Biology of Reproduction最新文献

The mechanistic target of rapamycin (mTOR) 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 mTOR signaling across gestation are poorly understood. We collected 81 human placental samples from 4-40 weeks' gestation to test the hypothesis that placental mTOR signaling activity increases over gestation and is activated in maternal obesity in early gestation. Proteins involved in upstream mTOR 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 mTOR 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.

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 (KO) mice, which exhibited asthenozoospermia and sterility. The sperm motility of Dnah3-KO mice significantly declined compared to wild-type mice. However, spermatozoa from Dnah3-KO mice displayed normal morphology in haematoxylin-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-KO 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.

The bovine conceptus elongates near Day 16 of development and releases interferon-tau (IFNT), disrupting the endometrial luteolytic mechanism to sustain luteal P4 and pregnancy. Conceptus factors other than IFNT 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 RNA 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: (1) no tissue (Control Med; n = 7), (2) mono-cultured conceptus (Conceptus; n = 9, 3) mono-cultured endometrium (Endo; n = 13), or (4) Endo-Conceptus Co-culture (n = 15). After 12 h, tissue RNA was 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 (DEG), respectively (P ≤ 0.01). More abundantly expressed endometrial DEG were associated with interferon signaling whereas more abundantly expressed conceptus DEG were associated with protein homeostasis and metabolism (FDR < 0.001). When Co-culture media where compared to Endo media, 288 more abundant protiens 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.

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 (IVP) 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 (ICM) 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 IVP embryos. Editing efficiency was ≥82%. Targeting IL6ST did not affect cleavage but reduced development to the 16-cell and blastocyst stages. A reduction in ICM cell numbers was detected, and a disorganization of the ICM 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 ICM cell development at the blastocyst stage. There also is evidence that these signals support the overall cellular organization of the blastocyst.

Chorionic trophoblast cells (CTCs) 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 CTCs dealt with two separate questions: (1) The necessity of highly enriched and defined media with inhibitors of oxidative stress and cell transition and their impact on growth and trophoblast phenotype, (2) The functional differences between CTCs and other placental trophoblast lineages of cells (placental cytotrophoblast cells [PTC], and extravillous trophoblast [EVT]). CTCs 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 GATA3, cytokeratin [CK] 7, and vimentin) were assessed. Syncytialization of cells (forskolin treatment) and invasive properties of CTCs (cell invasion assay) were tested and compared with PTCs and EVTs (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 CK7 or GATA3) compared to CTCs grown in complete and enriched media. The CTCs failed to syncytialize, contrasting with the high levels of membrane fusion observed in PTCs. Although CTCs express human leukocyte antigen G (HLA-G) like EVTs, they do not invade. CTCs require several specific constituents for in vitro growth and phenotype maintenance. CTCs are trophoblast lineage cells that barricade immune cell-enriched decidua without invading them. These properties support their location and function, which are distinct from PTCs and EVTs.

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.

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.

Gestation period in captive Yangtze finless porpoise (YFP) is a well-coordinated and dynamic process, involving both systemic and local alterations. The gut microbiota and its connection to fecal metabolites are crucial in supporting fetal development and ensuring maternal health during reproductive stages. This study evaluates changes in the gut microbiota and their correlation with fecal metabolites in captive YFPs during different reproductive stages. The results reveal that microbial community structure changed significantly during reproductive stages, while gut microbial diversity remained stable. The genus unclassified Peptostrptococcaceae, Corynebacterium, and norank KD4-96 were significantly greater in non-pregnancy (NP), Terrisporobacter was significantly greater in lactating (LL), and Clostridium was significantly higher in early-pregnancy (EP) compared to the other groups. The host fecal metabolome exhibited significant alterations during the reproductive stages. Indoxyl sulfate, octadecatrienoic acid, and methionyl-methionine were significantly higher in the NP; galactosylglycerol, chondroitin 6-sulfate, and lumichrome were significantly higher in the EP and mid-pregnancy (MP); and valylleucine and butyryl-l-carnitine were significantly higher in the LL. The altered metabolites were mostly concentrated in pathways associated with arachidonic acid metabolism (significantly altered in NP), leucine, valine, and isoleucine biosynthesis (significantly altered in EP and MP), and glycerophospholipid metabolism (significantly altered in LL compared to others stages). Additionally, we found a strong link between variations in the host metabolism and alterations in the fecal bacteria of captive YFP. In conclusion, this study provides detailed insights into host metabolic and fecal bacterial changes in captive YFP during reproduction stages, providing important knowledge for improving the reproductive management in the captive YFP.

Background: Recurrent spontaneous miscarriage is one of the complications during pregnancy. However, the pathogenesis of recurrent spontaneous miscarriage is far from fully elucidated.

Objective: Since the endocytic pathway is crucial for cellular homeostasis, our study aimed to explore the roles of endocytic recycling, especially EH domain containing 1, a member of the endocytic recycling compartment, in recurrent spontaneous miscarriage.

Study design: We first investigated the expression of the endocytic pathway member EH domain containing 1 in villi from the normal and recurrent spontaneous miscarriage groups. Then, we performed ribonucleic acid sequencing and experiments in villi, HTR8 cells and BeWo cells to determine the mechanisms by which EH domain containing 1-induced recurrent spontaneous miscarriage. Finally, placenta-specific EH domain containing 1-overexpressing mice were generated to investigate the recurrent spontaneous miscarriage phenotype in vivo.

Results: EH domain containing 1 was expressed in extravillous trophoblasts and syncytiotrophoblast in the villi. Compared with the control group, recurrent spontaneous miscarriage patients expressed higher EH domain containing 1. A high level of EH domain containing 1 decreased proliferation, promoted apoptosis, and reduced the migration and invasion of HTR8 cells by activating the TGFβ receptor 1-SMAD2/3 signaling pathway. The TGFβ receptor 1 antagonist LY3200882 partially reversed the EH domain containing 1 overexpression-induced changes in the cell phenotype. Besides, a high level of EH domain containing 1 also induced abnormal syncytialization, which disturbed maternal-fetal material exchanges. In a mouse model, placenta-specific overexpression of EH domain containing 1 led to the failure of spiral artery remodeling, excessive syncytialization, and miscarriage.

Conclusions: Increased expression of EH domain containing 1 impaired the invasion of extravillous trophoblasts mediated by the TGFβ receptor 1-SMAD2/3 signaling pathway and induced abnormal syncytialization of syncytiotrophoblast, which is at least partially responsible for recurrent spontaneous miscarriage.