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Volatile organic compounds (VOCs), such as benzene, toluene, ethylbenzene, and xylene (BTEX), are commonly used industrial chemicals and are associated with increased uterine smooth muscle contractility and preterm birth (PTB) accounting for over 2 million cases worldwide and 2.3% of American PTB’s. Previous studies showed that benzene and its metabolites function as endocrine disrupters effecting estrogen (E2) action through its interaction with the estrogen receptor (ER). E2 and ER action are critical components to the successful maintenance of normal gestational length through the regulation of uterine smooth muscle contractility. Therefore, we propose to examine the action of VOCs in promoting the incidence of PTB through impaired ER and E2 action and to define the precise molecular mechanism whereby the functional endpoint results in increased uterine smooth muscle contractility. We present novel preliminary data that demonstrate exposure to benzene and its metabolites promote alternate splicing and alternative promoter usage of the uterine ER, generating ER isoforms that promote precocious and enhanced uterine smooth muscle contractility. Briefly, exposure to benzene and its metabolites impair the adaptive uterine unfolded protein response (UPR), affecting the uterine m6A epitranscriptome both in vivo and in vitro. Our preliminary analyses demonstrate that UPR activation alone upregulates myometrial m6A readers and writers. In addition, we have recently published the critical regulatory nature of the uterine m6A epitranscriptome during pregnancy, whereby increased activity of myometrial hnRNPG, a m6A reader, directs myometrial alternative splicing events giving rise to ERΔ7 (dominant negative isoform of ERα, that lacks exon 7) (1). In this proposal, we will first test the hypothesis that alternative splicing and alternate promoter usage are crucial in the gestational selection of alternate ERα isoforms through posttranscriptional m6A methylation of uterine smooth muscle mRNA and that benzene alters this balance leading to PTB. Secondly we will examine the impact of benzene exposure in the modification of uterine smooth muscle m6A methylation on the global uterine smooth muscle pro-contractile proteome in vitro. Finally, we will generate a fetal female and adult reproductive developmental roadmap to compare in utero benzene treated and control untreated F1 females. We propose that our targeted analysis of benzene and benzene metabolite induced modification of myometrial m6A post-transcriptionally modified RNAs will allow important insight into the currently unresolved molecular mechanisms that allow for VOC exposure induced PTB. Completion of these aims will define the currently unresolved molecular mechanisms of environmental VOC exposure induced PTB, while also delivering novel drugable gene targets for the prediction and prevention of environmental VOC exposure induced PTB.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella, including mammalian sperm tails. Depletion of IFT27, a component of the IFT complex, in male germ cells results in infertility associated with disrupted sperm flagella structure and motility. Leucine zipper transcription factor-like 1 (LZTFL1) is an IFT27 associated protein. LZTFL1, also known as BBS17, is a Bardet-Biedl syndrome (BBS) associated protein. Patients carrying biallelic variants of LZTFL1 gene exhibit the common BBS phenotypes. The global Lztfl1 knockout mice showed abnormal growth rate and retinal degeneration, typical of BBS phenotype. However, it is not clear if Lztfl1 has a role in male fertility. The LZTFL1 protein is highly and predominantly expressed in mouse testis. During the first wave of spermatogenesis, the protein is only expressed during spermiogenesis phase from the round spermatid stage and displays a cytoplasmic localization with a vesicular distribution pattern. At the elongated spermatid stage, LZTFL1 is present in the developing flagella and appears also close to the manchette. Fertility of Lztfl1 knockout mice was significantly reduced and associated with low sperm motility and a high level of abnormal sperm (astheno-teratozoospermia). In vitro assessment of fertility revealed reduced fertilization and embryonic development when using sperm from homozygous mutant mice. In addition, we observed a significant decrease of the testicular IFT27 protein level in Lztfl1 mutant mice contrasting with a stable expression levels of other IFT proteins, including IFT20, IFT81, IFT88 and IFT140. Overall, our results support strongly the important role of LZTFL1 in mouse spermatogenesis and male fertility.

To examine the activation and consequence of uterine apoptotic caspase-3 action on day 1 post coitus (dpc) in the pregnant mouse. Previously we have demonstrated that pregnant uterine caspase-3 activation from mid to late gestation isolated to the myometrial compartment is largely non-apoptotic and controls uterine quiescence. We have also previously demonstrated that apoptotic caspase-3 activation, isolated to the endometrial compartment at term regulated endometrial prostaglandin synthesis METHODS: Uteri were isolated from pseudo pregnant (PsP) and non-ligated controls (GD), unilateral (UL) and bilateral ligated (BL) uterine horn mouse models at 1, 3 and 6 dpc. Uteri were examined for apoptotic indices, such as caspase-3 activation and TUNEL staining. Immunohistochemical analysis identified the site of uterine apoptotic caspase-3 activation. The truncated form of phospholipase A2 (tiPLA2) was examined as a measure of apoptotic caspase-3 mediated iPLA2 activation.We have identified the site and impact of uterine apoptotic caspase-3 activation utilizing uteri isolated from non-pregnant control animals at estrous and diestrous and control pregnant mice at 1-19 dpc. Our analysis determined that apoptotic caspase-3 and iPLA2 activation were limited to the endometrial compartments of the control and unilateral ligated uteri on 1dpc and were not found in the pseudo pregnant or bilateral ligated uterine horn or on 3 or 6 dpc in the control and unilateral ligated uteri.In this study we determined that uterine caspase-3 activation on 1 dpc, which is endometrial and apoptotic in nature, may play a potential role in regulating the previously reported pre-implantation surge in endometrial PGE2 synthesis through apoptotic caspase-3 mediated iPLA2 activation. Our data indicates that the presence of a conceptus on 1 dpc likely triggers an increase in endometrial apoptotic caspase-3 mediated iPLA2 activation. iPLA2 when activated causes the hydrolysis of fatty acids resulting in arachidonic acid release and production of PGE2, which in early pregnancy has been demonstrated to act in a leutoprotective manner, prolonging progesterone synthesis and promoting uterine receptivity.

Estrogen and estrogen action in the myometrium are necessary for a successful pregnancy. Estrogen is required for both the myometrial hyperplastic phase of gestation as well as the contractile response that leads to parturition at term. The excessive levels of circulating estrogens during pregnancy, pose a signalling conundrum. How do different tissues that have varying spatial and temporal needs for estrogen deal with this high estrogen ligand level? Insulin like growth factor (IGF1) and its receptor are important to the process of hyperplasia and are estrogen responsive. Although IGF1 is necessary for a successful pregnancy, myometrial production of IGF1 seems to be redundant for myometrial expansion. This review discusses the newly investigated functionality of estrogen receptor alpha splice isoforms in uterine myometrial contractility and quiescence while also covering the role of estrogen and IGF1 in uterine myometrial hyperplasia during pregnancy.

The objective of this study was to determine the consequence of uterine apoptotic caspase-3 activation on day 1 post coitus (dpc) in the pregnant mouse. We previously demonstrated that during pregnancy uterine caspase-3 activation isolated to the myometrial compartment is largely non-apoptotic and controls uterine quiescence. In this study we determined that uterine caspase-3 activation on 1 dpc may play a critical role in regulating endometrial PGE2 synthesis though iPLA2 activation. These analyses provide novel insight into the molecular mechanisms that regulate previously reported increases in endometrial PGE2 synthesis in very early pregnancy, that act to enhance uterine receptivity.We have identified the site and impact of that uterine apoptotic caspase-3 activation utilizing uteri isolated from non-pregnant control animals at estrous and diestrous and from control pregnant mice at 1-19 dpc. In addition, uteri were isolated from non-ligated controls (GD), unilateral (UL) and bilateral ligated (BL) uterine horn mouse models at 1, 3 and 6 dpc. Uteri were examined for apoptotic indices, such as caspase-3 activation and TUNEL staining. Immunohistochemical analysis was performed to identify the site of apoptotic caspase-3 activation. The presence of the truncated form of phospholipase A2 (tiPLA2) was examined as a measure of apoptotic caspase-3 mediated iPLA2 activation.Our analysis determined that apoptotic caspase-3 and iPLA2 activation were limited to the endometrial compartments of the control and unilateral uteri on 1dpc and were not found in the bilateral ligated uterine horn on 3 or 6 dpc. Our data indicates that the presence of a conceptus on 1 dpc triggers an increase in endometrial apoptotic caspase-3 mediated iPLA2 activation. iPLA2 when activated causes the hydrolysis of fatty acids resulting in arachidonic acid release and production of PGE2, which in early pregnancy has been demonstrated to act in a leutoprotective manner, prolonging progesterone synthesis and promoting uterine receptivity.

Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neural cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neural cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neural cells with consequences for glucocorticoid signaling.

Background Circulating estrogen (E2) levels are high throughout pregnancy and increase towards term, however its local tissue specific actions vary across gestation. For example, myometrial E2 regulated uterotonic action is disabled until term, whereas it's proliferative function is maintained in the breast. We have identified gestationally regulated splicing events, mediated by hnRNPG and modulated by E2 that generate alternatively spliced estrogen receptor alpha (ERα) variants (ERΔ7 and ERα46) in the myometrium. These variants allow for differential, gestationally regulated, modulation of the uterotonic action of E2. Methods Human myometrium isolated from preterm and term non-laboring and laboring pregnant women were analyzed for ERα isoforms and splice factor levels. Lentiviral mediated shRNA knockdown of hnRNPG and overexpression of ERΔ7 were performed in human myometrial (hTERT-HM) cells. Functional 3D collagen contraction assays were executed. Findings ERΔ7 acts as a dominant negative repressor of the uterotonic action of ERα66 and ERα46 isoforms through the regulation of the myometrial gap junction protein GJA1. Elimination of hnRNPG inhibits the generation of ERΔ7 while overexpression of ERΔ7 inhibited GJA1 expression. Moreover in vivo human myometrial hnRNPG levels decline at term in an E2 dependent manner resulting in a withdrawal of ERΔ7 levels and its tocolytic action at term. Interpretation Our findings implicate the unique role of ERΔ7 as a modulator of myometrial quiescence and define the mechanism of ERΔ7 generation, through hormonally regulated splicing events. Fund This study was supported by NIH OPRU U01 supplement (HD047905), University of Pittsburgh and Wayne State University Perinatal Research Initiative (USA).

A broad definition of preconditioning is “the preparation for a subsequent action.” Mounting evidence demonstrates that novel remote preconditioning paradigms, in which protective stimuli experienced locally can capacitate systemic tolerance and enhanced cell viability upon exposure to ensuing cellular insults, have been largely successful in the field of cardiovascular ischemia/reperfusion injury. To ensure successful protective preconditioning, some models (including the uterus) have been demonstrated to activate the unfolded protein response (UPR), which is a cellular stress response controlled at the level of the endoplasmic reticulum. However, in the context of remote preconditioning, activation of these intracellular molecular pathways must result in the extracellular transmission of adaptive signals to remote targets. In our recently published manuscript, we have described the activation of the UPR in the pregnant uterine myocyte to be associated with increased uterine myocyte quiescence and normal gestational length. We hypothesize that ubiquitous uterine gestational stresses experienced in every pregnancy, which have been demonstrated in other systems to activate the UPR, may induce a robust paracrine dissemination of a uterine secretome, for example, glucose-regulated protein 78, with preconditioning-like properties. Furthermore, we speculate that the gestational stress-induced uterine secretome acts to promote both local and systemic tolerance to the ensuing gestational insults, allowing for the maintenance of uterine quiescence. In this context, preterm labor may be the result of a pregnant uterus experiencing a stress it cannot accommodate or when it is unable to host an appropriate UPR resulting in insufficient preconditioning and a diminished local and systemic capacity to tolerate pregnancy-dependent increases in normal gestational stress. This is highly attractive from a clinical viewpoint as we ultimately aim to identify local and systemic adaptations that may serve as preconditioning stimuli for use as a strategy to restore appropriate preconditioning profiles to prolong uterine quiescence in pregnancy.

The elevated level of Steroidogenic Factor 1 (Nr5a1, Sf-1) expression in the male gonadal development pathway, post sex determination, implies a vital role in testis gonadal differentiation. In this study we generated Sertoli cell-specific Nr5a1 KO mice (SC-SF-1−/−) at E14.5, which coincides with testis development post sex determination, using the Amh-Cre mouse model. Analysis of SC-SF-1−/− (Sertoli cell specific Nr5a1 knockout) testes demonstrated apoptosis as early as E15. Further analysis revealed that SC-SF-1−/− gonads displayed lower MDM2 levels resulting in elevated TP53 levels, which we believe may lead to apoptosis of the Sertoli cell population, inferring the possibility that NR5A1 directly regulates MDM2 expression. By E15.5, the Sertoli cell and germ cell population declined in SC-SF-1−/− mice resulting in the disruption of seminiferous cords with limited cord structure remaining at E18.5. Due to the loss of Sertoli and germ cells, the testis weights of SC-SF-1−/− mice at 6-weeks were much reduced; however, SC-SF-1−/− seminal vesicles weights were comparable suggesting intact Leydig cell androgen production. We conclude that NR5A1 regulates the TP53 pathway during development, is essential for fetal Sertoli cell survival and controls the cell cycle of Sertoli cells during differentiation.

The prevention of apoptotic caspase 3 activation through biological preconditioning, mediated through the modulation of the unfolded protein response has been demonstrated to ameliorate multiple pathophysiologies. The maintenance of non-apoptotic caspase 3 activity by the unfolded protein response within the pregnant uterus has previously been proven to be critical in inhibiting uterine myocyte contractility during pregnancy. Here we report that the pregnant uterus utilizes an unfolded protein response-preconditioning paradigm to conserve myometrial caspase 3 in a non-apoptotic state in order to effectively inhibit uterine contractility thereby preventing the onset of preterm labor. In the absence of appropriate endogenous preconditioning during pregnancy, uterine caspase 3 is transformed from a non-apoptotic to an apoptotic phenotype. Apoptotic caspase 3 activation results in the precocious triggering of local uterine inflammatory signaling and prostaglandin production, consequently resulting in an increased incidence of preterm birth. These findings represent a paradigm shift in our understanding of how preconditioning promotes the maintenance of uterine non-apoptotic caspase 3 action during pregnancy preventing the onset of premature uterine contraction and therefore defining the timing of the onset of labor.

We previously identified myometrial caspase-3 (CASP3) as a potential regulator of uterine quiescence. We also determined that during pregnancy, the functional activation of uterine CASP3 is likely governed by an integrated endoplasmic reticulum stress response (ERSR) and is consequently limited by an increased unfolded protein response (UPR). The present study examined the functional relevance of uterine UPR-ERSR in maintaining myometrial quiescence and regulating the timing of parturition. In vitro analysis of the human uterine myocyte hTERT-HM cell line revealed that tunicamycin (TM)-induced ERSR modified uterine myocyte contractile responsiveness. Accordingly, alteration of in vivo uterine UPR-ERSR using a pregnant mouse model significantly modified gestational length. We determined that "normal" gestational activation of the ERSR-induced CASP3 and caspase 7 (CASP7) maintains uterine quiescence through previously unidentified proteolytic targeting of the gap junction protein, alpha 1(GJA1); however, surprisingly, TM-induced uterine ERSR triggered an exaggerated UPR that eliminated uterine CASP3 and 7 tocolytic action precociously. These events allowed for a premature increase in myometrial GJA1 levels, elevated contractile responsiveness, and the onset of preterm labor. Importantly, a successful reversal of the magnified ERSR-induced preterm birth phenotype could be achieved by pretreatment with 4-phenylbutrate, a chaperone protein mimic.

A broad definition of preconditioning is "the preparation for a subsequent action." Mounting evidence demonstrates that novel remote preconditioning paradigms, in which protective stimuli experienced locally can capacitate systemic tolerance and enhanced cell viability upon exposure to ensuing cellular insults, have been largely successful in the field of cardiovascular ischemia/reperfusion injury. To ensure successful protective preconditioning, some models (including the uterus) have been demonstrated to activate the unfolded protein response (UPR), which is a cellular stress response controlled at the level of the endoplasmic reticulum. However, in the context of remote preconditioning, activation of these intracellular molecular pathways must result in the extracellular transmission of adaptive signals to remote targets. In our recently published manuscript, we have described the activation of the UPR in the pregnant uterine myocyte to be associated with increased uterine myocyte quiescence and normal gestational length. We hypothesize that ubiquitous uterine gestational stresses experienced in every pregnancy, which have been demonstrated in other systems to activate the UPR, may induce a robust paracrine dissemination of a uterine secretome, for example, glucose-regulated protein 78, with preconditioning-like properties. Furthermore, we speculate that the gestational stress-induced uterine secretome acts to promote both local and systemic tolerance to the ensuing gestational insults, allowing for the maintenance of uterine quiescence. In this context, preterm labor may be the result of a pregnant uterus experiencing a stress it cannot accommodate or when it is unable to host an appropriate UPR resulting in insufficient preconditioning and a diminished local and systemic capacity to tolerate pregnancy-dependent increases in normal gestational stress. This is highly attractive from a clinical viewpoint as we ultimately aim to identify local and systemic adaptations that may serve as preconditioning stimuli for use as a strategy to restore appropriate preconditioning profiles to prolong uterine quiescence in pregnancy.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique — usually untranslated — first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

Our group has previously identified elevated levels of nonapoptotic active caspase 3 (CASP3) accompanied by increased prosurvival, antiapoptotic signaling in the pregnant mouse uterus during late gestation. We speculated that increased antiapoptotic signaling desensitized the pregnant uterine myocyte to the apoptotic action of uterine CASP3. This current study examines the mechanism by which the pregnant myocyte gains resistance to the apoptotic effects of increased uterine CASP3. Using both primary human pregnant fundal myometrial cultures and the telomerase-immortalized human uterine myocyte cell line (hTERT) as our model systems, uterine myocytes were exposed to UV irradiation and Fas ligand to stimulate both the intrinsic and extrinsic apoptotic pathways. Stimulation of either the intrinsic or extrinsic apoptotic pathways resulted in elevated levels of uterine myocyte CASP3. However, apoptotic cell death was restricted to CASP3 activated by intrinsic stimulation via UV light. In contrast Fas ligand-mediated CASP3 activation was accompanied by increased antiapoptotic signaling mimicking our in vivo observations in the pregnant mouse uterus. Using small interfering RNA to inhibit antiapoptotic signaling, we determined the ability of the human uterine myocyte to resist apoptotic cell death in the absence of the prosurvival, antiapoptotic signaling. Accordingly, suppression of antiapoptotic signaling specifically mediated by myeloid cell leukemia sequence 1 was sufficient to sensitize the uterine myocyte to undergo apoptotic cell death. These data demonstrate that elevated myeloid cell leukemia sequence 1 levels are sufficient to confer apoptotic resistance on the human uterine myocyte despite highly elevated levels of active CASP3.

Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11β-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11β-HSD1/H6PDH double-KO (DKO) mice, in which 11β-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11β-HSD1KO mice. Critically, in contrast to 11β-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11β-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.

The appropriate timing of the onset of labor is critical to a successful pregnancy, with potentially devastating consequences resulting to both the mother and child with the onset of preterm labor. In this study, we tested the central hypothesis that progesterone maintains uterine quiescence through regulation of active uterine caspase 3. Using the mouse as our model system, we examined, by Western blot analysis, levels of active caspase 3 and its association with the degradation of uterine contractile proteins during pregnancy. Our data demonstrate that caspase 3-specific cleavage fragments of uterine myocyte contractile proteins are elevated in late gestation. Prior to the onset of labor, active caspase 3 levels and fragmentation of the uterine myocyte contractile proteins decline. We postulate that uterine caspase 3 acts as an anticontractile agent maintaining uterine quiescence through degradation of uterine contractile proteins during late pregnancy. We propose that decreased progesterone action during the final days of pregnancy controls the timing of the onset of uterine contractions by removing the anticontractile action of the apoptotic protein caspase 3 locally in the pregnant myometrium.

Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells. The testes of adult gonad-specific SF-1 KO mice remained at the level of the bladder and were markedly hypoplastic, due at least partly to impaired spermatogenesis. Histological abnormalities of the testes were seen from early developmental stages and were associated with markedly decreased Leydig cell expression of two essential components of testosterone biosynthesis, Cyp11a and the steroidogenic acute regulatory protein. In females, the anti-Müllerian hormone type 2 receptor-Cre allele directed Cre expression to granulosa cells. Although wild-type and SF-1 KO ovaries were indistinguishable during embryogenesis and at birth, adult females were sterile and their ovaries lacked corpora lutea and contained hemorrhagic cysts resembling those in estrogen receptor α and aromatase KO mice. Collectively, these studies establish definitively that SF-1 expression in the gonads is essential for normal reproductive development and function.

Parturition is timed to begin only after the developing embryo is sufficiently mature to survive outside the womb. It has been postulated that the signal for the initiation of parturition arises from the fetus although the nature and source of this signal remain obscure. Herein, we provide evidence that this signal originates from the maturing fetal lung. In the mouse, secretion of the major lung surfactant protein, surfactant protein A (SP-A), was first detected in amniotic fluid (AF) at 17 days postcoitum, rising progressively to term (19 days postcoitum). Expression of IL-1β in AF macrophages and activation of NF-κB in the maternal uterus increased with the gestational increase in SP-A. SP-A stimulated IL-1β and NF-κB expression in cultured AF macrophages. Studies using Rosa 26 Lac-Z (B6;129S-Gt(rosa)26Sor) ( Lac-Z ) mice revealed that fetal AF macrophages migrate to the uterus with the gestational increase in AF SP-A. Intraamniotic (i.a.) injection of SP-A caused preterm delivery of fetuses within 6-24 h. By contrast, injection of an SP-A antibody or NF-κB inhibitor into AF delayed labor by >24 h. We propose that augmented production of SP-A by the fetal lung near term causes activation and migration of fetal AF macrophages to the maternal uterus, where increased production of IL-1β activates NF-κB, leading to labor. We have revealed a response pathway that ties augmented surfactant production by the maturing fetal lung to the initiation of labor. We suggest that SP-A secreted by the fetal lung serves as a hormone of parturition.

Targeted gene disruption has produced knockout (KO) mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 KO mice lacked adrenal glands and gonads, and also had impaired expression of gonadotropins in pituitary gonadotropes and marked structural abnormalities of the ventromedial hypothalamic nucleus (VMH). To define the roles of SF-1 within discrete sites of the hypothalamic-pituitary-steroidogenic organ axis, we have sought to make tissue-specific SF-1 KO mice (as reviewed here). We first used adrenal transplants to restore adrenal function in global SF-1 KO mice, providing a physiological form of a "VMH-specific" KO to study the roles of SF-1 in weight regulation. These adrenal-transplanted SF-1 KO mice became obese due to decreased locomotor activity, providing a novel model of hypothalamic obesity. Mice with a pituitary-specific KO of SF-1 mediated by the Cre-loxP recombination strategy exhibited hypogonadotropic hypogonadism, revealing essential roles of SF-1 in pituitary function in vivo. Ongoing studies seek to inactivate SF-1 in the brain or specific gonadal cell types, thereby defining its roles in development and function at these sites. In addition, we review our use of bacterial artificial chromosome transgenesis to develop a fluorescent marker for cells that express SF-1.

The formation of estrogens from androgens in all vertebrates is catalyzed by the "aromatase" complex, which consists of a membrane bound P450 enzyme, P450 aromatase (which binds the androgen substrate and inserts an oxygen into the molecule), and a flavoprotein (NADPH- cytochrome P450 reductase). Among vertebrates, the two major sites of aromatase expression are the brain and gonads. Given the importance of estrogen in reptile sex determination, we set out to examine whether P450arom was involved in the initiation and/or stabilization of sex determina- tion in turtles. We examined the expression of aromatase activity in the brain and gonads of two turtle species exhibiting temperature dependent sex determination (TSD), the diamondback terra- pin (Malaclemys terrapin), and the common snapping turtle (Chelydra serpentina). Estradiol when applied at stage 14 of the terrapin induces expression of aromatase in the gonad of embryos incu- bated at male temperatures (26.5°C). The level of expression is similar to that of a normal embry- onic ovary. When applied at stage 22, estradiol does not induce aromatase expression in the terrapin. The xenoestrogen, nonylphenol, sex reverses terrapin embryos at 26.5°C. Letrazole, a nonsteroidal aromatase inhibitor, suppresses aromatase activity in the brain at either incubation temperature. Ovotestes are produced by letrazole administration in the terrapin when incubated at 30.5°C. In the snapping turtle at stage 23, gonadal and brain aromatase activity in embryos incubated at female temperatures (30.5°C) is nearly half that exhibited in terrapin embryos at the same temperature. Moreover, letrazole administration suppresses aromatase expression to nearly basal levels. At male incubation temperatures (26.5°), brain aromatase expression is nearly three times higher than at female temperatures, while gonadal expression levels are nearly one third lower. However, the gonadal expression levels at male temperatures in the snapping turtle are nearly 25 times higher than that found in the terrapin. Estradiol administration elevates this level nearly three fold. These data suggest that is not merely the expression of aromatase that is important for ovarian development, but that the level of expression may be more important. J. Exp. Zool. 290:673- 690, 2001. © 2001 Wiley-Liss, Inc. It has been shown very clearly that estradiol (and other estrogens) will reverse the sex of males to females at male producing temperatures for turtles (Pieau, '74; Gutzke and Bull, '86; Bull et al., '88; Crews et al., '89; Crews et al., '91; Wibbels and Crews, '91, '95; Dorizzi et al., '91; Wibbels et al., '91, '93; Rhen and Lang, '94; Jeyasuria et al., '94; Pieau et al., '94; Crews et al., '95, '96) and alligators (Lance and Bogart, '92). In accordance with this finding, aromatase enzymatic activities are high in gonads at female producing tempera- ture while they are low at male temperatures (Desvages and Pieau, '92a; Pieau, '96). We have shown that the transcription of the aromatase mRNA (Jeyasuria et al., '95; Jeyasuria and Place, '98) corresponds with the aromatase activities measurements. However, we have also shown that aromatase is expressed in the brain of the terra- pin before the gonad begins differentiation (Jey- asuria and Place, '98). When one injects or applies steroids to the egg, the entire embryo is treated. Hence, one needs to examine all potential sites of

Sex determination in egg-laying amniotes may be fundamentally different from that of placental mammals. The mammalian ovary differentiates normally in the absence of estro- gen, whereas estrogen seems to be crucial for proper ovarian development in birds, reptiles, and lower vertebrates. Estrogens are produced normally by the biosynthetic conversion of androgens by the enzyme aromatase (CYP19), which is the sole mediator of this reaction. Aromatase inhibi- tors are capable of reversing females to males in turtles and chickens; therefore, a role for aromatase as the female sex determinant has been postulated for species in which sex determination is tem- perature-dependent. The entire aromatase coding sequence (1,509 base pairs) from adult terrapin ovaries was cloned, and Northern analysis indicates a single transcript (2.4 kb) for adult ovaries, whereas male and female brains express a 2.4-kb as well as a 9.6-kb transcript. Using a sensitive (attomole sensi- tivity) competitive RT-PCR technique, aromatase transcript abundance was quantified during embryonic development for embryos treated with and without estrogen. Aromatase is transcribed, well before the temperature-sensitive, (stage 12), at both male and female temperatures in the brain. There is a switch to lower aromatase transcript abundance in the female brain concurrent with an exponential rise of aromatase transcript in the putative ovary. Transcripts remain below the detection limits in the putative testes but exhibit female levels of aromatase transcript when treated with estrogen. Aromatase mRNA levels are generally reduced in the brain by estradiol application. On the basis of these findings, we have postulated a model based on the competition between 5α-reductase and P450 aromatase for androgen substrate in both the brain and the undifferentiated gonad to explain the TSD phenomenon in reptiles. J. Exp. Zool. 281:428n449, 1998. © 1998 Wiley-Liss, Inc.

In the diamondback terrapin, Malaclemys terrapin, males hatch at incubation temperatures below 28°C whereas females hatch at temperatures above 30°C. When estrogen is applied to the eggs at male temperatures early in development, females are produced. These data suggest that the enzyme necessary for estrogen synthesis (CYP19, aromatase) in the developing gonad plays a critical role in sex determination in these vertebrates. Accordingly, we have begun an examination of the role and regulation of the aromatase gene in sex determination in the diamond back terrapin, Malaclemys terrapin. We have obtained full-length cDNAs for terrapin ovarian aromatase. Using reverse transcription—polymerase chain reaction (RT-PCR) on mRNA from various tissues we have determined that aromatase is expressed in the female brain and ovary, whereas it is only expressed in the brain of the male. Brain expression of aromatase occurs before stage 15, the beginning of the temperature-dependent sex determining period. Ovarian expression occurs sometime later. To quantify expression levels, we have developed a competitive RT-PCR technique to study the ontogeny of aromatase transcript levels throughout development. The sensitivity of our assay (0.001–10 atmol of transcript) permits us to analyse individual embryonic adrenal/kidney/gonadal complexes without pooling samples. Female hatchlings (stage 26) brains express higher aromatase mRNA levels than male brains (381 ± 80 vs 202 ± 85 atmol/μg RNA, respectively). Similarly, ovaries express significantly higher aromatase mRNA levels than hatchling testes (352 ± 117 vs

Alternative promoter usage is typically associated with mRNAs with differing first exons that contain or consist entirely of a 5' untranslated region. The murine Bcrp1 (Abcg2) transporter has three alternative promoters associated with mRNAs containing alternative untranslated first exons designated as E1A, E1B, and E1C. The E1B promoter regulates Bcrp1 transcription in mouse intestine. Here, we report the identification and characterization of a novel Bcrp1 promoter and first exon, E1U, located upstream from the other Bcrp1 promoters/first exons, which is the predominant alternative promoter utilized in murine testis. Using in silico analysis we identified a putative steroidogenic factor-1 (SF-1) response element that was unique to the Bcrp1 E1U alternative promoter. Overexpression of SF-1 in murine TM4 Sertoli cells enhanced Bcrp1 E1U mRNA expression and increased Bcrp1 E1U alternative promoter activity in a reporter assay, whereas mutation of the SF-1 binding site totally eliminated Bcrp1 E1U alternative promoter activity. Moreover, expression of Bcrp1 E1U and total mRNA and Bcrp1 protein was markedly diminished in the testes from adult Sertoli cell-specific SF-1 knockout mice, in comparison to the testes from wild-type mice. Binding of SF-1 to the SF-1 response element in the E1U promoter was demonstrated by chromatin immunoprecipitation assays. In conclusion, nuclear transcription factor SF-1 is involved with the regulation of a novel promoter of Bcrp1 that governs transcription of the E1U mRNA isoform in mice. The present study furthers understanding of the complex regulation of Bcrp1 expression in specific tissues of a mammalian model.

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner.

There is considerable evidence that implicates oxidative stress in the pathophysiology of human pregnancy complications. However, the role and the mechanism of maintaining an antioxidant prosurvival uterine environment during normal pregnancy is largely unresolved. Herein we report that the highly active uterine unfolded protein response plays a key role in promoting antioxidant activity in the uterine myocyte across gestation. The unfolded protein response (UPR) senses the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and activates a signaling network that consists of the transmembrane protein kinase eukaryotic translation initiation factor 2 alpha kinase 3/PKR-like-ER kinase (EIF2AK3), which acts to decrease protein translation levels, allowing for a lowered need for protein folding during periods of ER stress. However, independent of its translational regulatory capacity, EIF2AK3-dependent signals elicit the activation of the transcription factor, nuclear factor erythroid 2-like 2 (NFE2L2) in response to oxidative stress. NFE2L2 binds to antioxidant response elements in the promoters of a variety of antioxidant genes that minimize the opportunities for generation of reactive oxygen intermediates. Our analysis demonstrates that in the absence of EIF2AK3, the uterine myocyte experiences increased levels of reactive oxygen species due to decreased NFE2L2 activation. Elevated levels of intracellular reactive oxygen species were observed in the EIF2AK3 null cells, and this was associated with the onset of apoptotic cell death. These findings confirm the prosurvival and antioxidant role of UPR-mediated EIF2AK3 activation in the context of the human uterine myocyte.

Sex determination in the diamondback terrapin, Malaclemys terrapin, is temperature-dependent. Eggs incubated at 31 degrees C, and above, hatch in approximately 45 days as females. Eggs incubated below 27 degrees C hatch in about 60 days as males. Sex is not reversible after hatching. Nest temperatures in the wild can be as low as 20 degrees C and as high as 37 degrees C with as much as a 10 degrees C diel cycle. The shortest incubation time measured in nature was 56 days and the longest approaching 120 days. Nests in our study site produced predominantly (> 95%) male hatchlings. Treatment of developing embryos with estrogen produces females at male producing temperatures while treatment with fadrozole (a nonsteroidal aromatase inhibitor) induces partial male-like gonads. Treatment with a steroidal aromatase inhibitor (4-hydroxyandrostenedione, 4-OHA) had no effect on sex determination. Both fadrozole and 4-OHA are potent competitive inhibitors (Ki approximately 40-50 nM) for terrapin in vitro aromatase activity. These findings are consistent with aromatase expression being a key step in sex determination of terrapins. We have cloned a partial single copy P-450 aromatase from the terrapin using a cDNA library constructed from ovarian mRNA. This partial clone is highly homologous to other vertebrate aromatases.

Preserving the uterus in a state of relative quiescence is vital to the maintenance of a successful pregnancy. Elevated cytoplasmic levels of uterine caspase 3 during pregnancy have been proposed as a potential regulator of uterine quiescence through direct targeting and disabling of the uterine contractile architecture. However, despite highly elevated levels of uterine caspase 3 during pregnancy, there is minimal evidence of apoptosis. This current study defines the mechanism whereby the pregnant uterine myocyte may harness the tocolytic activity of active caspases while avoiding apoptotic cell death. Using the pregnant mouse model, we have analyzed the uterus for changes in pro- and antiapoptotic signaling patterns associated with the advancing stages of pregnancy. Briefly, we have found that members of the IAP family, such as SURVIVIN and XIAP, and the Bcl2 family members, such as MCL1, are elevated in the uterine myocyte during late gestation. The IAP family members are the only endogenous inhibitors of active caspase 3, and MCL1 limits activation of caspase 3 by suppressing proapoptotic signaling. Elevated XIAP levels partner with SURVIVIN, resulting in increased levels of the antiapoptotic MCL1 via NFKB activation; these together have the potential to limit both the activity and level of active caspase 3 in the pregnant uterus as term approaches. We propose that modification of these antiapoptotic signaling partners allows the pregnant uterus to escape the apoptotic action of elevated active caspase 3 levels but also functions to limit the levels of active uterine caspase 3 near term.

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR−/−), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR−/− mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR−/− mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5' and 3' regulatory sequence. Insertion of an internal ribosome entry site and the EGFP gene 3' to AR allowed co-expression of AR and EGFP. Pronuclear injection of the BAC resulted in six founder mice that displayed EGFP production in appropriate AR expressing tissues. The six founder mice were mated into a Sertoli cell specific AR knockout (SCARKO) background in which spermatogenesis is blocked at the meiosis stage of germ cell development. The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR. Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR-null background. These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions. Transgenic mice expressing selective modifications of the AR-EGFP transgene may provide crucial information needed to elicit the molecular mechanisms by which AR acts in the testis and other androgen responsive tissues.

The local generation of active glucocorticoid by NADPH-dependent, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) oxoreductase activity, has emerged as an important factor in regulating hepatic glucose output and visceral adiposity. We have proposed that this NADPH is generated within the endoplasmic reticulum by the enzyme hexose-6-phosphate dehydrogenase. To address this hypothesis, we generated mice with a targeted inactivation of the H6PD gene. These mice were unable to convert 11-dehydrocorticosterone (11-DHC) to corticosterone but demonstrated increased corticosterone to 11-DHC conversion consistent with lack of 11beta-HSD1 oxoreductase and a concomitant increase in dehydrogenase activity. This increased corticosterone clearance in the knock-out mice resulted in a reduction in circulating corticosterone levels. Our studies define the critical requirement of hexose-6-phosphate dehydrogenase for 11beta-HSD1 oxoreductase activity and add a new dimension to the investigation of 11beta-HSD1 as a therapeutic target in patients with the metabolic syndrome.

We previously showed, using differential expression screening and in situ hybridization that Vanin-1, which encodes a glycosylphosphatidylinositol-linked membrane-associated pantetheinase, is expressed in a sex-specific manner during fetal gonad development in mice (Bowles, J., Bullejos, M., and Koopman, P. (2000) Genesis 27, 124-135). In the present study we investigate in detail the expression and regulation of Vanin-1 in the fetal testis. Vanin-1 is co-expressed with the transcription factors steroidogenic factor-1 (SF-1) and SOX9 in Sertoli cells and, at a lower level, with SF-1 in Leydig cells in developing testes. SF-1 is able to activate the transcription of the Vanin-1 promoter in in vitro reporter assays, and this activation is further augmented by SOX9. We found that SF-1 is able to bind to two sites in the Vanin-1 promoter, whereas SOX9 can bind to a single interposed site defined by DNA footprinting. Mutation of the SF-1 or SOX9 sites disrupts the binding of these factors and activation of transcription. The expression of Vanin-1 was abolished in Leydig cells of a mouse mutant lacking SF-1 in that cell type. Our findings account for the sex- and cell-type-specific expression of Vanin-1 in the developing mouse gonad in vivo, which we suggest is required to provide an appropriate environment for male germ cell development.

Axial skeletons of thirteen species representing a wide range of genera of Gorgonians were investigated using Young's modulus as a measure of stiffness and Torsion modulus as a measure of resistance to shear or twist. Atomic absorption spectroscopic determination of magnesium and calcium content as measures of mineralization were done. Relative quantities of calcareous material in the axial skeletons showed a strong linear correlation with Young's modulus and suggests an important role for calcareous material in the modulation of the mechanical properties of axoskeleton. Torsion moduli also showed a mathematical but non-linear relationship to calcareous content. Axis stiffness correlated well with zonation-related water movement. Stiffest axes occur in deeper water with no wave surge, most flexible in shallower water with moderate surge and intermediate stiffness in shallow, high energy habitats. An extremely high MgCO3 containing carbonate that may be a previously unreported biological structural material was found in the Plexauridae.