Unveiling the role of protein kinase A (PKA) activity in bovine oviductal epithelial cells: implications on apoptotic signaling pathways during the estrous cycle.

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70 kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.

Sperm hyperactivation in the uterus and oviduct: a double-edged sword for sperm and maternal innate immunity toward fertility.

In cattle, artificial insemination (AI) is a technique that allows breeding by depositing frozen-thawed and extended semen into the female reproductive tract. The semen contains sperm with various motility patterns including dead, progressive and hyperactivated. Sperm hyperactivation is high amplitude, asymmetrical beating of sperm tail which usually occurs in the oviduct as part of the capacitation process, but it can also be induced by cryopreservation. After insemination, sperm enter the uterine glands and trigger a pro-inflammatory response in the uterus. Hyperactivated sperm, stimulated by sperm-Toll-like receptor 2 (TLR2), penetrates the mucus and uterine glands more efficiently and enhances the immune response. This facilitates the clearance of excess and dead sperm from the uterus. Some sperm escape the immune response and reach the oviduct either before or after the immune response is initiated. In the oviduct, sperm bind to the epithelium and form a reservoir. This triggers an anti-inflammatory response and preserves the fertilization potential of sperm. Hyperactivation facilitates sperm detaching from the epithelium, swimming through the viscous mucus and cumulus cells, and penetrating the egg's zona pellucida. Sperm-TLR2 activation enhances Ca2+-influx and acrosome reaction, which enables sperm to penetrate and fertilize oocytes during in vitro fertilization. Altogether, post-AI in cattle, sperm and maternal immunity interact differentially depending upon the site of sperm hyperactivation - whether it occurs within the uterus or oviduct. Specifically, hyperactivated sperm that enter the uterus after AI or are triggered via sperm-TLR2 activation or other stimuli contribute to sperm-induced uterine inflammation. Such hyperactivated sperm may impede their capacity to ascend to the oviduct. Conversely, sperm that become hyperactivated within the oviduct modulate their interactions with the oviduct and oocytes, which is pivotal during fertilization process. Indeed, the location and timing of sperm hyperactivation partially via TLR2 activation are critical determinants of their different influence on fertility.

Preparation of sanguinarine/glabridin loaded antifungal double-layer nanoemulsion edible coating using arabic gum/WPI for forest frog's oviduct oil preservation.

Forest frog's oviduct oil (FFOO) is highly susceptible to microbial spoilage during storage, which causes serious safety concerns and economic losses. However, little information is available regarding the preservation of it up to now. The aim of this research is to understand the dominant microbial community of FFOO spoilage, and based on this, develop a kind of edible nanoemulsion coating for preserving FFOO. Microbial metagenomic analysis indicated that the Aspergillus genus increased significantly during storage. In the present study, gum arabic and whey protein isolate were chosen as the coating matrix, the natural compounds sanguinarine and glabridin were selected as antimicrobial agents to prepare double-layer nanoemulsion edible coating. When the ratio of sanguinarine and glabridin in the nanoemulsion was 1:3, it exhibited strongest storage stability and antifungal activity. The mycelial inhibition rate of 1:3 nanoemulsion against dominant microbial community (Aspergillus niger and Aspergillus glaucus) reached 88.89 ± 1.37 % and 89.68 ± 1.37 %, respectively. The experimental results indicated that the edible nanoemulsion coating not only had outstanding antifungal activity, but also had excellent fresh-keeping effect on FFOO. This nanoemulsion coating could be a promising and potential candidate for food preservation.

Common lizard primary oviduct cell culture: A model system for the genetic and cellular basis of oviparity and viviparity.

Reproduction by egg-laying (oviparity) or live-bearing (viviparity) is a genetically determined trait fundamental to the biology of amniotes. Squamates are an emerging model for the genetics of reproductive mode yet lack cell culture models valuable for exploring molecular mechanisms. Here, we report a novel primary culture model for reproductive biology: cell cultures derived from the oviduct tissues (infundibulum, uterus and vagina) of oviparous and viviparous common lizards (Lacertidae: Zootoca vivipara). We maintained and expanded these cultures for over 100 days, including repeated subculturing and successful revival of cryopreserved cells. Immunocytochemical investigation suggested expression of both epithelial and fibroblast-like proteins, and RNA sequencing of cultured cells as compared to in vivo oviduct tissue showed changes in gene expression in response to the cell culture environment. Despite this, we confirmed the maintenance of distinct gene expression patterns in viviparous and oviparous cells after 60+ days of cell culture, finding 354 differentially expressed genes between viviparous and oviparous cells. Furthermore, we confirmed the expression of 15 viviparity-associated candidate genes in cells maintained for 60+ days in culture. Our study demonstrates the feasibility and utility of oviduct cell culture for molecular analysis of reproductive mode and provides a tool for future genetic experiments.

Seasonal changes in vitamin A metabolism-related factors in the oviduct of Chinese brown frog (Rana dybowskii).

The oviduct of the Chinese brown frog (Rana dybowskii) expands during pre-brumation rather than the breeding period, exhibiting a special physiological feature. Vitamin A is essential for the proper growth and development of many organisms, including the reproductive system such as ovary and oviduct. Vitamin A is metabolized into retinoic acid, which is crucial for oviduct formation. This study examined the relationship between oviducal expansion and vitamin A metabolism. We observed a significant increase in the weight and diameter of the oviduct in Rana dybowskii during pre-brumation. Vitamin A and its active metabolite, retinoic acid, notably increased during pre-brumation. The mRNA levels of retinol binding protein 4 (rbp4) and its receptor stra6 gene, involved in vitamin A transport, were elevated during pre-brumation compared to the breeding period. In the vitamin A metabolic pathway, the mRNA expression level of retinoic acid synthase aldh1a2 decreased significantly during pre-brumation, while the mRNA levels of retinoic acid α receptor (rarα) and the retinoic acid catabolic enzyme cyp26a1 increased significantly during pre-brumation, but not during the breeding period. Immunohistochemical results showed that Rbp4, Stra6, Aldh1a2, Rarα, and Cyp26a1 were expressed in ampulla region of the oviduct. Western blot results indicated that Aldh1a2 expression was lower, while Rbp4, Stra6, RARα, and Cyp26a1 were higher during pre-brumation compared to the breeding period. Transcriptome analyses further identified differential genes in the oviduct and found enrichment of differential genes in the vitamin A metabolism pathway, providing evidences for our study. These results suggest that the vitamin A metabolic pathway is more active during pre-brumation compared to the breeding period, and retinoic acid may regulate pre-brumation oviductal expansion through Rarα-mediated autocrine/paracrine modulation.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken oviduct tract.

Utilizing publicly available RNA-seq data to screen for ideal reference genes is more efficient and accurate than traditional methods. Previous studies have identified optimal reference genes in various chicken tissues, but none have specifically focused on the oviduct (including the infundibulum, magnum, isthmus, uterus, and vagina), which is crucial for egg production. Identifying stable reference genes in the oviduct is essential for improving research on gene expression levels. This study investigated genes with consistent expression patterns in the chicken oviduct, encompassing both individual oviduct tract tissues and the entire oviduct, by utilizing multiple RNA-seq datasets. The screening results revealed the discovery of 100 novel reference genes in each segment of oviduct tissues, primarily associated with cell cycle regulation and RNA binding. Moreover, the majority of housekeeping genes (HKGs) showed inconsistent expression levels across distinct samples, suggesting their lack of stability under varying conditions. The stability of the newly identified reference genes was assessed in comparison to previously validated stable reference genes in chicken oviduct and commonly utilized HKGs, employing traditional reference gene screening methods. HERPUD2, CSDE1, VPS35, PBRM1, LSM14A, and YWHAB were identified to be suitable novel reference gene for different parts of the oviduct. HERPUD2 and YWHAB were reliable for gene expression normalization throughout the oviduct tract. Furthermore, overexpression and interference assays in DF1 cells showed LSM14A and YWHAB play a crucial role in cell proliferation, highlighting the importance of these newly reference genes for further research. Overall, this study has expanded the options for reference genes in RT-qPCR experiments in different segments of the chicken oviduct and the entire oviduct.

Generation of Oviductal Glycoprotein 1 Cre Mouse Model for the Study of Secretory Epithelial Cells of the Oviduct.

The epithelial cell lining of the oviduct plays an important role in oocyte pickup, sperm migration, preimplantation embryo development, and embryo transport. The oviduct epithelial cell layer comprises ciliated and nonciliated secretory cells. The ciliary function has been shown to support gamete and embryo movement in the oviduct, yet secretory cell function has not been well characterized. Therefore, our goal was to generate a secretory cell-specific Cre recombinase mouse model to study the role of the oviductal secretory cells. A knock-in mouse model, Ovgp1Cre:eGFP, was created by expressing Cre from the endogenous Ovgp1 (oviductal glycoprotein 1) locus, with enhanced green fluorescent protein (eGFP) as a reporter. EGFP signals were strongly detected in the secretory epithelial cells of the oviducts at estrus in adult Ovgp1Cre:eGFP mice. Signals were also detected in the ovarian stroma, uterine stroma, vaginal epithelial cells, epididymal epithelial cells, and elongated spermatids. To validate recombinase activity, progesterone receptor (PGR) expression was ablated using the Ovgp1Cre:eGFP; Pgrf/f mouse model. Surprisingly, the deletion was restricted to the epithelial cells of the uterotubal junction (UTJ) region of Ovgp1Cre:eGFP; Pgrf/f oviducts. Deletion of Pgr in the epithelial cells of the UTJ region had no effect on female fecundity. In summary, we found that eGFP signals were likely specific to secretory epithelial cells in all regions of the oviduct. However, due to a potential target-specific Cre activity, validation of appropriate recombination and expression of the gene(s) of interest is absolutely required to confirm efficient deletion when generating conditional knockout mice using the Ovgp1Cre:eGFP line.

Generation of a Wt1 conditional deletion, nuclear red fluorescent protein reporter allele in the mouse.

A Wt1 conditional deletion, nuclear red fluorescent protein (RFP) reporter allele was generated in the mouse by gene targeting in embryonic stem cells. Upon Cre-mediated recombination, a deletion allele is generated that expresses RFP in a Wt1-specific pattern. RFP expression was detected in embryonic and adult tissues known to express Wt1, including the kidney, mesonephros, and testis. In addition, RFP expression and WT1 co-localization was detected in the adult uterine stroma and myometrium, suggesting a role in uterine function. Crosses with Wnt7a-Cre transgenic mice that express Cre in the Müllerian duct epithelium activate Wt1-directed RFP expression in the epithelium of the oviduct but not the stroma and myometrium of the uterus. This new mouse strain should be a useful resource for studies of Wt1 function and marking Wt1-expressing cells.

Multi-omics analyses and machine learning prediction of oviductal responses in the presence of gametes and embryos.

The oviduct is the site of fertilization and preimplantation embryo development in mammals. Evidence suggests that gametes alter oviductal gene expression. To delineate the adaptive interactions between the oviduct and gamete/embryo, we performed a multi-omics characterization of oviductal tissues utilizing bulk RNA-sequencing (RNA-seq), single-cell RNA-sequencing (scRNA-seq), and proteomics collected from distal and proximal at various stages after mating in mice. We observed robust region-specific transcriptional signatures. Specifically, the presence of sperm induces genes involved in pro-inflammatory responses in the proximal region at 0.5 days post-coitus (dpc). Genes involved in inflammatory responses were produced specifically by secretory epithelial cells in the oviduct. At 1.5 and 2.5 dpc, genes involved in pyruvate and glycolysis were enriched in the proximal region, potentially providing metabolic support for developing embryos. Abundant proteins in the oviductal fluid were differentially observed between naturally fertilized and superovulated samples. RNA-seq data were used to identify transcription factors predicted to influence protein abundance in the proteomic data via a novel machine learning model based on transformers of integrating transcriptomics and proteomics data. The transformers identified influential transcription factors and correlated predictive protein expressions in alignment with the in vivo-derived data. In conclusion, our multi-omics characterization and subsequent in vivo confirmation of proteins/RNAs indicate that the oviduct is adaptive and responsive to the presence of sperm and embryos in a spatiotemporal manner.

Oral exposure to bisphenol S is associated with alterations in the oviduct proteome of an ovine model, with aggravated effects in overfed females.

BACKGROUND: Bisphenol S (BPS) is a substitute for bisphenol A in plastic manufacturing and, as a potential endocrine disruptor, may alter the physiology of the oviduct, in which fertilization and early embryo development take place in mammals. The objective of this study was to assess the effect of a daily dietary exposure to BPS combined with a contrasted diet on the oviduct fluid proteome using an ovine model. RESULTS: Eighty adult cyclic ewes were allotted to four groups (20/group): overfed (OF) consuming 50 µg/kg/day of BPS in their diet, underfed (UF) consuming 50 µg/kg/day of BPS, and non-exposed controls in each diet group. After three months, the mean body condition score, plasma levels of glucose and non-esterified fatty acids were significantly higher in OF than in UF females. The proteins in collected OF samples (50 µg) were analyzed by nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). Overall, 1563 proteins were identified, among which 848 were quantified. Principal component analysis of the data revealed a clear discrimination of samples according to the diet and a segregation between BPS-exposed and non-exposed females in overfed ewes. Hierarchical clustering of differentially abundant proteins (DAPs) identified two clusters of 101 and 78 DAPs according to the diet. Pairwise comparisons between groups revealed a stronger effect of BPS in OF than in UF females (70 vs. 24 DAPs) and a stronger effect of the diet in BPS-exposed than non-exposed females (56 vs. 36 DAPs). Functional analysis of DAPs showed an enrichment in metabolic processes, immune system, cell response to stress, and reproductive processes. CONCLUSIONS: This work highlights for the first time the important impact of BPS on the oviduct proteome, with larger effects seen in OF than UF females. These results, together with previous ones, raise health concerns for everyone and call for a greater regulation of BPS in the food industry.

Induction of in vivo-like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells.

We recently developed re-differentiated equine oviduct epithelial cell (REOEC) monolayers demonstrating various in vivo morphological characteristics, but lacking secondary ciliation. In this study, we evaluated the effects of fetal bovine serum, reproductive steroid hormones, Wnt- and Notch ligands and inhibitors, and different EOEC seeding densities, in both conventional wells and on microporous membranes, on EOEC morphology and, in particular, secondary ciliation. REOEC monolayers were assessed by confocal microscopy after combined staining of nuclei, cilia and the cytoskeleton. Only Wnt ligands, Notch inhibitors and oviduct explant cell concentration affected EOEC morphology. Undesirable epithelial-mesenchymal transition was observed in REOEC monolayers exposed to Wnt3a containing medium and Wnt ligand CHIR 99021. With respect to secondary ciliation, only the combined effect of oviduct explant cell concentration and Notch inhibition steered REOEC monolayers to in vivo-like ciliation patterns. De-differentiated EOECs, formed 10 days after oviduct explant cell seeding, were reseeded on inserts; only at initial oviduct explant cell concentrations of 1 and 5 x106 cells per well was the formation of REOEC monolayers with a high rate of diffuse ciliation supported. Within 1 month after air-liquid interface introduction, >40% and > 20% of the REOECs showed secondary cilia, respectively. At higher oviduct explant cell seeding densities secondary ciliation was not supported after re-differentiation. Additionally, Notch inhibition helped boost secondary ciliation rates to >60% in REOEC monolayers with diffuse ciliation only. These monolayers demonstrated higher clathrin expression under follicular phase conditions. Overall, the ciliated REOEC monolayers better resemble in vivo oviduct epithelial cells than previous models.

Effects of herbal dregs supplementation of Salvia miltiorrhiza and Isatidis Radix residues improved production performance and gut microbiota abundance in late-phase laying hens.

The present study was designed to evaluate the effect of a mixture of Chinese medicinal residues (CMRs) consisting of Salvia miltiorrhiza residues (SMR) and Isatidis Radix residues (IRR) on productive performance, egg quality, serum lipid and hormone levels, liver and blood antioxidant capacity, oviduct inflammation levels, and gut microbiota in the late-laying stage. A total of 288 fifty-four-week-old BaShang long-tailed hens were divided into four groups. The feed trial period was 8 weeks. The control group was fed the basic diet as a CCMR group, supplemented with 3, 4, and 6% for the experimental groups LCMR, MCMR, and HCMR. The egg production rate of the MCMR group was 8.1% higher than that of the CCMR group (p < 0.05). Serum triglyceride (TG) levels of hens of the CMR-supplemented group were significantly decreased than those of the CCMR group (p < 0.05). The group supplemented with different levels of CMR had significantly higher serum HDL-C levels compared with the control group (p < 0.05). Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were remarkably increased for the LCMR and MCMR groups and significantly decreased for the HCMR group compared to CCMR (p < 0.05). Serum and liver glutathione peroxidase (GSH-PX) activities were significantly increased, and malondialdehyde (MDA) levels were significantly decreased in the MCMR group compared to the CCMR group (p < 0.05). The expression levels of tubal inflammatory factor markers (IL-4, IL-1ß, TNF-α) in the MCMR and HCMR groups were consistent with the pathological findings of the sections. As for cecal microbiota, supplementation with CMR affected the alpha diversity of the cecum microbiome at the genus level. The Shannon index was significantly higher in the MCMR group than in the CCMR and HCMR groups (p < 0.05). Supplementation with different levels of CMR mainly regulated the ratio of intestinal Firmicutes to Bacteroidetes and the abundance of phyla such as Proteobacteria. In addition, CMR supplementation at different levels in the diet enriched lipid-metabolizing bacteria, such as Bacteroides and Ruminococcus_gnavus_group. Furthermore, according to linear discriminant analysis (LDA) effect size (LEfSe) analysis, the MCMR group showed an increase in the number of short-chain fatty acid-producing bacteria Romboutsia and fiber-degrading specialized bacteria Monoglobus. Therefore, supplementation of appropriate amounts of CMR to the diet of laying hens enhanced reproductive hormone levels, hepatic antioxidant capacity, and lipid metabolism, alleviated the levels of oviductal inflammatory factors, and modulated the abundance structure of bacterial flora to improve the late-laying performance and egg quality. The results of the current study showed that CMR is a beneficial feed supplement for chickens when added in moderation.

Sustainable production of multimeric and functional recombinant human adiponectin using genome-edited chickens.

BACKGROUND: Adiponectin (ADPN) plays a critical role in endocrine and cardiovascular functions, but traditional production methods, such as Escherichia coli and mammalian systems, have faced challenges in generating sufficiently active middle molecular weight (MMW) and high molecular weight (HMW) forms of recombinant human ADPN (hADPN). In our previous study, we proposed genome-edited chickens as an efficient platform for producing multimeric hADPN. However, the consistency of multimeric hADPN expression in this system across generations had not been further investigated. RESULTS: In this study, subsequent generations of ovalbumin (OVA) ADPN knock-in chickens showed stable multimeric hADPN production, yielding ~ 26% HMW ADPN (0.59 mg/mL) per hen. Comparative analysis revealed that egg white (EW)-derived hADPN predominantly consisted of hexameric and HMW forms, similar to serum-derived hADPN. In contrast, hADPN obtained from human embryonic kidney (HEK) 293 and High-Five (Hi-5) cells also exhibited the presence of trimers, indicating variability across different production systems. Furthermore, transcriptional expression analysis of ADPN multimerization-associated endoplasmic reticulum chaperone genes (Ero1-Lα, DsbA-L, ERP44, and PDI) indicated upregulation in the oviduct magnum of ADPN KI hens, suggesting the chicken oviduct magnum as the optimal site for HMW ADPN production. Lastly, the functional analysis demonstrated that EW-derived hADPN significantly reduced lipid droplets and downregulated lipid accumulation-related genes (LOX-1, AT1R, FAS, and FABP4) in human umbilical vein endothelial cells (HUVECs). CONCLUSION: In summary, stable and functional multimeric hADPN can be produced in genome-edited chickens even after generations. This highlights the potential of using chicken bioreactor for producing various high-value proteins.

Paraspeckles / CARM1 mediates the regulation of OEVs on cell differentiation during in vitro embryonic development of yak.

Mammalian embryos produced in vitro have poor embryo quality and low developmental ability compared with in vivo embryos. The main manifestations are the low number of blastocysts, the low ratio of the number of inner cell mass cells to the number of trophoblastic cells, and the high apoptosis rate of blastocysts, resulting in low embryo implantation rate. Therefore, optimizing in vitro culture conditions has become a key technology to im-prove the quality of preimplantation embryos. Oviduct Epithelial cells exosomes (OEVs) can be absorbed and internalized by embryos to improve the blastocyst rate and blastocyst quality of embryos in vitro. As a special nuclear structure, Paraspeckles are involved in the fate determination of mammalian early embryonic mammalian cells. However, the regulation of embryonic cell differentiation by OEVs remains unknown. We aimed to investigate the effects of OEVs on paraspeckle formation and cell fate determination in yak in vitro fertilization (IVF) of em-bryos. To simulate the in vivo oviduct environment after ovulation, we used follicular fluid exosomes (FEVs) to stimulate yak oviduct epithelial cells and collect OEVs. OEVs were added to the yak IVF embryo culture system. Paraspeckle formation, cell differentiation, and blastocyst quality in yak embryos were determined. Our results show that, development of yak embryos is unique compared to other bovine species, and OEVs can be used as a supplement to the in vitro culture system of yak embryos to improve embryonic development and blas-tocyst quality. And also Paraspeckles/CARM1 mediated the regulation of OEVs on cell differentiation during in vitro yak embryo production. These results provide new insights into the study of yak embryonic development and the role of OEVs in embryonic development.

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Oviductal extracellular vesicles miRNA cargo varies in response to embryos and their quality.

BACKGROUND: Increasing evidence points to an active role of oviductal extracellular vesicles (oEVs) in the early embryo-maternal dialogue. However, it remains unclear whether oEVs contribute to the recognition of the presence of embryos and their quality in the oviduct. Hence, we examined whether the molecular cargo of oEVs secreted by bovine oviduct epithelial cells (BOEC) differs depending on the presence of good (≥ 8 cells, G) or poor (< 8 cells, P) quality embryos. In addition, differences in RNA profiles between G and P embryos were analyzed in attempt to distinguish oEVs and embryonic EVs cargos. METHODS: For this purpose, primary BOEC were co-cultured with in vitro produced embryos (IVP) 53 h post fertilization as follows: BOEC with G embryos (BGE); BOEC with P embryos (BPE); G embryos alone (GE); P embryos alone (PE); BOEC alone (B) and medium control (M). After 24 h of co-culture, conditioned media were collected from all groups and EVs were isolated and characterized. MicroRNA profiling of EVs and embryos was performed by small RNA-sequencing. RESULTS: In EVs, 84 miRNAs were identified, with 8 differentially abundant (DA) miRNAs for BGE vs. B and 4 for BPE vs. B (P-value < 0.01). In embryos, 187 miRNAs were identified, with 12 DA miRNAs for BGE vs. BPE, 3 for G vs. P, 8 for BGE vs. GE, and 11 for BPE vs. PE (P-value < 0.01). CONCLUSIONS: These results indicated that oEVs are involved in the oviductal-embryo recognition and pointed to specific miRNAs with signaling and supporting roles during early embryo development.

Cell Heterogeneity Analysis Revealed the Key Role of Fibroblasts in the Magnum Regression of Ducks.

Duck egg production, like that of laying hens, follows a typical low-peak-low cycle, reflecting the dynamics of the reproductive system. Post-peak, some ducks undergo a cessation of egg laying, indicative of a regression process in the oviduct. Notably, the magnum, being the longest segment of the oviduct, plays a crucial role in protein secretion. Despite its significance, few studies have investigated the molecular mechanisms underlying oviduct regression in ducks that have ceased laying eggs. In this study, we conducted single-cell transcriptome sequencing on the magnum tissue of Shaoxing ducks at 467 days of age, utilizing the 10× Genomics platform. This approach allowed us to generate a detailed magnum transcriptome map of both egg-laying and ceased-laying ducks. We collected transcriptome data from 13,708 individual cells, which were then subjected to computational analysis, resulting in the identification of 27 distinct cell clusters. Marker genes were subsequently employed to categorize these clusters into specific cell types. Our analysis revealed notable heterogeneity in magnum cells between the egg-laying and ceased-laying ducks, primarily characterized by variations in cells involved in protein secretion and extracellular matrix (ECM)-producing fibroblasts. Specifically, cells engaged in protein secretion were predominantly observed in the egg-laying ducks, indicative of their role in functional albumen deposition within the magnum, a phenomenon not observed in the ceased-laying ducks. Moreover, the proportion of THY1+ cells within the ECM-producing fibroblasts was found to be significantly higher in the egg-laying ducks (59%) compared to the ceased-laying ducks (24%). Similarly, TIMP4+ fibroblasts constituted a greater proportion of the ECM-producing fibroblasts in the egg-laying ducks (83%) compared to the ceased-laying ducks (58%). These findings suggest a potential correlation between the expression of THY1 and TIMP4 in ECM-producing fibroblasts and oviduct activity during functional reproduction. Our study provides valuable single-cell insights that warrant further investigation into the biological implications of fibroblast subsets in the degeneration of the reproductive tract. Moreover, these insights hold promise for enhancing the production efficiency of laying ducks.

Expression and localization of apelin and apelin receptor protein in the oviduct of letrozole-induced hyperandrogenized mice.

Apelin and its receptor (APJ) are expressed in the reproductive organs of some mammalian females. The function of oviduct has also been suggested to be compromised in the hyperandrogenism condition. However, expression of apelin and APJ has not been shown in the oviduct of hyperandrogenized mice. Thus, the present study has investigated the localization and expression of apelin and APJ in the letrozole-induced hyperandrogenized mice oviduct. Histomorphometric analysis showed decreased lumen of oviduct in the hyperandrogenized mice. Our results showed elevated expression of APJ and decreased abundance of apelin in the hyperandrogenized mice oviduct. This finding suggests impaired apelin signaling in the oviduct of hyperandrogenized mice. The expression of androgen receptor was upregulated while estrogen receptors were downregulated in the hyperandrogenized mice. The expression of HSP70 was also downregulated along with increased expression of active caspase 3 and BAX and decreased expression of BCL2 in hyperandrogenized mice. Furthermore, the phosphorylation of phospho-Ser473-Akt and phospho-Thr308-Akt also showed differential levels in the oviduct of hyperandrogenized mice. Whether this differential phosphorylation of Akt was solely due to impaired apelin signaling in the oviduct, remains unclear. Moreover, increased androgen signaling and suppressed estrogen signaling coincides with elevated apoptosis. In conclusion, hyperandrogenized conditions could also impair the gamete transport and fertilization process due to apoptosis in the oviduct. However, further study would be required to unravel the exact role of apelin signaling in the oviduct in relation to apoptosis.

Single-cell transcriptomic profiling of the neonatal oviduct and uterus reveals new insights into upper Müllerian duct regionalization.

The upper Müllerian duct (MD) is patterned and specified into two morphologically and functionally distinct organs, the oviduct and uterus. It is known that this regionalization process is instructed by inductive signals from the adjacent mesenchyme. However, the interaction landscape between epithelium and mesenchyme during upper MD development remains largely unknown. Here, we performed single-cell transcriptomic profiling of mouse neonatal oviducts and uteri at the initiation of MD epithelial differentiation (postnatal day 3). We identified major cell types including epithelium, mesenchyme, pericytes, mesothelium, endothelium, and immune cells in both organs with established markers. Moreover, we uncovered region-specific epithelial and mesenchymal subpopulations and then deduced region-specific ligand-receptor pairs mediating mesenchymal-epithelial interactions along the craniocaudal axis. Unexpectedly, we discovered a mesenchymal subpopulation marked by neurofilaments with specific localizations at the mesometrial pole of both the neonatal oviduct and uterus. Lastly, we analyzed and revealed organ-specific signature genes of pericytes and mesothelial cells. Taken together, our study enriches our knowledge of upper MD development, and provides a manageable list of potential genes, pathways, and region-specific cell subtypes for future functional studies.