Depressive symptoms, body composition and bone mass in young adults: a prospective cohort study.

BACKGROUND: An association between depression and obesity is well recognised, but longitudinal studies of depressive symptoms in adolescents as a predictor of body composition are lacking. OBJECTIVE: We examined depressive symptoms at age 14, 17 and 20 years as predictors of lean, fat and bone mass at age 20 years in a birth cohort. SUBJECTS/METHODS: In 1161 participants (569 females) in the Western Australia Pregnancy Cohort (Raine) Study, depressive symptoms were assessed using the Beck Depression Inventory for Youth at age 14 and 17 years, and the Depression, Anxiety and Stress Scale 21 at age 20 years. Participants were further classified into two trajectories using latent class analysis: no/transient and persistent/recurrent depression. At age 20 years, lean body mass (LBM), fat body mass (FBM) and total body bone mass were measured by dual-energy X-ray absorptiometry. RESULTS: In females, accounting for age and lifestyle factors, depression scores at age 14 and 20 years were positively associated with body weight, body mass index (BMI), FBM and % FBM (r=0.110-0.184, P<0.05) but negatively correlated with % LBM (r=-0.120, P<0.05) at age 20 years. Females in the persistent/recurrent depression trajectory (n=99) had significantly higher body weight (+5.1 kg), BMI (+1.8 kg m-2), FBM (+3.9 kg) and % FBM (+2.2%) and significantly lower % LBM (-2.2%) at age 20 years than those with no/transient depression (n=470; all P<0.05). In males, depression scores at age 17 and 20 years were negatively associated with LBM but not weight or BMI, and depression trajectory was not a predictor of body composition at age 20 years. Depression scores and trajectories did not predict bone mass in either males or females. CONCLUSIONS: Depressive symptoms and persistent/recurrent depression in adolescence are predictors of greater adiposity at age 20 years in females, but not males, but do not predict bone mass in either gender.

Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors.

Gender differences in the relationships between lean body mass, fat mass and peak bone mass in young adults.

UNLABELLED: The relationships between fat mass and bone mass in young adults are unclear. In 1,183 young Australians, lean body mass had a strong positive relationship with total body bone mass in both genders. Fat mass was a positive predictor of total body bone mass in females, with weaker association in males. INTRODUCTION: Body weight and lean body mass are established as major determinants of bone mass, but the relationships between fat mass (including visceral fat) and peak bone mass in young adults are unclear. The aim of this study was to evaluate the associations between bone mass in young adults and three body composition measurements: lean body mass, fat mass and trunk-to-limb fat mass ratio (a surrogate measure of visceral fat). METHODS: Study participants were 574 women and 609 men aged 19-22 years from the Raine study. Body composition, total body bone mineral content (TBBMC), bone area and areal bone mineral density (TBBMD) were measured using DXA. RESULTS: In multivariate linear regression models with height, lean body mass, fat mass and trunk-to-limb fat mass ratio as predictor variables, lean mass was uniquely associated with the largest proportion of variance of TBBMC and TBBMD in males (semi-partial R(2) 0.275 and 0.345, respectively) and TBBMC in females (semi-partial R(2) 0.183). Fat mass was a more important predictor of TBBMC and TBBMD in females (semi-partial R(2) 0.126 and 0.039, respectively) than males (semi-partial R(2) 0.006 and 0.018, respectively). Trunk-to-limb fat mass ratio had a weak, negative association with TBBMC and bone area in both genders (semi-partial R(2) 0.004 to 0.034). CONCLUSIONS: Lean body mass has strong positive relationship with total body bone mass in both genders. Fat mass may play a positive role in peak bone mass attainment in women but the association was weaker in men; different fat compartments may have different effects.

Design, fabrication, and characterization of thermoplastic microlenses for fiber-optic probe imaging.

Microlens-ended fibers could find great usefulness in future biomedical applications, particularly in endoscopic imaging applications. In this context, this paper focuses on microlens-attached specialty optical fibers such as imaging fiber that can be used for probe imaging applications. Stand-alone self-aligned polymer microlenses have been fabricated by microcompression molding. The fabrication parameters have been optimized for different materials, such as poly(methyl methacrylate) (PMMA), polycarbonate (PC Lexan 123R), Zeonor 1060R (ZNR), and Topas COC. A comparison study of the focusing and spatial resolution of the fabricated lenses is performed prior to employing them for fiber-optic fluorescence imaging applications.

Differentiation of patient-specific void urine-derived human induced pluripotent stem cells to fibroblasts and skeletal muscle myocytes.

Cell-based therapy is a major focus for treatment of stress urinary incontinence (SUI). However, derivation of primary cells requires tissue biopsies, which often have adverse effects on patients. A recent study used human induced pluripotent stem cells (iPSC)-derived smooth muscle myocytes for urethral sphincter regeneration in rats. Here, we establish a workflow using iPSC-derived fibroblasts and skeletal myocytes for urethral tissue regeneration: (1) Cells from voided urine of women were reprogrammed into iPSC. (2) The iPSC line U1 and hESC line H9 (control) were differentiated into fibroblasts expressing FSP1, TE7, vinculin, vimentin, αSMA, fibronectin and paxillin. (3) Myogenic differentiation of U1 and H9 was induced by small molecule CHIR99021 and confirmed by protein expression of myogenic factors PAX7, MYOD, MYOG, and MF20. Striated muscle cells enriched by FACS expressed NCAM1, TITIN, DESMIN, TNNT3. (4) Human iPSC-derived fibroblasts and myocytes were engrafted into the periurethral region of RNU rats. Injected cells were labelled with ferric nanoparticles and traced by Prussian Blue stain, human-specific nuclear protein KU80, and human anti-mitochondria antibody. This workflow allows the scalable derivation, culture, and in vivo tracing of patient-specific fibroblasts and myocytes, which can be assessed in rat SUI models to regenerate urethral damages and restore continence.

A novel pathogenic variant of the LDLR gene in the Asian population and its clinical correlation with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a disease implicated with defects in either, Low density lipoprotein receptor gene (LDLR), Apolipoprotein B-100 gene (APOB), the Proprotein convertase subtilisin/kexin type 9 gene (PCSK9) or other related genes of the lipid metabolism pathway. The general characterization of heterozygous FH is by elevated low-density lipoprotein (LDL) cholesterol and early-onset cardiovascular diseases, while the more severe type, the homozygous FH results in extreme elevated levels of LDL cholesterol and usually death of an affected individual by early twenties. We present here a novel non-synonymous, missense mutation in exon 14 of the LDLR gene in two siblings of the Malay ethnicity discovered during an in-house genetic test. We postulate that their elevated cholesterol is due to this novel mutation and they are positive for homozygous FH. This is the first report of a C711Y mutation in patients with elevated cholesterol in Asia.

HER1 signaling mediates extravillous trophoblast differentiation in humans.

This study examines the role of HER1 signaling in the differentiation of proliferative extravillous trophoblast (EVT) into invasive EVT. Using the JAR choriocarcinoma cell line and placental villous explants as experimental models and immunohistochemical assessment of protein markers of EVT differentiation (downregulation of HER1 and Cx40 and upregulation of HER2 and alpha1 integrin), we show that the ability of decidual conditioned medium (DCM) to induce HER1/2 switching was abrogated in the presence of the HER1 antagonist, AG1478. Similarly, epidermal growth factor (EGF) treatment resulted in the downregulation of HER1 and an upregulation of HER2 expression, whereas co-incubation of EGF with AG1478 inhibited this response. However, EGF did not downregulate Cx40 or induce migration of EVT. In contrast, heparin-binding epidermal-like growth factor (HBEGF) stimulated dose-dependent JAR cell migration, which was inhibited by both AG1478 and AG825 (HER2 antagonist). Western blot analysis of HER1 activation demonstrated that HBEGF-mediated phosphorylation of the HER1 Tyr992 and Tyr1068 sites, while EGF activated the Tyr1045 site. Moreover, HBEGF induced a stronger and more sustained activation of both the mitogen-activated protein kinase and phosphoinositol 3 kinase (PIK3) signaling pathways. Migration assays using a panel of signaling pathway inhibitors demonstrated that the HBEGF-mediated migration was dependent on the PIK3 pathway. These results demonstrate that HBEGF-mediated HER1 signaling through PIK3 is an important component of EVT invasion.

Spatiotemporal expression of alpha(1), alpha(3) and beta(1) integrin subunits is altered in rat myometrium during pregnancy and labour.

Integrins are transmembrane extracellular matrix (ECM) receptors composed of alpha- and beta-subunits. Integrins can cluster to form focal adhesions and, because there is significant ECM remodelling and focal adhesion turnover in the rat myometrium during late pregnancy, we hypothesised that the expression of alpha(1), alpha(3) and beta(1) integrin subunits in the rat myometrium would be altered at this time to accommodate these processes. Expression of alpha(1) and beta(1) integrin subunit mRNA was significantly increased on Days 6-23 of pregnancy compared with non-pregnant (NP) and postpartum (PP) time points (P < 0.05). In contrast, alpha(3) integrin subunit mRNA expression was significantly increased on Days 14, 21 and 22 compared with NP, Day 10, 1 day PP and 4 days PP (P < 0.05). A relative gene expression study revealed that, of the integrins studied, the expression of beta(1) integrin mRNA was highest in pregnant rat myometrium. The alpha(1), alpha(3) and beta(1) integrin subunit proteins became immunolocalised to myocyte membranes in situ by late pregnancy and labour in both myometrial muscle layers. Increased alpha(1), alpha(3) and beta(1) integrin gene expression during gestation and the specific detection of these subunits in myocyte membranes during late pregnancy and labour may contribute to the cell-ECM interactions required for the development of a mechanical syncytium.

Pilot implementation of Bukhali: A preconception health trial in South Africa.

OBJECTIVES: This article describes the learnings from the pilot phase of the Healthy Life Trajectories Initiative, a preconception health trial for 18- to 25-year-old women in Soweto, South Africa. METHODS: The study compares two arms focussed on either physical and mental health (intervention; delivered by community health workers - 'Health Helpers') or standard of care plus (control; standard access to healthcare plus additional telephonic input on 'life skills'; delivered by call centre assistants). These are collectively referred to as Bukhali. Data on the pilot implementation of the Bukhali trial (n = 1655) were collected from (1) weekly team meetings, (2) two focus groups (one with the intervention team Health Helpers, n = 7; one with intervention participants, n = 8) and one paired interview with control call centre assistants (n = 2), (3) notes from eight debrief sessions with Health Helpers and (4) quantitative trial monitoring data. Qualitative data were thematically analysed. RESULTS: The findings clustered within three themes: (1) challenges for young women in Soweto, (2) priorities for young women in Soweto and (3) implementation challenges and perceptions of the intervention. Challenges were mostly related to tough socioeconomic circumstances and less prioritisation of living a healthier life. The priorities of employment and educational opportunities reflected the socioeconomic challenges, where health was not recognised as priority. The main challenge to participation and compliance with the trial was that young women in Soweto generally wanted a tangible and preferably financial and immediate benefit. Community peer sessions, despite being recommended by young women as part of the intervention development, were not successful. Many women also moved between multiple households within Soweto, which flagged concerns for a cluster trial and risk of contamination. CONCLUSION: Preconception health trials should consider socioeconomic challenges present in urban poor contexts. Learnings from the pilot phase significantly affected the design and implementation of the main Bukhali trial.

Urban young women's preferences for intervention strategies to promote physical and mental health preconception: A Healthy Life Trajectories Initiative (HeLTI).

This study aimed to qualitatively investigate young women's preferences for preconception intervention strategies to promote physical and mental health in a rapidly transitioning, urban setting. Four semi-structured focus group discussions were conducted with young women (n = 29, 18-24 years old) from Soweto, South Africa. Qualitative data were thematically analysed. Two main themes were identified: 1) challenges and needs of intervention beneficiaries; and 2) preferences for intervention strategies (content and delivery). The challenges participants mentioned could be classified as those relating to social pressure, identity, and socioeconomic circumstances. Mental health support appeared to be a greater need than physical health, and this featured in their preferences for intervention content, although a number of physical health topics were also mentioned (healthy eating and contraception). Participants had mixed preferences for intervention materials, ranging from printed to electronic and mobile resources. Their preferences for intervention activities ranged from educational sessions, to fun and interactive practical activities, and activities they could take home. Community health workers (CHWs) were the preferred agent of delivery for interventions, though participants emphasised the importance of CHWs having appropriate interpersonal skills and own life experience. Some women preferred one-on-one sessions with a CHW, while others preferred group sessions. While recognising the value of family sessions, young women were less enthusiastic about this approach. These findings provide valuable formative data for developing effective interventions to optimise young women's preconception health in urban Africa. These contextual realities should be acknowledged when addressing key physical and mental health issues facing young women.

Endocrine and paracrine regulation of birth at term and preterm.

We have examined factors concerned with the maintenance of uterine quiescence during pregnancy and the onset of uterine activity at term in an animal model, the sheep, and in primate species. We suggest that in both species the fetus exerts a critical role in the processes leading to birth, and that activation of the fetal hypothalamic-pituitary-adrenal axis is a central mechanism by which the fetal influence on gestation length is exerted. Increased cortisol output from the fetal adrenal gland is a common characteristic across animal species. In primates, there is, in addition, increased output of estrogen precursor from the adrenal in late gestation. The end result, however, in primates and in sheep is similar: an increase in estrogen production from the placenta and intrauterine tissues. We have revised the pathway by which endocrine events associated with parturition in the sheep come about and suggest that fetal cortisol directly affects placental PGHS expression. In human pregnancy we suggest that cortisol increases PGHS expression, activity, and PG output in human fetal membranes in a similar manner. Simultaneously, cortisol contributes to decreases in PG metabolism and to a feed-forward loop involving elevation of CRH production from intrauterine tissues. In human pregnancy, there is no systemic withdrawal of progesterone in late gestation. We have argued that high circulating progesterone concentrations are required to effect regionalization of uterine activity, with predominantly relaxation in the lower uterine segment, allowing contractions in the fundal region to precipitate delivery. This new information, arising from basic and clinical studies, should further the development of new methods of diagnosing the patient at risk of preterm labor, and the use of scientifically based strategies specifically for the management of this condition, which will improve the health of the newborn.

Inhibition of TGF-beta 3 restores the invasive capability of extravillous trophoblasts in preeclamptic pregnancies.

Preeclampsia, the major cause of maternal morbidity and mortality in developed countries, is associated with abnormalities of placenta function due to shallow invasion of the maternal decidua by trophoblasts. Data suggest that TGF-beta may play a role in inhibiting trophoblast outgrowth or invasion, or both. We report that placental TGF-beta 3 expression is high in early pregnancy but falls at around 9 weeks' gestation. This pattern is inversely correlated with trophoblast outgrowth and fibronectin synthesis, markers of early trophoblast differentiation toward an invasive phenotype. We demonstrate that TGF-beta 3 is overexpressed in preeclamptic placentae. In contrast to control placentae, explants from preeclamptic pregnancies fail to exhibit spontaneous invasion in vitro. Significantly, antisense-induced inhibition of TGF-beta 3 expression, and inhibition of TGF-beta 3 activity with antibodies, induces the formation of columns of trophoblast cells, which migrate out of the explant into the underlying Matrigel. To our knowledge, this is the first demonstration that the hypoinvasive placental phenotype characteristic of preeclampsia can be essentially normalized in vitro by biochemical manipulation. We speculate that a failure to downregulate expression of TGF-beta 3 at around 9 weeks' gestation results in shallow trophoblast invasion and predisposes the pregnancy to preeclampsia.

Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFbeta(3)

During early pregnancy, placentation occurs in a relatively hypoxic environment that is essential for appropriate embryonic development. Intervillous blood flow increases around 10 to 12 weeks of gestation and results in exposure of trophoblast cells to increased oxygen tension. Before this time, low oxygen appears to prevent trophoblast differentiation toward an invasive phenotype. Using human villous explants of 5-8 weeks' gestation, we found that low oxygen tension triggered trophoblast proliferation, fibronectin synthesis, alpha(5) integrin expression, and gelatinase A activity. These biochemical markers were barely detectable under oxic conditions. We therefore examined the placental expression of hypoxia-inducible factor-1 (HIF-1), a master regulator of oxygen homeostasis, and determined that expression of HIF-1alpha subunit during the first trimester of gestation parallels that of TGFbeta(3), an inhibitor of extravillous trophoblast differentiation. Expression of both molecules is high in early pregnancy and falls around 9 weeks of gestation, when placental pO(2) levels are believed to increase. Increasing oxygen tension induced a similar decrease in expression in cultured explants. Moreover, antisense inhibition of HIF-1alpha expression in hypoxic explants inhibited expression of TGFbeta(3), arrested cell proliferation, decreased alpha(5) expression and gelatinase A activity, and triggered biochemical markers of an invasive trophoblast phenotype such as alpha(1) integrin and gelatinase B expression. These data suggest that the oxygen-regulated early events of trophoblast differentiation are in part mediated by TGFbeta(3) through HIF-1 transcription factors.

Expression of the multidrug resistance P-glycoprotein, (ABCB1 glycoprotein) in the human placenta decreases with advancing gestation.

The multidrug resistance p-glycoprotein (P-gp), encoded by the ABCB1 gene, is a plasma membrane protein that actively extrudes a wide variety of substances from cells. Preliminary studies in mice have shown that the ABCB1/P-gp can protect the fetus from a number of toxic substances. ABCB1/P-gp is expressed in the human placenta and is potentially capable of protecting the fetus from a large number of drugs and toxins, including herbicides and pesticides. The protein can also extrude various steroids including certain glucocorticoids and may therefore play an important role in regulating fetal access of glucocorticoids. The aim of the present study was to examine the expression profile and cellular localization of ABCB1/P-gp in human placenta throughout gestation. We hypothesized that there would be gestational age-related changes in the expression of the protein. ABCB1/P-gp mRNA was measured by Real-Time PCR using specific probes in tissues obtained from 6 weeks gestation to term. ABCB1/P-gp mRNA levels in placental tissue obtained at 6-10 weeks (n=5) and 24-35 weeks (n=5) were significantly higher than in tissues obtained at term (38-41 weeks gestation) by elective C-section (n=6) or following labor (n=6). The profile of ABCB1/P-gp protein levels, quantified using Western analysis, demonstrated a similar decrease with advancing gestation. At all gestational ages ABCB1/P-gp was localized by immunohistochemistry to the syncytiotrophoblast. In term tissues, it appeared to be localized to some areas of the villi and not others. Together, these data indicate that with advancing gestation there is a decrease in the level of ABCB1/P-gp in the human placenta indicating that the fetus may be more susceptible to toxic insults in the latter part of gestation. Further, the reduction in ABCB1/P-gp expression may contribute to the increased transfer of maternal cortisol to the fetus that is known to occur in late gestation.

EGF modulates trophoblast migration through regulation of Connexin 40.

In this study we show that decidua conditioned media (DCM) downregulate Connexin 40 (C x 40) expression in extravillous trophoblast (EVT) outgrowths and can promote EVT differentiation to the invasive phenotype resulting in switching of integrin and EGF receptor expression. This suggests that growth factors secreted by the decidua, such as EGF, mediate trophoblast migration/invasion and may do so by modulating C x 40 expression and function. To test this hypothesis we have utilized migration assays using cell lines expressing C x 40. Migration assays were performed with Jeg-3, Jeg-3 overexpressing C x 40 (JpUHD) and JAR cells seeded on fibronectin-coated inserts with 8 microm pores and incubated in the absence or presence of serum-starved decidual cells. Cell migration was only observed in the presence of DCM. Conversely overexpression of C x 40 in Jeg-3 cells resulted in inhibition of cell migration as compared to wild-type control. Addition of DCM to cultured JAR cells resulted in the downregulation of C x 40 protein. EGF is known to stimulate trophoblast migration/invasion and was detected in DCM; therefore, we investigated the action of EGF on C x 40. EGF (10 ng/mL) resulted in the downregulation of C x 40 in the JAR cell line. However, EGF had no effect on JAR cell migration. We conclude that decidual secretion of growth factors, such as EGF, may act to prime trophoblast for migration/invasion through modulation of connexin expression and function.

ATP-binding cassette transporters in reproduction: a new frontier.

BACKGROUND: The transmembrane ATP-binding cassette (ABC) transporters actively efflux an array of clinically relevant compounds across biological barriers, and modulate biodistribution of many physiological and pharmacological factors. To date, over 48 ABC transporters have been identified and shown to be directly and indirectly involved in peri-implantation events and fetal/placental development. They efflux cholesterol, steroid hormones, vitamins, cytokines, chemokines, prostaglandins, diverse xenobiotics and environmental toxins, playing a critical role in regulating drug disposition, immunological responses and lipid trafficking, as well as preventing fetal accumulation of drugs and environmental toxins. METHODS: This review examines ABC transporters as important mediators of placental barrier functions and key reproductive processes. Expression, localization and function of all identified ABC transporters were systematically reviewed using PubMed and Google Scholar websites to identify relevant studies examining ABC transporters in reproductive tissues in physiological and pathophysiological states. Only reports written in English were incorporated with no restriction on year of publication. While a major focus has been placed on the human, extensive evidence from animal studies is utilized to describe current understanding of the regulation and function of ABC transporters relevant to human reproduction. RESULTS: ABC transporters are modulators of steroidogenesis, fertilization, implantation, nutrient transport and immunological responses, and function as 'gatekeepers' at various barrier sites (i.e. blood-testes barrier and placenta) against potentially harmful xenobiotic factors, including drugs and environmental toxins. These roles appear to be species dependent and change as a function of gestation and development. The best-described ABC transporters in reproductive tissues (primarily in the placenta) are the multidrug transporters p-glycoprotein and breast cancer-related protein, the multidrug resistance proteins 1 through 5 and the cholesterol transporters ABCA1 and ABCG1. CONCLUSIONS: The ABC transporters have various roles across multiple reproductive tissues. Knowledge of efflux direction, tissue distribution, substrate specificity and regulation of the ABC transporters in the placenta and other reproductive tissues is rapidly expanding. This will allow better understanding of the disposition of specific substrates within reproductive tissues, and facilitate development of novel treatments for reproductive disorders as well as improved approaches to protecting the developing fetus.

Regulation of transcripts encoding the myometrial gap junction protein, connexin-43, by estrogen and progesterone.

In the rat, transcripts encoding the myometrial gap junction protein, connexin-43 (Cx-43), increase dramatically with the onset of labor in association with an increase in the ratio of estrogen to progesterone in plasma. We examined whether the level of Cx-43 transcripts might be regulated by these steroids in the rat myometrium. Administration of progesterone to late pregnant rats abolished the more than 12-fold increase in transcripts seen in control rats at term and blocked delivery of the fetuses. Treatment of rats on day 15 of gestation (when Cx-43 mRNA levels are low) with the progesterone antagonist RU486 resulted in a significant (2.5-fold) increase in transcripts within 9 h, with the maximal (5.6-fold) increase occurring between 24-48 h, and preterm delivery occurring between 48-72 h. Administration of a single dose of 17 beta-estradiol (5 micrograms, sc) to nonpregnant rats resulted in a significant increase in Cx-43 transcripts within 3 h; these levels were increased 3-fold between 6-24 h, before falling to lower levels by 48 h. Progesterone (4 mg, sc) administration at the same time as estradiol significantly attenuated the estradiol response. Chronic estradiol administration (5 micrograms/12 h for 36 h) failed to maintain elevated levels of Cx-43 transcripts beyond 36 h. Administration of progesterone 12 h after estradiol prematurely reduced the level of Cx-43 transcripts. These data demonstrate that steroid hormones can modulate the level of steady state transcripts of Cx-43 during pregnancy in association with changes in uterine contractile activity. Furthermore, the data from the nonpregnant studies suggest that the levels of transcripts are regulated positively by estradiol and negatively by progesterone.

Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activator protein-1, c-fos.

Expression of the myometrial gap junction protein connexin-43 (Cx-43) increases dramatically with the onset of labor in association with an increase in the plasma estrogen to progesterone ratio. Moreover, we reported that the levels of messenger RNA (mRNA) and protein encoded by the Cx-43 gene are regulated positively by estrogen and negatively by progesterone in both pregnant and nonpregnant rats. However, although the Cx-43 gene has been reported to be responsive to estrogen, it does not contain a palindromic estrogen response element. It does, however, contain a consensus activator protein-1 (AP-1)-binding site that may be of significance, as estrogen has been reported to increase expression of the AP-1 proteins, Fos and Jun, in the uterus. Therefore, we tested the hypothesis that the increase in Cx-43 mRNA levels in the myometrium during labor and after estrogen administration is mediated indirectly through induction of trans-activating factors, of which Fos and Jun are putative candidates. Treatment of nonpregnant ovariectomized rats with cyclohexamide blocked the estrogen-induced increase in Cx-43 mRNA in the myometrium, suggesting that this action of estrogen requires newly synthesized protein. Estrogen treatment alone induced an increase in mRNA encoding c-fos and c-jun in nonpregnant rats, which preceded by 2-3 h an increase in Cx-43 mRNA. Labor was also associated with a coincident expression of c-fos and Cx-43 in the myometrium, although there was no change in c-jun mRNA levels during this period. These associative data were strengthened by our observation that in paradigms in which labor was induced prematurely by ovariectomy or blocked by treatment with progesterone, changes in c-fos expression were closely matched by changes in Cx-43 mRNA. These data support our hypothesis that estrogen (and, by extension, labor-)-induced increases in Cx-43 mRNA are mediated indirectly through newly synthesized trans-activating factors. Moreover, the temporal correlation between the expression of c-fos and Cx-43 and the presence of AP-1 cis-acting elements within the Cx-43 promoter suggest that c-fos may be one of these trans-activating proteins.

Focal adhesion signaling in the rat myometrium is abruptly terminated with the onset of labor.

The dramatic increase in uterine growth during late pregnancy and the generation of labor contractions require dynamic remodeling of myometrial smooth muscle-ECM interactions. In many tissues, such interactions are provided by focal adhesions; however, there are no data as to the expression of focal adhesion proteins or of focal adhesion signaling in the myometrium. In this study, we show that tyrosine phosphorylation of myometrial FAK (FAK-P-Tyr) and of its downstream substrate, paxillin, exhibited a >10-fold increase during late pregnancy (days 15-22 of pregnancy) with each exhibiting a dramatic fall in P-Tyr on day 23 in association with the onset of labor. These changes in FAK-P-Tyr were paralleled by changes in FAK enzyme activity. Activated ERK1 and ERK2 expression remained relatively unchanged from day 15 to day 23, but decreased markedly 1 day post partum. Treatment of late pregnant rats with progesterone prevented the fall in FAK-P-Tyr/enzyme activity on day 23, and also blocked the onset of labor. These data suggest that progesterone (which decreases at term) modulates myometrial FAK activity/focal adhesion signaling and that these changes may underlie the tremendous remodeling that must occur in order for this muscle to develop optimal contractile activity during labor.