Abstract

   In pregnant animals, communication between the mother and conceptus
   occurs via extracellular vesicles (EVs) that carry several biomolecules
   such as nucleic acids (miRNAs, mRNAs), proteins, and lipids. At the
   time of implantation, the endometrium undergoes several morphological
   and physiological changes, such as angiogenesis, apoptosis, and cell
   proliferation regulation at the implantation site, to attain a
   receptive state. This study was conducted to detect pregnancy-specific
   miRNAs derived from extracellular vesicles in the systemic circulation
   of Bubalus bubalis (water buffalo) and to assess their functional
   significance in the modulation of endometrial primary cells. The
   extracellular vesicles were isolated from the blood plasma using a
   precipitation-based method and further characterized by various methods
   such as Differential light scattering, Nanoparticle tracking assay,
   Western blot, and transmission electron microscopy. The relative
   expression of the selected extracellular vesicles associated miRNAs
   (EV-miRNA) at different intervals (days 15, 19, 25, and 30) post
   artificial insemination (AI) was analyzed using RT-qPCR, and expression
   of miR-195-5p was found to be significantly higher (P < 0.01) in
   pregnant animals on day 19 post AI (implantation window) as compared to
   day 15 post AI. The elevated expression might indicate the involvement
   of this miRNA in the maternal-conceptus cross-talk occurring during the
   implantation period. The KEGG pathway enrichment and Gene Ontology
   analyses of the miR-195-5p target genes revealed that these were mostly
   involved in the PI3-Akt, MAPK, cell cycle, ubiquitin-mediated
   proteolysis, and mTOR signaling pathways, which are related to the
   regulation of cell proliferation. Transfecting the in vitro cultured
   cells with miR-195-5p mimic significantly suppressed (P < 0.05) the
   expression of its target genes such as YWHAQ, CDC27, AKT-3, FGF-7,
   MAPK8, SGK1, VEGFA, CACAND1, CUL2, MKNK1, and CACAN2D1. Furthermore,
   the downregulation of the miR-195-5p target genes was positively
   correlated with a significant increase in the apoptotic rate and a
   decrease in the proliferation. In conclusion, the current findings
   provide vital information on the presence of EV miR-195-5p in maternal
   circulation during the implantation window indicating its important
   role in the modulation of buffalo endometrium epithelial cells via
   promoting cell death. Altogether, the milieu of miR-195-5p may serve as
   a novel and potential molecular factor facilitating the implantation of
   the early embryo during the establishment of pregnancy in buffaloes.
   Thus, miR-195-5p may be identified as a unique circulatory EV biomarker
   related to establishing pregnancy in buffaloes as early as day 19
   post-AI.

   Subject terms: Biotechnology, Cell biology, Developmental biology,
   Molecular biology

Introduction

   During pregnancy establishment, maternal-conceptus crosstalk provides
   essential signals for maternal recognition of pregnancy^[38]1.
   Successful pregnancy establishment depends on the exact timing of the
   maternal-conceptus crosstalk and synchronized transcriptional
   regulation between them. The placental cells release extracellular
   vesicles (EVs) that can cross the placental barrier between the mother
   and the conceptus to regulate various biological functions in the
   target cells^[39]2. EVs can enter physiological fluids such as plasma,
   urine, amniotic fluid, seminal plasma, milk, saliva, and uterine
   luminal fluid and then transmit their content to the target
   cells^[40]3. EVs secreted by the cells reflect the physiological state
   and function of the originating cells, such as the endometrium and
   primary trophoblast from the placenta^[41]4–[42]6. EVs contain several
   biomolecules such as lipids, proteins, mRNA, and other small non-coding
   RNAs such as miRNAs^[43]7.

   The miRNAs are a class of small (~ 22 nucleotides long) non-coding RNAs
   that negatively regulate gene expression by binding to the
   3′—untranslated region of target mRNAs^[44]8–[45]11 It has been
   observed that the abundance of miRNAs present within the EV differs in
   early pregnant, non-pregnant, and pregnancy loss^[46]12. The placental
   secreted miRNAs contribute to the circulating miRNA profile of the
   maternal blood and, thus, can be used as biomarkers for pregnancy
   detection^[47]13. During early pregnancy, i.e., the first trimester,
   the number of exosomes increases in the maternal circulation^[48]14.
   The association of miRNAs and exosomes increases the stability of these
   miRNAs, thereby preventing their degradation from RNase^[49]15,[50]16.
   During pregnancy, the extracellular vesicles associated miRNA
   (EV-miRNA) profile follows specific trends in different trimesters, at
   term and pre-term birth, which indicates changes in maternal and
   conceptus tissue^[51]17.

   The interaction between the uterus and the embryo is crucial for
   successful pregnancy development in mammals. During the implantation
   window, the endometrium attains a receptive state and undergoes several
   morphological and physiological changes, such as angiogenesis,
   apoptosis, and cell proliferation regulation at the implantation
   site^[52]18–[53]20. Numerous studies have shown that silencing the
   essential miRNA processing enzymes causes developmental arrest or even
   embryonic death^[54]21,[55]22. Placenta-derived miRNAs have been
   reported to be released into the maternal circulation after being
   packed into the EVs, thus providing intercellular communication between
   the mother and the conceptus^[56]23. Several studies have reported that
   the placenta-derived EV-miRNAs regulate the bi-directional interaction
   between endometrium and embryo at the time of implantation^[57]24.
   Placenta-derived EV-miRNAs present in the maternal circulation have
   been reported to protect the conceptus from the maternal immune
   response^[58]25. The functional roles of EV-miRNAs concerning pregnancy
   establishment have not been much explored. Therefore, to decipher the
   roles of EV-miRNAs in pregnancy establishment in water buffalo (Bubalus
   bubalis), the current study aims (i) To analyze the expression of
   pregnancy-specific EV-miRNAs present in the bloodstream of pregnant and
   non-pregnant buffaloes. (ii) Elucidating the role of
   pregnancy-associated EV-miRNA miR-195-5p, in the cell growth rate,
   proliferation, and apoptosis in the buffalo-cultured endometrial
   primary cells.

Results

Isolation and characterization of the EVs from blood plasma

   Small EVs were isolated from the blood plasma of three randomly
   selected Murrah buffaloes using a precipitation-based method. These
   isolated EVs were then pooled and characterized further. The isolated
   EVs were characterized using Differential light scattering (DLS),
   Nanoparticle tracking assay (NTA), Transmission electron microscopy
   (TEM), and Western blot. The particle size obtained through the NTA and
   DLS was found to be mean size of 102 + /− 1.9 nm and Z-average size
   91.3 nm, respectively (Fig. [59]1A–C), which lies within the expected
   size range of the small EVs, i.e., 30–150 nm^[60]26. The nanoparticle
   concentration was 2.55 × 10^11 ± 7.09 × 10^9 particles/ml in pooled
   blood plasma samples. Most of the nanoparticle concentration was found
   within the range of 40nm-150 nm, representing the small EV population.
   A video was obtained capturing the Brownian motion of the particles
   (Supplementary Video [61]S1). General morphology and ultrastructure of
   blood plasma-derived EVs were assessed using transmission electron
   microscopy (TEM), allowing the visualization of round cup shape
   vesicles with lipid bilayer structures with diameters varying between
   30 and 100 nm (Fig. [62]1 D). The presence of small EVs was also
   confirmed by Western blot using the EV protein markers, namely, a
   cluster of differentiation 63 (CD63), Tumor susceptibility gene101
   (TSG101), a cluster of differentiation CD9, and EV negative marker
   Calnexin (Fig. [63]1E) (Supplementary Fig. [64]S3A–D).

Figure 1.

   [65]Figure 1
   [66]Open in a new tab

   Characterization of extracellular vesicles. (A) Intensity-based Size
   distribution of buffalo blood plasma-derived EVs, as assessed by the
   Zetasizer nano zs particle sizer. Each curve shows means ± SD from
   three replicates in a representative experiment out of the three
   performed with similar results. (B) Nanoparticle Tracking Analysis
   (NTA) on the buffalo blood plasma-derived small EVs under
   100× dilutions. FTLA size per concentration graph, taken as five
   replicates (C) graph represents the averaged FTLA size per
   concentration (particles/ml) (D) Transmission Electron Microscopy
   (TEM). TEM image of EVs derived from buffalo blood plasma. EVs were
   negatively stained with 1% phosphotungstic acid after removing the
   extra moisture. (Magnification-250000×, Scale bar—50 nm, 120 kV) (E)
   Identification of the CD9, CD63 and TSG101 EV-specific protein markers
   by the western blot analysis of isolated pooled EVs samples from the
   blood plasma of Murrah buffalo. Full blots are shown in Supplementary
   Fig. [67]S3A–C.

Expression profile of the selected circulatory EV—miRNAs in pregnant and
non-pregnant buffaloes post different days of insemination

   The relative expression profiles of the selected panel of EV- miRNAs
   were generated using RT-qPCR at four different intervals post AI,
   namely, on days day 15, 19, 25, and 30, in three pregnant and three
   non-pregnant Murrah buffaloes. A dynamic expression of the candidate
   miRNAs on different days post-insemination was observed. The miR-195-5p
   exhibited a significant increase in the expression level from day 15 to
   19 (P < 0.0001) post-AI, with a sharp decrease on day 25. Furthermore,
   the expression of miR-195-5p was much higher in the pregnant animals
   compared to the non-pregnant animals on day 19. The expression of
   EV-associated miR-200a-3p and miR-27 was observed to be decreased on
   day 19 and day 25 compared to day 15 in pregnant animals. The
   expression of miR-1246 was decreased on day 19 but later increased on
   day 25 of pregnancy. The expression of miR-27 and miR-200a-3p increased
   on day 30 in pregnant animals (Fig. [68]2). In bovine species, day
   19–20 of pregnancy is referred to as the implantation window and
   trophoblast attachment begins during this phase. We wanted to explore
   the functional significance of increased expression of miRNA on day 19
   of pregnancy.

Figure 2.

   [69]Figure 2
   [70]Open in a new tab

   The pattern of expression of EV—miRNA in blood plasma. The Relative
   expression profiles of the EV-miRNA across the pregnant vs.
   non-pregnant buffalo on different days after insemination. Expression
   values were normalized to the mean of let-7 and miR-16a reference
   controls. Day 15 in pregnant and non-pregnant animals was chosen as the
   calibrator. Days post insemination is identified on the X-axis.
   (Pregnant animals (n = 3) and non-pregnant animals (n = 3) (*P < 0.05;
   **P < 0.01; and ****P < 0.0001).

In-silico target gene prediction of miR-195-5p

   The potential target genes for miR-195-5p were determined by in silico
   analysis using different miRNA target prediction tools, namely miRWalk,
   TargetScan, and miRmap. The clustering analysis results of all
   predicted miRNA target genes are presented in Fig. [71]3A,
   Supplementary Table [72]S3. It revealed a total of 196 target genes
   identified by a minimum of two databases after clustering through the
   Venn diagram ([73]https://bioinformatics.psb.ugent.be/webtools/Venn/).
   The number of targets predicted by the intersection of Target Scan (TS)
   miRmap (MM) and miRWalk was 12; the intersection of TS and MM revealed
   150 targets, the intersection of MM and MW revealed 42 targets, while
   the intersection of MW and TS revealed 28 targets.

Figure 3.

   [74]Figure 3
   [75]Open in a new tab

   (A) miRNA target prediction. Venn diagram of the number of miR-195-5p
   targets predicted by each tool (B) KEGG pathway enrichment analysis.
   Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of
   miR-195-5p (Top 10 highly gene enriched with significance (P < 0.05)
   target genes by Database for Annotation, Visualization and Integrated
   Discovery software. (Top 10). (C–E) Gene ontology. Gene ontology (GO)
   enrichment analysis for miR-195-5p targets in the category of
   biological processes. Gene ontology analysis of miRNA target genes
   according to biological process, cell component, and molecular
   function.

KEGG pathway enrichment and gene ontology analysis for miR-195-5p target
genes

   KEGG pathway enrichment for miR-195-5p target genes indicated that they
   are predominantly involved in the PI3K-Akt signaling pathway (FGF7,
   AKT3, FGF2 and VEGFA.; P = 0.031), Ubiquitin mediated proteolysis
   (CDC23, CUL2 and CDC27; P = 0.029), MAPK signaling pathway (FGF7,
   MAPK8, MKNK1 and AKT3; P = 0.019), Cell cycle (CDC23, YWHAQ, CHEK1 and
   CDC27; P  = 0.015) and mTOR signaling pathway (AKT3 and SGK1;
   P = 0.002) (Fig. [76]3B, Supplementary Table [77]S4). The GO analysis
   for miR-195-5p revealed that its target genes were enriched in
   biological processes (BPs) such as ubiquitin-dependent protein
   catabolic process (CUL2 and UBE4B; P = 0.006), cell division (CDC23 and
   CDC27; P = 0.011), mitotic cell cycle (WEE1 and MYB; P = 0.013)
   (Fig. [78]3C, Supplementary Table [79]S5). The molecular functions
   (MFs) of these genes were protein serine/threonine kinase activity
   (MAPK8, MKNK1, AKT3, CHEK1, and SGK1; p = 0.011), GTP binding (GLUD1
   and RRAGA; p = 0.018) and ubiquitin-protein ligase activity (WWP1 and
   BTRC; p = 0.009), (Fig. [80]3E, Supplementary Table [81]S5). The
   analysis of cellular component (CC) Gene Ontology terms revealed
   significant enrichments in specific cellular locations for the genes.
   Notably, FGF2, FGF7, and AKT3 were found to be significantly associated
   with cytosolic localization (P = 3.9e−04), whereas CHEK1, CDC27, and
   MKNK1 exhibited significant enrichment in the nucleus (P = 1.4e−03)
   (Fig. [82]3D, Supplementary Table [83]S5).

Isolation and molecular characterization of endometrial primary cells

   The cells isolated from the uterine horns contained most of endometrial
   epithelial cells (EECs) with some impurities of stromal cells.
   Nonetheless, seeding the isolated cell population for 12 h in a culture
   flask effectively separated the pure epithelial cells from the stromal
   cells. After isolation, EECs could be differentiated from stromal cells
   by their large and spherical appearance. The EECs remained in the
   suspension while the stromal cells got attached to the substratum
   (culture flask) after 12 h. Endometrial epithelial cells showed
   cuboidal or columnar morphologies^[84]27 and gradually became a
   squamous polygon, whereas stromal cells possessed a spindle shape. EECs
   had granular cytoplasm and big, centrally located nuclei (Supplementary
   Fig. [85]S2). In addition, immunocytochemistry (ICC) and PCR determined
   the purity of epithelial cells. Vimentin, cytokeratin18, and
   fibronectin are the characteristic markers for endometrial stromal
   cells, endometrial epithelial cells, and epithelium to mesenchymal
   transition cells, respectively, and these were used to screen the
   cultured cells. A strong signal for cytokeratin 18 and a faint signal
   for vimentin and fibronectin indicated that the cell population
   consisted predominantly of EECs (Fig. [86]4).

Figure 4.

   [87]Figure 4
   [88]Open in a new tab

   Primary endometrial cell identification. Isolated epithelial cells were
   stained with (A–C) epithelial cell marker cytokeratin18 and (D–F)
   stromal cell marker vimentin and (G–I) epithelial to mesenchymal
   transition marker Fibronectin to demonstrate that the majority of the
   cells are epithelial endometrial cells.

Expression profile of miR-195-5p target genes upon endometrial primary cells
transfection with miR-195-5p mimic

   To further investigate the role of miR-195-5p in early pregnancy
   establishment, the endometrial primary cells were transfected with the
   miR-195-5p mimic. The relative expression of the target genes between
   the cells transfected with miR-195-5p mimic and miR-NC (miRNA negative
   control) was determined upon transfection (n = 2). The relative
   expression profile was generated using qRT-PCR for the miR-195-5p
   target genes involved in the (i) cell cycle (YWHAQ, CDC27, CHEK1,
   CDC23, and SMAD2) (ii) PI3-Akt signaling (AKT-3, FGF-7, YWHAQ, MAPK8,
   SGK1, VEGFA, MKNK1, MAPK8, CACAND1, and MYB), (iii) Ubiquitin mediated
   proteolysis (CUL-2, CDC27, WWP1, BTRC, CDC23, and UBE4B) (iv) MAPK
   pathway (AKT-3, MKNK1, CACNA2D1, FGF-7, and MAPK8) and (v) mTOR
   signaling pathway (AKT-3 and SGK1). The relative expression of the
   miR-195-5p target genes such as YWHAQ, CDC27, AKT-3, FGF-7, MAPK8,
   SGK1, VEGFA, CACAND1, CUL2, MKNK1, and CACAN2D1 were found to be
   significantly (P < 0.05) suppressed. At the same time, no significant
   differences were observed in SMAD2, CHEK1, CDC23, MYB, WWP1, BTRC, and
   UBE4B. In addition to miR-195-5p target genes, the relative expression
   of CDK1, CDKN1A, CDKN1B, COP1, CREB1, BCL-2, CCNG2, RAF1, and MAPK1
   genes involved in the pathways as mentioned above were also measured.
   The relative expression of CDK1, COP1, BCL-2, and MAPK1 genes was found
   to be significantly decreased, while no significant changes were
   observed in the expression of CDKN1A, CDKN1B, CREB1, RAF1, GRB2, and
   CCNG2. The fold changes in the expression levels of the genes, as
   mentioned above, between the miR-NC and miR-195-5p transfected groups
   are given in Fig. [89]5, Supplementary Fig. [90]S4, and Table [91]1.

Figure 5.

   [92]Figure 5
   [93]Open in a new tab

   Relative expression patterns of miR-195-5p target genes. The relative
   expression profiles of the miR-195-5p target genes in the buffalo
   endometrial primary cells transfected with miR-195-5p mimic and miR-NC.
   Expression values were normalized to GAPDH (n = 2). Transfected cells
   are identified on the X-axis. Y –axis represents relative expression
   levels. *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001.

Table 1.

   Relative expression of the studied genes in terms of fold change.
   S. No Gene name Fold change Pathway
   1 CDK1 − 0.77 Cell cycle, PI3-Akt signaling
   2 YWHAQ − 0.63 Cell cycle, PI3-Akt signaling
   3 CDC27 − 0.64 Cell cycle, Ubiquitin mediated proteolysis,
   4 AKT-3 − 0.41 PI3-Akt signaling, MAPK pathway, mTOR signaling pathway
   5 FGF-7 − 0.52 PI3-Akt signaling, MAPK pathway
   6 COP-1 − 0.58 PI3-Akt signaling, Ubiquitin mediated proteolysis
   7 MAPK8 − 0.43 PI3-Akt signaling, MAPK pathway
   8 SGK1 − 0.36 PI3-Akt signaling, mTOR signaling pathway
   10 BCL-2 − 0.51 PI3-Akt signaling
   11 MAPK1 − 0.42 PI3-Akt signaling
   12 VEGFA − 0.29 PI3-Akt signaling
   13 CACNA2D1 − 0.37 MAPK pathway
   14 CUL2 − 0.49 Ubiquitin mediated proteolysis,
   15 MKNK1 − 0.88 PI3-Akt signaling, MAPK pathway,
   17 CHEK1 − 0.40 Cell cycle,
   18 CDKN1A − 0.46 Cell cycle, PI3-Akt signaling
   19 CDC23 − 0.48 Cell cycle, Ubiquitin mediated proteolysis
   20 CDKN1B − 0.34 Cell cycle
   22 MYB − 0.02 PI3-Akt signaling
   24 CREB1 − 0.17 PI3-Akt signaling
   25 RAF1 − 0.11 PI3-Akt signaling
   26 CCNG2 0.12 PI3-Akt signaling
   27 WWP1 − 0.15 Ubiquitin mediated proteolysis
   28 BTRC − 0.65 Ubiquitin mediated proteolysis
   29 UBE4B − 0.23 Ubiquitin mediated proteolysis
   30 GRB2 − 0.245 MAPK, PI3-Akt signaling
   31 SMAD2 0.5 Cell cycle
   [94]Open in a new tab

Quantification of cell proliferation and apoptosis after transfection of the
endometrial primary cells with miR-195-5p mimic

   Cell proliferation rate was measured post 48 h of miRNA transfection by
   BrdU incorporation to correlate the RT-qPCR results with cell
   proliferation and death. The cell proliferation rate was observed by
   calculating the mean of their optical densities. The mean optical
   density of the cells transfected with miR-195-5p was found to be
   significantly decreased (P < 0.05) compared to miR-NC transfected
   cells, or in other words, the BrdU incorporation into the proliferating
   cells was less in the miR-195-5p mimic transfected cells (n = 5)
   (Fig. [95]6C). To establish a correlation between the role of
   miR-195-5p and the regulation of apoptosis, the apoptotic rates were
   determined for the cells transfected with the miR-195-5p and miR-NC
   using flow cytometry-based Annexin V dead cell apoptosis detection kit.
   The data analysis revealed that the percentage of apoptotic was
   significantly higher (P < 0.01) in the miR-195-5p transfected cells
   compared to the cells transfected with miRNA negative control
   transfected cells (n = 4) (Fig. [96]6A,B).

Figure 6.

   [97]Figure 6
   [98]Open in a new tab

   (A) Cellular apoptosis assay with FCM. (A) Flow cytometry analysis with
   Annexin V-FITC/Propidium Iodide (PI) staining was performed to evaluate
   the percentage of apoptotic cells in endometrial primary cells
   transfected with miR-negative control and miR-195-5p mimic at a
   concentration of 10 pmol, 30 pmol (B) The percentage of apoptotic cells
   in the miR-195-5p transfected group were significantly increased
   compared with that of miRNA negative control (n = 4). (C) Cell
   proliferation assay. BrdU assay was used to detect the proliferation
   level of cells in the miR-NC and miR-195 transfected group (n = 5).
   *P < 0.05; **P < 0.01.

Discussion

   The aim of this study was to find the function of pregnancy-associated
   extracellular vesicle (EV) miRNAs in the maternal systemic circulation
   and assess their impact on shaping the uterine environment. In early
   pregnancy, there is a dynamic exchange of secretions between the
   developing conceptus and the endometrium, leading to intricate
   interactions that drive structural and functional changes in the
   endometrium. These changes enhance uterine receptivity for successful
   embryo implantation^[99]28,[100]29.

   Previous research has shown that circulatory miRNAs play a role in
   regulating pregnancy in cattle and buffalo. There is also emerging
   evidence suggesting the significance of EV-miRNAs in controlling embryo
   development, endometrial functions, and the communication between
   embryos and endometrium during pregnancy establishment^[101]30–[102]34.
   In this study, we identified placental-origin EV-miRNAs in the blood of
   pregnant buffalo and investigated the functional impact of miR-195-5p
   on endometrial cells in vitro. Previous studies have reported
   differential expression of circulating extracellular miRNAs, including
   miR-195-5p, miR-200a-3p, miR-27, and miR-1246, in the blood plasma of
   pregnant cattle and buffaloes^[103]31,[104]35, [105]36. Therefore, we
   examined the presence of these miRNAs within EVs derived from blood
   plasma and found that all four miRNAs were indeed present within these
   vesicles.

   The reason for the presence of these miRNAs on the EVs may be
   attributed to the fact that EVs prevent the RNase-mediated degradation
   of the miRNAs^[106]15,[107]16. We further investigated the differential
   expression of these EV-miRNAs among the pregnant and non-pregnant
   buffaloes concerning specific days post-artificial insemination. The
   expression of miR-195-5p was observed to be significantly high on day
   19. Thus, we selected this miRNA for further experiments. In bovine
   species, day 19–20 of pregnancy is referred to as the implantation
   window, and the trophoblast attachment also begins in this
   phase^[108]37. It has been reported that a decrease in the miR-195-5p
   levels causes defective differentiation and invasion of the
   trophoblastic cells thus, leading to abnormal placentation in
   humans^[109]38,[110]39. Therefore, miR-195-5p may have a crucial role
   in the implantation window of pregnancy. This miRNA may also diagnose
   pregnancy on day 19 due to its high expression.

   The uterus is a dynamic physiological system in which cellular
   proliferation, differentiation, and apoptosis occur in a temporal and
   cell-specific manner during pregnancy. Decidualization of the
   endometrium cells is a vital event for successful embryo implantation.
   Studies have shown that regulating the cell cycle is extremely
   important for the decidualization of the endometrium^[111]40. The
   endometrial cells cease proliferating and become differentiated in the
   pre-implantation period. At the implantation site, the Endometrial
   cells undergo apoptosis when they come in contact with
   trophoblast^[112]41,[113]42. Apoptosis is physiologically vital for
   optimal placental growth and development^[114]43–[115]45. Due to high
   levels of miR-195-5p at day 19 post AI in pregnant buffaloes, we wanted
   to investigate its involvement in regulating cell proliferation in the
   endometrial cells.

   KEGG pathway analysis identified that miR-195-5p's target genes are
   linked to key cell proliferation pathways vital for pregnancy
   establishment. The significant pathways include PI3K-Akt, Cell cycle,
   Ubiquitin-mediated proteolysis, MAPK, and mTOR signaling pathways. Gene
   Ontology analysis highlighted their involvement in Mitotic cell cycle,
   cell division, endocytosis, cell differentiation, and protein
   serine/threonine kinase activity. Specifically, the PI3-Akt pathway
   supports endometrial cell migration and decidualization during the
   implantation window^[116]46,[117]47. Ubiquitin-related proteins play
   crucial roles in endometrial remodeling, placental development, and
   embryo implantation^[118]48,[119]49. Inhibiting the mTOR pathway can
   induce cell death during implantation^[120]50. The MAP Kinase pathway
   regulates vital cellular functions such as proliferation,
   differentiation, apoptosis, and development^[121]51. Notably, the role
   of EV-miR-195-5p in modulating endometrial cells remains unexplored in
   existing literature.

   The RT-qPCR results indicate a significant decrease in the expression
   of specific genes involved in various pathways following transfection
   with the miR-195-5p mimic in endometrial primary cells. These pathways
   include PI3-Akt signaling, Ubiquitin-mediated processes, MAPK
   signaling, Cell Cycle regulation, and mTOR signaling. Notably,
   suppression of the anti-apoptotic BCL-2 gene promotes
   apoptosis^[122]52. AKT, particularly AKT-3, is directly influenced by
   PI3K and impacts cell proliferation, differentiation, and
   apoptosis^[123]53,[124]54. FGF-7 contributes to cell proliferation,
   differentiation, migration, and angiogenesis^[125]55. MAPKs regulate
   apoptosis through transcriptional and post-transcriptional mechanisms,
   sometimes acting as pro-apoptotic or anti-apoptotic in specific cell
   types^[126]56. YWHAQ protects cells from apoptosis by inhibiting p53
   and Bax in hepatocellular carcinoma^[127]57. CDK1 inhibition induces
   apoptosis in primary effusion lymphoma cells^[128]58. Genes like SGK1,
   COP1, and CDC27 promote cell proliferation and regulate apoptosis in
   cancer cells^[129]59–[130]62. VEGFA promotes vascular permeability and
   angiogenesis, affecting proliferation, migration, cell survival, and
   apoptosis inhibition in endothelial cells^[131]63. Cyclin D1 and CDKL1,
   both proto-oncogenes, regulate the G1 to S phase transition in the cell
   cycle, facilitating progression^[132]64,[133]65. In summary, the
   RT-qPCR results show that miR-195-5p mimic transfection significantly
   alters gene expression, particularly in apoptosis and cell cycle
   regulation pathways, revealing its multifaceted impact on endometrial
   primary cells.

   To investigate the link between miR-195-5p suppression and cell cycle
   regulation in endometrial primary cells, we conducted BrdU ELISA-based
   proliferation assays and flow cytometry-based apoptosis detection on
   cultured cells transfected with the miR-195-5p mimic. The transfection
   led to reduced cell proliferation (BrdU assay) and increased apoptosis
   (flow cytometry), consistent with previous findings indicating that
   elevated miR-195 levels decrease cell viability and proliferation while
   promoting apoptosis^[134]66,[135]67. This suggests miR-195-5p's
   involvement in modulating apoptotic and proliferation pathways that
   could impact embryo implantation. Endometrial tissue remodeling during
   implantation involves apoptotic cell death, rendering the uterus
   receptive to the embryo^[136]19,[137]20. Thus, our study sheds light on
   miR-195-5p's role in regulating these processes, potentially
   influencing embryo implantation. To the best of our knowledge, this is
   the first study highlighting the role of EV-borne miR-195-5p in
   governing apoptosis and cell proliferation in endometrial cells.

Conclusion

   In conclusion, the increased expression of miR-195-5p promotes
   apoptosis by regulating the expression of genes involved in cell
   proliferation and apoptosis at the initial stages of implantation
   (Fig. [138]7). This may have a profound role in promoting uterine
   receptivity; thus, modulation of miR-195-5p may enhance the chances of
   successful pregnancy in buffaloes. In addition, due to the high levels
   of miR-195-5p in the blood plasma of pregnant buffaloes at day 19
   post-AI, it can be used as a candidate miRNA for detecting a successful
   pregnancy.

Figure 7.

   [139]Figure 7
   [140]Open in a new tab

   Schematic diagram representation of the effect of Extracellular
   vesicle-associated miR-195-5p on the endometrium primary cell culture.

Methods

Ethics statement

   The animals employed in the study were reviewed and approved by the
   Institutional Animal Ethics Committee, ICAR-National Dairy Research
   Institute, Karnal, India. The study was conducted in compliance with
   ARRIVE guidelines and the relevant guidelines and regulations performed
   on all methods^[141]68.

Experimental design

   This study was conducted through several experiments.

   In experiment 1, Blood samples were collected from different animals
   categorized into two groups viz., pregnant and non-pregnant Murrah
   Buffaloes. For the characterization, EVs were isolated from the blood
   plasma of three different randomly selected Murrah buffaloes
   irrespective of their pregnancy status. EVs were characterized by the
   Differential light scattering method, Nano Sight, to determine the
   average concentration and size, western blotting (WB) to identify the
   presence of protein markers and visualized under transmission electron
   microscopy for morphological identification.

   In Experiment 2, miRNAs were chosen using previous Next-Generation
   Sequencing (NGS) results, which highlighted their elevated presence
   during the initial stages of pregnancy in buffaloes (Accession:
   PRJNA705293, ID: 705293) as documented in NCBI. Additionally, miR-1246
   was selected based on existing scholarly references. These specific