was revealed by immunoblotting. Furthermore, L-PRLR mRNA expression was demonstrated in primary isolated adipocytes. In mouse adipose tissue, the level of L-PRLR mRNA expression increased 2.3-fold during lactation compared with those in virgin and pregnant mice. In contrast, in the liver the expression of L-PRLR increased 3.4-fold during pregnancy compared with those in virgin and lactating mice. When comparing the levels of L-PRLR expression in virgin female and male mice, no difference was detected in adipose tissue. However, in virgin female liver the expression was 4.5-fold higher than that in male liver. As PRL up-regulates its own receptor in some tissues, we analyzed L-PRLR expression in PRL-transgenic female and male mice. In PRL-transgenic mice L-PRLR expression was significantly increased in both adipose tissue (1.4-fold in females and 2.4-fold in males) and liver (1.9-fold in females and 2.7-fold in males) compared with that in control mice. Furthermore, in female PRL-transgenic mice retroperitoneal adipose tissue was decreased in weight compared with that in control mice. However, no difference was detected when comparing the masses of parametrial adipose tissue. Our results suggest a direct role for PRL, mediated by PRLR, in modulating physiological events in adipose tissue.

Ovarian follicular atresia occurs throughout follicular development and involves apoptosis. In addition to regulation by various hormones and growth factors, ovarian granulosa cell apoptosis was shown to be dependent on transcription and translation since the spontaneous onset of DNA degradation in incubated rat granulosa cells was inhibited by actinomycin-D or cycloheximide. Using differential display of mRNA, five transcriptionally upregulated cDNA clones were isolated in the ovary after withdrawal of the anti-apoptotic factor, estrogen. Two of these estrogen-regulated genes, designated ARG-33 and ARG-40, were further characterized using Northern blot hybridizations. ARG-40 showed increased mRNA expression during apoptosis after estrogen withdrawal in the ovary, and after androgen withdrawal in the prostate, while ARG-33 was upregulated during apoptosis only after estrogen withdrawal. This suggests that the two cDNA clones are regulated by steroids, and may be involved in apoptosis in these tissues. ARG-40 showed high homology to cytochrome b, while ARG-33 was novel.

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of high density lipoprotein cholesterol. SR-BI is expressed at high levels in the ovary, indicating that it plays a role in the delivery of cholesterol as substrate for steroid hormone production. However, SR-BI also binds anionic phospholipids with high affinity and could therefore be involved in the recognition of apoptotic cells. In this study we have characterized the expression of SR-BI in rat ovarian follicles undergoing atresia. Atretic follicles with cells undergoing apoptosis were identified by in situ DNA end labeling, and SR-BI expression was determined by in situ hybridization and immunohistochemistry. SR-BI was expressed in thecal cells at all stages of follicular development, including atretic follicles, and in corpus luteum. Isolated apoptotic granulosa cells (but not viable granulosa cells) bound annexin V, indicating that they display anionic phospholipids on the cell surface. Transfection of COS-7 cells with an expression vector carrying the rat SR-BI complementary DNA resulted in increased binding to apoptotic granulosa cells (46 ± 2% of the SR-BI-expressing cells bound at least one granulosa cell compared with 24 ± 3% for the mock-transfected cells; P < 0.0001), whereas the binding to viable granulosa cells was unchanged. Apoptotic granulosa cells also bound to isolated thecal shells. We conclude that thecal cells of both nonatretic and atretic follicles express SR-BI. The location of SR-BI expression in the ovary supports a role of this receptor in the uptake of high density lipoprotein cholesterol. In addition, our data suggest that SR-BI mediates the recognition of apoptotic granulosa cells by the surrounding thecal cells and that it therefore may play a role in the remodeling of atretic follicles to secondary interstitial cells.

We have compared the expression of somatostatin receptor (sstr) subtypes with the outcome of somatostatin receptor scintigraphy and the effect of somatostatin receptor activation in patients with disseminated carcinoid tumours. Tumour tissues from nine patients with midgut carcinoids (ileal) and three patients with foregut carcinoids (gastric, thymic) were analysed using Northern blotting. Expression of somatostatin receptors was demonstrated in all tumours (12 out of 12), with all five receptor subtypes present in 9 out of 12 tumours. Somatostatin receptor scintigraphy using [111In]DTPA-D-Phe1-octreotide visualized tumours in all patients (12 out of 12). The 111In activity concentrations in tumour tissue (T) and blood (B) were determined in three tumours 1-7 days after injection of the radionuclide. The T/B 111In activity concentration ratios ranged between 32 and 651. Clinically, treatment with the long-acting somatostatin analogue octreotide resulted in marked symptom relief accompanied by a significant reduction in tumour markers, for example urinary-5-HIAA levels (28-71% reduction). Incubation of midgut carcinoid tumours in primary culture with octreotide (10 microM) resulted in a reduction in spontaneously secreted serotonin (45-71% reduction) and 5-HIAA (41-94% reduction). The results demonstrate that carcinoid tumours possess multiple somatostatin receptor subtypes and that somatostatin analogues such as octreotide, which preferentially bind to somatostatin receptor subtype 2 and 5, can be used in the diagnosis and medical treatment of these tumours. In the future, novel somatostatin analogues with subtype specific receptor profiles may prove to be of value for individualizing the treatment of disseminated carcinoid tumour disease. Images Figure 1

High density lipoprotein (HDL) participates in reverse cholesterol transport and in the delivery of cholesterol to steroid-producing tissues. Scavenger receptor class B type I (SR-BI) was recently shown to bind HDL and mediate internalization of its cholesterol content. We have cloned the rat homolog of this receptor, determined its chromosomal location, and examined its expression in rat tissues and in a model of follicular development, ovulation, and luteinization. The predicted protein contained two transmembrane domains, a leucine zipper motif, and a peroxisomal targeting sequence. The rat and human SR-BI genes were mapped to a region previously linked between rat and human chromosomes 12. SR-BI gene expression was detected in several rat tissues, with high levels in ovarian tissue, liver, and adrenal cortex, as determined by ribonuclease protection assay and in situ hybridization. A significant increase in SR-BI gene expression was detected in the late phase of corpus luteum formation, and transcripts were abundant in corpus luteum and in thecal cells at all stages of follicular development. In conclusion, the rat SR-BI complementary DNA predicted a protein with several conserved motifs, including a putative leucine zipper and a peroxisomal targeting sequence. The chromosomal locations of the rat and human SR-BI homologs suggest that this gene is a new member of a previously reported, conserved synteny group. SR-BI gene expression was high in steroid-producing tissues and in the liver, consistent with a role of this receptor in the uptake of HDL cholesterol.

It is likely that many paediatricians will find the Internet useful. The main benefits are probably the ease and speed of communication and immediate access to a few databases such as MEDLINE. It is also practical to integrate the import, processing, storage, and export of data into one's own computer. It is also possible that the Internet in all its forms will become an integrated part of our daily paediatric practice as a result of the increased usage of the Internet by patients, parents, and paediatricians.

Human ectopic pregnancy (EP) is a leading cause of pregnancy-related death, but the molecular basis underlying the onset of tubal EP is largely unknown. Female Dicer1 conditional knockout mice are infertile with dysfunctional Fallopian tube and have a different miRNA expression profile compared to wild-type mice, and we speculated that Dicer-mediated regulation of miRNA expression and specific miRNA-controlled targets might contribute to the onset of tubal EP. In the present study, we used microarray analysis and quantitative RT-PCR to examine the expression of miRNAs and core miRNA regulatory components in Fallopian tube tissues from women with EP. We found that the levels of DICER1, four miRNAs (let-7i, miR-149, miR-182, and miR-424), and estrogen receptor α distinguished the tubal implantation site from the non-implantation site. Computational algorithms and screening for interactions with the estrogen and progesterone receptor signaling pathways showed that the four miRNAs were predicted to target ten genes, including NEDD4, TAF15, and SPEN. Subsequent experiments showed differences in NEDD4 mRNA and protein levels between the implantation and non-implantation sites. Finally, we revealed that increases in smooth muscle cell NEDD4 and stromal cell TAF15, in parallel with a decrease in epithelial cell SPEN, were associated with tubal implantation. Our study suggests that changes in miRNA levels by the DICER-mediated miRNA-processing machinery result in aberrant expression of cell type-specific proteins that are potentially involved in the onset of tubal EP.

Background The transport of gametes as well as the zygote is facilitated by motile cilia lining the inside of the fallopian tube. Progesterone reduces the ciliary beat frequency within 30 minutes in both cows and mice. This rapid reduction suggest the involvement of a non-genomic signaling mechanism, although it is not known which receptors that are involved. Here we investigated the possible involvement of the classical progesterone receptor in this process. Method The ciliary beat frequency of mice fallopian tube was measured ex vivo using an inverted bright field microscope and a high speed camera. The effects of the agonists progesterone and promegestone and an antagonist, mifeprestone, were investigated in wildtype mice. The effect of progesterone was also investigated in mice lacking the classical progesterone receptor. Results Progesterone, as well as the more specific PR agonist promegestone, significantly reduced the CBF at concentrations of 10–100 nanomolar within 10–30 minutes. In the absence of progesterone, the PR antagonist mifeprestone had no effect on the ciliary beat frequency at a concentration of 1 micromolar. When ciliated cells were pre-incubated with 1 micromolar mifeprestone, addition of progesterone did not reduce the ciliary beat frequency. Accordingly, in ciliated cells from mice not expressing the classical progesterone receptor, exposure to 100 nanomolar progesterone did not reduce the ciliary beat frequency. Conclusions This is the first study to provide comprehensive evidence that the classical progesterone receptor mediates the rapid reduction of the tubal ciliary beat frequency by progesterone.

Ectopic pregnancy is a common reproductive disorder of unknown etiology and is a leading cause of maternal and fetal mortality. Because of the asymptomatic nature of early tubal ectopic pregnancy and the lack of specific biomarkers for early diagnosis, a better understanding of the complex cellular and molecular interactions that contribute to tubal ectopic pregnancy is required. DNA methylation is the most studied epigenetic process in various tissues and cells, and the goal of this article is to provide a brief review of recent work describing the potential mechanisms of DNA methylation and the biological function of such methylation in normal intrauterine pregnancy. Further, novel findings from our laboratory highlight the possible role of DNA methylation in human Fallopian tube dysfunction and suggest a possible correlation between methylation of estrogen receptor α in women and the occurrence of tubal ectopic pregnancies.

In rats with dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS), repeated low-frequency electrical stimulation of acupuncture needles restores whole-body insulin sensitivity measured by euglycemic hyperinsulinemic clamp. We hypothesized that electrical stimulation causing muscle contractions and manual stimulation causing needle sensation have different effects on insulin sensitivity and related signaling pathways in skeletal muscle and adipose tissue, with electrical stimulation being more effective in DHT-induced PCOS rats. From age 70 days, rats received manual or low-frequency electrical stimulation of needles in abdominal and hind limb muscle five times/wk for 4-5 wks; controls were handled but untreated rats. Low-frequency electrical stimulation modified gene expression (decreased Tbc1d1 in soleus, increased Nr4a3 in mesenteric fat) and protein expression (increased pAS160/AS160, Nr4a3 and decreased GLUT4) by western blot and increased GLUT4 expression by immunohistochemistry in soleus muscle; glucose clearance during oral glucose tolerance tests was unaffected. Manual stimulation led to faster glucose clearance and modified mainly gene expression in mesenteric adipose tissue (increased Nr4a3, Mapk3/Erk, Adcy3, Gsk3b), but not protein expression to the same extent; however, Nr4a3 was reduced in soleus muscle. The novel finding is that electrical and manual muscle stimulation affect glucose homeostasis in DHT-induced PCOS rats through different mechanisms. Repeated electrical stimulation regulated key functional molecular pathways important for insulin sensitivity in soleus muscle and mesenteric adipose tissue to a larger extent than manual stimulation. Manual stimulation improved whole-body glucose tolerance, an effect not observed after electrical stimulation, but did not affect molecular signaling pathways to the same extent as electrical stimulation. Although more functional signaling pathways related to insulin sensitivity were affected by electrical stimulation, our findings suggest that manual stimulation of acupuncture needles has a greater effect on glucose tolerance. The underlying mechanism of the differential effects of the intermittent manual and the continuous electrical stimulation remains to be elucidated.

Cryptorchidism is associated with histologic changes in the human testis apparent by 2 y of age. The mechanism accounting for these changes is still unknown. To clarify whether apoptosis plays a role in human cryptorchidism, we evaluated its occurrence in cryptorchid testes of 73 prepubertal boys, 43 of whom had received human chorionic gonadotropin (hCG) treatment. The histologic samples in our study included both scrotal and inguinal testes. Using anin situ apoptosis detection method, we were able to demonstrate that both interstitial cells and germ cells were affected and that the specific germ cells undergoing apoptosis were exclusively spermatogonia. Apoptosisin situ was further seen in both scrotal and inguinal testes; in scrotal testes the numbers of apoptotic spermatogonia were 170% of those seen in the cryptorchid testes (p < 0.05). Analysis of apoptotic DNA fragmentation from isolated DNA of a few selected biopsy samples served to validate our in situ findings. The amount of germ cell apoptosis analyzed during the 1st mo after hCG treatment was increased in both scrotal and inguinal testes compared with the amount before treatment (p< 0.001). But after the 1st mo it returned to the initial level, suggesting that hCG (and/or androgen) withdrawal increases germ cell apoptosis in the human testis. Our findings lead to the conclusion that apoptosis is a hormonally controlled, normal phenomenon in a human prepubertal testis and that cryptorchidism decreases its occurrence by reducing the number of germ cells capable of undergoing apoptosis.

It has become evident that apoptosis, an active form of cell 'suicide', plays an important role in the normal function of all tissues. A balance of cell proliferation and apoptosis is maintained in a healthy individual and any imbalance of the two processes could lead to pathological changes. In both sexes, massive apoptosis accounts for the demise of a majority of gonadal cells (ovarian granulosa cells and male germ cells) during reproductive life. Recent studies have indicated the important role of gonadotrophins as survival factors in both the ovary and the testis. Furthermore, intra-gonadal survival. factors in the ovary (oestrogens, insulin-like growth factor I, epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta, nitric oxide, etc.) and testis (androgens) have been shown to act in concert with the gonadotrophins. In contrast, several apoptotic factors (androgens, gonadotrophin-releasing hormone-like peptide and interleukin-6) may be important in inducing the demise of ovarian follicles. Understanding of the hormonal and cellular mechanisms responsible for gonadal cell apoptosis will provide new approaches for the treatment of gonadal degenerative conditions such as premature ovarian failure and cryptorchidism, as well as for the design of new contraceptive approaches.

A growing body of evidence suggests that growth hormone (GH) plays a role in regulating ovarian function by augmenting gonadotropin stimulation of granulosa cell differentiation and folliculogenesis. The majority of follicles in the mammalian ovary do not ovulate, but instead undergo a degenerative process (atresia) involving apoptotic cell death. The objective of the present study was to investigate the role of GH in regulating follicle apoptosis and to determine whether or not insulin-like growth factor-I (IGF-I) mediates GH action in this process. Preovulatory follicles obtained from eCG-primed rats were cultured for 24 h in serum-free conditions with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3' ends with [32P]dideoxy-ATP. Culture of preovulatory follicles resulted in a spontaneous onset of apoptotic DNA fragmentation that was suppressed by ovine GH (oGH) in a dose-dependent manner, reaching a maximum of 65% suppression. To rule out the effect of residual gonadotropin in the oGH preparation, follicles were also cultured with recombinant bovine growth hormone (rbGH). Like oGH, rbGH suppressed apoptotic DNA fragmentation. Our earlier study indicated that hCG and FSH treatment also suppress apoptosis in the present model system, but no additive effect of GH and either hCG or FSH on the suppression of apoptosis was observed. To determine whether the observed effect of GH action on follicle apoptosis is mediated by IGF-I, three types of studies were carried out.(ABSTRACT TRUNCATED AT 250 WORDS)

Background Several peripheral proteins that might be useful for detecting the presence of ectopic pregnancy (EP) have been evaluated, but none have been proven entirely useful in the clinic. We investigated the presence and the possible changes in circulating molecules that distinguish between normal intrauterine pregnancy (IUP) and tubal ectopic pregnancy. Methods Non-pregnant women during the menstrual cycle, women with IUP, and women with tubal EP after informed consent. Serum levels of 17β-estradiol (E2), progesterone (P4), testosterone (T), beta-human chorionic gonadotropin (β-hCG), vascular endothelial growth factor-A (VEGF-A), placental growth factor (PIGF), and a distintegrin and metalloprotease protein 12 (ADAM12) were analyzed. Receiver operating characteristic analysis was used to assess the diagnostic discrimination of EP and gestational age-matched IUP. Results E2, P4, PIGF, and ADAM12 levels increased and β-hCG decreased throughout IUP. E2 and VEGF-A levels were significantly different between women with tubal EP and IUP. However, using a serum β-hCG cut-off of less than 1000 mIU/mL, P4 was significantly lower in women with tubal EP compared to IUP. Although E2 was inversely correlated with VEGF-A in women in the early stages of IUP, E2 was not correlated with VEGF-A in women with EP prior to tubal surgery. There were no significant differences in either PIGF or ADAM12 alone between women with tubal EP or IUP. Although no significant correlations were seen between E2 and PIGF or P4 and ADAM12 in women in the early stages of IUP, E2 was positively correlated with PIGF and P4 was positively correlated with ADAM12 in women with EP prior to tubal surgery. Our studies defined associations but not causality. Conclusions Individual measurements of serum E2 or VEGF-A levels are strongly related to early pregnancy outcomes for women with IUP and EP, and pregnancy-associated E2 and VEGF-A levels provide diagnostic accuracy for the presence of tubal EP. This study demonstrates that correlation analysis of E2/VEGF-A and E2/PIGF serum levels may be able to distinguish a tubal EP from a normal IUP.

17β-estradiol, acting through estrogen receptors α and β, plays a fundamental role in the regulation of Fallopian tube cell homeostasis and in the modulation of normal tubal physiological processes. Fluctuations in E2 levels also play crucial roles in the initiation or progression of numerous human diseases. Fallopian tube malfunction often results in tubal ectopic pregnancy, which is one cause of maternal morbidity and mortality in women. Several factors have been proposed to be associated with increased risk of tubal ectopic pregnancy, but whether these factors are the cause of, or are merely symptoms of, such pregnancies remains unresolved due to the lack of knowledge in regards to the mechanisms by which embryos inadvertently implant in the Fallopian tube. This review summarizes recent findings, including data from our own laboratory, on E2 metabolism and estrogen receptor (ER) subtype expression within the Fallopian tube in humans and rodents. This review also outlines several important, unresolved questions in the field that, once addressed, could offer important clues into how E2/ER signaling contributes to the pathology of tubal function. A better understanding of the specific functions of estrogen receptor subtypes in vivo, as well as of the mechanism and consequences of receptor subtype interactions is critical to understanding their respective roles in Fallopian tube physiology and in the pathophysiology and etiology of tubal ectopic pregnancy.

Ectopic pregnancy (EP) is an enigmatic reproductive disorder. Although tubal EP is difficult to predict, several hypotheses about its etiology have been proposed. In retrospective case-control studies, smoking is associated with an increased rate of EPs in the fallopian tube. Studies of experimental animals in vivo and human fallopian tubal tissues in vitro have suggested mechanisms of fallopian tubal damage and dysfunction induced by nicotine and other smoking-related chemicals that may explain this association. However, the pathogenesis of smoking-induced modulation of implantation leading to tubal EP is largely unknown. Because cigarette/tobacco smoke adversely affects the success of intrauterine implantation, there is a great need to determine how embryo implantation occurs in the fallopian tube in female smokers of reproductive age.

Both low-frequency electro-acupuncture (EA) and manual acupuncture improve menstrual frequency and decrease circulating androgens in women with polycystic ovary syndrome (PCOS). We sought to determine whether low-frequency EA is more effective than manual stimulation in regulating disturbed oestrous cyclicity in rats with PCOS induced by 5α-dihydrotestosterone. To identify the central mechanisms of the effects of stimulation, we assessed hypothalamic mRNA expression of molecules that regulate reproductive and neuroendocrine function. From age 70 days, rats received 2 Hz EA or manual stimulation with the needles five times per week for 4-5 weeks; untreated rats served as control animals. Specific hypothalamic nuclei were obtained by laser microdissection, and mRNA expression was measured with TaqMan low-density arrays. Untreated rats were acyclic. During the last 2 weeks of treatment, seven of eight (88%) rats in the EA group had epithelial keratinocytes, demonstrating oestrous cycle change (P = 0.034 versus control rats). In the manual group, five of eight (62%) rats had oestrous cycle changes (n.s. versus control animals). The mRNA expression of the opioid receptors Oprk1 and Oprm1 in the hypothalamic arcuate nucleus was lower in the EA group than in untreated control rats. The mRNA expression of the steroid hormone receptors Esr2, Pgr and Kiss1r was lower in the manual group than in the control animals. In rats with 5α-dihydrotestosterone-induced PCOS, low-frequency EA restored disturbed oestrous cyclicity but did not differ from the manual stimulation group, although electrical stimulation lowered serum testosterone in responders, those with restored oestrus cyclicity, and differed from both control animals and the manual stimulation group. Thus, EA cannot in all aspects be considered superior to manual stimulation. The effects of low-frequency EA may be mediated by central opioid receptors, while manual stimulation may involve regulation of steroid hormone/peptide receptors.

GREATER THAN 99% of ovarian follicles undergo a degenerative process called atresia during reproductive life. Extending earlier morphological analysis, recent studies have demonstrated that apoptotic cell death is the molecular mechanism underlying follicle atresia. The use of DNA 3′-end-labeling methods allows quantitation and identification of internucleosomal degradation of DNA after gel fractionation as well as in situ analysis of specific cell types undergoing DNA fragmentation in histological sections. Using rats as the experimental model, gonadotropins, epidermal growth factor (EGF)/transforming growth factor-α (TGFα), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and estrogens have been identified as follicle survival factors capable of suppressing apoptotic DNA fragmentation, whereas androgens, interleukin-6 (IL-6), and GnRH are potential atretogenic factors. This review summarizes the historical background of studies on follicle atresia and selection as well as rec...

The causes of anxiety and depression in women with polycystic ovary syndrome (PCOS) remain elusive. To identify steps linking androgen signaling to the regulation of affective symptoms in vivo, we compared behavioral responses in female rats continuously exposed to DHT from puberty (a model of DHT-induced PCOS) and in rats exposed to DHT for 1 week. Continuous and 1 week of DHT exposure resulted in a general decrease in locomotor activity and time spent on the open arms in the elevated plus maze, indicating anxiety-like behavior. Rats with DHT-induced PCOS have increases in adiposity and circulating leptin levels accompanied by leptin resistance. One week of DHT exposure decreased androgen receptor (AR) expression in the hypothalamus and leptin synthesis and function in adipocytes; it also inhibited signal transducer and activator of transcription 3 (STAT3) and attenuated leptin activity by increasing levels of soluble leptin receptor, a leptin-binding protein, in the hypothalamus. This may affect the androgen-induced anxiety-related behavior in female rats. In conclusion, our results highlight the central role of androgens in behavioral function in female rats and suggest that androgens directly regulate the AR by decreasing its hypothalamic expression. Androgens also increase leptin synthesis in adipocytes, which drives central leptin signaling, and may regulate anxiety-related behaviors. Elucidating mechanisms by which androgens modulate female anxiety-like behavior may uncover useful approaches for treating women with PCOS who have symptoms of anxiety.

Recent studies have indicated that growth factors such as insulin-like growth factors (IGFs) increase the growth rate of cultured preimplantation embryos. We therefore hypothesized that the fallopian tube may produce IGFs which in turn participate in the regulation of preimplantation embryo development in vivo. In the present study we examined the expression of IGF-I in the fallopian tube. We demonstrated that IGF-I transcripts (7.0, 1.7, and 1.2-0.8 kilobases) were abundant in the fallopian tube. Immunoreactive IGF-I was most abundant in the epithelial cells in the fallopian tube, and IGF-I messenger RNA (mRNA) was detected in the luminal region of the fallopian tube. A solution hybridization assay was used to examine the regulation of IGF-I mRNA. The abundance of IGF-I transcripts changed markedly during the 4-day estrous cycle with the highest levels on the day of proestrus. The increase in IGF-I mRNA between the day of diestrus II and the day of proestrus was 4-fold (P0.01). The pattern of IGF-I mRNA expression in the fallopian tube resembled the pattern of ovarian estrogen production during the estrous cycle. The level of IGF-I mRNA decreased after hypophysectomy. The expression of IGF-I mRNA in the fallopian tube was dose-dependently regulated by estradiol, and a single sc injection of estradiol [5 micrograms/100 g body wt (BW)] increased the IGF-I mRNA in a time-dependent manner with a significant increase after 3 h (P0.01). The lowest estradiol dose tested (0.1 microgram/100 g BW) increased the expression after 6 h, whereas progesterone (5 micrograms/100 g BW) was ineffective. The presence of embryos in the fallopian tube did not statistically significantly influence the abundance of IGF-I transcripts as measured with a solution hybridization assay on RNA extracted from whole fallopian tubes. In order to determine possible targets for fallopian tube-derived IGF-I we examined the expression of IGF-I receptor mRNA. Northern blot analysis revealed that an 11-kilobase IGF-I receptor transcript was expressed in the fallopian tube. Using a reverse transcriptase-polymerase chain reaction, IGF-I receptor mRNA was also detected in the eight-cell but not two-cell preimplantation embryo. The present study demonstrates that IGF-I is produced in the fallopian tube and its expression is regulated by estradiol. Both the fallopian tube and the eight-cell preimplantation embryo express IGF-I receptors and are therefore potential target tissues.

The results of several reports indicate that GH can modulate ovarian function. In the present study, the expression of the growth hormone-receptor (GH-R) mRNA was studied in the rat ovary using an RNA probe corresponding to a part of the extracellular domain of the GH-R. The probe hybridized to two major transcripts with estimated sizes of 4.0 kb and 1.2 kb in RNA extracted from liver and ovary. Recently, these transcripts have been shown to encode the GH-R and the GH-binding protein (GH-BP). The ontogeny of the GH-R/GH-BP mRNA expression was studied using Northern blot analysis and a solution hybridization RNase protection assay. In the liver GH-R/GH-BP mRNA levels increased with age, while in the ovary, the levels decreased between 1 and 5 weeks of age. Hypophysectomy caused a decrease in GH-R/GH-BP gene expression in the ovary, an effect which could be partly reversed with a single injection of GH (2 mg/kg). No significant changes in the ovarian concentration of GH-R/GH-BP transcripts were seen during the estrus cycle. Using in situ hybridization GH-R/GH-BP transcripts were found to be most abundant in follicles. Northern blot analysis of RNA extracted from isolated granulosa cells and corpora lutea showed that both these compartments contained GH-R and GH-BP mRNA, although more abundant in granulosa cells. Immunoreactive GH-R was detected in granulosa cells of healthy follicles, corpus luteum, and in the germinal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism and insulin resistance, possibly reflecting defects in skeletal muscle and adipocyte insulin signaling. Low-frequency (2 Hz) electroacupuncture (EA) increases insulin sensitivity in female rats with dihydrotestosterone (DHT)-induced PCOS, but the mechanism is unclear. We hypothesized that low-frequency EA regulates mediators involved in skeletal muscle glucose uptake and metabolism and alters the lipid profile in rats with DHT-induced PCOS. To test this hypothesis, we implanted in prepubescent female rats 90-day continuous-release pellets containing DHT (PCOS). At 70 days of age, the rats were randomly subdivided into two groups: one received low-frequency EA (evoking muscle twitches) for 20–25 min five times/wk for 4–5 wk; the other did not. Controls were implanted with pellets containing vehicle only. All three groups were otherwise handled similarly. Lipid profile was measured in fasting blood samples. Insulin sensitivity was determined by euglycemic hyperinsulinemic clamp, soleus muscle protein expression of glucose transporter 4 (GLUT4), and phosphorylated and nonphosphorylated Akt, and Akt substrate of 160 kDa was determined by Western blot analysis and GLUT4 location by immunofluorescence staining. PCOS EA rats had normalized insulin sensitivity, lower levels of total high-density lipoprotein and low-density lipoprotein cholesterol, and increased expression of GLUT4 in different compartments of skeletal muscle compared with PCOS rats. Total weight and body composition did not differ in the groups. Thus, in rats with DHT-induced PCOS, low-frequency EA has systemic and local effects involving intracellular signaling pathways in muscle that may, at least in part, account for the marked improved insulin sensitivity.

Background The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations. Methods A method to assess tubal CBF of mice was developed. Fallopian tubes were dissected and the tissue was cut in small pieces. Tissue samples with moving cilia were located under an inverted bright field microscope and held still against the bottom of a petri dish by a motorized needle system. Images were acquired over 90 minutes at 35 degrees C with a high-speed camera and used for assessing changes in the CBF in response to the addition of hormone. Results The baseline CBF of the mouse fallopian tube was 23.3 +/- 3.8 Hz. The CBF was stable over at least 90 minutes allowing establishment of a baseline frequency, addition of hormone and subsequent recordings. Progesterone at concentrations of 20 micromolar and 100 nM significantly reduced the CBF by 10% and 15% respectively after 30 minutes compared with controls. Conclusions The present study demonstrates that the mouse, despite its small size, is a useful model for studying the fallopian tube CBF ex vivo. The rapid reduction in CBF by 100 nM progesterone suggests that gamete transport in the fallopian tube could be mediated by progesterone via a non-genomic receptor mechanism.

Adult female rats continuously exposed to androgens from prepuberty have reproductive and metabolic features of polycystic ovary syndrome (PCOS). We investigated whether such exposure adversely affects estrous cyclicity and the expression and distribution of gonadotropin-releasing hormone (GnRH), GnRH receptors, and corticotrophin-releasing hormone (CRH) in the hypothalamus and whether the effects are mediated by the androgen receptor (AR). We also assessed the effect of low-frequency electro-acupuncture (EA) on those variables. At 21 days of age, rats were randomly divided into three groups (control, PCOS, and PCOS EA; n = 12/group) and implanted subcutaneously with 90-day continuous-release pellets containing vehicle or 5alpha-dihydrostestosterone (DHT). From age 70 days, PCOS EA rats received 2-Hz EA (evoking muscle twitches) five times/week for 4-5 weeks. Hypothalamic protein expression was measured by immunohistochemistry and western blot. DHT-treated rats were acyclic, but controls had regular estrous cycles. In PCOS rats, hypothalamic medial preoptic AR protein expression and the number of AR- and GnRH-immunoreactive cells were increased, but CRH was not affected; however, GnRH receptor expression was decreased in both the pituitary and hypothalamus. Low-frequency EA restored estrous cyclicity within 1 week and reduced the elevated hypothalamic GnRH and AR expression levels. EA did not affect GnRH receptor or CRH expression. Interestingly, nuclear AR co-localized with GnRH in the hypothalamus. Thus, rats with DHT-induced PCOS have disrupted estrous cyclicity and an increased number of hypothalamic cells expressing GnRH, most likely mediated by AR activation. Repeated low-frequency EA normalized estrous cyclicity and restored GnRH and AR protein expression. These results may help explain the beneficial neuroendocrine effects of low-frequency EA in women with PCOS.

Prolactin is best known for its involvement in lactation, where it regulates mechanisms that supply nutrients for milk production. In individuals with pathological hyperprolactinemia, glucose and fat homeostasis have been reported to be negatively influenced. It is not previously known, however, whether prolactin regulates lipogenesis in human adipose tissue. The aim of this study was to investigate the effect of prolactin on lipogenesis in human adipose tissue in vitro. Prolactin decreased the concentration of malonyl-CoA, the product of the first committed step in lipogenesis, to 77+/-6% compared to control 100+/-5% (p=0.022) in cultured human adipose tissue. In addition, prolactin was found to decrease glucose transporter 4 ( GLUT4) mRNA expression, which may cause decreased glucose uptake. In conclusion, we propose that prolactin decreases lipogenesis in human adipose tissue as a consequence of suppressed malonyl-CoA concentration in parallel with decreased GLUT-4 expression. In the lactating woman, this regulation in adipose tissue may enhance the provision of nutrients for the infant instead of nutrients being stored in adipose tissue. In hyperprolactinemic individuals, a suppressed lipogenesis could contribute to an insulin resistant state with consequences for the health.

In this study, it was hypothesized that progesterone (P4) acts as a survival factor primarily by actions of the classic nuclear progesterone receptor (PGR) signaling pathway in rat periovulatory granulosa cells. Granulosa cells were isolated from immature female rats primed with equine chorionic gonadotropin/human chorionic gonadotropin and treated in vitro with PGR antagonists. As little as 10 nM of two different PGR antagonists (Org 31710 and RU 486) increased apoptosis measured as caspase 3/7 activity, which was reversed by cotreatment with the progestin R5020. Concurrently, P4 synthesis was decreased. Inhibition of P4 synthesis by cyanoketone similarly induced apoptosis but required greater inhibition of P4 synthesis than that seen after treatment with PGR antagonists. Therefore, the induction of apoptosis by PGR antagonists cannot be explained by decreased P4 synthesis alone. Low concentrations of R5020 also completely reversed the effects of cyanoketone. Inhibition of P4 synthesis was more effective in inducing apoptosis than treatment with PGR antagonists. However, cotreatment with PGR antagonists protected cells from the additional effects of cyanoketone, indicating partial agonist effects of the antagonists and a dominating role for PGR in P4-mediated regulation of apoptosis. Progesterone receptor membrane component 1 (PGRMC1) was expressed in granulosa cells; however, an anti-PGRMC1 antibody did not induce apoptosis in periovulatory granulosa cells. Neither anti-PGRMC1 nor P4 or cyanoketone affected apoptosis of immature granulosa cells. In conclusion, we show that P4 regulates apoptosis in periovulatory granulosa cells by acting via the classic nuclear receptor.

The effects of hypophysectomy and hormonal replacement therapy on GH receptor (GH-R) gene expression was studied in rat adipose tissue with a cRNA probe corresponding to the amino-terminal of the hepatic GH-R. Male Sprague-Dawley rats, 50-65 days of age, were used. In all fat depots tested (epididymal, retroperitoneal, and sc), two transcripts with an estimated size of 4.0 and 1.2 kilobases (kb), respectively, were detected. An intermediate-size transcript (2.6 kb) was sometimes observed. Also, isolated adipocytes and adipocyte precursor cells from the epididymal fat pad expressed these GH-R transcripts. The pituitary dependance of GH-R gene expression was analyzed in epididymal fat. Hypophysectomies were performed at 50 days of age, and the rats were then given replacement therapy with L-T4 (10 micrograms/kg.day) and hydrocortisone (400 micrograms/kg.day). Hypophysectomy decreased the abundance of both the 4.0 and the 1.2-kb transcripts, an effect that in part was restored by GH treatment. A solution hybridization RNase protection assay was then used to further characterize the effect of GH treatment of hypophysectomized rats on GH-R gene expression. A single injection of human GH (100 micrograms/rat) increased GH-R mRNA levels within 1 h, and maximal levels were reached between 3-12 h after the injection. The increase in GH-R mRNA levels was dose dependent and was observed also after prolonged treatment (1 or 5 mg/kg.day for 6 days) with bovine GH. These results confirm that GH-R mRNAs are present in rat adipose tissue from different fat depots. GH-R transcripts of the same estimated size were detected in isolated adipocytes and adipocyte precursor cells. Furthermore, the results show that there is a rapid and GH-dependent regulation of GH-R mRNA levels in adipose tissue.

The ontogeny and estrous cycle-dependent variation of insulin-like growth factor-I (IGF-I) gene expression was analyzed in the rat uterus. RNA extracted from rat uteri contained transcripts with estimated sizes of 7.0, 1.7, and 1.2-0.8 kb that were recognized by a 32P-labelled mouse IGF-I RNA probe. A solution hybridization RNase protection assay was used to measure the abundance of IGF-I mRNAs in uteri from rats of different ages. The highest levels were found in adult rats (p less than 0.01). The levels of IGF-I transcripts changed markedly during the estrous cycle with the highest levels at proestrus (p less than 0.01). There was an 8-fold increase in the abundance of IGF-I mRNA between diestrus-2 and proestrus. The corresponding livers had no significant variation of IGF-I gene expression during the estrous cycle, demonstrating a tissue-specific regulation of the IGF-I gene. The time and dose dependency of estrogen regulation of IGF-I gene expression was studied in hypophysectomized rats. The levels of IGF-I mRNA in the uterus decreased after hypophysectomy. A single s.c. injection of estradiol significantly increased the levels of IGF-I transcripts after 3 h (p less than 0.01). A low dose of estradiol (0.1 micrograms/100 g) increased the levels of IGF-I transcripts but progesterone in higher doses (5 micrograms/100 g) was without effect, indicating that the effect was specific for estradiol. However, the present study provides no information regarding whether this regulation is at the level of transcription or mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)

The anterior pituitary-derived hormone prolactin (PRL) signals through the PRL receptor (PRLR) and is important for female reproductive function in mammals. In contrast to the extensive studies of PRLR expression and regulation in human and mouse ovary and uterus, the mechanisms controlling the regulation of PRLR isoform expression in the fallopian tube are poorly understood. Because dynamic interaction of hormonal signaling in gonadal tissue and the pituitary or in gonadal tissues themselves in mammals suggests endocrine or paracrine regulation of PRLR expression, we questioned whether differential regulation of PRLR isoforms by PRL ovarian-derived estrogen (E(2)) and progesterone (P(4)) exists in the fallopian tube and pituitary of prepubertal female mice. Western blot analysis showed distinct molecular separation of PRLR isoforms in mouse and human fallopian tubes, and cellular localization was found in mouse and human tubal epithelia but not in mouse tubal smooth muscle cells. These data support the concept of an isoform- and cell type-specific expression of PRLR in human and mouse fallopian tubes. Moreover, expression of the long form of PRLR decreased after PRL treatment and increased after blockage of endogenous PRL secretion by bromocriptine (an inhibitor of PRL secretion) in a time-dependent manner in mouse fallopian tube. The opposite regulation was observed in the pituitary. Treatment with exogenous E(2) or P(4) led to changes in PRLR expression in the fallopian tube similar to those of PRL treatment. However, E(2) and P(4) did not affect PRLR expression in the pituitary. Estrogen had no effect on the long form of PRLR expression, whereas P(4) regulated the long form of PRLR in the fallopian tube, as did PRL. Taken together, the data from our comparative study provide evidence that PRLR can be regulated by an interplay of two different mechanisms, PRL or ovarian steroid hormones independently or in combination in a tissue-specific manner. Furthermore, we found that ovarian steroid hormones selectively suppress the expression of PRLR isoforms in mouse fallopian tubes. These findings may contribute to our understanding of the mechanisms controlling PRLR isoform expression in the fallopian tube (in addition to ovary and uterus), with implications for female reproduction.

The presented data demonstrate a dose-dependent inhibition of spontaneous meiosis of cumulus-enclosed rat oocytes by guanosine, hypoxanthine, and adenosine. The inhibition by adenosine was transient whereas guanosine and hypoxanthine exerted a persistent effect over 24 h of incubation. The order of potency of the substances was guanosine greater than hypoxanthine greater than adenosine and the inhibition was reversible. The inhibitory effect was reduced when the cumulus cells around the oocyte were removed. The inhibition during the first 12 h of incubation was potentiated by FSH. However, at 24 h of incubation FSH partially overcame the inhibitory effect by hypoxanthine but did not influence the inhibitory effect by guanosine. Also 8BrcAMP potentiated the inhibitory effect observed by guanosine, hypoxanthine, and adenosine, suggesting that the potentiating effect of FSH was mediated via cAMP. Our data demonstrate that adenosine, hypoxanthine, and guanosine synergized with FSH in inhibiting spontaneous rat meiosis, as previously shown in mouse. FSH could partially overcome the inhibitory effect exerted by hypoxanthine but did not counteract the inhibitory effect of guanosine.