Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization.
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ABSTRACT:
SUBMITTER: Qu Y
PROVIDER: S-EPMC8464547 | biostudies-literature | 2021 Oct
REPOSITORIES: biostudies-literature
Publications
Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization.
Qu Yongcun Y Chen Qi Q Guo Shanshan S Ma Chiyuan C Lu Yonggang Y Shi Junchao J Liu Shichao S Zhou Tong T Noda Taichi T Qian Jingjing J Zhang Liwen L Zhu Xili X Lei Xiaohua X Cao Yujing Y Li Wei W Li Wei W Plachta Nicolas N Matzuk Martin M MM Ikawa Masahito M Duan Enkui E Zhang Ying Y Wang Hongmei H
Protein & cell 20210301 10
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Project description:PurposeTo investigate whether in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), influence the embryo's development and its quality using the mouse as a model.MethodsAssisted fertilization was performed using ICSI and IVF. Fluorescent beads were adhered to the fertilization cone or place of previous sperm injection in the natural mated (NM), IVF and ICSI embryos, respectively. Embryo examination was carried out at the two-cell and blastocyst stage to determine the position of fluorescent bead. Protein expression was detected by fluorescence immunocytochemical staining and confocal microscopic imaging of blastocysts.ResultsIVF and ICSI embryos developed at rates comparable to NM group. Embryos show similar expression patterns of two transcription factors, Oct4 and Cdx2. The most preferred place for spermatozoa attachment was the equatorial site of the egg, whether fertilization occurred in vitro or under natural conditions. We also link the sperm entry position (SEP) to embryo morphology and the number of cells at the blastocyst stage, with no influence of the method of fertilization.ConclusionsIVF and ICSI, do not compromise in vitro pre-implantation development. Additional data, related to sperm entry, could offer further criteria to predict embryos that will implant successfully. Based on embryo morphology, developmental rate and protein expression level of key transcription factors, our results support the view that ART techniques, such as IVF and ICSI, do not perturb embryonic development or quality.
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Project description:Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm-oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con- and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of approximately 1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm-ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OF-exposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoprotein-heparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy.
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Project description:BackgroundWhen preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear.Methodology/principal findingsWe have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP(3) receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton.Conclusions/significanceOur findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.
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Project description:During transport through the oviduct, sperm interact with epithelial cells by attaching to specific glycans, a mechanism believed to select sperm and prolong their viability. An in vitro model of sperm-oviduct interactions was developed, consisting of a glass surface (either a slide or a coverslip) to which an oviduct glycan (sulfated Lewis X trisaccharide; suLeX) is coupled. The ability of porcine sperm to attach to suLeX-surfaces and detach in response to progesterone and mature cumulus-oocyte complexes (COCs) was validated. The suLeX-coverslip was adapted for in vitro fertilization (IVF), termed glycan-IVF, by allowing porcine sperm to first bind suLeX before transferring mature COCs. The glycan-IVF method produced a percentage of fertilized oocytes comparable to that of conventional IVF (75.1 vs. 72.0%). Finally, the ability of the suLeX-coverslip to maintain sperm fertilizing ability over time was assessed. After 24 h of incubation, fertilization by sperm bound to the suLeX-coverslip was sustained, compared to sperm with unmodified coverslips (12.0 vs. 1.0%, p < 0.05). Furthermore, the percentage of polyspermic zygotes was reduced in the suLeX-coverslip method (17.7 vs. 41.3%, p < 0.05). This study validated an in vitro model for studying sperm-oviduct interactions, with potential applications in assisted reproductive technologies.
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Project description:Salpingitis is a common cause for subfertility and infertility both in humans and animals. However, the effects of salpingitis on tubal function and reproductive success are largely unknown. Therefore we set out to investigate the effects of inflammation on sperm and oocyte transport and gameto-maternal interaction in the oviduct using the bovine as a model. For this purpose, oviducts revealing mild (n = 45), moderate (n = 55) and severe (n = 45) inflammation were obtained from cows immediately after slaughter and investigated by live cell imaging, histochemistry and scanning electron microscopy. Our studies showed that endometritis was always correlated with salpingitis. Moderate and severe inflammation caused a significant increase in the thickness of tubal folds (p < 0.05). Severe inflammation was characterized by luminal accumulations of mucus and glycoproteins, increased apoptosis, loss of tight junctions and shedding of tubal epithelial cells. The mean ciliary beat frequency (CBF) in the ampulla was significantly reduced as compared to the controls (p < 0.05). The higher the grade of inflammation, the lower was the CBF (p < 0.001). In severe inflammation, spermatozoa were stuck in mucus resulting in decreased sperm motility. Our results imply that tubal inflammation impairs proper tubal function and leads to reduced sperm fertilizing capacity.
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Project description:Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa's pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics.
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Project description:Background Fertilization is a prerequisite for successful human reproduction. The choice of clinical fertilization strategy is crucial and directly affects clinical outcomes. This study analyzes the most appropriate assisted reproductive technology (ART) strategy based on sperm parameters. Methods Semen samples were divided into six groups based on semen progressive motility (PR) and semen density (SD): HMLD (high motility-low density) (PR ≥32% and sperm density <15×106/mL, n=60), HMID (high motility-intermediate density) (PR ≥32% and 15×106/mL ≤ SD <30×106/mL, n=106), HMHD (high motility-high density) (PR ≥32% and SD ≥30×106/mL, n=1,009), LMLD (low motility-low density) (PR <32% and SD <15×106/mL, n=99), LMID (low motility-intermediate density) (PR <32% and 15×106/mL ≤ SD <30×106/mL, n=77), and LMHD (low motility-high density) (PR <32% and SD ≥30×106/mL, n=164). We analyzed hyaluronic acid binding (HAB) assay and acrosin activity, along with fertilization, embryonic development, and pregnancy outcomes, to demonstrate the correlation of sperm parameters with fertilization function. Results In the PR <32% groups, the rate of intracytoplasmic sperm injection (ICSI) treatment decreased with increasing sperm concentration. Specifically, approximately 10% of in vitro fertilization (IVF) treatment cycles required a rescue ICSI when sperm PR was <32% accompanied by SD ≥15×106/mL and PR ≥32% accompanied by SD <30×106/mL, which was significantly higher than HMHD group, P<0.001. Sperm acrosin activity and HAB ability were significantly higher in the groups with good sperm parameters, P<0.05. Conclusions The findings of this study suggest, fertilization ability of sperm is closely related to sperm motility and density. In clinical practice, IVF strategies should be refined based on male sperm parameters.
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Project description:When entering the oviduct for fertilisation, spermatozoa come into contact with the oviduct fluid (OF) and can bind to luminal epithelial cells in the isthmus to form a sperm reservoir. The objective of this study was to examine how the OF modulates sperm adhesion to the oviduct reservoir using an in vitro model of oviduct epithelial spheroids (OES). Bovine oviducts from a local slaughterhouse were used to collect OF and isthmic fragments for the in vitro incubation of OES. Compared to a non-capacitating control medium, the pre-ovulatory OF significantly decreased by 80-90% the density of spermatozoa bound to OES without affecting sperm motility, membrane integrity, or sperm-cilia interactions. This effect on sperm binding was reproduced with (1) OF from different cycle stages and anatomical regions of the oviduct; (2) OF fractions of more than 3 kDa; (3) modified OF in which proteins were denatured or digested and (4) heparan sulphate but not hyaluronic acid, two glycosaminoglycans present in the OF. In conclusion, the OF significantly decreased the number of spermatozoa that bind to oviduct epithelial cells without affecting sperm motility and this effect was due to macromolecules, including heparan sulphate.
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Project description:As the time of ovulation draws near, mouse spermatozoa move out of the isthmic reservoir, which is a prerequisite for fertilization. However, the molecular mechanism remains unclear. The present study revealed that mouse cumulus cells of oocytes-cumulus complexes (OCCs) expressed transforming growth factor-β ligand 1 (TGFB1), whereas ampullary epithelial cells expressed the TGF-β receptors, TGFBR1 and TGFBR2, and all were upregulated by luteinizing hormone (LH)/human chorionic gonadotropin (hCG). OCCs and TGFB1 increased natriuretic peptide type C (NPPC) expression in cultured ampullae via TGF-β signaling, and NPPC treatment promoted spermatozoa moving out of the isthmic reservoir of the preovulatory oviducts. Deletion of Tgfb1 in cumulus cells and Tgfbr2 in ampullary epithelial cells blocked OCC-induced NPPC expression and spermatozoa moving out of the isthmic reservoir, resulting in compromised fertilization and fertility. Oocyte-derived paracrine factors were required for promoting cumulus cell expression of TGFB1. Therefore, oocyte-dependent and cumulus cell-derived TGFB1 promotes the expression of NPPC in oviductal ampulla, which is critical for sperm migration in the oviduct and subsequent fertilization.
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