A light-controlled phospholipase C for imaging of lipid dynamics and controlling neural plasticity.

Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCß (opto-PLCß). Opto-PLCß uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCß triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCß can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCß offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.

All-optical presynaptic plasticity induction by photoactivated adenylyl cyclase targeted to axon terminals.

Intracellular signaling plays essential roles in various cell types. In the central nervous system, signaling cascades are strictly regulated in a spatiotemporally specific manner to govern brain function; for example, presynaptic cyclic adenosine monophosphate (cAMP) can enhance the probability of neurotransmitter release. In the last decade, channelrhodopsin-2 has been engineered for subcellular targeting using localization tags, but optogenetic tools for intracellular signaling are not well developed. Therefore, we engineered a selective presynaptic fusion tag for photoactivated adenylyl cyclase (bPAC-Syn1a) and found its high localization at presynaptic terminals. Furthermore, an all-optical electrophysiological method revealed rapid and robust short-term potentiation by bPAC-Syn1a at brain stem-amygdala synapses in acute brain slices. Additionally, bPAC-Syn1a modulated mouse immobility behavior. These results indicate that bPAC-Syn1a can manipulate presynaptic cAMP signaling in vitro and in vivo. The all-optical manipulation technique developed in this study can help further elucidate the dynamic regulation of various cellular functions.

State-dependent modulation of positive and negative affective valences by a parabrachial nucleus-to-ventral tegmental area pathway in mice.

Appropriately responding to various sensory signals in the environment is essential for animal survival. Accordingly, animal behaviors are closely related to external and internal states, which include the positive and negative emotional values of sensory signals triggered by environmental factors. While the lateral parabrachial nucleus (LPB) plays a key role in nociception and supports negative valences, it also transmits signals including positive valences. However, the downstream neuronal mechanisms of positive and negative valences have not been fully explored. In the present study, we investigated the ventral tegmental area (VTA) as a projection target for LPB neurons. Optogenetic activation of LPB-VTA terminals in male mice elicits positive reinforcement in an operant task and induces both avoidance and attraction in a place-conditioning task. Inhibition of glutamic acid decarboxylase (GAD) 65-expressing cells in the VTA promotes avoidance behavior induced by photoactivation of the LPB-VTA pathway. These findings indicate that the LPB-VTA pathway is one of the LPB outputs for the transmission of positive and negative valence signals, at least in part, with GABAergic modification in VTA.

Experience-dependent changes in affective valence of taste in male mice.

Taste plays an essential role in the evaluation of food quality by detecting potential harm and benefit in what animals are about to eat and drink. While the affective valence of taste signals is supposed to be innately determined, taste preference can also be drastically modified by previous taste experiences of the animals. However, how the experience-dependent taste preference is developed and the neuronal mechanisms involved in this process are poorly understood. Here, we investigate the effects of prolonged exposure to umami and bitter tastants on taste preference using two-bottle tests in male mice. Prolonged umami exposure significantly enhanced umami preference with no changes in bitter preference, while prolonged bitter exposure significantly decreased bitter avoidance with no changes in umami preference. Because the central amygdala (CeA) is postulated as a critical node for the valence processing of sensory information including taste, we examined the responses of cells in the CeA to sweet, umami, and bitter tastants using in vivo calcium imaging. Interestingly, both protein kinase C delta (Prkcd)-positive and Somatostatin (Sst)-positive neurons in the CeA showed an umami response comparable to the bitter response, and no difference in cell type-specific activity patterns to different tastants was observed. Meanwhile, fluorescence in situ hybridization with c-Fos antisense probe revealed that a single umami experience significantly activates the CeA and several other gustatory-related nuclei, and especially CeA Sst-positive neurons were strongly activated. Intriguingly, after prolonged umami experience, umami tastant also significantly activates the CeA neurons, but the Prkcd-positive neurons instead of Sst-positive neurons were highly activated. These results suggest a relationship between amygdala activity and experience-dependent plasticity developed in taste preference and the involvement of the genetically defined neural populations in this process.

Excitatory subtypes of the lateral amygdala neurons are differentially involved in regulation of synaptic plasticity and excitation/inhibition balance in aversive learning in mice.

The amygdala plays a crucial role in aversive learning. In Pavlovian fear conditioning, sensory information about an emotionally neutral conditioned stimulus (CS) and an innately aversive unconditioned stimulus is associated with the lateral amygdala (LA), and the CS acquires the ability to elicit conditioned responses. Aversive learning induces synaptic plasticity in LA excitatory neurons from CS pathways, such as the medial geniculate nucleus (MGN) of the thalamus. Although LA excitatory cells have traditionally been classified based on their firing patterns, the relationship between the subtypes and functional properties remains largely unknown. In this study, we classified excitatory cells into two subtypes based on whether the after-depolarized potential (ADP) amplitude is expressed in non-ADP cells and ADP cells. Their electrophysiological properties were significantly different. We examined subtype-specific synaptic plasticity in the MGN-LA pathway following aversive learning using optogenetics and found significant experience-dependent plasticity in feed-forward inhibitory responses in fear-conditioned mice compared with control mice. Following aversive learning, the inhibition/excitation (I/E) balance in ADP cells drastically changed, whereas that in non-ADP cells tended to change in the reverse direction. These results suggest that the two LA subtypes are differentially regulated in relation to synaptic plasticity and I/E balance during aversive learning.

Parabrachial-to-parasubthalamic nucleus pathway mediates fear-induced suppression of feeding in male mice.

Feeding behavior is adaptively regulated by external and internal environment, such that feeding is suppressed when animals experience pain, sickness, or fear. While the lateral parabrachial nucleus (lPB) plays key roles in nociception and stress, neuronal pathways involved in feeding suppression induced by fear are not fully explored. Here, we investigate the parasubthalamic nucleus (PSTN), located in the lateral hypothalamus and critically involved in feeding behaviors, as a target of lPB projection neurons. Optogenetic activation of lPB-PSTN terminals in male mice promote avoidance behaviors, aversive learning, and suppressed feeding. Inactivation of the PSTN and lPB-PSTN pathway reduces fear-induced feeding suppression. Activation of PSTN neurons expressing pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide enriched in the PSTN, is sufficient for inducing avoidance behaviors and feeding suppression. Blockade of PACAP receptors impaires aversive learning induced by lPB-PSTN photomanipulation. These findings indicate that lPB-PSTN pathway plays a pivotal role in fear-induced feeding suppression.

Localization of Ectopic Mediastinal Parathyroid Adenomas Using Indigo Carmine Injection for Surgical Management: A Preliminary Report.

An ectopic parathyroid adenoma (EPA) is a rare entity. The aim of this study was to report our experience in the preoperative localization and surgical management of EPAs. This was a multicenter retrospective study involving patients diagnosed with an EPA (three males and seven females) from January 2005 to November 2021. The clinical features, preoperative management, and surgical procedures were analyzed. A cervical neck ultrasound was performed in all patients and showed a focus in eight patients. Cervicothoracic enhanced computed tomography was performed in all patients and showed a focus in nine patients. The 99mTc-MIBI scintigraphy was performed in eight patients and showed uptake in six of them. We performed a neck dissection and thoracotomy in one patient, a thoracoscopy in one patient, surgery with a focused approach in seven patients, four of whom were injected with indigo carmine blue, and surgery with a bilateral approach in one patient. 1 h following the parathyroidectomy, the parathyroid hormone (PTH) concentration was decreased to 40-80% of the baseline value. Establishing a preoperative diagnosis of an EPA is challenging for the surgeon, despite the progress in the morphologic assessment. An intraoperative PTH assay and injection of indigo carmine have been shown to be valuable tools in the appropriate surgical management of an EPA.

Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis.

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.

DAF-16/FOXO promotes taste avoidance learning independently of axonal insulin-like signaling.

The avoidance of starvation is critical for the survival of most organisms, thus animals change behavior based on past nutritional conditions. Insulin signaling is important for nutritional state-dependent behavioral plasticity, yet the underlying regulatory mechanism at the cellular level remains unclear. Previous studies showed that insulin-like signaling is required for taste avoidance learning, in which the nematode Caenorhabditis elegans avoids salt concentrations encountered under starvation conditions. DAF-2c, a splice isoform of the DAF-2 insulin receptor, functions in the axon of the ASER sensory neuron, which senses changes in salt concentrations. In addition, mutants of a major downstream factor of DAF-2, the forkhead transcription factor O (FOXO) homolog DAF-16, show defects in taste avoidance learning. Interestingly, the defect of the daf-2 mutant is not suppressed by daf-16 mutations in the learning, unlike those in other phenomena, such as longevity and development. Here we show that multiple DAF-16 isoforms function in ASER. By epistasis analysis using a DAF-2c isoform-specific mutant and an activated form of DAF-16, we found that DAF-16 acts in the nucleus in parallel with the DAF-2c-dependent pathway in the axon, indicating that insulin-like signaling acts both in the cell body and axon of a single neuron, ASER. Starvation conditioning induces nuclear translocation of DAF-16 in ASER and degradation of DAF-16 before starvation conditioning causes defects in taste avoidance learning. Forced nuclear localization of DAF-16 in ASER biased chemotaxis towards lower salt concentrtions and this effect required the Gq/PKC pathway and neuropeptide processing enzymes. These data imply that DAF-16/FOXO transmits starvation signals and modulates neuropeptide transmission in the learning.

Synergistic effects of tumor necrosis factor-α and insulin-like growth factor-I on survival of human trophoblast-derived BeWo cell line.

OBJECTIVE: Trophoblast survival is regulated by cytokines and growth factors. While the pharmacological levels (10-100 ng/mL) of tumor necrosis factor (TNF)- α affect trophoblasts survival in vitro, the effects of the physiological levels (1-10 pg/mL) of TNF-α remain unknown. We investigated the effects of the physiological levels of TNF-α on proliferation and apoptosis of human trophoblast cells by using BeWo cells. Insulin-like growth factor (IGF)-I is also a potent regulator of trophoblast survival and has been known to exert synergistic effects with other hormones. The interaction of IGF-I and TNF-α on BeWo cells survival was also examined. METHODS: After incubating BeWo under the presence of TNF-α (10-105 pg/mL) and IGF-I (102 ng/mL), we assessed cell number by WST-1 assay and cell proliferation by BrdU uptake assay and immunocytochemistry with anti-Ki67 antibody. Apoptosis was evaluated by TUNEL assay and caspase-3, 8 activity assays. RESULTS: Under the presence of IGF-I, cell number, BrdU uptake, and Ki-67 expression of BeWo were dose-dependently enhanced by low TNF-α (10-102 pg/mL), while no such effects were detected without IGF-I. Higher levels of TNF-α (104-105 pg/mL) showed inhibiting effects on cell number and cell proliferation. The number of TUNEL positive cells were decreased and caspase activities were suppressed by lower levels (10-102 pg/mL) of TNF-α and IGF-I independently. Higher levels of TNF-α (104-105 pg/mL) showed promoting effects on apoptosis irrespective of IGF-I. CONCLUSION: The physiological levels of TNF-α and IGF-I had synergetic effects on enhancing cell proliferation and also independently inhibited apoptosis of Bewo cells.

Regulation of body size in Caenorhabditis elegans: effects of environmental factors and the nervous system.

Body size is one of the basic traits of animals and is regulated to adapt to the environment. Animals perceive environmental stimuli with sensory neurons, and signals from the nervous system alter the size of organs, thus regulating body size. The model animal Caenorhabditis elegans is particularly suited for genetic analysis of body size regulation, and has already contributed to the elucidation of various genetic pathways that regulate body size. In this review, we summarize the available literature regarding environmental factors that regulate body size and the role of the nervous system in such regulation. We discuss in detail a recent report on body size regulation by the neurotransmitter, dopamine.

In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicumIMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty acid synthase (FAS-II) system. In this study, we reported genetic evidence demonstrating that the FAS-I system is the source of the biotin precursor in vivo in the engineered biotin-prototrophic C. glutamicum strain. This study also uncovered the important physiological role of FasB in lipoic acid biosynthesis. Here, we present an FAS-I enzyme that functions in supplying the lipoic acid precursor, although its biosynthesis has been believed to exclusively depend on FAS-II in organisms. The findings obtained here provide new insights into the metabolic engineering of this industrially important microorganism to produce these compounds effectively.

Branched-chain amino acids regulate insulin-like growth factor-binding protein 1 (IGFBP1) production by decidua and influence trophoblast migration through IGFBP1.

STUDY QUESTION: Do branched-chain amino acids (BCAAs) influence the migration of human extravillous trophoblast (EVT) cells through changes in insulin-like growth factor-binding protein 1 (IGFBP1) production in decidual cells? STUDY FINDING: Decidua-derived IGFBP1 had a stimulating effect on migration of EVT. WHAT IS KNOWN ALREADY: IGFBP1 is abundantly secreted from human decidual cells and influences trophoblast migration in human placenta of early pregnancy. In hepatic cells, the expression of IGFBP1 is influenced by nutritional status and BCAAs regulate IGFBP1 production. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: This is a laboratory-based study using human decidual cells and trophoblast cells isolated from placental tissue of early pregnancy (n = 50) and grown as primary cultures. Production of IGFBP1 from decidual cells was examined by enzyme-linked immunosorbent assay and immunoblotting after incubation with or without BCAAs. EVT migration was evaluated using the media conditioned by decidual cells. The effect of conditioned media on phosphorylation of focal adhesion kinase (FAK) in EVT was also analyzed by immunoblotting. The same experiments were repeated in the presence of RGD peptide, which inhibits IGFBP1 binding to α5ß1 integrin. An EVT migration assay and the immunoblotting of phosphorylated FAK were also conducted with exogenous IGFBP1. The effect of the conditioned media on cytotrophoblast cell number was also assessed using WST-1 in a cell proliferation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Deprivation of BCAAs on decidual cells significantly suppressed IGFBP1 secretion (P < 0.05, versus BCAA+). Exogenous IGFBP1-stimulated EVT migration (P < 0.05) and phosphorylation of FAK (P < 0.05), and the RGD peptide inhibited these effects. EVT migration and phosphorylation of FAK were stimulated by the conditioned media, presumably by IGFBP1 in the media. RGD treatment abrogated the stimulating effects of conditioned media. The conditioned media deprived of BCAAs had suppressive effects on EVT migration (P < 0.05, versus BCAA+) and phosphorylation of FAK (P < 0.05, versus BCAA+). The conditioned media did not affect number of cytotrophoblast cells. LIMITATIONS, REASONS FOR CAUTION: The conclusions are based on in vitro experiments with human decidual cells and trophoblast cells isolated from placental tissue of early pregnancy, and we were unable to ascertain whether these mechanisms actually operate in vivo. We investigated the effect of decidua-derived IGFBP1 on EVT migration, however, we cannot completely rule out the possibility that endogenous IGF could also influence cell migration. WIDER IMPLICATIONS OF FINDINGS: Interruption of the BCAA supply to uterine decidual cells in early pregnancy may suppress EVT migration through reduced IGFBP1 secretion, which may be one of the pathophysiological conditions responsible for pre-eclampsia. LARGE SCALE DATA: None. STUDY FUNDING/ AND COMPETING INTERESTS: All funds were obtained through Kyorin University School of Medicine. The authors have no conflict of interest to declare.

Dopamine regulates body size in Caenorhabditis elegans.

The nervous system plays a critical role in the regulation of animal body sizes. In Caenorhabditis elegans, an amine neurotransmitter, dopamine, is required for the tactile perception of food and food-dependent behavioral changes, while its role in development is unknown. In this study, we show that dopamine negatively regulates body size through a D2-like dopamine receptor, DOP-3, in C. elegans. Dopamine alters body size without affecting food intake or developmental rate. We also found that dopamine promotes egg-laying, although the regulation of body size by dopamine was not solely caused by this effect. Furthermore, dopamine negatively regulates body size through the suppression of signaling by octopamine and Gq-coupled octopamine receptors, SER-3 and SER-6. Our results demonstrate that dopamine and octopamine regulate the body size of C. elegans and suggest a potential role for perception in addition to ingestion of food for growth.

CD34 and CD49f Double-Positive and Lineage Marker-Negative Cells Isolated from Human Myometrium Exhibit Stem Cell-Like Properties Involved in Pregnancy-Induced Uterine Remodeling.

Repeated and dramatic pregnancy-induced uterine enlargement and remodeling throughout reproductive life suggests the existence of uterine smooth muscle stem/progenitor cells. The aim of this study was to isolate and characterize stem/progenitor-like cells from human myometrium through identification of specific surface markers. We here identify CD49f and CD34 as markers to permit selection of the stem/progenitor cell-like population from human myometrium and show that human CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells exhibit stem cell-like properties. These include side population phenotypes, an undifferentiated status, high colony-forming ability, multilineage differentiation into smooth muscle cells, osteoblasts, adipocytes, and chondrocytes, and in vivo myometrial tissue reconstitution following xenotransplantation. Furthermore, CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells proliferate under hypoxic conditions in vitro and, compared with the untreated nonpregnant myometrium, show greater expansion in the estrogen-treated nonpregnant myometrium and further in the pregnant myometrium in mice upon xenotransplantation. These results suggest that the newly identified myometrial stem/progenitor-like cells influenced by hypoxia and sex steroids may participate in pregnancy-induced uterine enlargement and remodeling, providing novel insights into human myometrial physiology.

BMPR2 is required for postimplantation uterine function and pregnancy maintenance.

Abnormalities in cell-cell communication and growth factor signaling pathways can lead to defects in maternal-fetal interactions during pregnancy, including immunologic rejection of the fetal/placental unit. In this study, we discovered that bone morphogenetic protein receptor type 2 (BMPR2) is essential for postimplantation physiology and fertility. Despite normal implantation and early placental/fetal development, deletion of Bmpr2 in the uterine deciduae of mice triggered midgestation abnormalities in decidualization that resulted in abnormal vascular development, trophoblast defects, and a deficiency of uterine natural killer cells. Absence of BMPR2 signaling in the uterine decidua consequently suppressed IL-15, VEGF, angiopoietin, and corin signaling. Disruption of these pathways collectively lead to placental abruption, fetal demise, and female sterility, thereby placing BMPR2 at a central point in the regulation of several physiologic signaling pathways and events at the maternal-fetal interface. Since trophoblast invasion and uterine vascular modification are implicated in normal placentation and fetal growth in humans, our findings suggest that abnormalities in uterine BMPR2-mediated signaling pathways can have catastrophic consequences in women for the maintenance of pregnancy.

Ingestion of theanine, an amino acid in tea, suppresses psychosocial stress in mice.

The antistress effect of theanine (γ-glutamylethylamide), an amino acid in tea, was investigated using mice that were psychosocially stressed from a conflict among male mice in conditions of confrontational housing. Two male mice were housed in the same cage separated by a partition to establish a territorial imperative. When the partition was removed, the mice were co-housed confrontationally. As a marker for the stress response, changes in the adrenal gland were studied in comparison to group-housed control mice (six mice in a cage). Significant adrenal hypertrophy was observed in mice during confrontational housing, which was developed within 24 h and persisted for at least 1 week. The size of cells in the zona fasciculata of the adrenal gland, from which glucocorticoid is mainly secreted, increased (∼1.11-fold) in mice during confrontational housing, which was accompanied by a flattened diurnal rhythm of corticosterone and ACTH in blood. The ingestion of theanine (>5 µg ml(-1)) prior to confrontational housing significantly suppressed adrenal hypertrophy. An antidepressant, paroxetin, suppressed adrenal hypertrophy in a similar manner in mice during confrontational housing. In mice that ingested theanine, behavioural depression was also suppressed, and a diurnal rhythm of corticosterone and ACTH was observed, even in mice that were undergoing confrontational housing. Furthermore, the daily dose of theanine (40 µg ml(-1)) blocked the counteracting effects of caffeine (30 µg ml(-1)) and catechin (200 µg ml(-1)). The present study demonstrated that theanine prevents and relieves psychosocial stress through the modulation of hypothalamic-pituitary-adrenal axis activity.

Transforming growth factor ß receptor type 1 is essential for female reproductive tract integrity and function.

The transforming growth factor ß (TGFß) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFß type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFß ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1-mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1-mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.

Connective tissue growth factor is required for normal follicle development and ovulation.

Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-ß superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-ß family intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-ß and progesterone signaling cascades and is necessary for normal follicle development and ovulation.