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Follicle analysis pipeline with OvoPath. Description of the follicle component annotation outputs of scripts 1 to 6. QR code contains the link to the GitHub repository where the OvoPath graphical user interface and step-by-step instructions can be found. Representative drawings depict the follicle morphometric details (grey color highlights the specific area analyzed) and follicle classification stages obtained after using OvoPath.
Uterine infections cause ovarian dysfunction and infertility. The bacterial endotoxin, lipopolysaccharide, accumulates in the follicular fluid of dominant follicles of cows with uterine infections. Granulosa cells produce an innate inflammatory response to lipopolysaccharide, altering the follicular microenvironment of the oocyte. We hypothesized that developmental competence and embryo quality would be reduced when oocytes are matured in an inflammatory environment. Bovine mural granulosa cells were exposed to either 1 µg/mL of lipopolysaccharide or medium alone for 24 h to produce a conditioned medium. Inflammatory responses of mural granulosa cells were confirmed by increased expression of CXCL8, IL1B, IL6, and TNF. Bovine cumulus–oocyte complexes were matured for 22 ± 1 h in a medium supplemented with either 1 µg/mL of lipopolysaccharide, 10% v/v conditioned medium of granulosa cells treated with either lipopolysaccharide (LCM) or medium alone, or no supplementation. In addition, polymyxin B (20 µg/mL) was added to the maturation medium to sequester LPS. Following maturation, cumulus–oocyte complexes were fertilized and cultured for 7.5 days with no further treatment. Oocyte maturation using lipopolysaccharide or LCM impaired development to the blastocysts stage, reduced the number of total and CDX2-negative blastomeres, and increased TUNEL-positive cells in blastocysts. Polymyxin B could rescue these effects in the lipopolysaccharide group but not in the LCM group, indicating factors produced by granulosa cells and not lipopolysaccharide alone compromised oocyte development. These findings suggest that the inflammatory milieu produced by granulosa cells in response to lipopolysaccharide impairs oocyte competence and the quality of resultant blastocyst-stage embryos.
Summary Sentence
In vitro maturation of bovine oocytes under inflammatory conditions reduces developmental competence and alters embryo quality by increasing apoptosis and decreasing blastomere cell number.
This work explored whether bovine embryo development relies on signaling from the interleukin-6 (IL6) cytokine family. This was accomplished by interrupting IL6 signal transducer (IL6ST), the common beta-subunit receptor used by the IL6 family. One series of studies cultured in vitro–produced embryos with SC144, a pharmacological IL6ST inhibitor. Providing the inhibitor at a concentration that partially diminished IL6ST signaling reduced development to the 16-cell and blastocyst stages and reduced inner-cell-mass cell numbers. Inhibitor concentrations that completely blocked IL6ST signaling prevented blastocyst development. Another series of studies used CRISPR-Cas9 to disrupt IL6ST. Two electroporation approaches were used to introduce guide RNAs and Cas9 protein into one-cell in vitro–produced embryos. Editing efficiency was ≥82%. Targeting IL6ST did not affect cleavage but reduced development to the 16-cell and blastocyst stages. A reduction in inner-cell-mass cell numbers was detected, and disorganization of the inner cell mass was observed in approximately one-half of the IL6ST-targeted blastocysts. These observations indicate that embryo-derived IL6 family members that signal through IL6ST are needed to support normal in vitro bovine embryo development. These signals are needed by the 16-cell stage and for inner-cell-mass cell development at the blastocyst stage. There is also evidence that these signals support the overall cellular organization of the blastocyst.
Summary Sentence
IL6ST disruption affects in vitro bovine embryo development and specifically development after d 2 post-fertilization, and this disruption also decreases inner cell mass cell numbers in blastocysts.
Optimal embryonic development depends upon cell-signaling molecules released by the maternal reproductive tract called embryokines. The identity of specific embryokines that enhance the competence of the embryo for sustained survival is largely lacking. The current objective was to evaluate the effects of three putative embryokines in cattle on embryonic development to the blastocyst stage. The molecules tested were vascular endothelial growth factor A (VEGFA), C-X-C motif chemokine ligand 12 (CXCL12), and interleukin-6 (IL6). Molecules were added from day 4 to 7.5 of culture at 50 ng/mL (VEGFA and CXCL12) or 100 ng/mL (IL6). Endpoints were development to the blastocyst stage and transcript abundance for 94 specific genes involved in lineage commitment, epigenetic regulation, and other functions. Among the genes examined were eight whose transcript abundance has been related to embryo competence for survival after embryo transfer. None of the molecules increased the proportion of putative zygotes or cleaved embryos becoming blastocysts at day 7.5 of development. An embryo competence index based on a Bayesian multiple regression formula to weigh transcript abundance of the eight biomarker genes was not affected by treatment with VEGFA but was increased by both CXCL12 and IL6. The transcript abundance of 5 genes was modified by VEGFA, 19 by CXCL12, and 19 by IL6. A total of 11 genes were modified in a similar manner by CXCL12 and IL6. Most differentially expressed genes for CXCL12 and IL6 were downregulated, suggesting that the embryokines may promote a less energetically demanding metabolic state than would be the case in their absence.
Summary Sentence
Both C-X-C motif chemokine ligand 12 and interleukin-6 can act on the preimplantation bovine embryo to change gene expression in a manner consistent with increased competence for embryo survival.
Embryo implantation in the mare occurs just over one month after fertilization, coinciding with the production of chorionic gonadotropin. The factors that regulate this late implantation in the mare, and whether they are unique to horses or shared with more invasive embryo implantation in other species, remain poorly understood. This study aimed to determine and compare the transcriptome and subpopulations of endometrial cells before and after embryo implantation in the horse. Single-cell RNA sequencing was used to characterize the transcriptome of nearly 97,000 endometrial cells collected from biopsies of the endometrium at the beginning (day 33 of gestation) and after embryo implantation (day 42 of gestation) in mares. Sixteen immune and 24 non-immune cell clusters were identified, representing known major cell populations as well as novel subpopulations of horse immune cells such as resident innate lymphoid cells and mucosal-associated invariant T cells. Contrary to current knowledge, endometrial natural killer (eNK) cells were the most abundant endometrial leukocyte population during implantation in horses. Moreover, eNK cells not only expressed genes that may interact with fetal MHC I, such as LY49F, but also exert immunoregulatory functions independent of MHC I expression, such as CD96/TIGIT. Analogous to other species studied, upregulation of CXCR4 was found in several subpopulations of immune cells. Our results suggest that despite distinctive and later placentation compared with humans, horses share some key similarities in the mechanisms of embryo implantation.
Summary Sentence
Endometrial NK cells are the most abundant and antigen-presenting cells are the most responsive populations of immune cells in the equine endometrium across implantation. Additional key contributor cells include other innate lymphoid and mucosal-associated invariant T cells, which were defined for the first time in the horse.
The mammalian target of rapamycin (mTOR) signaling pathway is activated by luteinizing hormone in preovulatory follicle. However, its impact on ovulation remains inadequately explored. Utilizing in vivo studies and in vitro fertilization, we demonstrated that the negative effect of inhibition of mTOR signaling by rapamycin on oocyte quality during the ovulatory phase, with a notable decrease in the total cell count of blastocysts, a reduction in gastrula size, and fetal degeneration on the 16th day of gestation while not affecting ovulated oocyte count or granulosa cell luteinization. Mechanistically, our study elucidated that in the ovulatory phase, mTOR signaling inhibition enhances lipid consumption, mitochondrial membrane potential of oocytes, and ATP generation. As a result, embryos derived from these oocytes exhibit higher levels of reactive oxygen species, insufficient energy supply, and lower developmental potency. Furthermore, the impact of mTOR signaling on oocytes remains consistent across various species, and its inhibition has been demonstrated to enhance energy metabolism during the in vitro maturation process of bovine oocytes. These findings demonstrate the critical role of mTOR signaling during the ovulatory phase in balancing oocyte energy metabolism, enriching our understanding of the role of mTOR on ovulation regulation.
Summary Sentence
The mTOR pathway maintains energy metabolism balance in oocytes during the ovulatory phase. Its disruption elevates metabolism and lipid consumption, ultimately yielding poor-quality oocytes.
Di(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer known for its toxic effects on the male reproductive system. Green tea polyphenols (GTPs), recognized for their antioxidant and anti-inflammatory properties, have demonstrated protective effects on various organs, but the mechanisms by which GTPs mitigate DEHP-induced testicular damage remain unclear. Healthy male C57BL/6J mice were divided into five groups: control, DEHP, DEHP + GTP treatment, GTP, and oil groups. Testicular histopathological changes were assessed using hematoxylin–eosin (H&E), periodic acid-Schiff (PAS), and Masson staining. Ultrastructural alterations were examined through transmission electron microscopy. High-throughput sequencing was performed to analyze the expression of mRNA, miRNA, and lncRNA and construct an lncRNA–miRNA–mRNA regulatory network for identifying key regulatory axes. Mice in the DEHP group exhibited significant testicular damage, including reduced sperm count, mitochondrial deformation, and endoplasmic reticulum dilation. GTP treatment notably improved testicular structural integrity, restored sperm count, and alleviated mitochondrial and endoplasmic reticulum damage. Additionally, DEHP significantly increased activated CD8+ T cells, which were reduced with GTP treatment. High-throughput sequencing revealed that GTP treatment exerted protective effects through the regulation of six key lncRNA–miRNA–mRNA axes. GTPs significantly protect against DEHP-induced testicular damage, and the lncRNA–miRNA–mRNA regulatory axes play a potential role in this process.
Summary Sentence
Our study substantiates the remedial promise of GTPs in treating DEHP-induced testicular damage, highlighting the importance of the lncRNA–miRNA–mRNA regulatory axis.
Asthenozoospermia, a prevalent contributor to male infertility, exhibits a multifaceted pathogenesis. This study identified a significant downregulation in sperm dynein heavy chain 3 (DNAH3) protein levels in individuals with asthenozoospermia. To elucidate the role of DNAH3 in asthenozoospermia, we constructed Dnah3-knockout mice, which exhibited asthenozoospermia and sterility. The sperm motility of Dnah3-knockout mice significantly declined compared to wild-type mice. However, spermatozoa from Dnah3-knockout mice displayed normal morphology in hematoxylin and eosin staining and transmission electron microscopy analyses. Sperm metabolomics revealed that DNAH3 deficiency disturbed sperm energy metabolism, resulting in substantial reductions of L-palmitoylcarnitine and glycocholic acid. Notably, offspring were successfully obtained from Dnah3-knockout male mice through intracytoplasmic sperm injection. Collectively, these findings indicate that DNAH3 deficiency induces disturbances in energy metabolism, rather than abnormalities in sperm flagellar morphology, culminating in asthenozoospermia development. Our investigation provides valuable insights into understanding asthenozoospermia and offers guidance for clinical consultation.
Sentence summary
DNAH3 was significantly downregulated in individuals with asthenozoospermia. Moreover, DNAH3 deficiency was associated with disrupted energy metabolism rather than sperm flagellar morphology abnormalities, ultimately contributing to asthenozoospermia.
Katheryn D. Peterson, Trevor F. Freeman, Shankar P. Poudel, Susanta K. Behura, D. Kakhniashvili, Daniel L. Johnson, Tulio M. Prado, Lew G. Strickland, Jonathan E. Beever, Thomas E. Spencer, Daniel J. Mathew
The bovine conceptus elongates near Day 16 of development and releases interferon-tau, disrupting the endometrial luteolytic mechanism to sustain luteal P4 and pregnancy. Conceptus factors other than interferon tau modify local endometrial activities to support pregnancy; however, the microenvironment is largely uncharacterized. We utilized a bovine conceptus-endometrial culture system to elucidate the microenvironment in the form of gene transcripts and protein. Estrus synchronized heifers remained cyclic (13) or were inseminated (9) to produce Day 16 cyclic endometrium and elongating conceptuses, respectively. Conceptus sections and endometrium were then used to generate tissue cultures in 1 ml of medium: (i) no tissue (control med; n = 7), (ii) mono-cultured conceptus (conceptus; n = 9) (iii) mono-cultured endometrium (endo; n = 13), or (iv) endo-conceptus co-culture (n = 15). After 12 h, tissue gene transcripts were sequenced (RNA-Seq) and media underwent proteomic analysis (LC–MS/MS). Compared to conceptus and endo, co-cultured conceptus and endometrial tissue contained 3400 and 4575 differentially expressed genes, respectively (P ≤ 0.01). More abundantly expressed endometrial differentially expressed genes were associated with interferon signaling whereas more abundantly expressed conceptus differentially expressed genes were associated with protein homeostasis and metabolism (FDR < 0.001). When co-culture media where compared to endo media, 288 more abundant proteins were identified (P < 0.05). Biological processes related to these proteins included antigen presentation via MHC Class Ib and keratinization (FDR < 0.001). Within the mono-cultured conceptus and endometrial media, folate receptor alpha (FOLR1; P < 0.001) was identified as the most abundant secreted protein suggesting the reproductive tissues elicit a microenvironment supportive of conceptus growth involving folate metabolism.
Summary Sentence
Day 16 bovine conceptus and endometrial transcripts and proteins.
Chorionic trophoblast cells are one of the principal components of the fetal membrane and join with the decidua to form a feto–maternal interface. Recent success in isolating chorionic trophoblast cells dealt with two separate questions: (i) the necessity of highly enriched and defined media with inhibitors of oxidative stress and cell transition and their impact on growth and trophoblast phenotype, (ii) the functional differences between chorionic trophoblast cells and other placental trophoblast lineages of cells (placental cytotrophoblast cells, and extravillous trophoblast). Chorionic trophoblast cells were cultured either in defined media with various inhibitors or in media from which inhibitors were removed individually. Cellular morphology and growth (microscopy and crystal violet staining) and cellular and molecular biological features (immunofluorescence staining for GATA-binding protein 3, cytokeratin 7, and vimentin) were assessed. Syncytialization of cells (forskolin treatment) and invasive properties of chorionic trophoblast cells (cell invasion assay) were tested and compared with placental cytotrophoblast cells and extravillous trophoblasts (HTR8/SVneo), respectively. Removal of various growth-supporting agents from the media delayed cell growth and inclined towards cellular transition (increase in vimentin compared to cytokeratin 7 or GATA-binding protein 3) compared to chorionic trophoblast cells grown in complete and enriched media. The chorionic trophoblast cells failed to syncytialize, contrasting with the high levels of membrane fusion observed in placental cytotrophoblast cells. Although chorionic trophoblast cells express human leukocyte antigen G like extravillous trophoblasts, they do not invade. Chorionic trophoblast cells require several specific constituents for in vitro growth and phenotype maintenance. Chorionic trophoblast cells are trophoblast lineage cells that barricade immune cell-enriched decidua without invading them. These properties support their location and function, which are distinct from placental cytotrophoblast cells and extravillous trophoblasts.
Summary Sentence
Chorionic trophoblasts are distinct from placental trophoblasts and require a specific set of inhibitors to maintain their phenotype in vitro culture.
The mechanistic target of rapamycin system is vital to placental development, formation, and function. Alterations in this system in the placenta have been associated with altered fetal growth. However, changes in placental mechanistic target of rapamycin signaling across gestation are poorly understood. We collected 81 human placental samples from 4 to 40 weeks gestation to test the hypothesis that placental mechanistic target of rapamycin signaling activity increases over gestation and is activated in maternal obesity in early gestation. Proteins involved in upstream mechanistic target of rapamycin regulation and mTORC1/2 downstream signaling were quantified using immunoblotting in placentas of male or female fetuses. Readouts of mTORC1 activation, phospho-rpS6, and phospho-4EBP1 were highest in first trimester and decreased across gestation. Phosphorylation of AKT (308 and 473) increased over gestation. Interestingly, abundance of cytochrome c oxidase I and mitochondrial ATP synthase, key subunits of mitochondrial complexes III/IV and V, respectively, were elevated in first trimester obese placentas compared to control, but only in placenta from female fetuses. We suggest that the high placental mechanistic target of rapamycin signaling activity in early pregnancy may be related to the high anabolism and active trophoblast proliferation and invasion in the second half of the first trimester. In addition, we conclude that maternal obesity has only limited impact on this key placental signaling pathway across gestation in women.
Summary Sentence
Changes in human placental mTOR signaling across gestation differ primarily by gestational age, with only a few changes due to maternal obesity.
Economic losses in cattle farms are frequently associated with failed pregnancies. Some studies found that the transcriptomic profiles of blood and endometrial tissues in cattle with varying pregnancy outcomes display discrepancies even before artificial insemination (AI) or embryo transfer (ET). In the study, 330 samples from seven distinct sources and two tissue types were integrated and divided into two groups based on the ability to establish and maintain pregnancy after AI or ET: P (pregnant) and NP (nonpregnant). By analyzing gene co-variation and employing machine learning algorithms, the objective was to identify genes that could predict pregnancy outcomes in cattle. Initially, within each tissue type, the top 100 differentially co-expressed genes (DCEGs) were identified based on the analysis of changes in correlation coefficients and network topological structure. Subsequently, these genes were used in models trained by seven different machine learning algorithms. Overall, models trained on DCEGs exhibited superior predictive accuracy compared to those trained on an equivalent number of differential expression genes. Among them, the deep learning models based on differential co-expression genes in blood and endometrial tissue achieved prediction accuracies of 91.7% and 82.6%, respectively. Finally, the importance of DCEGs was ranked using SHapley Additive exPlanations (SHAP) and enrichment analysis, identifying key signaling pathways that influence pregnancy. In summary, this study identified a set of genes potentially affecting pregnancy by analyzing the overall co-variation of gene connections between multiple sources. These key genes facilitated the development of interpretable machine learning models that accurately predict pregnancy outcomes in cattle.
Summary Sentence
In cattle with different pregnancy outcomes, blood gene co-expression patterns exhibit more pronounced differences compared to those in the endometrium, which enables deep learning algorithms to accurately predict the outcomes.
The redox state of thiol groups derived from cysteine residues in proteins regulates cellular functions. Changes in the redox state of thiol groups in the epididymis are involved in sperm maturation. Furthermore, the redox state of thiol groups in proteins changes during the process of sperm capacitation. However, the effect of the redox state of thiol groups in sperm membrane proteins on the fertilization ability of sperm has not been studied. Therefore, in this study, we oxidized thiol groups in sperm membrane proteins using 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), which is a thiol-selective oxidizing agent, and examined the effect of oxidation of these thiol groups on the fertilization ability of sperm. Oocytes and sperm were obtained from C57BL/6 J mice, and Jcl:ICR mice were used as recipients for embryo transfer. Oxidation of the thiol groups by DTNB decreased the in vitro fertilization rate, and removal of the zona pellucida recovered the fertilization rate. DTNB treatment decreased the amplitude of the lateral head, which is an indicator of hyperactivation, and suppressed an increase in the intracellular calcium ion concentration, which is essential for hyperactivation. These findings suggest that oxidation of thiol groups in sperm membrane proteins can decrease the fertility of sperm by suppressing calcium ion influx and hyperactivation.
Summary Sentence
Oxidization of protein thiols in sperm membrane proteins by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) decreases the fertilization ability and motility of sperm and suppresses calcium influx.
Artificially induced haploidy is lethal in vertebrates, although it is useful for genetic screening and genome editing due to its single set of genomes. Haploid embryonic stem (ES) cell lines in mammals contribute to genetic studies and the production of gametes derived from haploid ES cells. In fish breeding, doubled haploids induced by artificially induced gynogenesis are used to generate isogenic gametes for cloning purposes. However, gametes have not been directly differentiated from artificially induced haploid cells, even though haploid ES cell lines have been established in medaka. Here, we aimed to demonstrate that fertile haploid sperm with identical genotypes could be differentiated in germline chimeras in zebrafish, wherein primordial germ cells (PGCs) derived from an inviable haploid embryo were transplanted into sterilized recipient embryos. Haploid spermatogonia differentiated from haploid PGCs in germline chimeras, and genome doubling occurred in haploid spermatogonia with one set of chromosomes, which could not pair with counterpart homologs to form bivalents during meiosis. Subsequently, meiosis produced isogenic haploid gametes because identical elements were exchanged between chromosomes doubled from the haploid set during recombination. Consequently, haploid PGCs can survive beyond embryogenesis and potentially differentiate into fertile sperm.
Summary Sentence
Artificially induced gynogenetic haploid primordial germ cells, when transplanted into sterile recipients, survived beyond embryogenesis in germline chimeras and differentiated into isogenic sperm via diploidization of their haploid genome in the germ cells.
The adult mammalian testis is filled with seminiferous tubules, which contain somatic Sertoli cells along with germ cells undergoing all phases of spermatogenesis. During spermatogenesis in postnatal mice, male germ cells undergo at least 17 different nomenclature changes as they proceed through mitosis as spermatogonia (=8), meiosis as spermatocytes (=6), and spermiogenesis as spermatids (=3). Adding to this complexity, combinations of germ cells at each of these stages of development are clumped together along the length of the seminiferous tubules. Due to this, considerable expertise is required for investigators to accurately analyze changes in spermatogenesis in animals that have spontaneous mutations, have been genetically modified (transgenic or knockout/knockin), or have been treated with pharmacologic agents. Here, we leverage our laboratory's expertise in spermatogenesis to optimize the open-source “Quantitative Pathology & Bioimage Analysis” software platform for automated analyses of germ and somatic cell populations in both the developing and adult mammalian testis.
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