Murine uterine gland branching is necessary for gland function in implantation.

Uterine glands are branched, tubular structures whose secretions are essential for pregnancy success. It is known that pre-implantation glandular expression of leukemia inhibitory factor (LIF) is crucial for embryo implantation; however, the contribution of uterine gland structure to gland secretions, such as LIF, is not known. Here, we use mice deficient in estrogen receptor 1 (ESR1) signaling to uncover the role of ESR1 signaling in gland branching and the role of a branched structure in LIF secretion and embryo implantation. We observed that deletion of ESR1 in neonatal uterine epithelium, stroma, and muscle using the progesterone receptor PgrCre causes a block in uterine gland development at the gland bud stage. Embryonic epithelial deletion of ESR1 using a Müllerian duct Cre line, Pax2Cre, displays gland bud elongation but a failure in gland branching. Reduction of ESR1 in adult uterine epithelium using the lactoferrin-Cre (LtfCre) displays normally branched uterine glands. Unbranched glands from Pax2Cre Esr1flox/flox uteri fail to express glandular pre-implantation Lif, preventing implantation chamber formation and embryo alignment along the uterine mesometrial-antimesometrial axis. In contrast, branched glands from LtfCre Esr1flox/flox uteri display reduced expression of ESR1 and glandular Lif resulting in delayed implantation chamber formation and embryo-uterine axes alignment but mice deliver a normal number of pups. Finally, pre-pubertal unbranched glands in control mice express Lif in the luminal epithelium but fail to express Lif in the glandular epithelium, even in the presence of estrogen. These data strongly suggest that branched glands are necessary for pre-implantation glandular Lif expression for implantation success. Our study is the first to identify a relationship between the branched structure and secretory function of uterine glands and provides a framework for understanding how uterine gland structure-function contributes to pregnancy success.

Attenuated retinoic acid signaling is among the early responses in mouse uterus approaching embryo attachment.

The uterus is transiently receptive for embryo implantation. It remains to be understood why the uterus does not reject a semi-allogeneic embryo (to the biological mother) or an allogeneic embryo (to a surrogate) for implantation. To gain insights, we examined uterine early response genes approaching embryo attachment on day 3 post coitum (D3) at 22 hours when blue dye reaction, an indication of embryo attachment, had not manifested in mice. C57BL/6 pseudo-pregnant (control) and pregnant mouse uteri were collected on D3 at 22 hours for microarray analysis. The self-assembling-manifold (SAM) algorithm identified 21,858 unique probesets. Principal component analysis indicated a clear separation between the pseudo-pregnant and pregnant groups. There were 106 upregulated and five downregulated protein-coding genes in the pregnant uterus with fold change (fc) >1.5 and q value <5%. Gene ontology (GO) analysis of the 106 upregulated genes revealed 38 significant GO biological process (GOBP) terms (P <0.05), and 32 (84%) of them were associated with immune responses, with a dominant natural killer (NK) cell activation signature. Among the top eight upregulated protein-coding genes, Cyp26a1 inactivates retinoic acid (RA) while Lrat promotes vitamin A storage, both of which are expected to attenuate RA bioavailability; Atp6v0d2 and Gjb2 play roles in ion transport and transmembrane transport; Gzmb, Gzmc, and Il2rb are involved in immune responses; and Tdo2 is important for kynurenine pathway. Most of these genes or their related pathways have functions in immune regulations. RA signaling has been implicated in immune tolerance and immune homeostasis, and uterine NK cells have been implicated in immunotolerance at the maternal-fetal interface in the placenta. The mechanisms of immune responses approaching embryo attachment remain to be elucidated. The coordinated effects of the early response genes may hold the keys to the question of why the uterus does not reject an implanting embryo.

Murine uterine gland branching is necessary for gland function in implantation.

Uterine glands are branched, tubular structures whose secretions are essential for pregnancy success. It is known that pre-implantation glandular expression of leukemia inhibitory factor (LIF) is crucial for embryo implantation, however contribution of uterine gland structure to gland secretions such as LIF is not known. Here we use mice deficient in estrogen receptor 1 (ESR1) signaling to uncover the role of ESR1 signaling in gland branching and the role of a branched structure in LIF secretion and embryo implantation. We observed that deletion of ESR1 in neonatal uterine epithelium, stroma and muscle using the progesterone receptor PgrCre causes a block in uterine gland development at the gland bud stage. Embryonic epithelial deletion of ESR1 using a mullerian duct Cre line - Pax2Cre, displays gland bud elongation but a failure in gland branching. Surprisingly, adult uterine epithelial deletion of ESR1 using the lactoferrin-Cre (LtfCre) displays normally branched uterine glands. Intriguingly, unbranched glands from Pax2Cre Esr1flox/flox uteri fail to express glandular pre-implantation Lif, preventing implantation chamber formation and embryo alignment along the uterine mesometrial-antimesometrial axis. In contrast, branched glands from LtfCre Esr1flox/flox uteri display reduced expression of glandular Lif resulting in delayed implantation chamber formation and embryo-uterine axes alignment but deliver a normal number of pups. Finally, pre-pubertal unbranched glands in control mice express Lif in the luminal epithelium but fail to express Lif in the glandular epithelium even in the presence of estrogen. These data strongly suggest that branched glands are necessary for pre-implantation glandular Lif expression for implantation success. Our study is the first to identify a relationship between the branched structure and secretory function of uterine glands and provides a framework for understanding how uterine gland structure-function contributes to pregnancy success.

Upregulation of FOXA2 in uterine luminal epithelium and vaginal basal epithelium of epiERα-/- (Esr1fl/flWnt7aCre/+) mice†.

Forkhead box protein A2 (FOXA2) is a pioneer transcription factor important for epithelial budding and morphogenesis in different organs. It has been used as a specific marker for uterine glandular epithelial cells (GE). FOXA2 has close interactions with estrogen receptor α (ERα). ERα binding to Foxa2 gene in the uterus indicates its regulation of Foxa2. The intimate interactions between ERα and FOXA2 and their essential roles in early pregnancy led us to investigate the expression of FOXA2 in the female reproductive tract of pre-implantation epiERα-/- (Esr1fl/flWnt7aCre/+) mice, in which ERα is conditionally deleted in the epithelium of reproductive tract. In the oviduct, FOXA2 is detected in the ciliated epithelial cells of ampulla but absent in the isthmus of day 3.5 post-coitum (D3.5) Esr1fl/fl control and epiERα-/- mice. In the uterus, FOXA2 expression in the GE appears to be comparable between Esr1fl/fl and epiERα-/- mice. However, FOXA2 is upregulated in the D0.5 and D3.5 but not PND25-28 epiERα-/- uterine luminal epithelial cells (LE). In the vagina, FOXA2 expression is low in the basal layer and increases toward the superficial layer of the D3.5 Esr1fl/fl vaginal epithelium, but FOXA2 is detected in the basal, intermediate, and superficial layers, with the strongest FOXA2 expression in the intermediate layers of the D3.5 epiERα-/- vaginal epithelium. This study demonstrates that loss of ERα in LE and vaginal basal layer upregulates FOXA2 expression in these epithelial cells during early pregnancy. The mechanisms for epithelial cell-type specific regulation of FOXA2 by ERα remain to be elucidated.

Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†.

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.

Synergistic effects of Co/CoO nanoparticles on imine-based covalent organic frameworks for enhanced OER performance.

The development of non-noble metal electrocatalysts toward the oxygen evolution reaction (OER) is a key challenge in advancing electrocatalytic water splitting, which is essential for the commercialization of clean and renewable energy. A covalent organic framework (COF) has a precise and controllable structure, high π-π conjugation, large surface area, and porosity and shows great potential as an OER electrocatalyst. However, the relative conductivity and inherent instability greatly limit the further improvement of its performance. Herein, imine-based COF-supported Co/CoO nanoparticles (Co/CoO@COF) were developed for the high-performance electrocatalytic OER. For the Co/CoO@COF catalyst, Co/CoO could form a conjugation effect with the COF, which can increase the electron cloud density of the delocalized large π bond, then improve the conductivity. The combination of Co/CoO and COF effectively enhances the structural stability of the catalyst and enriches the catalytic active sites. Under alkaline conditions, the Co/CoO@COF shows a very low overpotential of 278 mV at a current density of 10 mA cm-2, and a Tafel slope of 80.11 mV dec-1 which is better than that of commercial RuO2.

Varied effects of doxorubicin (DOX) on the corpus luteum of C57BL/6 mice during early pregnancy†.

Certain chemotherapeutic drugs are toxic to ovarian follicles. The corpus luteum (CL) is normally developed from an ovulated follicle for producing progesterone (P4) to support early pregnancy. To fill in the knowledge gap about effects of chemotherapy on the CL, we tested the hypothesis that chemotherapy may target endothelial cells and/or luteal cells in the CL to impair CL function in P4 steroidogenesis using doxorubicin (DOX) as a representative chemotherapeutic drug in mice. In both mixed background mice and C57BL/6 mice, a single intraperitoneal injection of DOX (10 mg/kg) on 0.5-day postcoitum (D0.5, postovulation) led to ~58% D3.5 mice with serum P4 levels lower than the serum P4 range in the phosphate buffer saline-treated control mice. Further studies in the C57BL/6 ovaries revealed that CLs from DOX-treated mice with low P4 levels had less defined luteal cords and disrupted collagen IV expression pattern, indicating disrupted capillary, accompanied with less differentiated luteal cells that had smaller cytoplasm and reduced StAR expression. DOX-treated ovaries had increased granulosa cell death in the growing follicles, reduced proliferating cell nuclear antigen-positive endothelial cells in the CLs, enlarged lipid droplets, and disrupted F-actin in the luteal cells. These novel data suggest that the proliferating endothelial cells in the developing CL may be the primary target of DOX to impair the vascular support for luteal cell differentiation and subsequently P4 steroidogenesis. This study fills in the knowledge gap about the toxic effects of chemotherapy on the CL and provides critical information for risk assessment of chemotherapy in premenopausal patients.

Uterine Luminal Epithelium as the Transient Gateway for Embryo Implantation.

The uterine luminal epithelium (LE) is the first maternal contact for an implanting embryo. Intrauterine fluid resorption, cessation of LE proliferation and apoptosis, and LE structural changes are prerequisites for establishing transient uterine receptivity for embryo implantation. Vesicle trafficking in the LE and receptor-mediated paracrine and autocrine mechanisms are crucial both for LE preparation and LE communications with the embryo and stroma during the initiation of embryo implantation. This review mainly covers recent in vivo studies in LE of mouse models from 0.5 days post-coitus (D0.5) to ∼D4 20 h when the trophoblasts pass through the LE layer for embryo implantation. The review is organized into three interconnected sections: preimplantation LE preparation for embryo attachment, embryo-LE communications, and LE-stroma communications.

Association of luteal cell degeneration and progesterone deficiency with lysosomal storage disorder mucolipidosis type IV in Mcoln1-/- mouse model†.

Transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1) (MCOLN1/Mcoln1) is a lysosomal counter ion channel. Mutations in MCOLN1 cause mucolipidosis type IV (MLIV), a progressive and severe lysosomal storage disorder with a slow onset. Mcoln1-/- mice recapitulate typical MLIV phenotypes but roles of TRPML1 in female reproduction are unknown. Despite normal mating activities, Mcoln1-/- female mice had reduced fertility at 2 months old and quickly became infertile at 5 months old. Progesterone deficiency was detected on 4.5 days post coitum/gestation day 4.5 (D4.5). Immunohistochemistry revealed TRPML1 expression in luteal cells of wild type corpus luteum (CL). Corpus luteum formation was not impaired in 5-6 months old Mcoln1-/- females indicated by comparable CL numbers in control and Mcoln1-/- ovaries on both D1.5 and D4.5. In the 5-6 months old Mcoln1-/- ovaries, histology revealed less defined corpus luteal cord formation, extensive luteal cell vacuolization and degeneration; immunofluorescence revealed disorganized staining of collagen IV, a basal lamina marker for endothelial cells; Nile Red staining detected lipid droplet accumulation, a typical phenotype of MLIV; immunofluorescence of heat shock protein 60 (HSP60, a mitochondrial marker) and in situ hybridization of steroidogenic acute regulatory protein (StAR, for the rate-limiting step of steroidogenesis) showed reduced expression of HSP60 and StAR, indicating impaired mitochondrial functions. Luteal cell degeneration and impaired mitochondrial functions can both contribute to progesterone deficiency in the Mcoln1-/- mice. This study demonstrates a novel function of TRPML1 in maintaining CL luteal cell integrity and function.

Dietary exposure to mycotoxin zearalenone (ZEA) during post-implantation adversely affects placental development in mice.

Zearalenone (ZEA) is a common food contaminant (ppb-ppm) derived from Fusarium fungi. With its estrogenicity and potential chronic exposure, ZEA poses a risk to pregnancy. Our previous studies implied post-implantational lethality by ZEA. Since a functional placenta is essential for fetal development and survival, it was hypothesized that ZEA may have adverse effects on placental development leading to post-implantational lethality. Exposure of young mice to 0, 0.8, 4, 10, and 40 ppm ZEA diets from gestation day 5.5 (D5.5) to D13.5 led to increased resorption of implantation sites, increased placental hemorrhage, decreased placental and fetal weights, proportionally reduced placental layers, and disorganized placental labyrinth vascular spaces in the 40 ppm ZEA group, as well as lipid accumulation in the labyrinth layer of all four ZEA treatment groups examined on D13.5. These data demonstrate adverse effects of ZEA on placental development.

Protein-polyphenol interactions enhance the antioxidant capacity of phenolics: analysis of rice glutelin-procyanidin dimer interactions.

Rice glutelin and procyanidins are often used in functional foods as sources of plant-based proteins and polyphenols, respectively, but little is currently known about the interactions between them. In our research, the interaction between rice glutelin and the B-type procyanidin dimer (PB2) was investigated. The presence of the PB2 decreased the α-helix and random coil structure of the rice protein and reduced its surface hydrophobicity. However, the PB2 did not adversely affect the functional performance of RG in emulsions. Conversely, the antioxidant capacity of the PB2 was enhanced in the presence of the rice protein. Fluorescence spectroscopy confirmed that the protein and PB2 formed molecular complexes, which were primarily the result of hydrophobic attractive forces. Molecular docking analysis provides insights into the nature of the interaction between the rice protein and PB2. This study provides valuable insights into the nature of the interactions between plant proteins and polyphenolic nutraceuticals.

Chemotherapeutic agent doxorubicin alters uterine gene expression in response to estrogen in ovariectomized CD-1 adult mice†.

Chemotherapy can potentially impair fertility in premenopausal cancer patients. Female fertility preservation has been mainly focused on the ovarian aspects and benefited greatly from assisted reproductive technologies, such as in vitro fertilization (IVF). The rate-limiting step for the success of IVF is embryo implantation in the uterus. Doxorubicin (DOX) is a widely used chemotherapeutic agent with ovarian toxicity. It remains unknown if the uterus is a direct target of DOX. To circumvent the indirect uterine effect from ovarian toxicity of DOX and to investigate potential long-term impact of DOX on the uterus, young adult ovariectomized CD-1 mice were given an intraperitoneal injection once with PBS or DOX (10 mg/kg, a human relevant chemotherapeutic dose), and 30 days later, each set of mice was randomly assigned into three groups and subcutaneously injected with oil, 17ß-estradiol (E2, for 6 h), and progesterone (P4, for 54 h), respectively. Uterine transcriptomic profiles were determined using RNA-seq. Principal component analysis of the uterine transcriptomes revealed four clusters from the six treatment groups: PBS-oil & DOX-oil, PBS-P4 & DOX-P4, PBS-E2, and DOX-E2, indicating that DOX treatment did not affect the overall uterine transcriptomic profiles in the oil and P4-treated mice but altered uterine responses to E2 treatment. DAVID analysis indicated that the top-affected gene cluster was "Glycoprotein". These data demonstrate that DOX can directly target the uterus and has a long-term impact on uterine responses to E2.

Comparing the binding interaction between ß-lactoglobulin and flavonoids with different structure by multi-spectroscopy analysis and molecular docking.

Four kinds of flavonoids (apigenin, naringenin, kaempferol, genistein) were skillfully selected to investigate the interaction between flavonoids and ß-lactoglobulin (ß-LG) by multi-spectroscopy analysis and molecular docking. Hydrogenation on C2C3 double bond weakened the affinity of apigenin for ß-LG and it's most obvious, followed by hydroxylation of C3 and position isomerism of phenyl ring B. The main interaction force for apigenin and naringenin binding to ß-LG (van der Waals forces and hydrogen bonds) was different from that of genistein and kaempferol (hydrophobic interactions). Circular dichroism and fluorescence experiments indicated that conformation of ß-LG became loose and surface hydrophobicity of ß-LG was reduced in the presence of flavonoids. Molecular docking indicated that flavonoids interacted with specific amino acid residues located on the outer surface of ß-LG. These findings can provide a deep understanding about the interaction mechanism between flavonoids and protein, and it may be valuable in dairy incorporation with flavonoids.

Seipin deficiency leads to increased endoplasmic reticulum stress and apoptosis in mammary gland alveolar epithelial cells during lactation.

Seipin is an integral endoplasmic reticulum (ER) membrane protein encoded by Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/Bscl2) gene. Most litters (59%) from Bscl2-/- dams mated with wild type (WT) (Bscl2+/+) males did not survive postnatal day 5 (PND5) and pups (Bscl2+/-) lacked milk in their stomachs. The survived litters had reduced pup survival rate at PND21. It was hypothesized that seipin was critical for lactation. Bscl2 was upregulated and highly detected in the lactation day 1 (LD1) WT mammary gland alveolar epithelial cells. LD1 Bscl2-/- mammary glands lacked adipocytes and alveolar clusters and had varied alveolar morphology: from interconnected mammary gland alveoli with dilated lumen and sloughed epithelial cells to undifferentiated mammary gland alveoli with unexpanded lumen. Comparable levels of whey acidic protein (WAP, a major component in rodent milk) staining and Nile Red lipid droplet staining between WT and Bscl2-/- LD1 alveolar epithelial cells indicated normal milk protein synthesis and lipid syntheses in LD1 Bscl2-/- mammary glands. Significantly reduced percentage of larger lipid droplets was detected in LD1 Bscl2-/- alveoli with unexpanded lumen. There was no obviously impaired proliferation detected by PCNA staining but increased apoptosis detected by cleaved caspase-3 staining in LD1 Bscl2-/- alveolar epithelial cells. Increased expression of protein disulfide isomerase and binding immunoglobulin protein in the LD1 Bscl2-/- mammary gland alveolar epithelial cells indicated increased ER stress. This study demonstrates increased ER stress and apoptosis in LD1 Bscl2-/- mammary gland alveolar epithelial cells and reveals a novel in vivo function of seipin in lactation.

Seipin deficiency leads to defective parturition in mice.

Seipin is an integral endoplasmic reticulum membrane protein encoded by Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/Bscl2) gene. Seipin deficiency results in lipodystrophy, diabetes, muscle hypertrophy, and male infertility in both human and mouse. Seipin function in female reproduction is unknown. Bscl2-/- dams had normal embryo implantation and body weight gain during pregnancy but reduced delivery rates from 2nd to 4th pregnancies and reduced numbers of pups delivered from 1st to 4th pregnancies. Characterization of first pregnancy revealed increased gestation period and parturition problems, including uterine prolapse, difficulty in delivery, undelivered fetuses, and undelivered tissues in Bscl2-/- females. Bscl2-/- uterine weight was comparable to control at 3 weeks old but significantly increased with myometrial hypertrophy at 10 months old. In situ hybridization revealed relatively low level of Bscl2 mRNA expression in myometrium throughout pregnancy and postpartum but high level of expression in uterine luminal epithelium, suggesting that systemic effect (e.g. elevated glucose and insulin levels) rather than local seipin-deficiency in myometrium might be a main contributing factor to myometrial hypertrophy. On near-term gestation day 18.5 (D18.5), Bscl2-/- females had normal levels of serum progesterone and 17ß-estradiol, indicating functional ovary and placenta. Proliferating Cell Nuclear Antigen (PCNA) staining showed minimal myometrial cell proliferation in both D18.5 Bscl2+/+ and Bscl2-/- uteri. There was strong LC3 immunostaining in Bscl2+/+ and Bscl2-/- peripartum myometrium and increased LC3 staining in Bscl2-/- peripartum uterine luminal epithelium, suggesting a potential role of seipin in regulating autophagy in uterine luminal epithelium but not myometrium. This study demonstrates an association of seipin with myometrium and parturition.

Deletion of RhoA in Progesterone Receptor-Expressing Cells Leads to Luteal Insufficiency and Infertility in Female Mice.

Ras homolog gene family, member A (RhoA) is widely expressed throughout the female reproductive system. To assess its role in progesterone receptor-expressing cells, we generated RhoA conditional knockout mice RhoAd/d (RhoAf/f-Pgr-Cre+/-). RhoAd/d female mice had comparable mating activity, serum luteinizing hormone, prolactin, and estradiol levels and ovulation with control but were infertile with progesterone insufficiency, indicating impaired steroidogenesis in RhoAd/d corpus luteum (CL). RhoA was highly expressed in wild-type luteal cells and conditionally deleted in RhoAd/d CL. Gestation day 3.5 (D3.5) RhoAd/d ovaries had reduced numbers of CL, less defined corpus luteal cord formation, and disorganized CL collagen IV staining. RhoAd/d CL had lipid droplet and free cholesterol accumulation, indicating the availability of cholesterol for steroidogenesis, but disorganized ß-actin and vimentin staining, indicating disrupted cytoskeleton integrity. Cytoskeleton is important for cytoplasmic cholesterol movement to mitochondria and for regulating mitochondria. Dramatically reduced expression of mitochondrial markers heat shock protein 60 (HSP60), voltage-dependent anion channel, and StAR was detected in RhoAd/d CL. StAR carries out the rate-limiting step of steroidogenesis. StAR messenger RNA expression was reduced in RU486-treated D3.5 wild-type CL and tended to be induced in progesterone-treated D3.5 RhoAd/d CL, with parallel changes of HSP60 expression. These data demonstrated the in vivo function of RhoA in CL luteal cell cytoskeleton integrity, cholesterol transport, StAR expression, and progesterone synthesis, and a positive feedback on StAR expression in CL by progesterone signaling. These findings provide insights into mechanisms of progesterone insufficiency.

Acidification of uterine epithelium during embryo implantation in mice.

Uterine luminal epithelium (LE) is essential for establishing uterine receptivity. Previous microarray analysis revealed upregulation of Atp6v0d2 in gestation day 4.5 (D4.5) LE in mice. Realtime PCR showed upregulation of uterine Atp6v0d2 starting right before embryo attachment ∼D4.0. In situ hybridization demonstrated specific uterine localization of Atp6v0d2 in LE upon embryo implantation. Atp6v0d2 encodes one subunit for vacuolar-type H+-ATPase (V-ATPase), which regulates acidity of intracellular organelles and extracellular environment. LysoSensor Green DND-189 detected acidic signals in LE and glandular epithelium upon embryo implantation, correlating with Atp6v0d2 upregulation in early pregnant uterus. Atp6v0d2-/- females had significantly reduced implantation rate and marginally reduced delivery rate from first mating only, but comparable number of implantation sites and litter size compared to control and comparable fertility to control from subsequent matings, suggesting a nonessential role of Atp6v0d2 subunit in embryo implantation. Successful implantation in both control and Atp6v0d2-/- females was associated with uterine epithelial acidification. No significant compensatory upregulation of Atp6v0d1 mRNA was detected in D4.5 Atp6v0d2-/- uteri. To determine the role of V-ATPase instead of a single subunit in embryo implantation, a specific V-ATPase inhibitor bafilomycin A1 (2.5 µg/kg) was injected via uterine fat pad on D3 18:00 h. This treatment resulted in reduced uterine epithelial acidification, delayed implantation, and reduced number of implantation sites. It also suppressed oil-induced artificial decidualization. These data demonstrate uterine epithelial acidification as a novel phenomenon during embryo implantation and V-ATPase is involved in uterine epithelial acidification and uterine preparation for embryo implantation.

Novel function of LHFPL2 in female and male distal reproductive tract development.

Congenital reproductive tract anomalies could impair fertility. Female and male reproductive tracts are developed from Müllerian ducts and Wolffian ducts, respectively, involving initiation, elongation and differentiation. Genetic basis solely for distal reproductive tract development is largely unknown. Lhfpl2 (lipoma HMGIC fusion partner-like 2) encodes a tetra-transmembrane protein with unknown functions. It is expressed in follicle cells of ovary and epithelial cells of reproductive tracts. A spontaneous point mutation of Lhfpl2 (LHFPL2(G102E)) leads to infertility in 100% female mice, which have normal ovarian development, ovulation, uterine development, and uterine response to exogenous estrogen stimulation, but abnormal upper longitudinal vaginal septum and lower vaginal agenesis. Infertility is also observed in ~70% mutant males, which have normal mating behavior and sperm counts, but abnormal distal vas deferens convolution resulting in complete and incomplete blockage of reproductive tract in infertile and fertile males, respectively. On embryonic day 15.5, mutant Müllerian ducts and Wolffian ducts have elongated but their duct tips are enlarged and fail to merge with the urogenital sinus. These findings provide a novel function of LHFPL2 and a novel genetic basis for distal reproductive tract development; they also emphasize the importance of an additional merging phase for proper reproductive tract development.

Deletion of Lysophosphatidic Acid Receptor 3 (Lpar3) Disrupts Fine Local Balance of Progesterone and Estrogen Signaling in Mouse Uterus During Implantation.

Lpar3 encodes LPA3, the third G protein-coupled receptor for lysophosphatidic acid (LPA). Lpar3(-/-) female mice had delayed embryo implantation. Their serum progesterone and estrogen levels were comparable with control on Gestation Day 3.5 (D3.5) at 1100 h. There was reduced cell proliferation in D3.5 and D4.5 Lpar3(-/-) stroma. Progesterone receptor (PGR) disappeared from D4.5 Lpar3(+/+) uterine luminal epithelium (LE) but remained highly expressed in D4.5 Lpar3(-/-) LE. Pgr and PGR- target genes but not estrogen receptor alpha (ERalpha [Esr1]) or ESR target genes, were upregulated in D4.5 Lpar3(-/-) LE. It was hypothesized that suppression of PGR activity in LE could restore on-time uterine receptivity in Lpar3(-/-) mice. A low dose of RU486 (5 µg/mouse) given on D3.5 at 900 h rescued delayed implantation in all pregnant Lpar3(-/-) females and significantly increased number of implantation sites compared to vehicle-treated pregnant Lpar3(-/-) females detected on D4.5. E2 (25 ng/mouse) had a similar effect as 5 µg RU486 on embryo implantation in Lpar3(-/-) females. However, when the ovaries were removed on late D2.5 to create an experimentally induced delayed implantation model, 25 ng E2 activated implantation in Lpar3(+/+) but not Lpar3(-/-) females detected on D4.5. These results demonstrate that deletion of Lpar3 leads to an increased ratio of progesterone signaling/estrogen signaling that can be optimized by low doses of RU486 or E2 to restore on-time implantation in Lpar3(-/-) females.